EP2473853A1 - NOVEL TUMOR MARKERS SELECTED FROM A GROUP OF cAMP-REGULATORY PATHWAY MEMBERS - Google Patents

NOVEL TUMOR MARKERS SELECTED FROM A GROUP OF cAMP-REGULATORY PATHWAY MEMBERS

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Publication number
EP2473853A1
EP2473853A1 EP10760034A EP10760034A EP2473853A1 EP 2473853 A1 EP2473853 A1 EP 2473853A1 EP 10760034 A EP10760034 A EP 10760034A EP 10760034 A EP10760034 A EP 10760034A EP 2473853 A1 EP2473853 A1 EP 2473853A1
Authority
EP
European Patent Office
Prior art keywords
tumor
group
tumor marker
expression
neoplastic disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10760034A
Other languages
German (de)
French (fr)
Inventor
Ralf Hoffmann
Edwin P. Romijn
Hugo M. Visser
Tim Hulsen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Koninklijke Philips NV
Original Assignee
Koninklijke Philips Electronics NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Koninklijke Philips Electronics NV filed Critical Koninklijke Philips Electronics NV
Priority to EP10760034A priority Critical patent/EP2473853A1/en
Publication of EP2473853A1 publication Critical patent/EP2473853A1/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention relates to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises a tumor marker selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAP13), High affinity cGMP-specific 35-cyclic phosphodiesterase 9A
  • AKAP6 A-kinase anchor protein
  • the present invention further relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage comprising affinity ligands for the expression products of the tumor markers, to corresponding methods, and to the use of said tumor markers for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage.
  • the present invention further relates to a corresponding immunoassay, to a method of identifying an individual for eligibility for a neoplastic disease therapy, as well as to a pharmaceutical composition based on the inhibition and/or activation of the expression of said tumor markers.
  • Neoplasms are abnormal masses of tissue as a result of neoplasia, i.e. an abnormal proliferation of cells. The growth of such clones of cells exceeds, and is
  • Neoplasms or neoplastic swellings usually cause lumps or tumors.
  • a neoplasm can be benign, potentially malignant (constituting a pre-cancer condition), or malignant (constituting a cancer condition or indication a cancer disease).
  • benign neoplasms include uterine fibroids and melanocytic nevi, which normally do not transform into cancer.
  • Potentially malignant neoplasms may include carcinoma in situ, which may not invade and destroy tissue, but can, given enough time, transform into a cancer.
  • the third type of neoplasms is a malignant neoplasm, i.e.
  • cancers in which a group of cells display uncontrolled growth, leading to the invasion of tissue and sometimes to metastasis.
  • These three malignant properties of cancers differentiate them from benign tumors and potentially malignant neoplasms, which are self- limited and do not invade or metastasize.
  • prostate cancer is the most common malignancy in European males.
  • an estimated 225,000 men were newly diagnosed with prostate cancer and about 83,000 died from this disease.
  • neoplastic diseases in particular with cancer development.
  • phosphodiesterase PDE7 has been shown to be linked to chronic lymphocytic leukemia.
  • early developmental stages e.g. between benign tumors, premalignant states and early malignant states of a neoplastic disease
  • marker molecule available for early developmental stages, e.g. between benign tumors, premalignant states and early malignant states of a neoplastic disease.
  • maspin whose up-regulation is believed to precede the transition from premalignant prostate lesions to prostate cancer.
  • PSA prostate-specific antigen
  • the present invention addresses this need and provides means and methods which allow the diagnosis and detection of a neoplastic disease, in particular neoplastic diseases of the prostate.
  • cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP- dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAP13), High affinity cGMP-specific 35- cyclic phosphodiesterase 9A (PDE9A), cAMP-dependent protein kinase type I-beta regulatory subunit (PRKAR1B), and cAMP-specific 35-cyclic phosphodiesterase 4C
  • AKAP6 A-kinase anchor protein 6
  • CNBG3 cyclic nucleotide-gated cation channel beta-3
  • KGP2 cGMP- dependent protein kinase 2
  • AKAPl A-kinase anchor protein 1
  • novel tumor markers were significantly down-regulated or up-regulated when comparing samples from patients having a less progressed stage of a neoplastic disease to samples from patients having a more progressed stage of the disease. Therefore, these markers are considered as markers for the prediction of neoplastic disease, in particular for the prediction of early cancer forms as well as for transitions into or between malignant cancer forms.
  • tumor markers may further serve as a decision tool for the stratification of certain neoplasm or cancer surveillance regimes, as well as for the prognosis and monitoring of neoplasm or cancer progression. Diagnostic methods and uses based on the tumor markers of the present invention can thus advantageously be employed for (i) detecting and diagnosing neoplastic disease forms, in particular forms of neoplastic diseases of prostate cancer, (ii)
  • prognosticating neoplastic disease forms in particular neoplastic diseases of the prostate
  • monitoring of neoplastic disease progression towards more progressed disease stages e.g. early malignant cancer forms, in particular of prostate cancer
  • distinguishing between less and more progressed forms of neoplastic diseases in particular less and more progressed forms of neoplastic diseases of the prostate.
  • pharmaceutical compositions based on these tumor markers will provide novel therapeutic avenues in the treatment of cancer, in particular prostate cancer.
  • the present invention thus relates in a first aspect to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8 or all tumor markers selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta- 3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1
  • AKAP6 A-kinase anchor protein 6
  • CNBG3 cyclic nucleotide-gated cation channel beta- 3
  • KGP2 cGMP-
  • AKAPl A-kinase anchor protein 9
  • AKAPl 3 A-kinase anchor protein 13
  • PDE9A High affinity cGMP-specific 35-cyclic phosphodiesterase 9A
  • PRKARIB cAMP-dependent protein kinase type I-beta regulatory subunit
  • PDE4C phosphodiesterase 4C
  • the group of tumor markers consists of AKAP6 and/or CNGB3 and/or KGP2 and/or AKAPl and/or AKAP9 and/or AKAPl 3 and/or PDE9A and/or PRKARIB and/or PDE4C.
  • the expression of the marker(s) is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section P), R), T) or V) of Table 1.
  • the expression of the marker(s) is decreased (down-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section Q), S), U) or W) of Table 1.
  • the p-value of the expression modification is 0.2 or lower, as indicated in section L) or M) of Table 1.
  • the present invention relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand for the expression product(s) or protein(s) of a tumor marker or a group of tumor markers as defined above.
  • the present invention relates to a method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the step of determining the level of a tumor marker or a group of tumor markers as defined above, in a sample.
  • the determining step of said method is accomplished by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of the tumor marker or group of tumor markers as defined above.
  • said method comprises the additional step of comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease.
  • said method is a method of graduating cancer, comprising the steps of:
  • control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease
  • step (c) deciding on the stage or developmental status of a neoplastic disease based on the results obtained in step (b).
  • the present invention relates to the use of said tumor marker or a group of tumor markers as defined above as a marker for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease.
  • the present invention relates to an immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or for detecting, diagnosing, monitoring or prognosticating the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the steps of (a) testing in a sample obtained from an individual for the expression of a tumor marker or a group of tumor markers as defined above;
  • step (d) deciding on the presence, stage or risk of recurrence of a neoplastic disease or the progression of the neoplastic based on the results obtained in step (c), wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or group of tumor markers as defined above.
  • the present invention relates to a method of identifying an individual for eligibility for a neoplastic disease therapy comprising:
  • step (c) classifying the levels of expression of step (a) relative to levels of step
  • the present invention relates to a method of identifying an individual for eligibility for a neoplastic disease therapy comprising:
  • step (c) classifying the levels of expression of step (a) relative to levels of step
  • the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:
  • step (c) determining the difference in expression of said up-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said up-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b);
  • step (d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an increased level of expression of an up-regulated tumor marker or group of tumor markers as defined above.
  • the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:
  • step (c) determining the difference in expression of said down-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said down-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b);
  • step (d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an decreased level of expression of an down-regulated tumor marker or group of tumor markers as defined above.
  • said immunoassay or said method as mentioned above comprises the additional step of determining the level of prostate specific antigen (PSA).
  • PSA prostate specific antigen
  • the present invention relates to a pharmaceutical composition for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of an up-regulated tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of:
  • the present invention relates to a pharmaceutical composition for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the decreased (down-regulated) expression of adown-regulated tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of:
  • the present invention relates to a vaccine for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of a tumor marker selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAPl 3), High affinity cGMP-specific 35-cyclic
  • AKAP6 A-kinase anchor protein 6
  • CNBG3 cyclic nucleotide-gated cation channel beta-3
  • PDE9A phosphodiesterase 9A
  • PPvKAPvlB cAMP-dependent protein kinase type I-beta regulatory subunit
  • PDE4C cAMP-specific 35-cyclic phosphodiesterase 4C
  • Table 3 comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 3, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) of Table 3, or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) of Table 3, or any fragment thereof.
  • the present invention relates to the use of a tumor marker or a group of tumo markers as defined above for identifying pharmaceutically active agents useful in the treatment or prevention of a neoplastic disease.
  • the present invention relates to the use of a tumor marker or a group of tumo markers as defined above for identifying pharmaceutically active agents useful in the treatment or prevention of prostate cancer.
  • An especially preferred embodiment of the present invention relates to the above mentioned tumor marker or group of tumor markers, the above mentioned
  • said less progressed stage of the neoplastic disease as mentioned in the context of the above defined tumor marker or group of tumor markers, the above defined compositions, the above defined methods, the above defined uses, the above defined immunoassays, the above defined pharmaceutical compositions or the above mentioned vaccine is a healthy prostate tissue, and said more progressed stage is a benign prostate tumor.
  • said less progressed stage of the neoplastic disease as mentioned in the context of the above defined tumor marker or group of tumor markers, the above defined compositions, the above defined methods, the above defined uses, the above defined immunoassays, the above defined pharmaceutical compositions or the above mentioned vaccine is a benign prostate tumor, and said more progressed stage of the neoplastic disease is a prostate cancer of stage ⁇ T2 (UICC 2002 classification).
  • said less progressed stage of the neoplastic disease as mentioned in the context of the above defined tumor marker or group of tumor markers, the above defined compositions, the above defined methods, the above defined uses, the above defined immunoassays, the above defined pharmaceutical compositions or the above mentioned vaccine is a ⁇ T2 (UICC 2002 classification) prostate cancer with a Gleason score ⁇ 6, and said more progressed stage of the neoplastic disease is a > T2 (UICC 2002 classification) prostate cancer and/or a prostate cancer with a Gleason score > 7.
  • said less progressed stage of the neoplastic disease as mentioned in the context of the above defined tumor marker or group of tumor markers, the above defined compositions, the above defined methods, the above defined uses, the above defined immunoassays, the above defined pharmaceutical compositions or the above mentioned vaccine is a >T2 (UICC 2002 classification) prostate cancer
  • said more progressed stage of the neoplastic disease is a > T3 (UICC 2002 classification) prostate cancer with serum PSA ⁇ 10 ng/ml.
  • Fig. 1 depicts a list of clinical samples used for the experiments described the present application.
  • Fig. 2 shows the statistical distribution of sample collection dates (A) and age distribution (B) of clinical samples.
  • Fig. 3 indicates the statistical distribution of PSA values of the clinical
  • Fig. 4 indicates the statistical distribution of prostate volumes (A) or total size of cancer tissue in the biopsy core (B) of the clinical samples.
  • Fig. 5 shows the statistical distribution of total size of non-cancerous tissue in the biopsy core (A) or primary Gleason score groups 3 and 4 (B) and secondary Gleason score groups 3 and 4 (C) of the clinical samples.
  • Fig. 6 shows a corresponding Bradford standard curve using bovine serum albumin (BSA).
  • Fig. 7 shows an exemplary strong cation exchange chromatography (SCX)- Chromatogram of sample clean-up to remove unbound iTRAQ-labels.
  • Fig. 8 shows an exemplary chromatogram of a normal RPLC clean-up
  • Fig. 9 depicts an exemplary chromatogram of a high pH RPLC for final separation of peptides.
  • tumor markers as depicted in Table 1 are differentially expressed, depending on the stage of progression of a neoplastic disease when testing samples of patients.
  • tumor markers are statistically significant down-regulated or up-regulated when comparing samples from a less progressed stage to a more progressed stage of a neoplastic disease.
  • the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates a deviation from the indicated numerical value of ⁇ 20 %, preferably ⁇ 15 %, more preferably ⁇ 10 %, and even more preferably ⁇ 5 %.
  • the term “comprising” is not limiting.
  • the term “consisting of is considered to be a preferred embodiment of the term “comprising of .
  • a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.
  • first”, “second”, “third” or “(a)”, “(b)”, “(c)”, “(d)” etc. relate to steps of a method or use there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.
  • a first aspect of the present invention pertains to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8 or all tumor markers selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAP13), High affinity cGMP-specific 35-cyclic
  • AKAP6
  • NB: a2 healthy tissue
  • b2 benign prostate neoplastic disease or benign prostate tumor
  • cl local prostate cancer, insignificant disease corresponding to a clinical tumor stage ⁇ T2 and Gleason Score ⁇ 6 and Prostate Cancer Volume ⁇ 0.5 ml
  • d2 advanced prostate cancer disease, with a level of PSA >10 ng/ml, corresponding to a clinical tumor stage > T3 (UICC 2002 classification)
  • marker or "tumor marker”, as used herein, relates to a gene, genetic unit or sequence (a nucleotide sequence or amino acid or protein sequence) as defined in Table 1, whose expression level is modified, i.e. decreased or increased, in a neoplastic cell, or in a neoplastic tissue, in particular a cancerous cell or cancerous tissue, or in any type of sample comprising neoplastic, in particular cancerous cells or neoplastic, in particular cancerous tissues or portions or fragments thereof, in comparison to a control level or state.
  • the term also refers to any expression product of said genetic unit or sequence or variants or fragments thereof, as well as homologues or derivatives thereof.
  • the term specifically refers to the genes, genetic units, sequences, proteins, protein sequences, homologues and/or derivatives thereof indicated as marker #1 to #9 in Table 1, having the nucleotide or amino acid sequences of SEQ ID NOs: 1 to 18, respectively, wherein the uneven numbers indicate the nucleotide sequences and the following even numbers the corresponding amino acid sequences.
  • the term also comprises corresponding genomic sequences as indicated in section F) of Table 1.
  • the term additionally comprises any known or yet unknown isoform (either as mRNA molecule or transcript or in the form of a polypeptide or protein), splice variant or corresponding derivative which can be derived from said genomic sequence.
  • isoforms or splice variants would either be known by the person skilled in the art or could be retrieved with the help of suitable techniques, software tools etc. from databases or information depositories.
  • the presence, size, form and/or identity of isoforms or splice variants may additionally be detected, determined and/or calculated with suitable tools known to the person skilled in the art.
  • intron and/or exon sequences and/or boundaries within said genomic sequences would also be known to the person skilled in the art. It is envisaged by the present invention that correspondingly identified intron and exon boundaries may be respected or used during the course of marker detection etc. as described herein.
  • a “marker” or “tumor marker” according to the present invention may also comprises nucleotide sequences showing a high degree of homology to a marker molecule as indicated in Table 1, in particular to the nucleotide sequence indicated in section D) of Table 1. Furthermore, a “marker” or “tumor marker” according to the present invention may comprise amino acid sequences or protein sequences showing a high degree of homology to a marker molecule as indicated in Table 1, in particular to the amino acid sequence indicated in section E) of Table 1.
  • Nucleic acid sequences according to the present invention may be, for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the sequence as set forth in section D) of Table 1
  • amino acid sequences may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section E) of Table 1
  • nucleic acid sequences encoding amino acid sequences may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%), 96%o, 97%), 98%o or 99%> identical to the sequence as set forth in section E) of Table
  • amino acid sequences may be encoded by nucleic acid sequences being at least 75%>, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 9
  • tumor marker gene or “marker gene” as used herein thus relates to the gene encoding the tumor marker mentioned in Table 1.
  • the term relates to a gene expressing a tumor marker protein as indicated in Table 1, e.g. specific exon
  • the term also relates to DNA molecules derived from mRNA transcripts encoding a tumor marker as indicated in Table 1 , preferably cDNA molecules.
  • a “gene”, “genetic unit” or a “nucleotide sequence” is a nucleic acid sequence which may be transcribed under certain physiological or biochemical conditions.
  • the transcribed nucleic acid may further (but must not necessarily) be translated under certain physiological or biochemical conditions into a polypeptide, e.g. when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence may be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • polypeptide are used herein to designate a produced or naturally occurring polypeptide or a recombinant polypeptide corresponding to the tumor marker as mentioned in Table 1.
  • the term "protein” according to the present invention is to be seen as being interchangeably with the term "polypeptide”.
  • the polypeptides or proteins may be encoded by any of the above mentioned nucleic acid molecules.
  • the polypeptides or proteins may further be glycosylated or may be non-glycosylated or may otherwise by modified.
  • polypeptides or proteins may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • neoplastic refers to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, as well as any of a number of characteristic structural and/or molecular features.
  • a “neoplastic cell” is accordingly understood as a cell having specific structural properties, lacking differentiation and in many instances, being capable of invasion and metastasis.
  • neoplastic neoplastic disease
  • neoplastic disease state etc.
  • neoplastic further also relates in the context of the present invention to a neoplastic disease state as defined herein below.
  • non-neoplastic relates in the context of the present invention to a condition in which neither benign nor malign proliferation can be detected. Suitable means for said detection are known in the art.
  • progression of a neoplastic disease relates to a switch between different stages of development or genesis of a neoplastic disease and principally refers to a situation in which the neoplastic disease becomes worse and/or spreads in the body. Explicitly encompassed in this term is also the transition of healthy tissue and/or cells into a benign tumor, the transition of a benign tumor into a malignant
  • lesion/growth/hyperplasia or cancer due to uncontrolled, abnormal proliferation of cells and featuring any of a number of characteristic structural and/or molecular features, as well as the transition between stages or forms of malignant tumors or cancers, e.g. of prostate cancer.
  • any changes that are associated with a worsening of the disease i.e. be it the nature of the transformation (healthy to benign tumor/lesion/growth/hyperplasia, benign tumor/lesion/growth/hyperplasia to malignant tumor or cancer), aggressiveness of the cancer, the localization of the tumor and or /cancerous cells, the occurrence of metastases, presentation of additional or more profound clinical symptoms, recurrence of a tumor after treatment, decreased survival rate, are typically translated into a progression of stages of a given neoplastic disease.
  • any worsening of the disease can thus be translated into a switch into the next stage of a given neoplastic disease.
  • Any worsening of a neoplastic disease beyond the stage of a benign tumor, a benign lesion, benign growth, or hyperplasia is understood as a "cancer".
  • Different classifications exist for such switches, e.g. stages 0 and I to IV of the TNM classification, preferably the TNM classification system for prostate cancer as defined herein below, or any other stage or sub-stage of any suitable graduating or scoring system, starting from a healthy condition up to a terminal cancer scenario.
  • switches are accompanied by a modification of the expression level of the tumor marker or group of tumor markers according to Table 1 , preferably an increase or decrease of the expression level in a test sample in comparison to a previous test sample from the same individual, a test sample from an individual having been diagnosed with a certain type of a neoplastic disease as defined herein above and/or a stage or state of a neoplastic disease as defined herein above, or a value derivable from an information depository on expression data etc.
  • Gleason score Progression of a neoplastic disease as defined herein above may further be determined, checked, crosschecked or independently be diagnosed etc. according to the "Gleason score".
  • Gleason score typically a grade is assigned to the most common tumor pattern, and a second grade to the next most common tumor pattern. The two grades are added together to get a Gleason score.
  • the Gleason grade is also known as the
  • the Gleason pattern or Gleason sum may range from 1 to 5, with 5 having the worst prognosis.
  • the Gleason score typically ranges from 2 to 10, with 10 having the worst prognosis.
  • Methods to apply the Gleason score system, corresponding assessment techniques etc. would be known to the person skilled in the art.
  • the scoring system may be used, for example, in order to verify, check or fine-tune the diagnosis, detection, indication of stages or monitoring according to the present invention.
  • the term "less progressed stage” as used herein, relates to an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, is/are known or from the same subject at an earlier point in time, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 2 years, 3 years, 4 years, 5 years, 10 years before etc. ("expression level of a less progressed stage”).
  • the term may also refer to an expression level corresponding to the stage of a neoplastic disease as defined herein above or a form of a neoplastic disease, whose disease state or stage is known.
  • disease state relates to any state or type of cellular or molecular condition between a non-neoplastic or noncancerous cell state and/or healthy condition on the one hand and a terminal cancerous cell state on the other hand.
  • the term thus, includes benign tumor forms as well as malignant tumor forms.
  • the term includes different early proliferation/developmental stages or levels of tumor development in the organism between a non-cancerous cell state and (including) an advanced malignant cancerous cell state.
  • the above mentioned stages may include all stages of the histological grading as per the guidelines of the American Joint Commission on Cancer. As per their standards, one grading possibility is:
  • Such developmental stages may also include all stages of the TNM (Tumor, Node, Metastasis) classification system of malignant tumors as defined by the UICC, e.g. stages 0 and I to IV.
  • the stages may in particular include all TNM stages of prostate cancer as defined herein below.
  • the term also includes stages before TNM stage 0, e.g.
  • a less progressed stage may be a healthy state.
  • a less progressed sate may also be a stage of malignant tumor development, e.g. malignant prostate cancer stages.
  • Corresponding expression levels or information about the expression level(s) of (a) less progressed stage(s) may be derived, for example, from experimental approaches or from a database of expression patterns or expression levels from previously tested subjects, tissues or cells or from any suitable source of information known to the person skilled in the art.
  • the expression level of a less progressed stage can be determined from a reference sample derived from a subject who has been diagnosed to suffer from a certain cancer, and wherein the stage and development has been determined.
  • Correspondingly obtained values and information may also be combined, normalized and statistically processed according to any suitable technique or method known to the person skilled in the art.
  • more progressed stage relates to the reflection of any changes that are associated with a worsening of the disease as defined herein above, e.g. the aggressiveness of the cancer, the nature of the transformation (benign to malignant), the localization of the tumor and or /cancerous cells, the occurrence of metastases, the presentation of additional or more profound clinical symptoms, recurrence of a tumor after treatment, decreased survival rate, the modification of the expression of bio- or tumor markers, e.g. of tumor marker known to the person skilled in the art like PSAin comparison to a corresponding less progressed stage as defined herein above.
  • the aggressiveness of the cancer e.g. the aggressiveness of the cancer, the nature of the transformation (benign to malignant), the localization of the tumor and or /cancerous cells, the occurrence of metastases, the presentation of additional or more profound clinical symptoms, recurrence of a tumor after treatment, decreased survival rate, the modification of the expression of bio- or tumor markers, e.g. of
  • the term relates to a worsened disease state of a neoplastic disease as defined herein above, e.g. cancer or a malignant tumor in comparison to a less progressed stage as defined herein above.
  • a neoplastic disease as defined herein above, e.g. cancer or a malignant tumor
  • the more progressed stage may be any higher or more advanced stage, e.g. a stage of any one of stage I, II, III or IV.
  • a "more progressed stage" as used herein may be the next worse stage if starting from a less progressed stage as defined herein above.
  • the more progressed stage may be a stage I etc.
  • the "next worse stage” may be reflected by any of the known staging and/or grading systems known to the person skilled in the art.
  • the "next worse stage” refers to the staging system provided by the UICC 2002 classification, more preferably the TNM classification for prostate cancer provided herein below.
  • the staging or grading of a tissue may optionally or additionally be determined, checked, crosschecked or independently be diagnosed by classical staging methods known to the person skilled in the art, e.g. via histological approaches, imaging methods etc.
  • a more progressed stage may be determined by a comparison of the expression level of a tumor marker as indicated in Table 1 to a control level or control state of the same tumor marker.
  • control level (or "control state"), as used herein, relates to an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, e.g. cancerous, non-cancerous, having a tumor, having no tumor, and whose disease stage(s) as defined herein above is/are known.
  • control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of the gene(s) of the tumor marker or group of tumor markers according to Table 1 in samples from subjects whose disease state is known.
  • control level can be derived from a database of expression levels or patterns from previously tested subjects or cells.
  • control level may be multiple control levels whose control levels are determined from multiple reference samples. The control level may accordingly be derived from experimental approaches or from a database of expression levels from previously tested subjects, tissues or cells or from any suitable source of information known to the person skilled in the art.
  • control level can be determined from a reference sample derived from a subject who has been diagnosed to suffer from a certain neoplastic disease as defined herein above, preferably cancer, and wherein the stage and development of the neoplastic disease has been determined, as well as from healthy individuals.
  • control level determined from a biological sample that is known not to be neoplastic is called "normal control level".
  • the control level can be from a neoplastic biological sample, e.g. a sample from a subject for which the neoplastic disease as defined herein above, preferably cancer, or prostate cancer, was diagnosed independently, it may be designated as "neoplastic control level".
  • reference samples may comprise material derived from cell lines, e.g. immortalized cancer cell lines, or be derived from tissue xenografts.
  • material derived from prostate cancer cell lines or material derived from tissue xenografts with human prostate tissue, in particular with benign and tumor- derived human prostate tissue may be comprised in a reference sample according to the present invention.
  • cancer cell lines to be used comprise cells lines PC346P, PC346B, LNCaP, VCaP, DuCaP, PC346C, PC3, DU145, PC346CDD, PC346Flul,
  • PC346Flu2 examples of xenografts which may be used comprise PC295, PC310, PC-EW, PC82, PC133, PC135, PC324 and PC374. Preferably an entire panel of cell lines and xenografts may be used, e.g. the human PC346 panel.
  • values and information may also be combined, normalized and statistically processed according to any suitable technique or method known to the person skilled in the art.
  • a control level By comparing a control level to a measured expression level a modification of the expression may be registered, which may accordingly be used for the determination of the more progressed stage of a neoplastic disease as defined herein above.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more different control levels may be determined or assessed. Accordingly, the less progressed stage of a neoplastic disease as defined herein above may be determined by assessing the outcome of such comparison process.
  • said comparison process comprises the determination of control levels in a sample of an individual.
  • said comparison process comprises the determination of control levels in a sample of an individual afflicted with a neoplastic disease, e.g. cancer.
  • said comparison process comprises the determination of control levels obtained from a sample of an individual afflicted with a neoplastic disease, e.g. cancer, wherein the sample is representative of stages of the histological four-, three-, two-layer grading as per the guidelines of the American Joint Commission on Cancer, as defined herein above.
  • control levels determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state is/are known to be prostate tumors, e.g. benign prostate tumors, or prostate cancer, more preferably prostate cancer of stage ⁇ T2 (UICC 2002 classification) or of stage >T3 (UICC 2002 classification), or prostate cancer of stage >T3 (UICC 2002 classification) and showing a serum level of PSA of about ⁇ 10 ng/ml.
  • a control level derived from a healthy tissue or based on expression values from healthy samples or healthy patients e.g. patients who have independently been analyzed and were found to not suffer from any cancer or tumor, e.g. form prostate tumors or cancer.
  • expression level refers to the amount of any transcript and/or protein derivable from a defined number of cells or a defined tissue portion, preferably to the amount of a transcript and/or protein obtainable in a standard nucleic acid (e.g. RNA) or protein extraction procedure. Suitable extraction methods are known to the person skilled in the art.
  • modified or “modified expression level” in the context of the present invention thus denotes a change in the expression level.
  • Expression levels are deemed to be “changed” when the gene expression of the tumor marker or group of tumor markers according to Table 1, e.g.
  • a control level or the expression level of a less progressed stage differs by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level or the expression level of a less progressed stage, as defined herein above, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level or the expression level of a less progressed stage as defined herein above.
  • modified as used throughout the specification relates preferably to a decrease or down-regulation or an increase or up-regulation of the expression level of the tumor marker or group of tumor markers according to Table 1 or a complete inhibition of the expression of the tumor marker or group of tumor markers according to Table 1 if a test sample is compared to a control level or the expression level of a less progressed stage as defined herein above.
  • the expression of the tumor marker(s) or group of tumor markers is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in any of sections P), R), T) and/or V) of Table 1.
  • “increased expression level” or “up-regulated expression level” or “increase of expression level” in the context of the present invention thus denotes a raise in the expression level of the tumor marker or group of tumor markers according to Table 1 between a situation to be analyzed, e.g. a situation derivable from a patient's sample, and a reference point, which could either be a control level derivable from any suitable tumor, e.g. a prostate tumor, or neoplastic disease stage known to the person skilled in the art, e.g. a healthy state, a benign tumor stage or the expression of a less progressed stage as defined herein above.
  • a situation to be analyzed e.g. a situation derivable from a patient's sample
  • a reference point which could either be a control level derivable from any suitable tumor, e.g. a prostate tumor, or neoplastic disease stage known to the person skilled in the art, e.g. a healthy state, a benign tumor stage
  • an up-regulation may be present between a healthy tissue (e.g. indicated as pool a2 in Table 1) and a benign tumor tissue (e.g. indicated as pool b2 in Table 1).
  • a benign tumor tissue e.g.
  • an up-regulation may be present between a local cancer showing insignificant disease parameters (e.g. indicated as pool cl in Table 1) and a local cancer showing significant disease parameters (e.g. indicated as pool c2 in Table 1).
  • an up-regulation may be present between a local cancer showing significant disease parameters (e.g. indicated as pool c2 in Table 1) and an advanced cancer disease (e.g. indicated as pool dl in Table 1). Expression levels are deemed to be "increased" when the gene expression of the tumor marker or group of tumor markers according to Table 1, e.g.
  • a control level differs by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%), 40%), 50%), or more than 50%> from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level or the expression level of a less progressed stage as defined herein above.
  • the tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5 or all tumor markers indicated with a "+” in section P) of Table 1, at least 1, 2, 3, 4 or all tumor markers indicated with a "+” in section R) of Table 1, at least 1, 2, 3 or all tumor markers indicated with a "+” in section T) of Table 1, at least 1, 2, 3, 4 or all tumor markers indicated with a "+” in section V) of Table 1.
  • the tumor marker or group of tumor markers comprises the first 1, 2, 3, 4, 5, 6 tumor markers indicated with a "+” in section P) of Table 1, the first 1, 2, 3, 4, 5 tumor markers indicated with a "+” in section R) of Table 1, the first 1, 2, 3, 4 tumor markers indicated with a "+” in section T) of Table 1, the first 1, 2, 3, 4, 5 tumor markers indicated with a "+” in section V) of Table 1.
  • the expression of the tumor marker(s) or group of tumor markers is decreased (down-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in any of sections Q), S), U) and/or W) of Table 1.
  • the term "reduced” or “reduced expression level” or “down-regulated expression level” or “decrease of expression level” thus denotes a reduction of the expression level of the tumor marker or group of tumor markers according to Table 1 between a situation to be analyzed, e.g.
  • a situation derivable from a patient's sample, and a reference point which could either be a control level derivable from any suitable prostate tumor or neoplastic disease stage or cancer stage known to the person skilled in the art, e.g. a healthy state, a benign tumor stage or the expression of a less progressed stage as defined herein above.
  • a decrease or down-regulation between continuous steps in cancer development e.g. next worse steps as mentioned herein above.
  • a down-regulation may be present between a healthy tissue (e.g. indicated as pool a2 in Table 1) and a benign tumor tissue (e.g. indicated as pool b2 in Table 1).
  • a down-regulation may be present between a benign tumor tissue (e.g.
  • a down-regulation may be present between a local cancer showing insignificant disease parameters (e.g. indicated as pool cl in Table 1) and a local cancer showing significant disease parameters (e.g. indicated as pool c2 in Table 1).
  • a down-regulation may be present between a local cancer showing significant disease parameters (e.g. indicated as pool c2 in Table 1) and an advanced cancer disease (e.g. indicated as pool dl in Table 1).
  • Expression levels are deemed to be “reduced” or “down- regulated” when the gene expression of the tumor marker or group of tumor markers according to Table 1 decreases by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level or the expression level of a less progressed stage as defined herein above.
  • the tumor marker or group of tumor markers comprises at least 1 , 2, or all tumor markers indicated with a "+” in section Q) of Table 1, at least 1, 2, 3 or all tumor markers indicated with a "+” in section S) of Table 1, at least 1, 2, 3, 4 or all tumor markers indicated with a "+” in section U) of Table 1, at least 1, 2, 3 or all tumor markers indicated with a "+” in section W) of Table 1.
  • the tumor marker or group of tumor markers comprises the first 1, 2, 3 tumor markers indicated with a "+” in section Q) of Table 1, the first 1, 2, 3, 4 tumor markers indicated with a "+” in section S) of Table 1, the first 1, 2, 3, 4, 5 tumor markers indicated with a "+” in section U) of Table 1, the first 1, 2, 3, 4 tumor markers indicated with a "+” in section W) of Table 1.
  • the group of tumor marker may comprise markers which are up-regulated and marker which are down-regulated, preferably at least one marker with an increased expression level in the more progressed stage and one marker with a decreased expression level in the more progressed stage.
  • the group of tumor marker may comprise at least one tumor marker indicated with a + in section P), R), T) or V) of Table 1 and at least one tumor marker indicated with a + in section Q), S), U) or W) of Table 1.
  • the tumor marker or group of tumor markers may comprises at least 1, 2, 3, 4, 5 or all tumor markers indicated with a "+” in section P) of Table 1 or at least 1, 2, 3, 4 or all tumor markers indicated with a "+” in section R) of Table 1 or at least 1, 2, 3 or all tumor markers indicated with a "+” in section T) of Table 1 or at least 1, 2, 3, 4 or all tumor markers indicated with a "+” in section V) of Table 1 and at least 1, 2, or all tumor markers indicated with a "+” in section Q) of Table 1, or at least 1, 2, 3 or all tumor markers indicated with a "+” in section S) of Table 1, or at least 1, 2, 3, 4 or all tumor markers indicated with a "+” in section U) of Table 1, or at least 1, 2, 3 or all tumor markers indicated with a "+” in section W) of Table 1 , wherein combinations of markers indicated with a "+” from sections P) and Q), R) and S), T) and U) as well as V) and W), respectively,
  • the group of tumor markers comprises or consists of AKAP6 and/or CNGB3 and/or KGP2 and/or AKAP1 and/or AKAP9 and/or AKAP13 and/or PDE9A and/or PRKAR1B and/or PDE4C, as indicated in Table 1.
  • the group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the tumor markers of Table 1.
  • the group may further comprise any sub-grouping or combinations of these markers.
  • the group of tumor markers comprises those tumor markers which show a p-value of the expression modification of 0.03, 0.04, 0.05, 0.06, 0,07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 016, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.3, 0.4, 0.5, 0.6 or lower as indicated in section K) of Table 1.
  • p-value is a measure of the probability that a variate would assume a value greater than or equal to the observed value strictly by chance and is expressed by the following term: P (z > z 0 bserved). Thus, in the context of the present invention, the p-value may be seen as a measure of statistical significance.
  • the group of tumor markers comprises least 2 tumor markers corresponding to tumor marker #1 to #2 of Table 1, to tumor marker #2 to #3 of Table 1, to tumor marker #3 to #4 of Table 1, to tumor marker #5 to #6 of Table 1, to tumor marker #6 to #7 of Table 1, to tumor marker #7 to #8 of Table 1 or to tumor marker #8 to #9 of Table 1, or at least 4 tumor markers corresponding to tumor marker #1 to #4 of Table 1, to tumor marker #2 to #5 of Table 1, to tumor marker #3 to #6 of Table 1, to tumor marker #4 to #7 of Table 1, to tumor marker #5 to #8 of Table 1 or to tumor marker #6 to #9 of Table 1.
  • the group of tumor marker according to the present invention may also comprise tumor markers #1 , #3, #5, #7 and #9 of Table 1 , #2, #4, #6, #8, and #9 of Table 1 , #2, #4, #7, #9 and #1 of Table 1, #1, #3, #4, #6 and #8 of Table 1, #5, #4, #9, and # 2 of Table 1, etc.
  • the present invention relates to the use of a tumor marker or group of tumor markers as defined herein above as a marker for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed of a neoplastic disease to a more progressed stage of a neoplastic disease, or prognosticating the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease.
  • diagnosing a cancer disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease means that a subject or individual may be considered to be suffering from a more progressed neoplastic disease as defined herein above, e.g. a more progressed prostate cancer, when the expression level of a tumor marker or the group of tumor markers of the present invention is modified, e.g. increased/up-regulated or reduced/down-regulated, compared to the expression level of a less progressed disease state as defined herein above, or compared to a control level as defined herein above.
  • An expression level may be deemed to be modified, when the expression level of a tumor marker or group of tumor markers as defined herein above differs by, for example, between about 1% and 50%, e.g. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% or 40% from the expression level of a less progressed disease state or from a control level as defined herein above, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to such a less progressed disease state or control level.
  • the modification may be an increase or a reduction of said expression level.
  • an additional similarity in the overall gene expression pattern of a group of tumor markers according to the present invention between a sample obtained from a subject and a control sample or a sample corresponding to a less progressed disease state of a neoplastic disease as defined herein above as described herein above may indicate that the subject is suffering from a more progressed disease stage of the neoplastic disease as defined herein above.
  • the diagnosis may be combined with the elucidation of additional cancer biomarker expression levels, in particular prostate cancer biomarkers. Suitable biomarkers, in particular prostate cancer biomarkers, would be known to the person skilled in the art. For example, the expression of biomarkers like PSA may be tested.
  • detecting a neoplastic disease associated with a progression from a less progressed stage to a more progressed stage means that the presence of a neoplastic disease or disorder as defined herein above in an organism, which is associated with a more progressed stage may be determined or that such a neoplastic disease or disorder as defined herein above may be identified in an organism.
  • the determination or identification of a more progressed neoplastic disease or disorder as defined herein above may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers of the present invention in a sample from a patient or individual to be analyzed and the expression level of a control level as defined herein above, wherein said control level corresponds to the expression level of said more progressed neoplastic disease or disorder as defined herein above.
  • a more progressed stage of a neoplastic disease as defined herein above may be detected if the expression level of the tumor marker or group of tumor markers is similar to an expression level of a more progressed stage of the neoplastic disease as defined herein above.
  • the expression level of the more progressed stage of the neoplastic disease as defined herein above may be independently established, e.g. from sample depositories, value databases etc. as mentioned herein above.
  • grade of a neoplastic disease associated with a progression from a less progressed stage to a more progressed stage means that the clinical stage, phase, grade or any other suitable sub-step of features related to the neoplastic disease as defined herein above, such as the progression defined herein above, the transition from a benign to a malignant tumor, the grade of malignancy, the grade of tissue damage to noncancerous tissue, the grade of the extent of tumor growth, the grade of aggressiveness of a tumor, the grade of metastasizing and all other useful and suitable parameters of a cancerous disease or disorder in an organism may be determined in an organism.
  • the graduating of a more progressed stage of the neoplastic disease as defined herein above may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1 of the present invention in a sample from a patient or individual to be analyzed and a control level as defined herein above, or the expression level of a less progressed stage as defined herein above of the neoplastic disease as defined herein above.
  • the graduating of a more progressed stage may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1 of the present invention in a sample from a patient or individual to be analyzed and any of the cancer cells, cancer tissues, tumor biopsies, or the cancerous control levels mentioned above.
  • the graduating of a more progressed stage of the neoplastic disease as defined herein above may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1 of the present invention in a sample from a patient or individual to be analyzed and the above mentioned cancer cells, cancer tissues, tumor biopsies, or the neoplastic control levels being derived from or representative for a less progressed stage of the neoplastic disease as defined herein above.
  • the graduating of a more progressed stage of the neoplastic disease as defined herein above, preferably cancer may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1 of the present invention in a sample from a patient or individual to be analyzed and the above mentioned cancer cells, cancer tissues, tumor biopsies, or the neoplastic control levels being derived from or representative for a more progressed stage of the neoplastic disease as defined herein above, preferably cancer.
  • accompaniment of a diagnosed or detected, more progressed neoplastic disease or disorder as defined herein above e.g. during a treatment procedure or during a certain period of time, typically during 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other period of time.
  • accompaniment means that states of disease as defined herein above and, in particular, changes of these states of disease may be detected by comparing the expression level of the tumor marker or group of tumor markers of the present invention in a sample to a control level as defined herein above or to the expression level of an established, e.g.
  • the established, e.g. independently established, cancer cell or cell line giving rise to a control level may be derived from samples corresponding to different stages of neoplastic development, e.g. the stages as mentioned herein above.
  • the term relates to the accompaniment of a diagnosed more progressed prostate cancer.
  • prognosticating a neoplastic disease associated with a progression form a less progressed stage to a more progressed stage refers to the prediction of the course or outcome of a diagnosed or detected more progressed stage of the neoplastic disease as defined herein above, e.g. during a certain period of time, during a treatment or after a treatment.
  • the term also refers to a determination of chance of survival or recovery from the disease, as well as to a prediction of the expected survival time of a subject.
  • a prognosis may, specifically, involve establishing the likelihood for survival of a subject during a period of time into the future, such as 6 months, 1 year, 2 years, 3 years, 5 years, 10 years or any other period of time.
  • progression from a less progressed stage to a more progressed stage relates to a switch between different stages of the development or genesis of a neoplastic disease, as defined herein above.
  • Such a progression may be a development in small steps, e.g. from a certain stage to the next, or may alternatively be a development skipping one or more such steps, e.g. from stage I to stage III of the TNM classification.
  • a progression from a less progressed stage to a more progressed stage may be considered as being detected or diagnosed if the expression level of a tumor marker or group of tumor marker according to the present invention is modified, e.g.
  • the modification may be detected over any period of time, preferably over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years, i.e. the value indicated above may be calculated by comparing the expression level of the tumor marker or group of tumor markers at a first point in time and at a second point in time after the above indicated period of time.
  • progression from a less progressed stage to a more progressed stage relates to a switch from a benign prostate tumor state to a malignant prostate cancer state, as defined herein.
  • test samples from other individuals may be used, e.g. test samples of healthy individuals.
  • test samples of healthy individuals e.g. test samples of healthy individuals.
  • the present invention relates to the diagnosis and detection of a predisposition for developing a more progressed stage of the neoplastic disease as defined herein above.
  • a "predisposition for developing a more progressed stage of the neoplastic disease as defined herein above, preferably cancer" in the context of the present invention is a state of risk of developing a more progressed stage neoplastic disease as defined herein above.
  • a predisposition for developing a more progressed stage may be present in cases in which the expression level of the tumor marker or group of tumor marker of the present invention as defined herein above is above a normal control level as defined herein above, i.e.
  • a reference expression level derived from tissues or samples of a subject which are evidently healthy derived from tissues or samples of a subject which are evidently healthy.
  • the term "above” in this context relates to an expression level of the tumor marker of group of tumor markers which is increased by about 2% to 20% in comparison to such a control level, preferably increased by about 15%.
  • a predisposition for developing a more progressed stage of the neoplastic disease as defined herein above, e.g. cancer in the context of the present invention may be given in situations in which the expression level of the tumor marker or group of tumor markers as defined herein above is above a normal control level and in which further, alternative cancer markers, e.g. PSA, show no modification of expression level or the expression pattern in a less progressed stage of the neoplastic disease as defined herein above. Suitable further cancer markers are known to the person skilled in the art.
  • a predisposition for a more progressed stage of the neoplastic disease as defined herein above, e.g. cancer may be considered as being diagnosed or detected if one of the above depicted situations is observed.
  • a group of tumor markers according to the present invention comprises tumor markers of Table 1 which show an increase of the expression level and, at the same time, other tumor markers of Table 1 which show a reduction of the expression level, the increase and the reduction of the corresponding tumor markers may be calculated
  • control nucleic acids e.g. housekeeping genes whose expression levels are known not to differ depending on the neoplastic or non-neoplastic state of the cell.
  • control genes include inter alia ⁇ -actin, glycerinaldehyde 3-phosphate dehydrogenase (GAPDH), 18s R A, ubiquitin C, cytochrome CI, ribosomal protein PI or PBGD. Particularly preferred is the use of GAPDH.
  • diagnosis and
  • tumor markers or groups of tumor markers may accordingly be used clinically in making decisions concerning treatment modalities, including therapeutic intervention or diagnostic criteria such as a surveillance for the disease.
  • an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease.
  • the present invention may be used to detect neoplastic cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.
  • a subject or individual to be diagnosed, monitored or in which a more progressed neoplastic disease as defined herein above, a progression towards such a neoplastic disease as defined herein above, or a predisposition of such a neoplastic disease as defined herein above, is to be detected or prognosticated according to the present invention is an animal, preferably a mammal, more preferably a human being. Also preferred is the use of molecular imaging tools as known to the person skilled in the art, e.g.
  • MRI magnetic resonance imaging
  • MPI magnetic photon resonance imaging
  • a tumor marker or group of tumor markers according to the present invention may be used as a marker for diagnosing, detecting, monitoring or prognosticating pre-malignant growths, swellings and lesions, malignant tumors, malignant cancers, metastases and the like or the progression towards more progressed states of such neoplastic disease and disorders as defined above, for example malignant, hormone- sensitive prostate cancer and/or the progression towards more progressed cancer states, in approaches like MRI or MPI that allow for online detection of the diagnostic marker within a human subject.
  • the present invention relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand for the expression product(s) or protein(s) of said tumor marker or a group of tumor markers as defined above.
  • nucleic acid affinity ligand for the expression product of a tumor marker or group of tumor markers refers to a nucleic acid molecule being able to specifically bind to a transcript or a DNA molecule derived the nucleic acid molecules of said tumor marker or a group of tumor markers as defined above, preferably to the nucleotide sequences, (DNA sequence(s)) depicted in section D) of Table 1 or to the complementary nucleotide sequences (DNA sequence(s)) of the sequence(s) depicted in section D) of Table 1 or a corresponding RNA molecule.
  • the nucleic acid affinity ligand may also be able to specifically bind to a DNA sequence being at least 75%, 80%>, 85%, 90%>, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section D) of Table 1 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section E) of Table 1 or to any fragments of said sequences.
  • peptide affinity ligand for the protein of a tumor marker or a group of tumor markers refers to a peptide molecule being able to specifically bind to the proteins of the tumor marker or group of tumor markers according to Table 1.
  • the peptide molecule may preferably be able to specifically bind to a protein or polypeptide comprising the amino acid sequence(s) as set forth in section E) of Table 1.
  • the peptide affinity ligand may also be able to specifically bind to a protein or polypeptide comprising an amino acid sequence encoded by a DNA sequence being at least 75%, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section E) of Table 1 or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section E) of Table 1 or to any fragments of said sequences.
  • peptide in the context of the affinity ligand of the present invention refers to any type of amino acid sequence comprising more than 2 amino acids, e.g.
  • polypeptide structures polypeptide structures, protein structures or functional derivatives thereof. Furthermore, the peptide may be combined with further chemical moieties or functionalities.
  • expression product refers to a transcript or an mR A molecule of the tumor marker or group of tumor markers according to Table 1 generated by the expression of the corresponding genomic sequence according to Table 1. More preferably, the term relates to a processed transcript of the tumor marker or group of tumor markers according to Table 1 as defined herein above, e.g. via the sequence(s) as set forth in sections D) and/or F) of Table 1. A person skilled in the art would know how to determine the identity, size, length and any other useful parameter of transcripts of tumor markers according to the present invention based on the information provided in said section F) of Table 1.
  • protein of a tumor marker or group of tumor markers also refers to any polypeptide, protein, in particular to the polypeptides or proteins as set forth in section E) of Table 1 or any domain, epitope, oligopeptide, or peptide derivable therefrom.
  • the composition of the present invention may comprise one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid and/or peptide affinity ligands selected from the group consisting of a set of oligonucleotides specific for the expression product of the tumor marker or group of tumor markers according to Table 1 , a probe specific for the expression product of the tumor marker or group of tumor markers according to Table 1 , an aptamer specific for the expression product or for the protein of the tumor marker or group of tumor markers according to Table 1, an antibody specific for the protein the tumor marker or group of tumor markers according to Table 1 and an antibody variant specific for the protein of the tumor marker or group of tumor markers according to Table 1.
  • nucleic acid and/or peptide affinity ligands selected from the group consisting of a set of oligonucleotides specific for the expression product of the tumor marker or group of tumor markers according to Table 1 , a probe specific for the expression product of the tumor marker or group
  • composition of the present invention may, for example, comprise a set of oligonucleotides specific for the expression product the tumor marker or group of tumor markers according to Table 1 and/or a probe specific for the expression product of the tumor marker or group of tumor markers according to Table 1.
  • oligonucleotide specific for the expression product the tumor marker or group of tumor markers according to Table 1 refers to a nucleotide sequence which is complementary to the sense- or antisense-strand of the tumor marker or group of tumor markers according to Table 1.
  • the oligonucleotide is
  • the oligonucleotide sequence may also be complementary to a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section D) of Table 1 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section E) of Table 1.
  • the oligonucleotide may have any suitable length and sequence known to the person skilled in the art, as derivable from the sequence(s) shown in section D) of Table 1 or its complement.
  • the oligonucleotide may have a length of between 8 and 60 nucleotides, preferably of between 10 and 35 nucleotides, more preferably a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides.
  • Oligonucleotide sequences specific for the expression product of the tumor marker or group of tumor markers according to Table 1 may be defined with the help of software tools known to the person skilled in the art.
  • oligonucleotide sequences may be complementary to genomic sequences localized in (an) exon(s) of the gene(s) encoding for the tumor marker or group of tumor markers according to Table 1.
  • oligonucleotide usable as a forward primer may be localized at the boundary between exonic and intronic sequences. Such boundary positions may be determined with the help of any suitable tool, based on the information provided in section F) of Table 1.
  • probe specific for the expression product of the tumor marker or group of tumor markers according to Table 1 means a nucleotide sequence which is complementary to the sense- or antisense-strand of the tumor marker or group of tumor markers according to Table 1.
  • the probe is complementary to the DNA sequence(s) depicted in section D) of Table 1 or to the complementary DNA sequence of the sequence(s) shown in section D) of Table 1.
  • the probe sequence may also be complementary to a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o or 99% identical to the sequence(s) as set forth in the sequences indicated in section D) of Table 1 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as shown in section E) of Table 1.
  • the probe may have any suitable length and sequence known to the person skilled in the, as derivable from the sequence(s) shown in section D) or its/their complement.
  • the probe may have a length of between 6 and 300 nucleotides, preferably of between 15 and 60 nucleotides, more preferably a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.
  • Probe sequences specific for the expression product of the tumor marker or group of tumor markers according to Table 1 may be defined with the help of software tools known to the person skilled in the art.
  • the composition of the present invention may additionally or alternatively comprise an aptamer specific for the expression product or protein of the tumor marker or group of tumor markers according to Table 1.
  • aptamer specific for the expression product of the tumor marker or group of tumor markers according to Table 1 refers to a short nucleic acid molecule, e.g. RNA, DNA, PNA, CNA, HNA, LNA or ANA or any other suitable nucleic acid format known to the person skilled in the art, being capable of specifically binding to the tumor marker or group of tumor markers according to Table 1, preferably the DNA molecule with (a) sequence(s) as shown in section D) of Table 1. More preferably, the nucleic acid aptamer molecule may specifically bind to a DNA sequence(s) shown in section D) of Table 1 or a double stranded derivative thereof.
  • the nucleic acid aptamer according to the present invention may also bind to an RNA molecule corresponding to the transcript(s) of the tumor marker or group of tumor markers according to Table 1, preferably an RNA molecule corresponding to the DNA sequence(s) as shown in section D) of Table 1.
  • the nucleic acid aptamer may further be capable of specifically binding to a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as shown in section D) of Table 1 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the sequence(s) as shown in section D) of Table 1 or RNA molecules corresponding to these sequences.
  • a nucleic acid aptamer according to the present invention may further be combined with additional moieties, e.g. with interacting portions like biotin or enzymatic functionalities like ribozyme elements.
  • aptamer specific for the protein of the tumor marker or group of tumor markers according to Table 1 refers to (a) short peptide(s) capable of interacting and specifically binding the protein(s) of the tumor marker or group of tumor markers according to Table 1.
  • the peptide aptamer(s) may preferably be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (the) amino acid sequence(s) as shown in section E) of Table 1.
  • the peptide aptamer(s) may also be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (an) amino acid sequence(s) encoded by (a) DNA sequence(s) being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o or 99%) identical to the sequence(s) as shown in section D) of Table 1 or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%>, 85%, 90%>, 91%>, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in shown in section E) of Table 1.
  • (a) peptide aptamer(s) is/are a variable peptide loop, comprising for example 10 to 20 amino acids.
  • the peptide aptamer(s) may preferably be attached at one or both ends to a scaffold structure.
  • the scaffold structure may be any molecule, preferably a protein, which has good solubility properties. Suitable scaffold molecules would be known to the person skilled in the art.
  • a preferred scaffold molecule to be used in the context of the present invention is the bacterial protein thioredoxin-A.
  • the aptamer peptide loop may preferably be inserted within a reducing active site of the scaffold molecule.
  • staphylococcal protein A and domains thereof and derivatives of these domains, such as protein Z or lipocalins may be used as scaffold structures in the context of the present invention.
  • Peptide aptamers may be generated according to any suitable method known to the person skilled in the art, e.g. via yeast two-hybrid approaches.
  • the composition may comprise, or may additionally comprise, an antibody or a group of antibodies specific for the expression product or protein of a tumor marker or group of tumor markers as defined herein, e.g. specific for one or more of the protein(s) of the tumor marker according to Table 1.
  • an antibody which specifically binds to an expression product, protein or peptide comprising the amino acid sequence as indicated in section E) of Table 1 , or a part or fragment of said sequence.
  • such an antibody specifically binds to a protein or polypeptide having the amino acid shown in section E) of Table 1 , or any derivative, fragment etc. thereof as defined herein above.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i. e. molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any type (e. g., IgG, IgE, IgM, IgD, IgA and IgY), class (e. g., IgGl, IgG2, IgG3, lgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind.
  • Preferred epitopes according to the present invention are amino acids 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, 191-200, 201-210 etc. or any other specific stretch of amino acids of a protein of the tumor markers of the present invention, preferably of the sequence as shown in section E) of Table 1. Further comprised are all other suitable epitopes, which can be recognized, determined, described and subsequently be employed according to methods known to the person skilled in the art.
  • the term "specific for the tumor marker according to Table 1" as used herein refers to the immunospecific detection and binding of an antibody to an antigenic epitope as defined herein above.
  • the term “specifically binding” excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens, in particular with antigens comprising the same antigenic epitope detected by the present antibody.
  • antibodies of the invention include polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, Fab' fragments, fragments produced by a Fab expression library, F(ab')2, Fv, disulfide linked Fv, minibodies, diabodies, scFv, sc(Fv)2, whole
  • immunoglobulin molecules small modular immunopharmaceuticals binding-domain immunoglobulin fusion proteins, camelized antibodies, V HH containing antibodies, anti- idiotypic (anti-Id) antibodies (including, e. g., anti-Id antibodies to antibodies of the invention) and epitope-binding fragments of any of the above.
  • the antibodies are human antigen-binding antibody fragments of the present invention and include Fab, Fab' and F (ab')2, Fv, single-chain Fvs (scFv), sc(Fv)2, single-chain antibodies, disulfide- linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
  • the antibodies according to the invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine (e. g., mouse and rat), donkey, monkey, rabbit, goat, guinea pig, camel, horse, or chicken.
  • the antibodies according to the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
  • Antibodies of the present invention may also be described or specified in terms of their cross-reactivity.
  • the present invention relates to antibodies that do not bind any other analog, ortholog, or homo log of a polypeptide of the present invention.
  • antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are included in the present invention.
  • the present invention also envisages an antigen and/or an epitope which is detected, recognized or specifically bound by an antibody against a tumor marker protein as defined in Table 1.
  • antigens or epitopes may preferably be derived from a tumor marker or group of tumor marker sequences as depicted in Table 1.
  • the antigen or epitope sequences encompassed by the present invention may, for example, comprise, have, or alternatively consist of amino acids 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, 191- 200, 201-210 etc.
  • the antigen or epitope sequences encompassed by the present invention may, for example, comprise, have, or alternatively consist of amino acids 1-10, 2-11, 3-12, 4- 13, 5-14, 6-15, 7-16, 8-17, 9-18, 10-19, 11-20, 12-21, 13-22, 14-23, 15-24, 16-25, 17-26, 18- 27, 19-28, 20-29, 21-30, 22-31, 23-32, 24-33, 25-34, 26-35, 27-36, 28-37, 29-38, 30-39, 31- 40, 32-41, 33-42, 34-43, 35-44, 36-45, 37-46, 38-47, 39-48, 30-49, 41-50, 42-51, 43-52, 44- 53, 45-54, 46
  • the epitope or antigen according to the present invention may further comprise or consist of additional amino acid stretches or fragments of any length or derived from any position, e.g. N-terminus, C-terminus, middle, specific domain, specified exposed region etc. of the protein or expression product of the tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, preferably of the sequence as shown in section E) of Table 1.
  • epitopes or antigens comprised in or comprising fragments or stretches of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 270, 300, 350 or 400 amino acids of the protein or expression product of the tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, preferably of the sequence as shown in section E) of Table 1.
  • the epitope or antigen of the present invention may further be present on extracellular domains, intracellular domains, sterically exposed domains, surface regions, exposed surface regions, interaction domains etc. of any of the tumor marker or group of tumor markers as defined herein, e.g. as derivable from Table 1, preferably of the sequence as shown in section E) of Table 1.
  • Such domains or regions and their position as well as their amino acid composition would be known to the person skilled in the art or could be deduced with known software or database tools, in particular in view of the information provided in sections E) of Table 1.
  • the person skilled in the art may, for example, use three dimensional modeling tools or structure information associated with the Entre Gene ID in order to identify such domains or regions.
  • epitope sequences e.g. in the form of a fusion construct or the like.
  • epitope sequences that bear disease-specific sequence mutations.
  • Such contructs may be used for immunological purposes, e.g. for the preparation of vaccines, the elicitation of antibodies etc.
  • the present inventin thus also encompasses the medical use of correspondingly prepared vaccines, pharmaceutical compositions comprising such vaccines, the vaccines themselves, methods of treatment involving the vaccines etc.
  • the present invention encompasses a screening method to identify antigen or epitope sequences bound by an antibody against a tumor marker protein as defined in Table 1. Further envisaged are the epitope sequences and/or antigen sequences derived from a screening procedure.
  • the antibodies of the invention include derivatives which are modified, for instance by the covalent attachment of any type of molecule to the antibody such that said covalent attachment does not prevent the antibody from specifically binding to the epitope or from generating an anti-idiotypic response.
  • modifications are glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • Chemical modifications may be carried out by known techniques, including specific chemical cleavage, acetylation, formylation etc.
  • the derivative may contain one or more non-classical amino acids.
  • Antibodies may be produced according to any suitable method known to the person skilled in the art. Monoclonal antibodies of defined specificity may be produced using, for instance, the hybridoma technology developed by Kohler and Milstein (Kohler and Milstein, 1976, Eur. J. Immunol, 6: 511-519).
  • the antibody or fragment thereof as defined herein above may be biotinylated or labeled.
  • said label is a radioactive label, an enzymatic label, a fluorescent label, a chemiluminescent or a bio luminescent label.
  • antibodies may also be labeled or combined with fluorescent polypeptides, e.g. green fluorescent protein (GFP) as well as derivates thereof known to the person skilled in the art.
  • GFP green fluorescent protein
  • a polynucleotide encoding an antibody may be generated from a nucleic acid from a suitable source.
  • a nucleic acid molecule encoding the antibody or fragment thereof as defined herein above may be used for recombinant antibody expression.
  • expression vectors contain the antibody coding sequences and appropriate transcriptional and translational control signals.
  • the vectors may either comprise coding sequences for the variable heavy chain or the variable light chain or for both.
  • Such vectors may also include the nucleotide sequence encoding the constant regions of the antibody molecule.
  • mammalian cells more preferably Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus may be used as an effective expression system for antibodies.
  • the present invention relates to a cell that produces the antibody or fragment thereof as defined herein above.
  • a cell may be a hybridoma cell as defined herein above or a cell which expresses a nucleic acid molecule encoding an antibody according to the present invention.
  • Particularly preferred are cells or cell lines which stably express the antibody molecule.
  • antibodies of the present invention or fragments thereof can be fused to any heterologous polypeptide sequence, preferably to those defined herein above, e.g. in order to facilitate antibody purification or to provide target means for the antibody.
  • antibodies against the tumor marker according to Table 1 in particular against a protein having an amino acid sequence as indicated in section E) of Table 1 may be comprised in the composition or may be used diagnostically.
  • An affinity ligand as described herein above, may be labeled with various markers or may be detected by a secondary affinity ligand, labeled with various markers, to allow detection, visualization and/or quantification.
  • This can be accomplished using any suitable labels, which can be conjugated to the affinity ligand capable of interaction with the expression product(s) of the tumor marker or group of tumor markers according to Table 1 or the corresponding protein(s) or to any secondary affinity ligand, using any suitable technique or methods known to the person skilled in the art.
  • the term "secondary affinity ligand” refers to a molecule which is capable of binding to the affinity ligand as defined herein above (i.e.
  • a "primary affinity ligand” if used in the context of a system with two interacting affinity ligands).
  • the binding interaction is preferably a specific binding.
  • labels that can be conjugated to a primary and/or secondary affinity ligands include fluorescent dyes or metals (e.g. fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g. rhodopsin), chemiluminescent compounds (e.g. luminal, imidazole) and bio luminescent proteins (e.g. luciferin, luciferase), haptens (e.g. biotin).
  • fluorescent dyes or metals e.g. fluorescein, rhodamine, phycoerythrin, fluorescamine
  • chromophoric dyes e.g. rhodopsin
  • chemiluminescent compounds e.g. luminal,
  • an affinity ligand to be used as a probe in particular a probe specific for the expression product(s) as defined herein above, may be labeled with a fluorescent label like 6-FAM, HEX, TET, ROX, Cy3, Cy5, Texas Red or Rhodamine, and/or at the same time with a quenching label like TAMRA, Dabcyl, Black Hole Quencher, BHQ-1 or BHQ-2.
  • a fluorescent label like 6-FAM, HEX, TET, ROX, Cy3, Cy5, Texas Red or Rhodamine
  • a quenching label like TAMRA, Dabcyl, Black Hole Quencher, BHQ-1 or BHQ-2.
  • Affinity ligands may also be labeled with enzymes (e.g.
  • radioisotopes e.g. 3 H, 14 C, 32 P, 33 P, 35 S, 125 I, n C, 13 N, 15 0, 18 F, 64 Cu, 62 Cu, 124 I, 76 Br, 82 Rb, 68 Ga or 18 F
  • particles e.g. gold
  • the different types of labels may be conjugated to an affinity ligand using various chemistries, e.g. the amine reaction or the thiol reaction.
  • chemistries e.g. the amine reaction or the thiol reaction.
  • other reactive groups than amines and thiols can also be used, e.g. aldehydes, carboxylic acids and glutamine.
  • nucleic acid affinity ligand or peptide affinity ligand of the present invention may be modified to function as a contrast agent, e.g. as an imaging contrast agent.
  • contrast agent refers to a molecular compound that is capable of specifically interacting with the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and which can be detected by an apparatus positioned outside the human or animal body.
  • contrast agents are suitable for use in magnetic resonance imaging (MRI) or magnetic photon imaging (MPI).
  • MRI magnetic resonance imaging
  • MPI magnetic photon imaging
  • Specifically interacting refer to the property of a molecular compound to preferentially interact with the tumor marker or group of tumor markers according to Table 1 on the cell surface of cells being present within the human or animal body over other proteins that are expressed by such cells.
  • Preferred contrast agents which may also be designated as contrast agent compositions will be capable of specifically detecting molecules having the nucleotide sequence(s) shown in section D) of Table 1 or the amino acid sequence(s) shown in section E) of Table 1 or derivatives or homologous variants thereof as defined herein above.
  • contrast agents are aptamers specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 as well as antibodies specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • Contrast agents aside from their property of being capable of specifically recognizing the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 will in addition typically comprise a further molecule which is detectable by the specific detection technology used.
  • modified to function thus refers to any suitable modifications known to the person skilled in the art, which may be necessary in order to allow the use of the contrast agent in molecular imaging methods, in particular in MRI or MPI.
  • fluorescent spectroscopy is used as a detection means, such molecules may comprise fluorophores as detectable marker molecules that can be excited at a specific wavelength.
  • a radioactive label e.g. a radioisotope as described herein above may be employed.
  • the contrast agents such as the above described antibodies may comprise a marker molecule which is detectable by MPJ.
  • detectable labels include e.g. USPIOS and 19Fluor.
  • a composition may additionally comprise accessory ingredients like PCR buffers, dNTPs, a polymerase, ions like bivalent cations or monovalent cations, hybridization solutions, secondary affinity ligands like, e.g. secondary antibodies, detection dyes and any other suitable compound or liquid necessary for the performance of a detection based on any of the affinity ligands or contrast agents as defined herein above, which is known to the person skilled in the art.
  • accessory ingredients like PCR buffers, dNTPs, a polymerase, ions like bivalent cations or monovalent cations, hybridization solutions, secondary affinity ligands like, e.g. secondary antibodies, detection dyes and any other suitable compound or liquid necessary for the performance of a detection based on any of the affinity ligands or contrast agents as defined herein above, which is known to the person skilled in the art.
  • the present invention relates to the use of a nucleic acid or peptide affinity ligand for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, for the preparation of a composition for diagnosing, detecting, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage to a more progressed stage or the progression from a less progressed stage to a more progressed stage or a predisposition for a more progressed stage, as described herein above.
  • the composition is preferably for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed cancer stage to a more progressed cancer stage or the progression from a less progressed cancer stage to a more progressed cancer stage or a predisposition for a more progressed cancer stage in an individual, e.g. a patient or subject to be analyzed or examined.
  • the present invention relates to the use of a set of oligonucleotides specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or a probe specific for the expression product(s) the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably specific for a nucleic acid sequence having a sequence as indicated in section D) of Table 1 or being complementary to such a sequence, for the preparation of a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage, as described herein above.
  • the present invention relates to the use of an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably a protein having an amino acid sequence as shown in section E) of Table 1, for the preparation of a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease, as described herein above.
  • the set of oligonucleotides is preferably for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease in an individual, e.g. a patient or subject to be analyzed or examined.
  • the present invention relates to the use of an antibody specific for protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or an antibody variant specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 for the preparation of a composition for diagnosing, detecting, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of said neplastsic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease as described herein above.
  • composition as defined herein above is a diagnostic composition.
  • the present invention relates to a diagnostic kit for diagnosing, detecting, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease, comprising a set of oligonucleotides specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a probe specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an antibody specific
  • the diagnostic kit of the present invention contains one or more agents allowing the specific detection of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • the agents or ingredients of a diagnostic kit may, according to the present invention, be comprised in one or more containers or separate entities. The nature of the agents is determined by the method of detection for which the kit is intended. Where detection at the mR A expression level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, i.e.
  • the agents to be comprised may be a set of oligonucleotides specific for the expression product(s) of said tumor marker or group of tumor markers and/or a probe specific for the expression product(s) of said tumor marker or group of tumor markers, which may be optionally labeled according to methods known in the art, e.g. with labels described herein above.
  • an aptamer specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be comprised.
  • the agents to be comprised may be antibodies or compounds containing an antigen-binding fragment of an antibody or antibody variants specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, as described herein above.
  • an aptamer specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be comprised.
  • a diagnostic kit may comprise a contrast agent as defined herein above.
  • a diagnostic kit of the present invention contains detection reagents for expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • detection reagents comprise, for example, buffer solutions, labels or washing liquids etc.
  • the kit may comprise an amount of a known nucleic acid molecule or protein, which can be used for a calibration of the kit or as an internal control.
  • a diagnostic kit for the detection of expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may comprise accessory ingredients like a PCR buffers, dNTPs, a polymerase, ions like bivalent cations or monovalent cations, hybridization solutions etc.
  • a diagnostic kit for the detection of protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may also comprise accessory ingredients like secondary affinity ligands, e.g. secondary antibodies, detection dyes and any other suitable compound or liquid necessary for the performance of a protein detection based known to the person skilled in the art. Such ingredients are known to the person skilled in the art and may vary depending on the detection method carried out.
  • kit may comprise an instruction leaflet and/or may provide information as to the relevance of the obtained results.
  • the present invention relates to a method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprising at least the step of determining the level of a tumor marker or a group of tumor markers as defined above, in a sample.
  • determining the level of a tumor marker or group of tumor markers refers to the determination of the presence or amount of expression product(s) of the tumor marker or tumor marker or group of tumor markers as mentioned herein above or according to Table 1, e.g. (a) transcript(s) of the tumor marker or group of tumor markers according to Table 1, and/or the determination of the presence and/or amount of (a) protein(s) of the tumor marker or group of tumor markers according to Table 1.
  • level of the tumor marker or group of tumor markers according to Table 1 thus means the presence or amount of (an) expression product(s) of the tumor marker or group of tumor markers according to Table 1, e.g. (a) transcript(s) of the tumor marker or group of tumor markers according to Table 1, and/or the determination of the presence or amount of (a) protein(s) of the tumor marker or group of tumor markers according to Table 1.
  • the determination of the presence or amount of (an) expression product(s) of the tumor marker or group of tumor markers according to Table 1, e.g. (a) transcript(s) of the tumor marker or group of tumor markers according to Table 1 or (a) protein(s) of the tumor marker or group of tumor markers according to Table 1 may be accomplished by any means known in the art.
  • the determination of the presence or amount of the expression products of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, e.g. transcript(s) and/or of protein(s) of the markers mentioned in Table 1 , comprising for instance sequences as depicted in sections D) and E) of Table 1 is accomplished by the measurement of nucleic acid or protein levels or by the determination of the biological activity of said tumor marker or group of tumor markers.
  • the measurement of the nucleic acid level of the expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be assessed by separation of nucleic acid molecules (e.g.
  • R A or cDNA obtained from the sample in agarose or polyacrylamide gels, followed by hybridization with tumor marker specific oligonucleotide probes as defined herein above, e.g. oligonucleotide probes comprising fragments of the sequences indicated in section D) of Table 1, or complementary sequences thereof.
  • the expression level may be determined by the labeling of nucleic acid obtained from the sample followed by separation on a sequencing gel. Nucleic acid samples may be placed on the gel such that patient and control or standard nucleic acid are in adjacent lanes. Comparison of expression levels may be accomplished visually or by means of a densitometer. Methods for the detection of mRNA or expression products are known to the person skilled in the art. Typically, Northern blot analysis may be used for such a purpose.
  • nucleic acid level of the expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be detected in a DNA array or microarray approach.
  • sample nucleic acids derived from subjects to be tested are processed and labeled, preferably with a fluorescent label.
  • nucleic acid molecules may be used in a hybridization approach with immobilized capture probes corresponding to the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 of the present invention or known biomarker or cancer marker genes.
  • Suitable means for carrying out microarray analyses are known to the person skilled in the art.
  • a DNA array or microarray comprises immobilized high- density probes to detect a number of genes.
  • the probes on the array are complementary to one or more parts of the sequence of the marker gene, or to the entire coding region of the marker gene.
  • any type of tumor marker associated polynucleotide may be used as probe for the DNA array, as long as the polynucleotide allows for a specific distinction between the tumor marker expression and the expression of other genes.
  • cDNAs, PCR products, and oligonucleotides are useful as probes.
  • a fragment comprising 5'- or 3 '-portions of the tumor markers or group of tumor markers as mentioned herein above or according to Table 1, e.g. of the sequences indicated in section D) of Table 1 may be used as a probe.
  • the DNA array or microarray may comprise probes of one or more of the tumor marker of Table 1, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 etc. or all of the tumor markers or any combination of said markers.
  • any type of fragment or sub-portion of any of the markers sequences may be combined with any further fragment or sub-portion of any of the markers sequences of Table 1.
  • the determination of the expression of tumor markers according to Table 1 also the determination of the expression of other genes, e.g. additional biomarker or cancer marker genes is envisaged by the present invention.
  • a DNA array- or microarray-based detection method typically comprises the following steps: (1) Isolating mRNA from a sample and optionally converting the mRNA to cDNA, and subsequently labeling this RNA or cDNA. Methods for isolating RNA, converting it into cDNA and for labeling nucleic acids are described in manuals for micro array technology. (2) Hybridizing the nucleic acids from step 1 with probes for the marker genes.
  • the nucleic acids from a sample can be labeled with a dye, such as the fluorescent dyes Cy3 (red) or Cy5 (blue). Generally a control sample is labeled with a different dye.
  • a marker gene can be represented by two or more probes, the probes hybridizing to different parts of a gene. Probes are designed for each selected marker gene. Such a probe is typically an oligonucleotide comprising 5-50 nucleotide residues. Longer DNAs can be synthesized by PCR or chemically. Methods for synthesizing such oligonucleotides and applying them on a substrate are well known in the field of micro-arrays. Genes other than the marker genes may be also spotted on the DNA array.
  • a probe for a gene whose expression level is not significantly altered may be spotted on the DNA array to normalize assay results or to compare assay results of multiple arrays or different assays.
  • the nucleic acid level of expression of the tumor marker or group of tumor markers according to Table 1 may be detected in a quantitative RT-PCR approach, preferably in a real-time PCR approach following the reverse transcription of the mRNA transcript(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • a transcript is reverse transcribed into a cDNA molecule according to any suitable method known to the person skilled in the art.
  • a quantitative or real-time PCR approach may subsequently be carried out based on a first DNA strand obtained as described above.
  • Taqman or Molecular Beacon probes as principal FRET -based probes of this type may be used for quantitative PCR detection.
  • the probes preferably probes of the tumor marker or group of tumor markers according to Table 1 as defined herein above, serve as internal probes which are used in conjunction with a pair of opposing primers that flank the target region of interest, preferably a set of oligonucleotides specific for the tumor marker or group of tumor markers according to Table 1 as defined herein above.
  • the probe may selectively bind to the products at an identifying sequence in between the primer sites, thereby causing increases in FRET signaling relative to increases in target frequency.
  • a Taqman probe to be used for a quantitative PCR approach may comprise (a) oligonucleotide(s) derived from the tumor marker or group of tumor markers according to Table 1 as defined above of about 22 to 30 bases that is labeled on both ends with a FRET pair.
  • the 5' end will have a shorter wavelength fluorophore such as fluorescein (e.g. FAM) and the 3' end is commonly labeled with a longer wavelength fluorescent quencher (e.g. TAMRA) or a non- fluorescent quencher compound (e.g. Black Hole Quencher).
  • the probes to be used for quantitative PCR in particular the probes tumor marker or group of tumor markers as mentioned herein above or according to Table 1, have no guanine (G) at the 5' end adjacent to the reporter dye in order to avoid quenching of the reporter fluorescence after the probe is degraded.
  • a Molecular Beacon probe to be used for a quantitative PCR approach according to the present invention preferably uses FRET interactions to detect and quantify a PCR product, with each probe having a 5' fiuorescent-labeled end and a 3' quencher-labeled end.
  • This hairpin or stem-loop configuration of the probe structure comprises preferably a stem with two short self-binding ends and a loop with a long internal target-specific region of about 20 to 30 bases.
  • Alternative detection mechanisms which may also be employed in the context of the present invention are directed to a probe fabricated with only a loop structure and without a short complementary stem region.
  • An alternative FRET -based approach for quantitative PCR which may also be used in the context of the present invention is based on the use of two hybridization probes that bind to adjacent sites on the target wherein the first probe has a fluorescent donor label at the 3 ' end and the second probe has a fluorescent acceptor label at its 5' end.
  • the measurement of protein levels of the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or of any fragments, homologues or derivates derived thereof may be carried out via any suitable detection technique known in the art.
  • the protein level of tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and derivatives thereof may be determined immunologically, e.g. by using an antibody specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, preferably an antibody as defined herein above.
  • antibody variants or fragments as defined herein above may be used.
  • the present invention also envisages the use of peptide affinity ligands like aptamers specific for the protein(s) of the tumor marker or group of tumor markers according to Table 1 as defined herein above.
  • Determination of the protein levels of the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 can be accomplished, for example, by the separation of proteins from a sample on a polyacrylamide gel, followed by identification of the protein(s) of tumor marker or group of tumor markers as mentioned herein above or according to Table 1 using specifically binding antibodies in a Western blot analysis.
  • proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection.
  • protein samples are analyzed by mass
  • antibodies specific for (a) protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be placed on a support and be immobilized. Proteins derived from samples or tissues to be analyzed may subsequently be mixed with the antibodies. A detection reaction may then be carried out, e.g. with a second affinity ligand as defined herein above, preferably with a specific antibody.
  • Immunological tests which may be used in the context of the present invention, in particular for the diagnostic purposes of the present invention, include, for example, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassay like RIA (radio-linked immunoassay), ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fiuorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence
  • immunoassays Such assays are routine and well known to the person skilled in the art.
  • binding affinity of an antibody to an antigen and the off-rate of an antibody- antigen interaction may be determined by competitive binding assays.
  • the binding affinity of an antibody to an antigen and the off- rate of an antibody- antigen interaction may be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3 H or 125 I) with a suitable antibody in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off- rates may be determined from the data by any suitable analysis approach, e.g. by a scatchard plot analysis.
  • Competition with a second antibody may also be determined using radioimmunoassays.
  • the antigen may be incubated with a suitable antibody conjugated to a labeled compound (e.g., 3 H or 125 I) in the presence of increasing amounts of an unlabeled second antibody.
  • aptamers specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be used in a method of detecting proteins of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • Such aptamers may preferably be labeled in order to allow the detection of a protein- ligand interaction.
  • the determination of the biological activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be carried out by employing molecular or enzymatic assays specific to the corresponding function or functions of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, e.g. of a tumor marker protein having a sequence as indicted in section E) of Table 1. Suitable techniques would be known to the person skilled in the art.
  • An inhibition of the activity may be carried out by any suitable means known to the person skilled in the art, preferably via the use of suitable antisense nucleotides, siR A molecules or miRNA molecules, more preferably via specifically hybridizing antisense nucleotides, specific siR A or miR A molecules, e.g. as described herein below.
  • the biological activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be tested with the help of suitable enzymatic reactions or tests for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , known to the person skilled in the art, or by employing specific inhibitors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • suitable enzymatic reactions or tests for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 known to the person skilled in the art, or by employing specific inhibitors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • the use of such inhibitors may, for example, be combined with an enzymatic readout system.
  • Typical inhibitors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 to be used comprise antisense molecules, siRNA molecules or miRNA molecules.
  • the level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may also be detected in methods involving histological or cell- biological procedures.
  • visual techniques such as light microscopy or
  • immunofluoresence microscopy as well as flow cytometry or luminometry may be used.
  • (a) protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 in a cell may, for instance, be detected or determined by removing cells to be tested from samples as defined herein above. Also tissue sections or biopsy samples may be used for these methods. Subsequently, affinity ligands for the tumor marker or group of tumor markers as mentioned herein above or according to
  • Table 1 may be applied, preferably antibodies or aptamers.
  • affinity ligands are labeled, preferably with fluorescent labels as defined herein above.
  • Typical methods to be used comprise fluorometric, luminometric and/or enzymatic techniques. Fluorescence is normally detected and/or quantified by exposing fluorescent labels to light of a specific wavelength and thereafter detecting and/or quantifying the emitted light of a specific wavelength. The presence of a luminescently tagged affinity ligand may be detected and/or quantified by luminescence developed during a chemical reaction. Detection of an enzymatic reaction is due to a color shift in the sample arising from chemical reaction.
  • the level of the tumor marker or group of tumor markers according to Table 1 may be determined by suitable molecular imaging techniques, e.g. magnetic resonance imaging (MRI) or magnetic photon imaging (MPI), and/or by using suitable contrast agents, e.g. contrast agents as defined herein above.
  • suitable molecular imaging techniques e.g. magnetic resonance imaging (MRI) or magnetic photon imaging (MPI)
  • suitable contrast agents e.g. contrast agents as defined herein above.
  • a method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, or the progression form a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprises the additional step of comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease.
  • the expression level of the tumor marker or the group of tumor markers of a less progressed stage of the same neoplastic disease may be an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, is/are known or from the same subject at an earlier point in time or an expression level corresponding to a stage of said neoplastic disease or form of said neoplastic disease, whose disease state or stage is known including different
  • comparing refers to any suitable method of assessing, calculating, evaluating or processing of data.
  • the determination of the presence or amount of (an) expression product(s), e.g. (a) transcript(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or of (a) protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, is accomplished by the measurement of nucleic acid or protein levels or by the determination of the biological activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • the expression level(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be determined by a method involving the detection of an mRNA encoded by the genes encoding for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , or the according cDNA sequence, e.g. the sequences indicated in section D) of Table 1, the detection of the protein(s) encoded by the transcript of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or the detection of the biological activity of the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, e.g. of the proteins having the sequences indicated in section E) of Table 1.
  • the measurement of the nucleic acid level of expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be assessed by separation of nucleic acid molecules as described herein above.
  • a decision on the presence or stage of a more progressed stage of the neoplastic disease as defined herein above, or the progression towards said stage of said neoplastic disease may be based on the results of the comparison step.
  • a more progressed stage of said neoplastic disease may be diagnosed or prognosticated or a progression towards said more progressed stage of said neoplastic disease may be diagnosed or prognosticated in said method according to the corresponding definitions provided herein above in the context of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • the above mentioned method is a method of graduating a neoplastic disease, comprising the steps of
  • control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease
  • step (c) deciding on the stage or developmental status of a neoplastic disease based on the results obtained in step (b).
  • the determination of the level of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be carried out according to steps as defined herein above. Preferably, the determination may be carried out as measurement of a nucleic acid level or protein level according to the herein above described options for such measurements. In one embodiment, steps a), b), c) and/or d) of this method of diagnosis may be performed outside the human or animal body. A more progressed stage of a neoplastic disease may be graduated in said method according to the corresponding definitions provided herein above in the context of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • the present invention relates to a method of data acquisition comprising at least the steps of:
  • control level refers to the expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or other suitable markers in a control or the expression level of said marker(s) in a less progressed stage of a neoplastic disease, as defined herein above.
  • the status, nature, amount and condition of the control level may be adjusted according to the necessities.
  • a comparison of the expression to a control level may be carried out according to any suitable method of assessing, calculating, evaluating or processing of data and particularly aims at the detection of differences between two data sets.
  • a statistical evaluation of the significance of the difference may further be carried out. Suitable statistical methods are known to the person skilled in the art. Obtained data and information may be stored, accumulated or processed by suitable informatics or computer methods or tools known to the person skilled in the art and/or be presented in an appropriate manner in order to allow the practitioner to use the data for one or more subsequent deduction or conclusion steps.
  • the present invention relates to a method of detecting, diagnosing, monitoring or prognosticating a neoplastic disease as defined herein above associated with a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprising at least the steps of:
  • step (d) deciding on the presence of a neoplastic disease as defined herein above in particular of a more progressed stage of said neoplastic disease or on the progression of said neoplastic disease, in particular from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease based on the results obtained in step (c).
  • steps a), b), c) and/or d) of this method of diagnosis may be performed outside the human or animal body.
  • the testing for expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be carried out according to steps as defined herein above.
  • the testing may be carried out as measurement of nucleic acid or protein levels of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or by determining the biological activity of said tumor markers, more preferably according to the herein above described options for such measurements.
  • the testing may be carried out in an individual, i.e. in vivo, or outside the individual, i.e. ex vivo or in vitro.
  • the present invention relates to an immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, or for detecting, diagnosing, graduating, monitoring or prognosticating the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprising at least the steps (a) testing in a sample obtained from an individual for the expression of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ;
  • step (d) deciding on the presence, stage or risk of recurrence of said neoplastic disease or the progression of said neoplastic disease based on the results obtained in step (c), wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or a group of tumor markers as defined above.
  • risk of recurrence refers to a likelihood or probabilty assessment regarding the chances or the probability that a subject or individual may be afflicted with or may be developing a similar or the same neoplastic disease as defined herein above, preferably cancer, comparable to the one that the subject or individual has been treated for or diagnosed for, based on the expression modification of the tumor marker or a group of tumor markers as defined above.
  • a risk of recurrence may, for example, be present when a tumor marker or group of turmor markers as defined herein above shows a modified expression level, e.g.
  • an increased or decreased expression level in comparison to a less progressed stage, in particular a non-malignant stage, benign tumor stage or a healthy stage, although histological markers, cell shapes etc. or other tumor biomarker, e.g. PSA, show no modification, or a different modification, e.g. an opposite modification or a less pronounced increase or decrease of expression level(s).
  • a less progressed stage in particular a non-malignant stage, benign tumor stage or a healthy stage, although histological markers, cell shapes etc. or other tumor biomarker, e.g. PSA, show no modification, or a different modification, e.g. an opposite modification or a less pronounced increase or decrease of expression level(s).
  • the testing for expression of the tumor marker or group of tumor markers according to Table 1 may be carried out according to steps as defined herein above.
  • the testing may be carried out as measurement of protein levels of the tumor marker or group of tumor markers according to Table 1 , more preferably according to the herein above described options for such measurements.
  • controls or control samples controls as defined herein above may be used.
  • the testing steps may be based on the use of an antibody specifically binding to a tumor marker according to Table 1 as laid out above, e.g. (a) commercially available antibody/antibodies against (a) protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • a neoplastic disease as defined herein above in particular a more progressed cancer stage may be diagnosed or prognosticated or a progression of a neoplastic disease as defined herein above, in particular from a less progressed to a more progressed stage of said neoplastic disease, may be diagnosed or prognosticated in said immunoassay according to the corresponding definitions provided herein above in the context of the tumor marker or group of tumor markers according to Table 1 as marker(s) for said neoplastic disease.
  • the present invention relates to a method of identifying an individual for eligibility for a neoplastic disease therapy comprising:
  • step (c) classifying the levels of expression of step (a) relative to levels of step
  • the present invention relates to a method of identifying an individual for eligibility for a neoplastic disease therapy comprising:
  • step (c) classifying the levels of expression of step (a) relative to levels of step
  • the level of a tumor marker or group of tumor markers may be determined on the nucleic acid, protein or activity level as described herein above. Preferred is the determination of the amount of tumor marker protein and/or transcript(s).
  • the level of a reference gene in a sample may be determined.
  • the term "reference gene” as used herein refers to any suitable gene, e.g. to any steadily expressed and continuously detectable gene, gene product, expression product, protein, peptide or protein variant in the organism of choice.
  • the term also includes gene products such as expressed proteins, peptides, polypeptides, as well as modified variants thereof.
  • the invention hence also includes reference proteins derived from a reference gene. Also encompassed are all kinds of transcripts derivable from the reference gene as well as modifications thereof or secondary parameters linked thereto. Alternatively or additionally, other reference parameters may also be used for reference purposes, e.g. metabolic concentrations, cell sizes etc.
  • Testing for the expression of a reference gene may be carried out in the same sample used for the determination of the tumor marker or group of tumor markers of the invention. If the testing is carried out in the same sample, a single detection or a multiplex detection approach may be performed.
  • concentration of primers and/or probe oligonucleotides may be modified.
  • concentration and presence of further ingredients like buffers, ions etc. may be modified, e.g. increased or decreased in comparison to manufacturers' indications.
  • the testing for the expression of a reference gene may be carried out in a different sample, preferably a control sample.
  • a control sample may be a control sample from the same individual as the test sample, or a control sample derived from a different source or individual.
  • the control sample may further be either a sample derived from the same tissue, preferably prostate tissue, or be derived from a different tissue type. Examples of preferred alternative tissue types are stromal prostate tissue, bladder epithelial tissue and urethra epithelial tissue.
  • the testing of the test sample for the expression of a reference gene and the testing of control sample for the expression of a tumor marker or group of tumor markers may be combined.
  • control sample may also be tested for the expression of the reference gene.
  • the obtained expression results may be compared and/or averaged or normalized according to any suitable statistical method known to the person skilled in the art.
  • the term further means that the expression in a test sample for a tumor marker and the expression in the same test sample for a reference gene are compared, e.g. after normalization against a further gene as normalization reference. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the reference gene, an increased expression in comparison to the reference gene, or an reduced expression in comparison to the reference gene.
  • an individual may be considered to be eligible for a neoplastic disease therapy when the expression level of an up- regulated tumor marker as defined herein above, or as indicated in section P), R), T) or V) of Table 1, or the expression level of a group of tumor markers as defined herein above is increased.
  • the expression level is deemed to be "increased" when the tumor marker gene expression, or the expression of the group of tumor marker genes in the test sample is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding tumor marker gene expression, or to the expression of the corresponding group of tumor markers in a control sample, or elevated at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the tumor marker expression, or the expression of the group of tumor markers in a control sample; or when the tumor marker or group of tumor marker gene expression is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the expression of a reference gene in a control sample, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at
  • the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes.
  • additional genes or markers e.g. housekeeping genes.
  • the amount of protein present, or the amount of detecting antibodies present may be measured.
  • the above indicated parameters also indicate an increase of expression.
  • an individual may also be considered to be eligible for a neoplastic disease therapy when the expression level of a down-regulated tumor marker as defined herein above, or as indicated in section Q), S), U) or W) of Table 1, or the expression level of a group of tumor markers as defined herein above is decreased.
  • the expression level is deemed to be "decreased" when the tumor marker gene expression, or the expression of the group of tumor marker genes in the test sample is lowered by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding tumor marker gene expression, or to the expression of the corresponding group of tumor markers in a control sample, or lowered at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the tumor marker expression, or to the expression of the group of tumor markers in a control sample; or when the tumor marker or group of tumor marker gene expression is lowered by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the expression of a reference gene in a control sample, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least
  • the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes.
  • additional genes or markers e.g. housekeeping genes.
  • the amount of protein present, or the amount of detecting antibodies present may be measured.
  • the above indicated parameters also indicate a decrease of expression.
  • the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:
  • step (c) determining the difference in expression of said up-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said up-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b);
  • step (d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an increased level of expression of an up-regulated tumor marker or group of tumor markers as defined above.
  • an immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:
  • step (c) determining the difference in expression of said down-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said down-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b);
  • step (d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an decreased level of expression of an down-regulated tumor marker or group of tumor markers as defined above.
  • the testing of the expression of a tumor marker or group of tumor markers may preferably be carried out via the determination of the amount of tumor marker protein or the determination of the tumor marker protein/expression product activity level. Preferred is the determination of the amount of tumor marker proteins or peptides with the help of specific antibodies binding said tumor marker protein or expression product.
  • the immunoassay may be carried out with any other suitable agent or be combined with the determination of other entities.
  • the assay may be combined with the detection of the presence or amount of nucleic acids, or enzymatic testing methods as described herein.
  • the level of a reference gene as described herein above in a sample may be determined. Testing for the expression of a reference gene may be carried out in the same sample used for the determination of the tumor marker or group of tumor markers. If the testing is carried out in the same sample, a single detection or a parallel or multiplex detection approach may be performed. Preferably, for a parallel or multiplex detection differently labeled primary or secondary antibodies may be used.
  • the testing for the expression of a reference gene may be carried out in a different sample, preferably a control sample.
  • a control sample may be a control sample from the same individual as the test sample, or a control sample derived from a different source or individual.
  • the control sample may further be either a sample derived from the same tissue, preferably prostate tissue, or be derived from a different tissue type. Examples of preferred alternative tissue types are stromal prostate tissue, bladder epithelial tissue and urethra epithelial tissue.
  • the testing of the test sample for the expression of a reference gene and the testing of control sample for the expression of a tumor marker or group of tumor markers may be combined.
  • control sample may also be tested for the expression of the reference gene.
  • the obtained expression results may be compared and/or averaged or normalized according to any suitable statistical method known to the person skilled in the art.
  • determining the difference in expression of a tumor marker or a group of tumor markers of step (a) and the expression of a tumor marker or group of tumor markers and/or the reference gene in step (b) means that the expression in a test sample for the tumor marker or group of tumor markers and the expression in a control sample for the tumor marker or group of tumor markers are compared, e.g. after
  • test sample is indicated as providing a similar expression as the control sample, an increased expression in comparison to the control sample, or an reduced expression in comparison to the control sample.
  • the term further means that alternatively or additionally the expression in a test sample for a tumor marker or group of tumor markers and the expression in the same test sample for a reference gene are compared, e.g. after normalization against a further gene as normalization reference.
  • test sample is indicated as providing a similar expression as the reference gene, or a difference in the expression. The difference may be either an increased expression in comparison to the reference gene, or an reduced expression in comparison to the reference gene.
  • neoplastic disease therapy means that an individual is identified as pertaining to a group of similar individuals, whose optimal therapy form is a neoplastic disease therapy, preferably a therapy against an earliy prostate cancer form in accordance with the outcome of the expression test as described herein above, in particular in accordance with encountered difference in the tumor marker or group of tumor marker expression level and a reference gene or the tumor marker or group of tumor marker expression level in different samples.
  • an individual may be identified as pertaining to a group of similar individuals whose optimal therapy form is a neoplastic disease therapy when the expression levels of the up-regulated tumor marker or group of tumor markers as defined herein above, or as indicated in section P), R), T) or V) of Table 1 , are increased.
  • the expression level is deemed to be "increased" when the gene expression of the up-regulated tumor marker or group of tumor marker as defined herein above, or as indicated in section P), R), T) or V) of Table 1, is elevated in the test sample by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to said tumor marker or group of tumor marker expression in a control sample, or at least elevated 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to said tumor marker or group of tumor marker expression in a control sample; or when the tumor marker or group of tumor marker gene expression is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the expression of a reference gene in a control sample, or is elevated at least 0.1 fold, at least 0.2 fold, at least 1
  • an individual may also be identified as pertaining to a group of similar individuals whose optimal therapy form is a neoplastic disease therapy when the expression levels of the down-regulated tumor marker or group of tumor marker as defined herein above, or as indicated in section Q), S), U) or W) of Table 1, are decreased.
  • the expression level is deemed to be "decreased" when the gene expression of the down-regulated tumor marker or group of tumor markers as defined herein above, or as indicated in section Q), S), U) or W) of Table 1, is lowered in the test sample by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to said tumor marker or group of tumor marker expression in a control sample, or at least lowered 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to said tumor marker or group of tumor marker expression in a control sample; or when the tumor marker or group of tumor marker gene expression is lowered by, for example, 5%>, 6%>, 7%>, 8%>, 9%>, 10%>, 15%>, 20%>, 25%o, 30%), 40%), 50%), or more than 50%> in comparison to the expression of a reference gene in a control sample, or is lowered at
  • neoplastic disease therapy refers to any suitable neoplastic disease therapy, preferably prostate cancer related therapy known to the person skilled in the art, and preferably includes surgical castration by removal of the testes as the main organ of male sex hormone production, chemical castration by e.g., suppression of generation of androgens or by inhibition of the androgen receptor activity, cytotoxic, chemotherapy, targeted therapy (e.g. targeting cellular proteins with chemical molecules to suppress or stimulate their activity), radiation therapy (External Beam Radiation Therapy, Brachytherapy), Cryotherapy, focal therapies like HIFU ablation (High Frequency
  • Ultrasound ablation or thermal ablation or any type of combination therapy of at least two of the above mentioned treatment forms either in direct combination, or used in a subsequent form.
  • an individual considered to be eligible for neoplastic disease therapy due to an increased expression of a tumor marker or group of tumor marker of the present invention may be deemed to be suffering from a neoplastic disease or be prone to develop a neoplastic disease in the future, e.g. within the next 1 to 24 months.
  • a correspondingly identified or stratified individual may be treated with a pharmaceutical composition according to the present invention, e.g. as defined herein below.
  • a correspondingly identified individual may be treated with a pharmaceutical composition according to the present invention in combination with an additional cancer therapy.
  • additional cancer therapy refers to any types of cancer therapy known to the person skilled in the art. Preferred are cancer therapy forms known for prostate cancer. The term includes, for example, all suitable forms of chemotherapy, radiation therapy, surgery, antibody therapies etc.
  • a correspondingly identified or stratified individual may also be treated solely with one or more cancer therapies such as a chemotherapy, radiation therapy, surgery, antibody therapies etc.
  • cancer therapies typically used for prostate cancer.
  • the classification method for eligibility or the immunoassay for stratification as described herein above may also be used for monitoring the treatment of an individual, e.g. an individual being classified as suffering from a neoplastic disease.
  • the monitoring process may be carried out as expression determination or protein detection over a prolonged period of time, e.g. during or after treatment sessions, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months, or 1, 2, 3 or more years.
  • the determination steps may be carried out in suitable intervals, e.g. every week, 2 weeks, 3 weeks, every month, 2 months, 3 months, 6 months, 12 months etc.
  • any treatment scheme as mentioned herein may be adjusted, e.g. enforced or attenuated, or altered in any suitable manner in correspondence with the results of the monitoring process.
  • the reference gene is a housekeeping gene.
  • housekeeping genes include inter alia ⁇ -actin, glycerinaldehyde 3-phosphate dehydrogenase (GAPDH), porphobilinogen deanimase (PBGD), and ribosomal protein PI .
  • GPDH glycerinaldehyde 3-phosphate dehydrogenase
  • PBGD porphobilinogen deanimase
  • ribosomal protein PI ribosomal protein PI .
  • any other suitable gene may be used as a house-keeping gene, as long as the gene shows an expression or transcription on a steady, non-modified level, in particular during different stages of neoplastic disease development, more preferably during different stages of prostate cancer development.
  • Expression data of a house-keeping gene may be obtained from one or more samples of the same individual or from more individuals, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 1000, 5.000, 10.000 or more. Expression data may
  • normalization and/or comparison with GAPDH or PBGD may preferably be used for the methods of identifying or the immunoassays for discriminating or stratifying individuals.
  • Corresponding determination steps may either be carried out in separate reactions, or, particularly preferred in multiplex reactions.
  • concentration of primers and/or probe oligonucleotides may be modified.
  • concentration and presence of further ingredients like buffers, ions etc. may be modified, e.g. increased or decreased in comparison to manufacturers' indications.
  • the method of identifying an individual for eligibility for a neoplastic disease therapy based on the expression of a tumor marker or group of tumor markers or the immunoassay for stratifying an individual or cohort of individuals as described herein above may further be combined with one or more similar identification methods, based on the expression of one or more different biomarkers.
  • PSA prostate specific antigen
  • the level of PSA in blood is encountered to be of a range of about 2 to 5 or more ng/ml, preferably of about 2.2 to 4.8 ng/ml or more, 2.4 to 4.4 ng/ml or more, 2.6 to 4.2 ng/ml ore more or 2.8 to 4.0 ng/ml or more, more preferably of about 2.5 to 4 ng/ml or more, an individual may be considered to be suffering from a neoplastic disease, or be likely to develop a neoplastic disease in the near future, i.e. within the next 1, 2, 3, 4, 5, 6, 12, 14, 48 months.
  • the testing for expression of a tumor marker or group of tumor markers may be carried out according to steps as defined herein above.
  • the detection or measurement of the tumor marker, or of the group or combination of markers as defined herein, i.e. derivable from Table 1, may further be used to monitor the potential re-currence of a neplastic disease, in partiuclar a neoplastic disease of the prostate, after a primary treatment, e.g. surgery, or radiotheary.
  • the tumor marker detection or measurement may be performed every 3, or every 6, or every 12 months after the patient has been treated. If the expression level of the tumor marker, or group of tumor markers as defined herein is going to pass a threshold as defined herein, e.g. as derivable form Table 1 or Table 7, by 5%, 10%, 15%, 20%, 25%, or 30% or more, further
  • the diagnosing, detecting, monitoring or prognosticating as mentioned above is to be carried out on a sample obtained from an individual.
  • sample obtained from an individual relates to any biological material obtained via suitable methods known to the person skilled in the art from an individual, as laid out above.
  • the sample used in the context of the present invention should preferably be collected in a clinically acceptable manner, more preferably in a way that nucleic acids (in particular R A) or proteins are preserved.
  • the biological samples may include body tissues and/or fluids, such as blood, or blood components like serum or plasma, sweat, sputum or saliva, semen and urine, as well as feces or stool samples.
  • the biological sample may contain a cell extract derived from or a cell population including an epithelial cell, preferably a neoplastic, e.g. cancerous epithelial cell or an epithelial cell derived from tissue suspected to be neoplastic, e.g. cancerous.
  • the sample used in the context of the present invention should preferably be collected in a clinically acceptable manner, more preferably in a way that nucleic acids (in particular RNA) or proteins are preserved.
  • the biological sample may contain a cell population derived from a glandular tissue, e.g. the sample may be derived from the prostate of a male individual.
  • cells may be purified from obtained body tissues and fluids if necessary, and then used as the biological sample. Samples, in particular after initial processing, may be pooled. However, also non-pooled samples may be used.
  • the content of a biological sample may also be submitted to an enrichment step.
  • a sample may be contacted with ligands specific for the cell membrane or organelles of certain cell types, e.g. prostate cells, functionalized for example with magnetic particles.
  • the material concentrated by the magnetic particles may subsequently be used for detection and analysis steps as described herein above or below.
  • biopsy or resections samples may be obtained and/or used.
  • samples may comprise cells or cell lysates.
  • cells e.g. tumor cells
  • cells may be enriched via filtration processes of fluid or liquid samples, e.g.
  • a sample may be a tissue sample, a urine sample, a biopsy sample, a urine sediment sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a semen sample, or a sample comprising circulating tumor cells.
  • a subject or individual to be diagnosed, monitored or in which a neoplastic disease as defined herein above, a progression of said neoplastic disease or predisposition for said neoplastic disease is to be detected or prognosticated according to the present invention is an animal, preferably a mammal, more preferably a human being.
  • the obtaining step of a sample may be included as a first or additional step in any of the herein mentioned methods, uses or approaches.
  • the present invention relates to a pharmaceutical composition relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one element selected from the group of: (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (e) a miR A specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned here
  • a compound directly inhibiting the activity of a tumor marker refers to a compound which is capable of decreasing the activity of a tumor marker according to Table 1.
  • a compound may be any direct interactor of the tumor marker according to Table 1, which has negative influence on the catalytic activity of the tumor marker or group of tumor markers according to Table 1.
  • Such a compound may preferably be an antagonist of the catalytic activity of the tumor marker according to Table 1 , e.g. of a tumor marker protein having a sequence as indicated in section E) of Table 1.
  • a compound indirectly inhibiting the activity of a tumor marker refers to a compound which is capable of decreasing the activity of the tumor marker or group of tumor markers according to Table 1 by an interaction with a direct interactor of the tumor marker according to Table 1 ("indirect interactor") or via an indirect working pathway not involving an interaction with of the tumor marker according to Table 1.
  • Such a compound may be any direct interactor of an interactor of the tumor marker according to Table 1, e.g. of a tumor marker protein having a sequence as indicated in section E) of Table 1.
  • the effect conveyed by the direct interactor of an interactor of the tumor marker according to Table 1 may be either negative if the interactor of the tumor marker or group of tumor markers according to Table 1 itself has a negative effect on the activity of the tumor marker according to Table 1 , or negative, if the interactor of the tumor marker according to Table 1 has a positive effect on the activity of said tumor marker.
  • negatively working indirect integrators may provoke a modification of the binding behavior of directly binding proteins, leading to a decreased activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • negatively working indirect interactors may have an inhibitory effect on activators of the tumor marker according to Table 1. Examples of such interactors are enzymatic activities degrading activators of the tumor marker according to Table 1, or proteins capable of binding and quenching activators of the tumor marker according to Table 1.
  • such interactors may positively modulate activities leading to a degradation of the tumor marker according to Table 1, e.g. proteinases. Further examples and their implementation would be known to the person skilled in the art.
  • an indirect inhibition of the activity of the tumor marker according to Table 1 may be conveyed by compounds deactivating, interfering or disrupting the expression of the endogenous gene(s) of said tumor markers.
  • compounds deactivating, interfering or disrupting the expression of the endogenous gene(s) of said tumor markers are specific interactors of transcription factors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 that inhibit and/or preclude binding of transcription factors and the basal transcription machinery to the promoters of the tumor marker according to Table 1, specific destabilizing activities of the mR A(s) of the tumor marker according to Table 1 or factors inhibiting the splicing factors specific for the tumor marker according to Table 1. Further examples and their implementation would be known to the person skilled in the art.
  • nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker refers to any nucleic acid capable of expressing a mutant form of a naturally occurring protein or polypeptide of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • the term refers to a nucleic acid encoding (a) variant(s) of the tumor marker according to Table 1, which comprises an antimorphic modification, in particular which adversely affects the tumor marker according to Table 1 of the invention.
  • an antimorphic modification in particular which adversely affects the tumor marker according to Table 1 of the invention.
  • such a behavior may occur if the antimorphic variant can interact with the tumor marker according to Table 1 but blocks some aspect of its function.
  • such variants may comprise or lack specific domains of the tumor marker according to Table 1, e.g. one or more protein-protein interacting or dimerization domains, complex assembly domains, one or more membrane-associated domains etc.
  • This is particularly of importance in a protein that functions as a dimer or multimer. If, for example, one part of that protein complex is mutant in some functional aspect of the multimer but is still able to form the multimer it may have a dominant effect on the other wildtype portions of the complex, and a negative effect if the mutation prevents the complex from carrying out its normal function.
  • a dominant-negative form can specifically block the action of the wild-type tumor-marker from which it was derived.
  • Tests to identify dominant negative variants include appropriate genetic screenings, for instance readout-systems based on the expression of nucleic acids comprising the nucleotide sequence as indicated in section D) of Table 1 and/or functional assays of the proteins and or polypeptides of the invention comprising the amino acid sequence as indicated in section E) of Table 1 or derivatives thereof.
  • miRNA A specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 refers to a short single-stranded R A molecule of typically 18-27 nucleotides in length, which regulate gene expression of one or more of the tumor marker according to Table 1.
  • miRNAs are encoded by genes from whose DNA they are transcribed but are not translated into a protein. In a natural context miRNAs are first transcribed as primary transcripts or pri-miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the
  • Microprocessor complex consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). After integration into an active RISC complex, miRNAs may base pair with their complementary mRNA molecules and inhibit translation or may induce mRNA degradation by the catalytically active members of the RISC complex, e.g. argonaute proteins. Mature miRNA molecules are typically at least partially
  • miRNAs according to the present invention may be 100% complementary to their target sequences. Alternatively, they may have 1, 2 or 3 mismatches, e.g. at the terminal residues or in the central portion of the molecule. miRNA molecules according to the present invention may have a length of between about 18 to 27 nucleotides, e.g. 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides. Preferred are 21 to 23 mers. miRNAs having 100% complementarity may preferably be used for the degradation of nucleic acids according to the present invention, whereas miRNAs showing less than 100% complementarity may preferably be used for the blocking of translational processes.
  • antisense molecule of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 refers to nucleic acids corresponding to the sequences indicated in section D) of Table 1 or the complementary strand thereof.
  • the antisense molecule of the invention comprises a sequence complementary to at least a portion of a tumor marker expression product according to the present invention. While antisense molecules complementary to the coding region sequence of tumor marker expression products may be used, those complementary to the transcribed and untranslated region are preferred. Generally, antisense technology can be used to control, i.e. reduce or abolish gene expression through antisense DNA or RNA, or through triple-helix formation. In one embodiment, an antisense molecule may be generated internally by the organism, for example intracellularly by transcription from an exogenous sequence. A vector or a portion thereof may be transcribed, producing an antisense nucleic acid of the invention.
  • Such a vector would contain a sequence encoding the antisense nucleic acid of the invention.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense molecule.
  • Corresponding vectors can be constructed by recombinant DNA technology methods known to the person skilled in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells, e.g. vectors as defined herein above.
  • the antisense molecule may be separately administered.
  • the 5' coding portion of a nucleic acid according to the present invention e.g. of the sequence indicated in section D) of Table 1 may be used to design an antisense RNA or DNA oligonucleotide of from about 6 to 50 nucleotides in length.
  • the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides in length.
  • the antisense nucleic acids of the invention typically comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest.
  • absolute complementarity although preferred, is not required.
  • a person skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • antisense molecules complementary to the 5' end of the transcript e.g., the 5' untranslated sequence up to and including the AUG initiation codon may be used in for the inhibition of translation.
  • sequences complementary to the 5' end of the transcript, e.g., the 5' untranslated sequence up to and including the AUG initiation codon may be used in for the inhibition of translation.
  • An antisense molecule according to the present invention may be DNA or
  • An antisense molecule preferably an antisense olignucleotide or any further antisense nucleic acid molecule according to the present invention or a siR A molecule according to the present invention or any other ncR A molecule according to the present invention as defined herein above can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the molecule may include other appended groups such as peptides (e.
  • the molecule may accordingly be conjugated to another molecule, e. g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense molecule or antisense oligonucleotide, miR A- or siRNA molecule may comprise at least one modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethyl-aminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methyl guanine, 3 -methyl cytosine, 5-methylcytosine, N6-adenine, 7- methyl guanine, 5-methylaminomethyluracil, 5-methoxya
  • the molecule may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2- fluoroarabinose, xylulose, and hexose.
  • the molecule comprises alternatively or additionally at least one modified phosphate backbone, e.g. a
  • phosphorothioate a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense molecule e.g. the antisense oligonucleotide may be an alpha-anomeric oligonucleotide, i.e. an oligonucleotide which forms specific double-stranded hybrids with complementary RNA in which the strands run parallel to each other.
  • siRNA specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 refers to a particular type of antisense- molecules, namely small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway to negatively regulate gene expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • RNAi RNA interference
  • These siRNA molecules can vary in length and may be between about 18-28 nucleotides in length, e.g. have a length of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides.
  • the molecule has a length of 21, 22 or 23 nucleotides.
  • siRNA molecule may contain varying degrees of complementarity to their target mRNA, preferably in the antisense strand.
  • siRNAs may have unpaired overhanging bases on the 5' or 3' end of the sense strand and/or the antisense strand.
  • the term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.
  • the siRNA may be double-stranded wherein the double-stranded siRNA molecule comprises a first and a second strand, each strand of the siRNA molecule is about 18 to about 23 nucleotides in length, the first strand of the siRNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA via RNA interference, and the second strand of said siRNA molecule comprises nucleotide sequence that is complementary to the first strand.
  • siRNA molecules may be obtained according to methods of identifying antagonists as described herein.
  • aptamer specific for the expression product or specific for the protein of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 refers to (a) short peptide(s) capable of interacting and specifically binding the protein(s) of the tumor marker according to Table 1.
  • the peptide aptamer(s) may preferably be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (the) amino acid sequence(s) as indicated in section E) of Table 1.
  • the peptide aptamer(s) may also be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (an) amino acid sequence(s) encoded by (a) DNA sequence(s) being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as indicated in section D) of Table 1 , or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as indicated in section E) of Table 1.
  • (a) peptide aptamer(s) is/are a variable peptide loop, comprising for example, 10 to 20 amino acids.
  • the peptide aptamer(s) may preferably be attached at one or both ends to a scaffold structure.
  • the scaffold structure may be any molecule, preferably a protein, which has good solubility properties. Suitable scaffold molecules would be known to the person skilled in the art.
  • a preferred scaffold molecule to be used in the context of the present invention is the bacterial protein thioredoxin-A.
  • the aptamer peptide loop may preferably be inserted within a reducing active site of the scaffold molecule.
  • staphylococcal protein A and domains thereof and derivatives of these domains, such as protein Z or lipocalins may be used as scaffold structures in the context of the present invention.
  • Peptide aptamers may be generated according to any suitable method known to the person skilled in the art, e.g. via yeast two-hybrid approaches.
  • the above mentioned peptide aptamer is capable to bind to a protein or polypeptide of the invention corresponding to the sequences indicated in section E) of Table 1 and to reduce the biological activity and/or the enzymatic activity of these/this protein(s) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or by at least 98% or 99% when compared to a control level obtained from an untreated sample.
  • the compound may be a natural compound, e.g. a secondary metabolite or an artificial compound, which has been designed and generated de novo.
  • a small molecule is capable of blocking the interaction between the tumor marker protein(s) and its interactor(s).
  • peptidomimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 refers to a small protein-like chain designed to mimic a peptide and capable of binding (a) protein(s) of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • a peptidomimetic may arise from a modification of an existing peptide, e.g. a peptide or peptide aptamer as defined herein above, in order to alter the molecule's properties.
  • a peptidomimetic may arise from a modification which changes the molecule's stability or binding capability. These modifications typically involve changes to the peptide that will not occur naturally.
  • a peptidomimetic according to the present invention may have altered peptide backbones or may comprise non-natural amino acids.
  • Methods and techniques for the preparation of peptidomimetics as well as assays for the testing of peptidomimetics are known to the person skilled in the art.
  • a pharmaceutical composition according to the present invention may also comprise an antibody specific for the protein(s) of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an antibody variant specific for said protein, e.g. an antibody or antibody variant as defined herein above.
  • such an antibody or antibody fragment may be capable of inhibiting the biological activity and/or enzymatic activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • the antibody or fragment thereof as defined herein above may be conjugated to a therapeutic or cytotoxic agent.
  • therapeutic agent refers to any compound, drug, small molecule or medicament, which is able to confer a therapeutic effect to a cell, a tissue or the entire organism. Examples of such agents are known to the person skilled in the art.
  • cytotoxic agent refers to any compound, drug, small molecule which is able to confer a toxic effect to a cell or a tissue.
  • Such agents may, for example, comprise compounds which activate endogenous cytotoxic effector systems, as well as radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death.
  • the term may also include radioisotopes known in the art, additional antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, R Ase, alpha toxin, ricin, abrin,
  • Pseudomonas exotoxin A diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin.
  • the term also refers to cytotoxic produgs.
  • cytotoxic prodrug is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound.
  • Cytotoxic prodrugs that may be used according to the invention include glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,
  • daunorubicin and phenoxyacetamide derivatives of doxorubicin.
  • the pharmaceutical composition may comprise, or may additionally comprise, an antibody or a group of antibodies specific for the expression product or protein of a tumor marker or group of tumor markers as defined herein, e.g. specific for one or more of the protein(s) of the tumor marker according to Table 1.
  • an antibody which specifically binds to an expression product, protein or peptide comprising the amino acid sequence as indicated in section E) of Table 1, or a fragment of said amino acid sequence, e.g. a peptide of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids length.
  • the present invention relates to a pharmaceutical composition for use in, or for the treatment or prevention of a neoplastic disease, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of a tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of: (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and
  • Such pharmaceutical composition preferably comprises elements as defined herein above.
  • a pharmaceutical composition may comprise elements or ingredients as defined herein above according to the up-regulation indication provided in any of sections P), R), T) or V) of Table 1.
  • a pharmaceutical composition may preferably comprise any of the above mentioned elements (a) to j) specifically for tumor markers which are indicated with a "+" in any of sections P), R), T) or V) of Table 1.
  • a pharmaceutical comprising at least one element selected from the group of: (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and (e) a miRNA inhibitor specific for a miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 refers to a compound which is capable of increasing the activity of one or more of the tumor marker according to Table 1.
  • a compound may be any direct interactor of the tumor marker according to Table 1 , which has positive influence on the catalytic activity of the tumor marker according to Table 1.
  • Such a compound may preferably be an agonist of the catalytic activity of the tumor marker according to Table 1.
  • a compound indirectly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 refers to a compound which is capable of increasing the activity of the tumor marker according to Table 1 by an interaction with a direct interactor of the tumor marker according to Table 1 ("indirect interactor") or via an indirect working pathway not involving an interaction with of the tumor marker according to Table 1.
  • Such a compound may be any direct interactor of an interactor of the tumor marker according to Table 1, e.g. of the protein(s) of a tumor marker or group of tumor markers according to section E) of Table 1.
  • the effect conveyed by the direct interactor of an interactor of the tumor marker according to Table 1 may be either positive if the interactor of the tumor marker according to Table 1 itself has a positive effect on the activity of the tumor marker according to Table 1, or negative, if the interactor of the tumor marker according to Table 1 has a negative effect on the activity of the tumor marker according to Table 1.
  • such positively working indirect integrators may provoke a modification of the binding behavior of directly binding proteins, leading to an increased activity of the tumor marker according to Table 1.
  • negatively working indirect interactors may have an inhibitory effect on inhibitors of the tumor marker according to Table 1. Examples of such interactors are enzymatic activities degrading inhibitors of the tumor marker according to Table 1, or proteins capable of binding and quenching inhibitors of the tumor marker according to Table 1.
  • such interactors may inhibit activities leading to a degradation of the tumor marker according to Table 1, e.g. proteinase inhibitors. Further examples and their implementation would be known to the person skilled in the art.
  • an indirect stimulation of the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be conveyed by compounds activating, protecting or sustaining the expression of the endogenous gene(s) of the tumor marker according to Table 1.
  • compounds activating, protecting or sustaining the expression of the endogenous gene(s) of the tumor marker according to Table 1.
  • examples of such compounds are specific transcription factors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, specific stabilizing activities of the mR A(s) of the the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or splice factors specific for the the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Further examples and their implementation would be known to the person skilled in the art.
  • the "protein of a tumor marker” comprised in the pharmaceutical composition may be a protein of the tumor marker according to Table 1 as defined herein above.
  • it may be a protein being encoded by splice variant of the tumor marker according to Table 1. More preferably it may have the amino acid sequence as set forth in section E) of Table 1.
  • the "protein of a tumor marker" as used in this context also comprises amino acid sequences being at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%) or 99% identical to the sequences as set forth in section E) of Table 1 and amino acid sequences being encoded by nucleotide sequences being at least 60%>, 70%>, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as indicated in section D) of Table 1.
  • the homologous variants of the tumor marker according to Table 1 may additionally or alternatively have a similar or identical localization pattern as the tumor marker according to Table 1 within a cell or within a tissue type.
  • biologically active equivalent of a tumor marker refers to a protein of the tumor marker according to Table 1 which is capable of performing all or a majority of the individual functions of the tumor marker according to Table 1.
  • biologically active equivalents of the tumor marker according to Table 1 may additionally or alternatively have a similar or identical localization pattern as the tumor marker according to Table 1 within a cell or within a tissue type.
  • Biologically active equivalents of the tumor marker according to Table 1 may also comprise variants of the tumor marker according to Table 1 as defined herein above.
  • the proteins of the tumor marker according to Table 1 or biologically active equivalents of the tumor marker according to Table 1 according to the present invention may be produced recombinantly by any suitable method known to the person skilled in the art.
  • the present invention thus, also encompasses methods for the production of the tumor marker proteins according to Table 1 or biologically active equivalents of the tumor markers according to Table 1.
  • the present invention contemplates vectors containing the polynucleotides encoding the tumor markers according to Table 1 or biologically active equivalents of the tumor markers according to Table 1 as defined herein above, host cells, and the production of the tumor markers according to Table 1 or biologically active equivalents of the tumor markers according to Table 1 by recombinant techniques.
  • a suitable vector may be, for example, a phage, plasmid, viral, or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the vectors may preferably comprise one or more of the nucleotide sequences indicated in section D) of Table 1.
  • Polynucleotides encoding the tumor markers according to Table 1 or biologically active equivalents of the tumor markers according to Table 1 may be joined to a vector or carrier containing a selectable marker for propagation in a host.
  • a corresponding polynucleotide insert may be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, or the PSA promoter. Other suitable promoters are known to the person skilled in the art.
  • the expression constructs may further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be
  • polypeptides or proteins may be glycosylated or may be non-glycosylated or may otherwise by modified.
  • polypeptides or proteins may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the polypeptide, protein or peptide may be modified by acetylation, pegylation, hesylation, formylation, phosphorylation, amidation, derivatization by known
  • polypeptide, peptide or variant may contain one or more non-classical amino acids.
  • tumor marker proteins according to Table 1 or biologically active equivalents of the tumor markers according to Table 1 of the invention can be chemically synthesized using techniques known in the art, e.g. by using a peptide synthesizer.
  • nucleic acid encoding and expressing the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 comprised in the pharmaceutical composition as defined herein above refers to any suitable carrier element, e.g. as described herein above, comprising an expressable gene of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • a carrier element may comprise the sequence(s) as indicated in section E) of Table 1.
  • Such a carrier element may also comprises nucleotide sequences showing a high degree of homology to the tumor markers according to Table 1, e.g.
  • nucleic acid sequences being at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as indicated in section D) of Table 1 or nucleic acid sequences encoding amino acid sequences being at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%o or 99% identical to the sequence(s) as indicated in section E) of Table 1.
  • the carrier may comprise the genomic sequence of the tumor marker according to Table 1.
  • biologically active equivalents of the tumor markers according to Table 1 as defined herein above may be comprised in a carrier of the present invention.
  • the polynucleotide encoding the tumor marker according to Table 1 may preferably be joined to a vector containing a selectable marker for propagation in a human cell.
  • the polynucleotide insert may be operatively linked to a PSA promoter.
  • nucleic acids encoding and expressing the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be provided via living therapeutics.
  • living therapeutic means that the tumor markers according to Table 1 or biologically active equivalents of the tumor markers according to Table 1 as defined herein above are expressed in any suitable live carrier.
  • the present invention relates to corresponding polynucleotides which are suitable for expression in a living cell.
  • the present invention also relates to vectors containing such polynucleotides, appropriate host cells, and the production of polypeptides by recombinant techniques in said host cells.
  • live carrier relates to any appropriate living host cell or virus known to the person skilled in the art.
  • appropriate hosts include, but are not limited to, bacterial cells such as Escherichia coli or Lactobacillus, fungal cells, such as yeast cells, protozoa, insect cells, or animal cells.
  • the term relates to attenuated bacteria, attenuated fungal cells or attenuated protozoa.
  • appropriate viruses include viruses of the group of adenoviruses, retrovirues or lentiviruses, preferably attenuated viruses of the group of adenoviruses, retroviruses or lentiviruses.
  • probiotic bacterial cells in particular probiotic Escherichia coli or Lactobacillus cells may be used. More preferably, cells of Escherichia coli Nissle 1973 and even more preferably cells of Lactobacillus casei or Lactobacillus zeae 393 may be used.
  • the "miRNA inhibitor specific for miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 comprised in the pharmaceutical composition as defined herein above refers to a nucleic acid molecule encoding a nucleic acid sequence complementary to a miRNA or microRNA molecule of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • the term “complementary” as used herein refers to a perfect complementary between the miRNA inhibitor nucleic acid (sense molecule) and the miRNA (antisense molecule) without any mismatch, as well as situations in which the nucleic acid contains any base mismatches and/or additional or missing nucleotides in comparison to the miRNA molecule.
  • the two molecules comprise one or more base mismatches or differ in their total numbers of nucleotides (due to additions or deletions).
  • the "complementary" miRNA inhibitor nucleic acid molecule comprises at least ten contiguous nucleotides showing perfect complementarity with a sequence comprised in the miRNA molecule.
  • miRNA inhibitor nucleic acid molecules are naturally occurring DNA- or RNA-molecules or synthetic nucleic acid molecules comprising in their sequence one or more modified nucleotides which may be of the same type or of one or more different types.
  • such a miRNA inhibitor nucleic acid molecule comprises at least one ribonucleotide backbone unit and at least one deoxyribonucleotide backbone unit.
  • the miRNA inhibitor nucleic acid molecule may contain one or more modifications of the RNA backbone into 2'-0-methyl group or 2'-0-methoxyethyl group (also referred to as "2'-0-methylation”), which prevented nuclease degradation in the culture media and, importantly, also prevented endonucleolytic cleavage by the RNA-induced silencing complex nuclease, leading to irreversible inhibition of the miRNA.
  • Another possible modification which is functionally equivalent to 2'-0- methylation, involves locked nucleic acids (LNAs) representing nucleic acid analogs containing one or more LNA nucleotide monomers, as defined herein above.
  • Another class of silencers of miRNA expression to be used in the context of the present invention comprises chemically engineered oligonucleotides named "antagomirs", which represent single-stranded RNA molecules conjugated to cholesterol.
  • the molecules may comprise between 19 and 25 nucleotides.
  • the molecule comprises 20, 21, 22, 23 or 24 nucleotides. More preferably, the molecule comprises 23 nucleotides (further details may be derived from Krutzfeldt et al, 2005, Nature, 438: 685-689).
  • miRNA inhibitors as defined herein above may be provided in the form of expression vectors to be introduced into tissue or cells.
  • such vectors may also be introduced in living therapeutics as defined herein above.
  • RNAs may be produced from transgenes provided in the form of trans fection or transient expression vectors or carriers.
  • competitive miRNA inhibitors may be provided as transcripts expressed from strong promoters, containing more than one, preferably multiple, tandem binding sites to a microRNA of interest.
  • a demethylation agent may be comprised in the pharmaceutical composition according to the present invention.
  • the term "demethylation agent” as used herein refers to an agent capable of demethylating chromatine structures, preferably promoter regions, more preferably the promoter(s) of the tumor marker according to Table 1.
  • demethylation agents examples include 5-aza-2'-deoxycytidine and 5-azacytidine, which reactivate genes inappropriately silenced by structural chromatin changes that involve DNA methylation and which can reverse these changes and, therefore, restore principal cellular pathways. This typically results in gene re-expression and reversion of some aspects of the transformed state.
  • 5-azacytidine and 5-aza-2'-deoxycytidine typically inactivate DNA cytosine C5- methyltransferases through the formation of stable complexes between the 5-aza-2'- deoxycytidine residues in DNA and the enzyme, thereby mimicking a stable transition state intermediate when bound to the methyltransferase enzyme.
  • a further agent which may be comprised in a pharmaceutical composition according to the present invention, either per se or in combination with 5-aza-2'- deoxycytidine and/or 5-azacytidine, is trichostatin A (TSA).
  • TSA trichostatin A
  • the present invention relates to a pharmaceutical composition for use in, or for the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, wherein said neoplastic disease implies the decreased (down-regulated) expression of a tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of: (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid
  • Such pharmaceutical composition preferably comprises elements as defined herein above.
  • a pharmaceutical composition may comprise elements or ingredients as defined herein above according to the down-regulation indication provided in any of sections Q), S), U) or W) of Table 1.
  • a pharmaceutical composition may preferably comprise any of the above mentioned elements (a) to (e) specifically for tumor markers which are indicated with a "+" in any of sections Q), S), U) or W) of Table 1.
  • a pharmaceutical composition according to the present invention may further comprise additional compounds being active against neoplastic and/or cancer cells.
  • the pharmaceutical composition may further comprise hormone-inhibitors, preferably anti-androgens or androgen antagonists like spironolactone, cyproterone acetate, flutamide, nilutamide, bicalutamide, ketoconazole, finasteride or dutasteride.
  • the skilled person is aware of the fact that also a situation in which a given tumor marker of the invention is up-regulated in a neoplastic disease as defined herein above, e.g. in cancer while in parallel another tumor marker of the invention is down-regulated. It is thus an aim of the present invention to provide also pharmaceutical compositions wherein the pharmaceutical composition contains any combination of such elements as laid out above, e.g. the compounds, proteins, dominant negative proteins, nucleic acids, miR As, siRNAs, antisense RNAs, aptamers, antibodies, peptidomimetics and small molecules, and wherein the composition contains said elements being capable of down-regulating at least one tumor marker according to Table 1 and up-regulating at least one other tumor marker according to Table 1.
  • the pharmaceutical composition contains any combination of such elements as laid out above, e.g. the compounds, proteins, dominant negative proteins, nucleic acids, miR As, siRNAs, antisense RNAs, aptamers, antibodies, peptidomimetics and small molecules, and wherein the composition
  • the pharmaceutical in another preferred embodiment of the invention relates to the pharmaceutical compositions as laid out in the present description for the treatment of a neoplastic disease as defined herein above, preferably cancer, more preferably prostate cancer.
  • compositions is for the manufacture of a medicament for the treatment of a neoplastic disease, preferably cancer, more preferably prostate cancer.
  • the pharmaceutical compositions and medicaments of the present invention are capable of reducing the tumor volume of a given tumor, e.g. a prostate carcinoma, by at least 5%, 10%, 20%, 30%>, 40%>, 50%, 60%, 70%, 80%, 90%, or at least 95% when compared to an untreated control.
  • the present invention also envisages screening procedures and methods for the identification of an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, a compound directly stimulating or modulating the activity of the tumor marker according to Table 1 , an agonist of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 enzymatic activity, a miR A inhibitor specific for miR A(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, an antagomir, a demethylation agent specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , or a peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • Such screening procedures may comprise the steps of (a) producing cells which express the tumor marker or group of tumor markers according to Table 1 as a polypeptide either as secreted protein or on the cell membrane or as intracellular component, (b) contacting the polypeptide produced in step (a) with a test sample potentially containing an interacting molecule, e.g.
  • an aptamer specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, an agonist of enzymatic activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, and (c) indentifying an interacting molecule by observing binding and/or inhibition or modulation of the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • such screening procedures may comprise the steps of (a) contacting a test sample potentially containing a directly or indirectly interacting molecule, e.g. an aptamer specific for the transcript(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a miRNA inhibitor specific for miRNA(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , an antagomir, a demethylation agent specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 with one or more cells which express the tumor marker according to Table 1 as (a) transcript(s), (b) detecting the expression level of said sequence; and (c) indentifying an interacting molecule by observing binding or a modulation or reduction of the expression level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • the present invention also encompasses an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to
  • Table 1 an agonist of the enzymatic activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a miR A inhibitor specific for miRNA(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , an antagomir, a demethylation agent specific for of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and a
  • peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 obtainable or obtained by a screening procedure or method as described herein above.
  • the present invention relates to a pharmaceutical composition as defined herein above for the treatment or prevention of a neoplastic disease, preferably cancer.
  • the present invention relates to the use of (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (e) a miRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (g) a siRNA specific for a tumor marker or
  • the present invention relates to the use of (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and (e) a miR A inhibitor specific for a miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 for the preparation of a pharmaceutical composition for the neoplastic disease as defined herein above, preferably cancer, preferably for the treatment or prevention of a neoplastic disease as defined here
  • the present invention relates to a method of treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, in particular the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, comprising the administration of (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group
  • the present invention relates to a method of treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, in particular the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, comprising the administration of (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above above above
  • a pharmaceutical composition according to the present invention may be administered to a patient, subject or individual with the help of various delivery systems known to the person skilled in the art, e.g., via encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction may be topical, enteral or parenteral and may include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, inhalational, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e. g., oral mucosa, rectal and intestinal mucosa, etc.) or by inhalation and may be administered together with other biologically active agents. Administration can be systemic or local. A preferred method of local administration is by direct injection.
  • the pharmaceutical composition may be delivered directly to internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the site of interest.
  • the pharmaceutical composition may also be administered to disease sites at the time of surgical intervention.
  • the composition can be delivered in a controlled release system.
  • the pharmaceutical composition is in a form, which is suitable for oral, local or systemic administration.
  • the pharmaceutical composition is administered locally, orally or systemically.
  • the pharmaceutical composition comprises a therapeutically effective amount of the ingredients of the pharmaceutical composition of the present invention as defined herein above and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such a carrier is pharmaceutically acceptable, i.e. is non-toxic to a recipient at the dosage and concentration employed.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition of the invention can be formulated as neutral or salt forms.
  • the pharmaceutical composition may be administered directly or in combination with any suitable adjuvant known to the person skilled in the art.
  • the composition of the present invention can be administered to an animal, preferably to a mammal.
  • "Mammal” as used herein is intended to have the same meaning as commonly understood by one of ordinary skill in the art. Particularly, “mammal” encompasses human beings.
  • administered means administration of a therapeutically effective dose of the aforementioned composition.
  • therapeutically effective amount is meant a dose that produces the effects for which it is administered, preferably this effect is induction and enhancement of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
  • the exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
  • concentration of the active ingredients or compounds of a pharmaceutical composition according to the present invention may be further adjusted to the intended dosage regimen, the intended usage duration, the exact amount and ratio of all ingredients of the composition and further factors and parameter known to the person skilled in the art.
  • the active agents or compounds according to the present invention may be administered alone or in combination with other treatments.
  • the pharmaceutical composition of the present invention may be administered in combination with an anti-hormone treatment, e.g. an anti-androgen treatment.
  • composition of the present invention can also comprise any suitable preservative known to the person skilled in the art.
  • the preparations according to the invention may also comprise compounds, which have an antioxidative, free-radical scavenger, antierythematous, antiinflammatory or antiallergic action, in order to supplement or enhance their action.
  • active components of the pharmaceutical composition as defined herein above may be fused to a suitable carrier protein, e.g. to Ig Fc receptor proteins or polymeric Ig receptors.
  • a suitable carrier protein e.g. to Ig Fc receptor proteins or polymeric Ig receptors.
  • the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or biologically active equivalents thereof as defined herein above may be provided as fusion proteins.
  • the fusion partner may be provided at the N- or C-terminus.
  • composition according to the present invention is to be administered in the form of a live cell or living therapeutic as defined herein above
  • transformed and prepared cells may be administered to a patient in any suitable form known to the person skilled in the art.
  • living therapeutics may be administered in the form of a composition comprising a microorganism, e.g. a Lactobacillus as described above, in an amount between 10 ⁇ to 101 cells, preferably 10 ⁇ to 10 ⁇ cells.
  • the ratio between two or more ingredients in the pharmaceutical composition or medicament may be suitably adjusted according to the skilled person's knowledge.
  • Suitable assays may optionally be employed to help identify optimal ratios and/or dosage ranges for ingredients of pharmaceutical compositions of the present invention.
  • the precise dose and the ratio between the ingredients of the pharmaceutical composition as defined herein above to be employed in the formulation will, inter alia, depend on the route of administration, and the exact type of disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses or ingredient ratios may be extrapolated from dose-response curves derived from in vitro or (animal) model test systems.
  • a typical dose can be, for example, in the range of 0.001 to 1000 ⁇ g; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the present invention relates to a medical kit for the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising at least one element selected from the group consisting of (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1
  • kits that can be used in the context of the administration of the pharmaceutical composition as defined herein above.
  • a kit according to the present invention may be used for the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease.
  • the ingredients of a medical kit may, according to the present invention, be comprised in one or more containers or separate entities. They may preferably be formulated as pharmaceutical compositions or medicaments, more preferably they may be formulated as has been described herein above in the context of the pharmaceutical compositions of the present invention, e.g. they may comprise suitable pharmaceutical carriers etc. Particularly preferred are formulations for topical administration as mentioned herein above in the context of pharmaceutical compositions of the invention.
  • the medical kit according to the present invention may optionally also comprise a documentation which indicates the use or employment of the medical kit and its components.
  • instructions comprised in the medical kit of the present invention may comprise recommended treatment options, dosage regimens etc.
  • the medical kit may also comprise an instruction leaflet and/or may provide additional information on the use, dosage etc.
  • the medical kit of the present invention may be administered to a patient according to any suitable dosage regimen known to the person skilled in the art.
  • the medical kit or kit components may preferably be given once a week, more preferably 2 times, 3 times, 4 times, 5 times or 6 times a week and most preferably daily and or 2 times a day or more often, unless otherwise indicated.
  • the dosages may be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., several times a day.
  • a response to the treatment may be monitored using herein described methods and further methods known to those skilled in the art and dosages may accordingly be optimized, e.g., in time, amount and/or composition. Progress can be monitored by periodic assessment.
  • the medical kit is employed in co-therapy approaches, i.e. in co-administration with other medicaments or drugs, for example antibiotics, antiviral medicaments or IgG or IgA immunoglobulins, anticancer medicaments and, preferably, anti-hormone medicaments, more preferably anti- androgens as mentioned herein above.
  • medicaments or drugs for example antibiotics, antiviral medicaments or IgG or IgA immunoglobulins, anticancer medicaments and, preferably, anti-hormone medicaments, more preferably anti- androgens as mentioned herein above.
  • the present invention relates to a vaccine comprising the expression product or protein, or any fragment thereof, of an up-regulated tumor marker or group of tumor markers as defined herein, or as indicated in section P), R), T) or V) of Table 1, or in sections F), G), H) or I) of Table 3.
  • the vaccine may comprise a protein or antigen, having, comprising or consisting of an amino acid sequence as defined in section E) of Table 3, or any fragment, variant, derivative or modified form thereof, having a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 300, 350 or 400 amino acids or the entire length or any other suitable length.
  • Table 3 :
  • the vaccine may preferably comprise a nucleic acid molecule encoding a tumor marker of the present invention or a group of tumor markers as defined above, e.g. a nucleic acid molecule comprising a nucleotide sequence as indicated in section D) of Table 3, and/or a vector comprising said nucleic acid molecule, a host cell comprising said vector, an antibody as defined herein, or a CTL specific for an antigen as defined herein.
  • a nucleic acid molecule encoding a tumor marker of the present invention or a group of tumor markers as defined above, e.g. a nucleic acid molecule comprising a nucleotide sequence as indicated in section D) of Table 3, and/or a vector comprising said nucleic acid molecule, a host cell comprising said vector, an antibody as defined herein, or a CTL specific for an antigen as defined herein.
  • a vaccine according to the present invention may, for example, comprise polypeptides or proteins of varying length comprising the or comprised in the amino acids as indicated in section E) of Table 3, or a nucleotide sequence encoding such a polypeptide, an expression vectors capable of expressing the polypeptide or comprising said nucleic acid or fragments thereof, e.g. DNA plasmid vectors, viral vectors etc., host cells expressing such a
  • polypeptide preferably host cells expressing the polypeptide at the surface of the cell, or secrete the polypeptide.
  • These components or ingredients may be present either separately or in combination or in any sub-grouping or sub-combination of the mentioned items.
  • one, two, three or more different tumor marker antigens or proteins of the present invention may be present either separately or in combination.
  • the present invention relates to a vaccine for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of a tumor marker selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAPl 3), High affinity cGMP-specific 35-cyclic
  • AKAP6 A-kinase anchor protein 6
  • CNBG3 cyclic nucleotide-gated cation channel beta-3
  • PDE9A phosphodiesterase 9A
  • PRKARIB cAMP-dependent protein kinase type I-beta regulatory subunit
  • PDE4C cAMP-specific 35-cyclic phosphodiesterase 4C
  • Table 3 comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 3, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) of Table 3, or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) of Table 3, or any fragment thereof.
  • the expression product, polypeptide or antigen comprised in the vaccine may comprise one epitope or various epitopes, e.g. a MHC I and/or a MHC II epitope or two or more copies of each or combinations thereof.
  • Corresponding, i.e. encoding nucleic acid molecules may be provided, preferably in the form of DNA or RNA molecules, e.g. DNA vectors or expression vectors.
  • Such vectors may preferably be DNA plasmids or viral vectors.
  • Vectors, in particular viral vectors may be capable of replication or replication- impaired or non-replicating.
  • non-replicating or “replication-impaired” as used herein means not capable of replication to any significant extent in the majority of normal mammalian cells or normal human cells.
  • Viruses which are non-replicating or replication- impaired may have become so naturally (i.e. they may be isolated as such from nature) or artificially e.g. by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication.
  • Suitable viral vectors for use in a vaccine according to the present invention include non-replicating adenoviruses such as El deletion mutants, vectors based on herpes virus and Venezuelan equine encephalitis virus (VEE).
  • VEE Venezuelan equine encephalitis virus
  • Suitable bacterial vectors include recombinant BCG and recombinant Salmonella and Salmonella transformed with plasmid DNA.
  • Alternative suitable non- viral vectors include lipid-tailed peptides known as lipopeptides, peptides fused to carrier proteins such as KLH either as fusion proteins or by chemical linkage.
  • a vaccinia virus vector such as MVA or NYVAC may be used.
  • MVA vaccinia strain modified virus ankara
  • MVA is a replication impaired vaccinia strain with a good safety record. In most cell types and normal human tissues, MVA does not replicate; limited replication of MVA is observed in a few transformed cell types such as BHK21 cells.
  • Alternatives to vaccinia vectors include pox virus vectors, e.g. avipox vectors such as fowl pox or canarypox vectors. Particularly suitable as an avipox vector is a strain of canarypox known as ALVAC, and strains derived therefrom.
  • CTL means "cytotoxic T lymphocyte” and refers in particular to a CD4+ T lymphocyte, a CD8+ T lymphocyte or a natural killer cell. Preferably, the term refers to a CD8+ T lymphocyte or a natural killer cell.
  • a CTL may be genertad according to any suitable approach for the generation of a CTL, which specifically detects an antigen according to the present invention, preferably a protein having or comprised in a sequence as indicted in section E) of Table 3. Subsequently, such a CTL may perform a cytotoxic reaction, e.g. via the release of the cytotoxins perforin and granulysin, or induce an appoptotic cell reaction, e.g.
  • the making of CTLs typically comprises the presentation of antigens, preferably of MHC I specific antigenic peptides, to T cells, preferably to CD8+ T cells.
  • the antigen or antigenic peptide may be presented by a dendritic cell (DC), more preferably by a MHC I molecule present on a dendritic cell.
  • DC dendritic cell
  • T lymphocytes may subsequently be selected, enriched and/or expanded according to suitable methods known to the person skilled in the art.
  • T cells to be used for the production of CTLs according to the present invention may be derived from lymphoid tissue, preferably they may be obtained from a peripheral blood mononuclear cell (PBMC) cell fraction.
  • PBMCs may be extracted from whole blood using ficoll.
  • PBMC may be extracted from whole blood using a hypotonic lysis. Any other suitable method known to the person skilled in the art may also be used.
  • PBMCs to be used may be derived from blood obtained from blood donors.
  • autologous PBMCs may be used.
  • any suitable method known to the person skilled in the art may be used.
  • the present invention relates to such CTLs, as well as to a method of making CTLs specific for the antigen or tumor marker expression product or protein, or group of tumor marker expression products or proteins of the present invention, e.g. comprising the amino acid sequence as indicated in section E) of Table 3, which comprises the step of stimulating autologous T cells in vitro with dendritic cells loaded with a peptide derived from the antigen or a fragment thereof.
  • This procedure may be carried out according to any suitable procedure known in the art.
  • non-adherent PBMCs may be co-cultered in suitable medium, e.g. in human serum, preferably in Aim-V medium supplemented with 10% pooled human serum, with mature dendritic cells preincubated with peptides derived from the antigen of the present invention.
  • suitable medium e.g. in human serum, preferably in Aim-V medium supplemented with 10% pooled human serum, with mature dendritic cells preincubated with peptides derived from the antigen of the present invention.
  • suitable medium e.g. in human serum, preferably in Aim-V medium supplemented with 10% pooled human serum
  • mature dendritic cells preincubated with peptides derived from the antigen of the present invention.
  • the co-cultivation may be carried out according to suitable parameters, e.g. for 7- 10 days.
  • the preincubation with the peptides may also be carried out according to any suitable parameters known to the person skilled in the art.
  • T cells may be collected and preferably restimulated with dendritic cells loaded with peptides derived from the antigen.
  • the restimulation may preferably be carried out once in a week.
  • the medium may be supplemented with additional factors, e.g. with IL-2, IL-7 and/or IL-15.
  • T-cell lines may be established by limiting-dilutions.
  • T-cell line clones may be expanded in T-cell medium comprising IL-2, IL-7 and/or IL-15, e.g. during 2 weeks.
  • the CTLs may additionally be tested for their biological activity according to known methods, e.g. a cytotoxicity test.
  • CTLs may be stored or further expanded or be used for the preparation of medicaments of pharmaceutical compositions. Any suitable deviation from this protocol based on the knowledge of the skilled person is also envisaged by the present invention.
  • an epitope to be comprised in a vaccine may be of varying length. It may be a B-cell, T-cell, MHC I specific, or MHC II specific epitope. It may preferably have a length of about 8 to 10 amino acids in the case of a MHC I specific epitope e.g. 8, 9 or 10 amino acids, it may preferably have a length of about 13 to 17 amino acids in the case of a MHC II epitope, e.g. 13, 14, 15, 16 or 17 amino acids.
  • the epitope or antigen may be capable of eliciting B-cell or T-cell immune responses.
  • the epitope or antigen may alternatively be capable of eliciting CTL or cytotoxic reactions.
  • the presentation by MHC II molecules may be required.
  • the presentation by MHC I molecules may be required.
  • Antigens, peptides or epitopes as defined herein may also stimulate NK-cells (natural killer cells) that are effective tumor killing cells and may be used for such an approach.
  • Peptides or epitopes according to the present invention e.g. as defined herein, may also stimulate dendritic cells for enhancing antigenic stimulation of lymphocytes and may be used for such an approach.
  • the activation of cells e.g. T-cells, NK cells or dendritic cells, may be tested with suitable tests known to the person skilled in the art.
  • an ELISPOT assay as known to the person skilled in the art may be used.
  • the epitopes of the present invention may be present in a peptide, polypeptide, protein, polyprotein or particle comprising one, two or more epitopes, or as a recombinant string of epitopes or in the context of the native target antigen, or in the form of a mixture or combination of the mentioned entities.
  • polyprotein refers to two or more proteins which may be the same, or preferably different, linked together. Particularly preferred in this embodiment is a recombinant proteinaceous particle such as a Ty virus-like particle (VLP).
  • VLP Ty virus-like particle
  • epitope string refers to a juxtaposition or combination of one or more epitopes, e.g. of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more epitopes.
  • Such a string may comprise solely MHC I specific epitopes or MHC II specific epitopes or a combination of both.
  • the epitopes in a string of epitopes or of multiple epitopes may be linked together without intervening sequences so that unnecessary nucleic acid and/or amino acid material is avoided.
  • the epitopes in a string of epitopes or of multiple epitopes may be linked together with intervening sequences.
  • Such intervening sequences may have a length of 1 to 10 amino acids, preferably 2 to 5 amino acids. They may comprise amino acids without influence on the overall structure of the string, e.g. glycine.
  • antigens or fragments of antigens may be presented in a string like manner, e.g. linked together. These strings may or may not have intervening sequences as mentioned above.
  • the string of epitopes or antigens or multiple epitopes may include one or more epitopes recognised by T helper cells, to augment the immune response generated by the epitope or antigen string.
  • T helper cell epitopes are ones, which are active in individuals of different HLA types, for example T helper epitopes from tetanus (against which most individuals will already be primed).
  • T helper epitopes from tetanus againstst which most individuals will already be primed.
  • Particularly preferred is a combination of three T helper epitopes and an epitope according to the present invention.
  • the epitope string may also include one or more B cell epitopes for stimulating B cell responses and antibody production. Suitable T helper and B cell epitopes are known to the person skilled in the art.
  • the present invention relates to a vaccine as defined herein, or a CTL as defined herein, for the treatment or prevention of a neoplastic disease, preferably for the treatment or prevention of a cancer disease, more preferably of a cancer disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastsic or cancer disease.
  • a vaccine according to the present invention is for the treatment or prevention of a neoplastic or cancer disease, it may preferably comprise, in addition to antigens or epitopes as mentioned above, additional antigens or epitopes derived from or representing known tumor associated antigens (TAAs). Furthermore the string of epitopes or multiple epitopes of a corresponding vaccine may include one or more epitopes derived from or representing a known tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • TAAs which may be included in vaccines of the present invention are MAGE antigen, a SSX antigen family member, NY-ESO-1, Melan-A/MART-1, gplOO, tyrosinase, tyrosinase-related protein 1 (TRP1), TRP2, CEA, PSA, Her2/neu, p53, MUC1, PRAME, sarcosin (N-methylglycin), CA-125 (Carbophydrate antigen- 125) or survivin.
  • TRP1 tyrosinase-related protein 1
  • TRP2 tyrosinase-related protein 1
  • CEA tyrosinase-related protein 1
  • PSA Her2/neu
  • p53 Her2/neu
  • MUC1 Her2/neu
  • PRAME sarcosin
  • CA-125 Carbophydrate antigen- 125
  • survivin The sequence and identity of these and further suitable TAAs would be known to
  • the present invention envisages a method of inducing an immune response in an individual comprising administering to said subject a therapeutically effective amount of an antigen, vector, epitope etc. as mentioned above.
  • the term "immune response” refers to a therapeutic immune response beneficial for the subject or individual. Such an immune response may be a passive or active immunization, or it may be a short-term or long-term immunoprotection.
  • the immune response is an immune response against a neoplastic disease, e.g. a cancer disease.
  • the term "immune response against a neoplastic disease” means that a neoplastsic cell or tissue, e.g.
  • a cancerous cell or tissue may be attacked by components of the immune system, e.g. by CTLs or antibodies, that such a cell or tissue may be reduced in its size or modified in its structure by the mentioned entities or that such cell or tissue may be eliminated by the mentioned entities.
  • the present invention also envisages a method of identifying an individual for eligibility for neoplastic disease therapy or treatment or a corresponding immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease as defined above, wherein the detection of an increased level of expression of a tumor marker or group of tumor markers as described herein above, leads to, or is used for the preparation of a vaccine or of a method of vaccination comprising an antigen or epitope linked to the detected, increased tumor marker or group of tumor markers of the present invention.
  • the present invention thus also encompasses a personalized vaccine or method for vaccination based on the outcome of a method of identifying an individual for eligibility for a neoplastic disease therapy, or a corresponding immunoassay for stratifying an individual or cohort of individuals with such a disease.
  • the term "linked to” as used herein means that the tumor marker or group of tumor markers encountered to be increased according to parameters as defined herein may be used in the form of a protein sequences, e.g. comprising or comprised in the amino acid sequence as indicated in sections E) of Table 3, or in the form of a nucleotide sequence, e.g. comprising or comprised in the nucleic acid sequence as indicated in section D) of Table 3.
  • antigens or nucleic acid molecules may be provided in different suitable forms, e.g. as full length sequence, or in the form of fragments, in combination with one or more tumor marker s as defined herein, or in combination with known markers or TAA as described above.
  • a vaccine according to the present invention or vaccine compounds or ingredients as defined herein above may be given, e.g. in the course of a method for immunization or method of treatment of the present invention, once or more than one time, e.g. 2, 3, 4, 5, 6 or more times, preferably 2, 3 or 4 times according to any suitable vaccination scheme known in the art.
  • a vaccine or vaccine ingredient are given more than once a prime and boost administration may be pursued, e.g. a "prime” administration is followed by one or more "boosts" to achieve the desired effects.
  • the same composition or vaccine ingredient can be administered as the prime and as the one or more boosts.
  • different compositions or vaccine ingredients can be used for priming and boosting.
  • priming and boosting compositions or vaccines may comprise different combinations of vaccine ingredients, e.g. first a combination of a DNA plasmid and peptide or polypeptide, followed a DNA plasmid or vice versa etc.
  • prime and boost compositions may be different in terms of vectors to be used.
  • the priming composition may be a viral vector and the boosting composition may also be a viral vector, however derived from a different virus.
  • a prime composition may comprise a DNA or plasmid vector and the boost composition may comprise a viral vector, or vice versa.
  • prime boost schemes in which at least one of the vectors is replication-impaired or non-replicating.
  • a preferred carrier for a vaccine is a molecule that does not itself induce the production of antibodies harmful to the individual receiving the vaccine.
  • Suitable carriers are typically large, slowly metabolized macro molecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactive virus particles.
  • Such carriers are well known to those of ordinary skill in the art.
  • the antigen of the present invention e.g. an antigen comprising or comprised in a tumor marker protein or expression product as defined in Section E) of Table 3 may be conjugated to carrier elements such as a bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc.
  • the vaccine used according to the invention may also be provided in frozen, freeze-dried or lyophilized form, which may be thawed, or reconstituted, respectively, when needed.
  • the vaccine may comprise or be mixed with at least one suitable adjuvant.
  • Adjuvants which are preferred for vaccines comprise 1018 ISS, aluminium salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, Amplivax, AS 15, BCG, CP-870893, CpG7909, CyaA, dSLIM, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V,
  • Adjuvants may be combined in any suitable form and amount with the pharmaceutical composition, kit or vaccine of the present invention.
  • the use of an adjuvant may be adjusted to the concrete purpose of the treatment. Such a use may vary depending on the target cell or tissue, the administration way, treatment scheme etc.
  • a vaccine or immunological formulation may contain the immunogenic active substance at any suitable concentration, preferably at low concentrations, such as in an immunogenic amount ranging from 0.01 ⁇ g to 10 mg.
  • a suitable immunogenic dose may be chosen, e.g. in the range of from 0.01 ⁇ g to 750 ⁇ g, preferably 100 ⁇ g to 500 ⁇ g.
  • the vaccine according to the present invention may be provided in the form of a depot vaccine which is to be delivered to the organism over an extended period of time may.
  • the amount of ingredients may be higher such as from at least 1 mg to up to more than 10 mg.
  • a vaccine usually may be provided, for example, in ready-to-use syringes having a volume of from 0.01 to 1 ml, preferably 0.1 to 0.75 ml, of the concentrated solution, or suspension, respectively.
  • Vaccines of the present invention may be administered to a subject or individual by any suitable method, preferably via injection using either a conventional syringe or a gene gun, such as the Accell® gene delivery system. Delivery of DNA into cells of the epidermis is particularly preferred as this mode of administration provides access to skin-associated lymphoid cells and provides for a transient presence of DNA in the recipient. Both, nucleic acids and/or peptides and/or antibodies can be injected either subcutaneously, epidermally, intradermally, intramucosally such as nasally, rectally and vaginally, intraperitoneally, intravenously, orally or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, needle-less injection,
  • auxiliary agents for the vaccine formulation, e.g. an adsorbate or a suspended mixture of vaccine ingredient with the auxiliary agent is administered.
  • the vaccine is administered as a solution, or liquid vaccine, respectively, in an aqueous solvent.
  • tumor markers as described herein may be over- expressed or down-regulated upon progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease.
  • pharmaceutically active agents that down-regulate or increase expression of such tumor markers can be used to specifically treat such neoplatic diseses, including neoplastic diseases of the prostate.
  • compositions comprising such active agents have been described in detail above.
  • knowledge of over-expression or down-regulation of tumor markers can also be used to screen molecular libraries of potentially pharmaceutically active agents for identifying pharmaceutically active agents that may be used to specifically treat or prevent the neoplatic diseases escribed herein such as neoplatic diseases of the prostate.
  • the present invention in one embodiment thus relates to the use of a tumor marker or a group of tumor markers as described herein for identifying pharmaceutically active agents useful in the treatment or prevention of a neoplastic disease.
  • the present invention relates to the use of a tumor marker or a group of tumor markers as described herein for identifying pharmaceutically active agents useful in the treatment or prevention of a neoplastic diease of the prostate.
  • a tumor marker or a group of tumor markers as described herein for identifying pharmaceutically active agents useful in the treatment or prevention of a neoplastic diease of the prostate.
  • the present invention relates to the use of a tumor marker or a group of tumor markers as described herein for identifying pharmaceutically active agents useful in the treatment or prevention of prostate cancer.
  • the present invention thus e.g. also relates to a method for identifying pharmaceutically active agents which can be used to treat or prevent progression from a less progressed stage of a neoplastic disease or cancer stage to a more progressed stage of a neoplastic disease or cancer stage comprising at least the steps of:
  • step b) comparing the expression level of step b) and d);
  • step a) one may use a cell obtained from a healthy human being of from a patient suffering from a disease such as a cancer.
  • a disease such as a cancer.
  • One may also use a cultured cell line. In a preferred embodiment, these cells and cell lines are outside the human body
  • step c) one may subject such cells and cell lines to libraries of potentially pharmaceutically active agents.
  • libraries may be small molecule libraries, aptamer libraries, antibody libraries, siR A libraries, peptide libraries and the like.
  • Determining expression levels in steps b) and d) may be undertaken as is common in the art.
  • the potentially pharmaceutically active agents can then be classified as an agent that either increases or decreases expression of a tumor marker or a group of tumor markers as described herein. Such agents may be used directly to treat or prevent progression from a less progressed cancer stage to a more progressed cancer stage or they may be used as hits or lead compounds for further drug development.
  • the accordingly encountered active agents are also envisaged as part of the present invention.
  • An especially preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • neoplastic disease as defined herein above is a neoplastic disease of the prostate.
  • said neoplastic disease of the prostate is a benign prostate tumor, a malignant prostate tumor or prostate cancer.
  • a "malignant prostate tumor" is understood to encompass prostate cancer and vice versa.
  • neoplastic disease of the prostate refers to different stages and developmental transitions starting form a healthy prostate tissue and/or cells over benign tumor/lesion/growth/hyperplasia, to cancers of the prostate gland.
  • a “neoplastic disease”, “prostate tumor” or “prostate cancer” as used herein thus denotes a developmental stage of prostate tissue which can be classified according to the TNM classification by the International Union against Cancer (UICC).
  • UICC International Union against Cancer
  • the terms relates to a classification of prostate tissue pursuing the following T - Primary Prostate Tumor classification schedule:
  • Tic Tumor identified by needle biopsy (e.g., because of elevated PSA) T2. Tumor confined within prostate (Tumor found in one or both lobes by needle biopsy, but not palpable or visible by imaging, is classified as Tic)
  • Tumor involves one half of one lobe or less
  • Tumor involves more than half of one lobe, but not both lobes
  • T2c Tumor involves both lobes
  • T3 Tumor extends through the prostatic capsule (Invasion into the prostatic apex, or into (but not beyond) the prostate capsule, is not classified as T3, but as T2)
  • Tumor invades seminal vesicle(s) T4. Tumor is fixed or invades adjacent structures other than seminal vesicles: bladder neck, external sphincter, rectum, levator muscles, or pelvic wall Nl . Tumor invades regional lymph node(s)
  • T categories are physical examination, imaging, endoscopy, biopsy, and biochemical tests
  • N categories are physical examination and imaging tests
  • M categories are physical examination, imaging, skeletal studies, and biochemical tests
  • stages I to IV of prostate cancer correspond to the following scheme:
  • Stage II Tla; NO; MO; G2, 3-4, or
  • the term "benign prostate tumor” as used herein particularly refers to a prostate tumor which lacks all three of the malignant properties of a cancer, i.e. does not grow in an unlimited, aggressive manner, does not invade surrounding tissues, and does not metastasize.
  • a benign prostate tumor implies a mild and non-progressive prostate neoplastic or swelling disease lacking the invasive properties of a cancer.
  • benign prostate tumors are typically encapsulated, and thus inhibited in their ability to behave in a malignant manner.
  • a benign tumor or a healthy condition may be determined by any suitable, independent molecular, histological or physiological method known to the person skilled in the art.
  • prostate cancer in contrast thereto the term "prostate cancer” as used herein relates to a cancer of the prostate gland in the male reproductive system, which occurs when cells of the prostate mutate and begin to multiply out of control.
  • prostate cancer is linked to an elevated level of prostate-specific antigen (PSA).
  • PSA prostate-specific antigen
  • the term “prostate cancer” relates to a cancer showing PSA levels above 4.0.
  • the term relates to cancer showing PSA levels above 2.0.
  • PSA level refers to the concentration of PSA in the blood in ng/ml.
  • a "prostate cancer” as used herein may further have one of the following grade of Gleason score: Grade 1 (the cancerous prostate closely resembles normal prostate tissue.
  • the glands are small, well- formed, and closely packed), Grade 2 (the tissue still has well- formed glands, but they are larger and have more tissue between them), Grade 3 (the tissue still has recognizable glands, but the cells are darker. At high magnification some of these cells have left the glands and are beginning to invade the surrounding tissue), Grade 4 (the tissue has few recognizable glands. Many cells are invading the surrounding tissue), Grade 5 the tissue does not have recognizable glands. There are often just sheets of cells throughout the surrounding tissue).
  • the grading typically follows the Gleason grading as established by the ASCP.
  • prostate cancer is further linked to an elevated level of prostate-specific antigen (PSA).
  • PSA prostate-specific antigen
  • the term "prostate cancer” relates to a cancer showing PSA levels above 4.0. In another embodiment the term relates to cancer showing PSA levels above 2.0.
  • PSA level refers to the concentration of PSA in the blood in ng/ml.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said less progressed stage of the neoplastic disease is a healthy prostate tissue.
  • healthy prostate tissue refers to a tissue which shows none of the above mentioned characteristics of benign or maligant tumors, in particular of benign or malignant prostate tumorand represents physiologically normally active gland structures.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said more progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said less progressed stage of the neoplastic disease is a healthy prostate tissue and said more progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above.
  • Particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section P) of Table 1. Further, particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section Q) of Table 1.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • insignificant disease parameters refers to a clinical tumor stage ⁇ T2 and/or a Prostate Cancer Volume ⁇ 0.5 ml and/or a Gleason Score
  • ⁇ 6 preferably to a clinical tumor stage ⁇ T2 and a Gleason Score ⁇ 6 and/or a Prostate Cancer Volume ⁇ 0.5 ml, more preferably to a clinical tumor stage ⁇ T2 and a Gleason Score
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is of stage > T2 (UICC 2002 classification) as defined herein above.
  • the more progressed stage of prostate cancer may imply a stage T2, T2a, T2b or T2c, preferably T2, or higher (UICC 2002 classification) as defined herein above.
  • a further preferred embodiment of the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above, and said more progressed stage of the neoplastic disease is a prostate cancer of stage ⁇ T2 (UICC 2002 classification) as defined herein above.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said less progressed stage of prostate cancer is a local prostate cancer with insignificant disease parameters.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • significant disease parameters refers to a clinical tumor stage > T2 and/or a Gleason Score > 6 and/or a Prostate Cancer Volume > 0.5 ml, alternatively to a clinical tumor stage > T2 and a Gleason Score > 6 and/or a Prostate Cancer Volume > 0.5 ml, alternatively also to a clinical tumor stage > T2 and a Prostate Cancer Volume > 0.5 ml and a Gleason Score > 6.
  • the term "insignificant disease parameters" may refer to a clinical tumor stage ⁇ T2 and a Gleason Score ⁇ 6 and a Prostate Cancer Volume ⁇ 0.5 ml and the term “significant disease parameters” refers to a clinical tumor stage > T2 or a Gleason Score > 6 or a Prostate Cancer Volume > 0.5 ml.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is of stage > T2 (UICC 2002 classification) as defined herein above.
  • the more progressed stage of prostate cancer may imply a stage T2, T2a, T2b or T2c, preferably T2, or higher (UICC 2002 classification) as defined herein above.
  • a further preferred embodiment of the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above, and said more progressed stage of the neoplastic disease is a prostate cancer of stage ⁇ T2 (UICC 2002 classification) as defined herein above.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said less progressed stage of prostate cancer is a local prostate cancer with insignificant disease parameters.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • significant disease parameters refers to a clinical tumor stage > T2 and/or a Gleason Score > 6 and/or a Prostate Cancer Volume > 0.5 ml, alternatively to a clinical tumor stage > T2 and a Gleason Score > 6 and/or a Prostate Cancer Volume > 0.5 ml, alternatively also to a clinical tumor stage > T2 and a Prostate Cancer Volume > 0.5 ml and a Gleason Score > 6.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said less progressed stage of prostate cancer is a local prostate cancer with insignificant disease parameters, as defined herein above and wherein said more progressed stage of prostate cancer is a local prostate cancer with significant disease parameters, as defined herein above.
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer has a Gleason score ⁇ 6.
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is of stage ⁇ T2 (UICC 2002 classification).
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is of stage ⁇ T2 (UICC 2002 classification) and has a Gleason score ⁇ 6.
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer has a tumor volume of between about ⁇ 0.05 and about 3.0ml, e.g.
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer can be any one of the clinically classifiable stages, grades, and/or sub-steps as defined herein above, wherein said less progressed stage of prostate cancer has a tumor volume of between about ⁇ 0.05 ml and about 3.0 ml, e.g.
  • Prostate Cancer Volume refers to a calculated or measured volume of a prostate tumor or prostate swelling, which may be based on the assessment of the extension of the tumor/s welling in three dimensions and a subsequent determination of the volume. For example, such an assessment may employ suitable morphometric reconstruction techniques, as known to the person skilled in the art. Preferably, such a measurement follows any suitable method of cancer volumetric calculations known to the person skilled in the art, for example guidance as provided in D'Amico et al., Urology, 1997 Mar;49(3):385-91.
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is of stage ⁇ T2 (UICC 2002 classification) and has a Gleason score ⁇ 6, and has a tumor volume of ⁇ 0.5 ml.
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer has a Gleason score >6.
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is of stage > T2 (UICC 2002 classification) as defined herein above and/or a prostate cancer with a Gleason Score >7.
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is of stage > T2 (UICC 2002 classification) as defined herein above and/or a prostate cancer with a Gleason Score >6.
  • the less progressed stage of prostate cancer may imply a Gleason score of 1, 2, 3, 4, 5, or 6, preferably 6.
  • the less progressed stage of prostate cancer may imply a stage of Tl, Tla, Tib, Tlc,T2, T2, T2a, T2b or T2c, preferably T2 (UICC 2002 classification).
  • the less progressed stage of prostate cancer may imply a Gleason score of 1, 2, 3, 4, 5, or 6, preferably 6 and a stage of Tl, Tla, Tib, Tlc,T2, T2, T2a, T2b or T2c, preferably T2 (UICC 2002
  • the more progressed stage of prostate cancer may imply a Gleason score of 7, 8, 9 or 10, preferably 7.
  • the more progressed stage of prostate cancer may imply a stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification).
  • the more progressed stage of prostate cancer may imply a Gleason score of 7, 8, 9 or 10, preferably 7 and a stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification).
  • An even more preferred embodiment of the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc, wherein said less progressed stage of prostate cancer is of stage ⁇ T2 (UICC 2002 classification) as defined herein above and has a Gleason score ⁇ 6, and said more progressed stage of prostate cancer is of stage >T2 (UICC 2002 classification), as defined herein above and has a Gleason score >7.
  • Particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section T) of Table 1. Further, particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section U) of Table 1.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said less progressed stage of prostate cancer is a local prostate cancer with significant disease parameters, as defined herein above.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said more progressed stage of prostate cancer is an advanced prostate cancer disease with a level of PSA of about ⁇ 10 ng/ml.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said less progressed stage of prostate cancer is a local prostate cancer with significant disease parameters and wherein said more progressed stage of prostate cancer is an advanced prostate cancer disease with a level of PSA of about ⁇ 10 ng/ml, e.g. 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 9.5 ng/ml or any other value between about 0.5 up to 9.9999 ng/ml.
  • a level of PSA of about ⁇ 10 ng/ml, e.g. 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 9.5 ng/ml or any other value between about
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is of stage > T2 (UICC 2002 classification).
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer shows a level of PSA of about ⁇ 10 ng/ml, e.g. 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 9.5 ng/ml or any other value between about 0.5 up to 9.9999 ng/ml.
  • a level of PSA of about ⁇ 10 ng/ml, e.g. 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/m
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer shows a level of PSA of about ⁇ 10 ng/ml, e.g.
  • a further particularly preferred embodiment relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer can be any one of the clinically classifiable stages, grades, and/or sub-steps as defined herein above, wherein said more progressed stage of prostate cancer shows a serum level of PSA of >2 ng/ml, > 2.5 ng/ml, >3 ng/ml,> 3.5 ng/ml, >4 ng/ml, >4.5 ng/ml,> 5 ng/ml, >5.5 ng/ml, >6 ng/ml, >7 ng/ml, >8 ng/ml, >9 ng/ml, >9.5 ng/ml.
  • a further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is of stage >T3 (UICC 2002 classification) as defined herein above.
  • the less progressed stage of prostate cancer may imply a stage of T2, T2a, T2b or T2c, preferably T2a,but not T3 or higher pre-treatment.
  • the more progressed stage of prostate cancer may imply a stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification).
  • the more progressed stage of prostate cancer may imply a level of PSA of about ⁇ 10 ng/ml, e.g. 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 9.5 ng/ml or any other value between about 0.5 up to 9.9999 ng/ml, and a stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification).
  • a stage of PSA of about ⁇ 10 ng/ml, e.g. 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 9.5 ng/ml or any other value between about 0.5 up to
  • An even more preferred embodiment of the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc, wherein said less progressed stage of prostate cancer is of stage > T2 but not T3 or higher pre-treatment (UICC 2002 classification) as defined herein above, and said more progressed stage of prostate cancer is of stage > T3 (UICC 2002 classification) as defined herein above, with serum PSA ⁇ 10 ng/ml as defined herein above.
  • Particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section V) of Table 1. Further, particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section W) of Table 1.
  • the clinical samples were human plasma drawn and prepared under standardized conditions. Prepared plasma samples were stored in aliquots at -80°C until use.
  • Group 1 men were selected based on very low PSA values ( ⁇ 0.5 ng/ml), presumably healthy (i.e., no cancer).
  • Group 2 men were selected based on PSA levels >3 ng/ml, but negative biopsy ((i.e., presumably men with a benign prostate disease).
  • Group 3 men were selected based on PSA levels >3 ng/ml and cancer positive biopsy and pre-treatment stages T2 or less.
  • Group 4 men were selected on signs of clinical advanced cancer disease (clinical stage >T3).
  • Pool 1 (pool name: al) 15 individual samples, patient group 1 (healthy, controls)
  • Pool 2 (pool name: a2) 15 individual samples, patient group 1 (healthy, controls)
  • Pool 3 (pool name: bl) 15 individual samples, patient group 2 (benign prostate disease)
  • Pool 4 (pool name: b2) 15 individual samples, patient group 2 (benign prostate disease)
  • Pool 5 (pool name: cl) 15 individual samples, patient group 3A (local prostate disease, insignificant disease)
  • Pool 6 (pool name: c2): 15 individual samples, patient group 3B (local prostate disease, significant disease)
  • Pool 7 (pool name: dl): 15 individual samples, patient group 4 (advanced disease, PSA ⁇ 10 ng/ml)
  • Pool 8 (pool name: d2): 15 individual samples, patient group 4 (advanced disease, PSA >10 ng/ml).
  • FIG. 1 A corresponding list of clinical samples is provided in Fig. 1.
  • BSA Bovine Serum Albumin
  • the standards were measured after 20 minutes of incubation.
  • First step 4 ml sample per tube. Centrifugation for 15 minutes with 4000 rpm at 4°C. 3 ml sample were added to the filter tube. Centrifugation for 12 minutes with 4000 rpm at 4°C. 2 ml sample were added to the filter tube. Centrifugation for 15 minutes with 4000 rpm at 4°C. 3 ml of 100 mM TEAB were added to the filter tube. Centrifugation for 20 minutes with 4000 rpm at 4°C. 3 ml 100 mM of TEAB were added to the filter tube.
  • Samples were dried using a speedvac. The samples were dissolved in 300 ⁇ 1M TEAB (total sample volume app. 500 ⁇ ). Samples were sonicated for 5 minutes in an ultrasonic bath to dissolve all proteins.
  • the iTRAQ-8-plex Kit (25 Units) was thawed. This kit contains 8 labels and every label consists in 25 units. 1 Unit is intended for 50 ⁇ g proteins. There is one label for each sample. Half of the label was used since samples contain 625 ⁇ g total protein.
  • the samples contain (in total) 500 ⁇ 1 M TEAB and urea in water.
  • the percentage organic phase in the sample must be greater than 70%. Therefore the sample must be diluted with IPA.
  • Each label is dissolved in app. 558 ⁇ IPA. The half of that was used (279 ⁇ ), so 887 ⁇ pure IPA was added to the label. 100 ⁇ IPA was used to wash all tubes, so there is only 787 ⁇ IPA added to the label.
  • Sample pool 1 was pipetted into one 2 ml tube. Both tubes were rinsed with 100 ⁇ IPA and this was added to the 2 ml tube. The volume was app. 600 ⁇ . 787 ⁇ IPA were pipetted in a separate tube. 279 ⁇ of label 113 were mixed with the 787 ⁇ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
  • Sample pool 2 was pipetted into one 2 ml tube. The tube was rinsed with 100 ⁇ IPA and this was added to the 2 ml tube. The volume was app. 600 ⁇ . 787 ⁇ IPA were pipetted in a separate tube. 279 ⁇ of label 114 were mixed with the 787 ⁇ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
  • Sample pool 3 was pipetted into one 2 ml tube. Both tubes were rinsed with 100 ⁇ IPA and this was added to the 2 ml tube. The volume was app. 600 ⁇ . 787 ⁇ IPA were pipetted in a separate tube. 279 ⁇ of label 115 were mixed with the 787 ⁇ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
  • Sample pool 4 was pipetted into one 2 ml tube. The tube was rinsed with 100 ⁇ IPA and this was added to the 2 ml tube. The volume was app. 600 ⁇ . 792 ⁇ IPA were pipetted in a separate tube. 274 ⁇ of label 116 were mixed with the 792 ⁇ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
  • Sample pool 5 was pipetted into one 2 ml tube. The tube was rinsed with 100 ⁇ IPA and this was added to the 2 ml tube. The volume was app. 600 ⁇ . 810 ⁇ IPA were pipetted in a separate tube. 256 ⁇ of label 117 were mixed with the 810 ⁇ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
  • Sample pool 6 was pipetted into one 2 ml tube. The tube was rinsed with 100 ⁇ IPA and this was added to the 2 ml tube. The volume was app. 600 ⁇ . 740 ⁇ IPA were pipetted in a separate tube. 326 ⁇ of label 118 were mixed with the 740 ⁇ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
  • Sample pool 7 was pipetted into one 2 ml tube. The tube was rinsed with 100 ⁇ IPA and this was added to the 2 ml tube. The volume was app. 600 ⁇ . 790 ⁇ IPA were pipetted in a separate tube. 276 ⁇ of label 119 were mixed with the 790 ⁇ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
  • Sample pool 8 was pipetted into one 2 ml tube. The tube was rinsed with 100 ⁇ IPA and this was added to the 2 ml tube. The volume was app. 600 ⁇ . 823 ⁇ IPA were pipetted in a separate tube. 243 ⁇ of label 121 were mixed with the 823 ⁇ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
  • the samples were incubated for 2 hours at room temperature. After two hours samples were placed in a -80°C freezer.
  • Buffers Buffer A: 25 % ACN in H 2 0, pH 2,5 (Formic acid, app. 2 ml per liter).
  • Buffer B Buffer A + 1 M KC1 (74.5 gram in 1 liter).
  • Buffer B means 0.75 M KC1.
  • Samples were dissolved in 50 ⁇ Buffer A. Volume was app. 140 ⁇ . 100 ⁇ Buffer A were added to reach a volume of 240 ⁇ . 240 ⁇ were injected. 1 -minute fractions were collected. SCX-fractions 20 through 32 (from 20 minutes to 33 minutes) were collected in one tube for IEF. Samples were stored in a -80°C freezer.
  • Buffers Buffer A: H 2 0 + 0.1 % TFA.
  • Buffer B ACN + 0.1 % TFA.
  • SCX samples were thawed and speedvacced. Subsequently, samples were dissolved in buffer A., mixed and spun. The samples were sonicated for 5 minutes in an ultrasonic bath to dissolve all peptides. The samples were mixed and spun. 300 ⁇ were injected. The fractions were collected in a well plate. An example of a chromatogram is shown in Figure 8. All samples were placed in a -80°C freezer.
  • the 3100 OFFGEL fractionator was prepared (24 strip, pH 3 - 10). The general protocol of the 3100 OFFGEL fractionator was followed. The Peptide Offgel stock solution was used (for PEPTIDE IEF).
  • the eight iTRAQ labeled samples were thawed. Each sample was dissolved in
  • the fractions were dried in the Speed Vac and placed in the -80°C freezer.
  • Buffers 20 mM NH 4 FA, pH 10 (lOx solution) 12.5 gram NH 4 OH (28%-solution) + 900 ml H 2 0 + 1.62 ml FA (99%- solution).
  • Buffer A Stock solution was diluted lOx with H 2 0.
  • Buffer B Stock solution was diluted lOx with ACN.
  • Buffer A was purged for 5 minutes (5 ml/min).
  • Buffer B was purged for 5 minutes (5 ml/min).
  • the IEF fractions were dried in the speedvac for 16 hours
  • IEF 2 fraction 3 and 4 together. l-A-6, 250 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • IEF 3 fraction 5 and 6 together. l-A-7, 250 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • IEF 4 fraction 7 and 8 together. l-A-8, 250 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • IEF 5 fraction 9 and 10 together. l-A-9, 250 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • IEF 6 fraction 11 and 12 together. l-A-10, 250 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • IEF 7 fraction 13 and 14 together. 1-A-l 1, 250 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • IEF 8 fraction 15 and 16 together. 1-A-l, 250 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • IEF 9 fraction 17 and 18 together. l-A-2, 250 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • IEF 10 fraction 19 and 20 together. l-A-3, 250 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • IEF 11 fraction 21 and 22 together. l-A-4, 325 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • IEF 12 fraction 23 and 24 together. l-A-5, 350 ⁇
  • the High pH fractions 1-35 were stored in the -80°C freezer.
  • the fractions 1 through 34 of IEF 1 were thawed. Volume was reduced by using the speedvac.
  • IEF 1 A fractions 2 - 8- 14- 20- 26- 32
  • IEF 1 B fractions 3 - 9- 15 - 21 - 27- 33
  • IEF 1 C fractions 4 - 10 - 16 -22 -28 -34
  • IEF 1 D fractions 5 - 11 - 17 -23 -29
  • IEF 1 E fractions 6 - 12 -18- 24- -30
  • the samples were dried with the speedvac.
  • the dry samples were placed in the -80°C freezer.
  • the fractions 1 through 34 of IEF 2 were thawed. Volume was reduced by using the speedvac.
  • IEF 2 A fractions 2 - 8- 14- 20- 26- 32
  • IEF 2 B fractions 3 - 9- 15 - 21 - 27- 33
  • IEF 2 C fractions 4 - 10 - 16 -22 -28 -34
  • IEF 2 D fractions 5 - 11 - 17 -23 -29
  • IEF 2 E fractions 6 - 12 -18- -24- -30

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Abstract

The present invention relates to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises a tumor marker selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAP1), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAP13), High affinity cGMP-specific 35-cyclic phosphodiesterase 9A (PDE9A), cAMP-dependent protein kinase type I-beta regulatory subunit (PRKAR1B), and cAMP-specifϊc 35-cyclic phosphodiesterase 4C (PDE4C). The present invention further relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage comprising affinity ligands for the expression products of the tumor markers, to corresponding methods, and to the use of said tumor markers for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage. The present invention further relates to a corresponding immunoassay, to a method of identifying an individual for eligibility for a neoplastic disease therapy, as well as to a pharmaceutical composition based on the inhibition and/or activation of the expression of said tumor markers.

Description

NOVEL TUMOR MARKERS SELECTED FROM A GROUP OF cAMP-REGULATORY PATHWAY MEMBERS
FIELD OF THE INVENTION
The present invention relates to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises a tumor marker selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAP13), High affinity cGMP-specific 35-cyclic phosphodiesterase 9A
(PDE9A), cAMP-dependent protein kinase type I-beta regulatory subunit (PRKARIB), and cAMP-specific 35-cyclic phosphodiesterase 4C (PDE4C). The present invention further relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage comprising affinity ligands for the expression products of the tumor markers, to corresponding methods, and to the use of said tumor markers for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage. The present invention further relates to a corresponding immunoassay, to a method of identifying an individual for eligibility for a neoplastic disease therapy, as well as to a pharmaceutical composition based on the inhibition and/or activation of the expression of said tumor markers.
BACKGROUND OF THE INVENTION
Neoplasms are abnormal masses of tissue as a result of neoplasia, i.e. an abnormal proliferation of cells. The growth of such clones of cells exceeds, and is
uncoordinated with, that of the normal tissues around it. Neoplasms or neoplastic swellings usually cause lumps or tumors. Generally, a neoplasm can be benign, potentially malignant (constituting a pre-cancer condition), or malignant (constituting a cancer condition or indication a cancer disease). Typically, benign neoplasms include uterine fibroids and melanocytic nevi, which normally do not transform into cancer. Potentially malignant neoplasms may include carcinoma in situ, which may not invade and destroy tissue, but can, given enough time, transform into a cancer. The third type of neoplasms is a malignant neoplasm, i.e. a cancer in which a group of cells display uncontrolled growth, leading to the invasion of tissue and sometimes to metastasis. These three malignant properties of cancers differentiate them from benign tumors and potentially malignant neoplasms, which are self- limited and do not invade or metastasize.
Among men, the three most commonly diagnosed cancers are prostate, lung and colorectal cancer in developed countries. Particularly prostate cancer is the most common malignancy in European males. In 2002 in Europe, an estimated 225,000 men were newly diagnosed with prostate cancer and about 83,000 died from this disease.
Several different proteins or peptides have been associated with neoplastic diseases, in particular with cancer development. For instance, phosphodiesterase PDE7 has been shown to be linked to chronic lymphocytic leukemia. Yet, for many cancer types or cancer progression forms, in particular for early developmental stages, e.g. between benign tumors, premalignant states and early malignant states of a neoplastic disease, there is no adequate marker molecule available. One of the few examples of an early cancer marker is maspin, whose up-regulation is believed to precede the transition from premalignant prostate lesions to prostate cancer.
Despite these developments, the detection of cancer is still largely based on the use of traditional medical approaches, e.g. breast-self examination, Pap tests or digital rectal examination in the case of prostate cancer. A further diagnostic perspective for prostate cancer is based on the determination of prostate-specific antigen (PSA). However, PSA is not prostate cancer-specific and can be raised due to other circumstances, leading to a large number of false-positives. Furthermore, there will be an unpredictable number of false- negatives who later develop prostate cancer in the presence of a "normal" PSA test.
Therefore, there is a need for the provision of a new and effective, alternative diagnosis perspective for the detection, monitoring and prognostication of neoplastic diseases, in particular of early cancer forms, as well as for transitions into or between malignant cancer forms, in particular of prostate cancer. SUMMARY OF THE INVENTION
The present invention addresses this need and provides means and methods which allow the diagnosis and detection of a neoplastic disease, in particular neoplastic diseases of the prostate.
The above objective is accomplished by the recognition of novel tumor markers selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP- dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAP13), High affinity cGMP-specific 35- cyclic phosphodiesterase 9A (PDE9A), cAMP-dependent protein kinase type I-beta regulatory subunit (PRKAR1B), and cAMP-specific 35-cyclic phosphodiesterase 4C
(PDE4C). Using tissue or bodily fluid samples of patients representing different progression stages of neoplastic diseases these novel tumor markers could be identified and were shown to be differentially expressed, depending on the stage of progression of the neoplastic disease. In particular, it was demonstrated by the present inventors that the novel tumor markers are significantly down-regulated or up-regulated when comparing samples from patients having a less progressed stage of a neoplastic disease to samples from patients having a more progressed stage of the disease. Therefore, these markers are considered as markers for the prediction of neoplastic disease, in particular for the prediction of early cancer forms as well as for transitions into or between malignant cancer forms. These tumor markers may further serve as a decision tool for the stratification of certain neoplasm or cancer surveillance regimes, as well as for the prognosis and monitoring of neoplasm or cancer progression. Diagnostic methods and uses based on the tumor markers of the present invention can thus advantageously be employed for (i) detecting and diagnosing neoplastic disease forms, in particular forms of neoplastic diseases of prostate cancer, (ii)
prognosticating neoplastic disease forms, in particular neoplastic diseases of the prostate, (iii) monitoring of neoplastic disease progression towards more progressed disease stages, e.g. early malignant cancer forms, in particular of prostate cancer, and (iv) distinguishing between less and more progressed forms of neoplastic diseases, in particular less and more progressed forms of neoplastic diseases of the prostate. Finally, pharmaceutical compositions based on these tumor markers will provide novel therapeutic avenues in the treatment of cancer, in particular prostate cancer.
The present invention, thus relates in a first aspect to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8 or all tumor markers selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta- 3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1
(AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAPl 3), High affinity cGMP-specific 35-cyclic phosphodiesterase 9A (PDE9A),cAMP-dependent protein kinase type I-beta regulatory subunit (PRKARIB), and cAMP-specific 35-cyclic
phosphodiesterase 4C (PDE4C), indicated in Table 1.
In a preferred embodiment of the present invention the group of tumor markers consists of AKAP6 and/or CNGB3 and/or KGP2 and/or AKAPl and/or AKAP9 and/or AKAPl 3 and/or PDE9A and/or PRKARIB and/or PDE4C.
In another preferred embodiment of the present invention the expression of the marker(s) is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section P), R), T) or V) of Table 1.
In another preferred embodiment the expression of the marker(s) is decreased (down-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section Q), S), U) or W) of Table 1.
In another preferred embodiment of the present invention the p-value of the expression modification is 0.2 or lower, as indicated in section L) or M) of Table 1.
In a further aspect the present invention relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand for the expression product(s) or protein(s) of a tumor marker or a group of tumor markers as defined above.
In yet another aspect the present invention relates to a method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the step of determining the level of a tumor marker or a group of tumor markers as defined above, in a sample.
In a preferred embodiment the determining step of said method is accomplished by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of the tumor marker or group of tumor markers as defined above.
In another preferred embodiment of the present invention said method comprises the additional step of comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease.
In yet another preferred embodiment of the present invention said method is a method of graduating cancer, comprising the steps of:
(a) determining the level of a tumor marker or group of tumor markers as defined above in a sample by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers, and
(b) comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease; and
(c) deciding on the stage or developmental status of a neoplastic disease based on the results obtained in step (b).
In a further aspect the present invention relates to the use of said tumor marker or a group of tumor markers as defined above as a marker for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease.
In yet another aspect the present invention relates to an immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or for detecting, diagnosing, monitoring or prognosticating the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the steps of (a) testing in a sample obtained from an individual for the expression of a tumor marker or a group of tumor markers as defined above;
(b) testing in a control sample for the expression of the same tumor marker or group of tumor markers as in (a);
(c) determining the difference in expression of the tumor marker or group of tumor markers of steps (a) and (b); and
(d) deciding on the presence, stage or risk of recurrence of a neoplastic disease or the progression of the neoplastic based on the results obtained in step (c), wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or group of tumor markers as defined above.
In another aspect the present invention relates to a method of identifying an individual for eligibility for a neoplastic disease therapy comprising:
(a) testing in a sample obtained from an individual for the expression of an up-regulated tumor marker or group of tumor markers as defined above;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said up-regulated tumor marker or group of tumor markers as defined above;
(c) classifying the levels of expression of step (a) relative to levels of step
(b); and
(d) identifying the individual as eligible to receive a neoplastic disease therapy where the individual's sample is classified as having an increased level of expression of an up-regulated tumor marker or group of tumor markers as defined above.
In another aspect the present invention relates to a method of identifying an individual for eligibility for a neoplastic disease therapy comprising:
(a) testing in a sample obtained from an individual for the expression of a down-regulated tumor marker or group of tumor markers as defined above;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said down-regulated tumor marker or group of tumor markers as defined above;
(c) classifying the levels of expression of step (a) relative to levels of step
(b); and
(d) identifying the individual as eligible to receive a neoplastic disease therapy where the individual's sample is classified as having a decreased level of expression of a down-regulated tumor marker or group of tumor markers as defined above. In yet another aspect the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:
(a) testing in a sample obtained from an individual for the expression of an up-regulated tumor marker or group of tumor markers as defined above;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said up-regulated tumor marker or group of tumor markers as defined above;
(c) determining the difference in expression of said up-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said up-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b); and
(d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an increased level of expression of an up-regulated tumor marker or group of tumor markers as defined above.
In yet another aspect the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:
(a) testing in a sample obtained from an individual for the expression of a down-regulated tumor marker or group of tumor markers as defined above;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said down-regulated tumor marker or group of tumor markers as defined above;
(c) determining the difference in expression of said down-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said down-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b); and
(d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an decreased level of expression of an down-regulated tumor marker or group of tumor markers as defined above.
In a preferred embodiment said immunoassay or said method as mentioned above comprises the additional step of determining the level of prostate specific antigen (PSA). In an additional aspect the present invention relates to a pharmaceutical composition for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of an up-regulated tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of:
(a) a compound directly inhibiting the activity of a tumor marker as defined above, preferably an antagonist of said tumor marker enzymatic activity;
(b) a compound indirectly inhibiting the activity of a tumor marker as defined above;
(c) a dominant negative form of a protein of a tumor marker as defined above or a biologically active equivalent thereof;
(d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker as defined above;
(e) a miR A specific for a tumor marker as defined above;
(f) an antisense molecule of a tumor marker as defined above;
(g) a siR A specific for a tumor marker as defined above;
(h) an aptamer specific for the expression product of a tumor marker as defined above or for the protein of a tumor marker as defined above;
(i) a small molecule or peptido mimetic capable of specifically binding to the protein of a tumor marker as defined above; and
(j) an antibody specific for the protein of a tumor marker as defined above and/or an antibody variant specific for the protein of a tumor marker as defined above.
In another aspect the present invention relates to a pharmaceutical composition for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the decreased (down-regulated) expression of adown-regulated tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of:
(a) a compound directly stimulating or modulating the activity of a tumor marker as defined above, preferably an agonist of said tumor marker enzymatic activity;
(b) a compound indirectly stimulating or modulating the activity of a tumor marker as defined above; (c) a protein of a tumor marker as defined above or a biologically active equivalent thereof;
(d) a nucleic acid encoding and expressing a protein of a tumor marker as defined above; and
(e) a miR A inhibitor specific for a miR A of a tumor marker as defined above.
In a further aspect the present invention relates to a vaccine for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of a tumor marker selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAPl 3), High affinity cGMP-specific 35-cyclic
phosphodiesterase 9A (PDE9A), cAMP-dependent protein kinase type I-beta regulatory subunit (PPvKAPvlB), and cAMP-specific 35-cyclic phosphodiesterase 4C (PDE4C) as indicated in sections F), G), H) or I) of Table 3, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 3, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) of Table 3, or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) of Table 3, or any fragment thereof.
In another aspect the present invention relates to the use of a tumor marker or a group of tumo markers as defined above for identifying pharmaceutically active agents useful in the treatment or prevention of a neoplastic disease.
In a preferred embodiment the present invention relates to the use of a tumor marker or a group of tumo markers as defined above for identifying pharmaceutically active agents useful in the treatment or prevention of prostate cancer.
An especially preferred embodiment of the present invention relates to the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, the above mentioned pharmaceutical compositions, or the above mentioned vaccine wherein said neoplastic disease is a neoplastic disease of the prostate. In a particularly preferred embodiment of the present invention said less progressed stage of the neoplastic disease as mentioned in the context of the above defined tumor marker or group of tumor markers, the above defined compositions, the above defined methods, the above defined uses, the above defined immunoassays, the above defined pharmaceutical compositions or the above mentioned vaccine, is a healthy prostate tissue, and said more progressed stage is a benign prostate tumor.
In a further particularly preferred embodiment of the present invention said less progressed stage of the neoplastic disease as mentioned in the context of the above defined tumor marker or group of tumor markers, the above defined compositions, the above defined methods, the above defined uses, the above defined immunoassays, the above defined pharmaceutical compositions or the above mentioned vaccine is a benign prostate tumor, and said more progressed stage of the neoplastic disease is a prostate cancer of stage < T2 (UICC 2002 classification).
In a further particularly preferred embodiment of the present invention said less progressed stage of the neoplastic disease as mentioned in the context of the above defined tumor marker or group of tumor markers, the above defined compositions, the above defined methods, the above defined uses, the above defined immunoassays, the above defined pharmaceutical compositions or the above mentioned vaccine is a < T2 (UICC 2002 classification) prostate cancer with a Gleason score < 6, and said more progressed stage of the neoplastic disease is a > T2 (UICC 2002 classification) prostate cancer and/or a prostate cancer with a Gleason score > 7.
In yet another particularly preferred embodiment of the present invention said less progressed stage of the neoplastic disease as mentioned in the context of the above defined tumor marker or group of tumor markers, the above defined compositions, the above defined methods, the above defined uses, the above defined immunoassays, the above defined pharmaceutical compositions or the above mentioned vaccine is a >T2 (UICC 2002 classification) prostate cancer, and said more progressed stage of the neoplastic disease is a > T3 (UICC 2002 classification) prostate cancer with serum PSA < 10 ng/ml.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 depicts a list of clinical samples used for the experiments described the present application.
Fig. 2 shows the statistical distribution of sample collection dates (A) and age distribution (B) of clinical samples. Fig. 3 indicates the statistical distribution of PSA values of the clinical
samples with maximally 20ng/ml (A) or maximally 200ng/ml.
Fig. 4 indicates the statistical distribution of prostate volumes (A) or total size of cancer tissue in the biopsy core (B) of the clinical samples.
Fig. 5 shows the statistical distribution of total size of non-cancerous tissue in the biopsy core (A) or primary Gleason score groups 3 and 4 (B) and secondary Gleason score groups 3 and 4 (C) of the clinical samples.
Fig. 6 shows a corresponding Bradford standard curve using bovine serum albumin (BSA).
Fig. 7 shows an exemplary strong cation exchange chromatography (SCX)- Chromatogram of sample clean-up to remove unbound iTRAQ-labels.
Fig. 8 shows an exemplary chromatogram of a normal RPLC clean-up
analysis to remove salts introduced in the SCX clean-up step.
Fig. 9 depicts an exemplary chromatogram of a high pH RPLC for final separation of peptides.
DETAILED DESCRIPTION OF EMBODIMENTS
It was shown that tumor markers as depicted in Table 1 are differentially expressed, depending on the stage of progression of a neoplastic disease when testing samples of patients. In particular, it was demonstrated by the present inventors that tumor markers are statistically significant down-regulated or up-regulated when comparing samples from a less progressed stage to a more progressed stage of a neoplastic disease.
Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.
Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given.
As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise.
In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, and even more preferably ±5 %. It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of is considered to be a preferred embodiment of the term "comprising of .
If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.
Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. relate to steps of a method or use there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.
It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
As has been set out above, a first aspect of the present invention pertains to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8 or all tumor markers selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAP13), High affinity cGMP-specific 35-cyclic phosphodiesterase 9A (PDE9A),cAMP- dependent protein kinase type I-beta regulatory subunit (PRKARIB), and cAMP-specific 35- cyclic phosphodiesterase 4C (PDE4C), which are indicated in the following Table 1.
Table 1:
Table 1 cont.
NB: a2 = healthy tissue
b2 = benign prostate neoplastic disease or benign prostate tumor
cl = local prostate cancer, insignificant disease corresponding to a clinical tumor stage < T2 and Gleason Score < 6 and Prostate Cancer Volume < 0.5 ml
c2 = local prostate cancer, significant disease corresponding to a clinical tumor stage > T2 (UICC 2002 classification); and/or Gleason Score > 6; and/or Prostate Cancer Volume > 0.5 ml dl = advanced prostate cancer disease, with a level of PSA <10 ng/ml, corresponding to a clinical tumor stage > T3 (UICC 2002 classification)
d2= advanced prostate cancer disease, with a level of PSA >10 ng/ml, corresponding to a clinical tumor stage > T3 (UICC 2002 classification)
The term "marker" or "tumor marker", as used herein, relates to a gene, genetic unit or sequence (a nucleotide sequence or amino acid or protein sequence) as defined in Table 1, whose expression level is modified, i.e. decreased or increased, in a neoplastic cell, or in a neoplastic tissue, in particular a cancerous cell or cancerous tissue, or in any type of sample comprising neoplastic, in particular cancerous cells or neoplastic, in particular cancerous tissues or portions or fragments thereof, in comparison to a control level or state.
The term also refers to any expression product of said genetic unit or sequence or variants or fragments thereof, as well as homologues or derivatives thereof. The term specifically refers to the genes, genetic units, sequences, proteins, protein sequences, homologues and/or derivatives thereof indicated as marker #1 to #9 in Table 1, having the nucleotide or amino acid sequences of SEQ ID NOs: 1 to 18, respectively, wherein the uneven numbers indicate the nucleotide sequences and the following even numbers the corresponding amino acid sequences. The term also comprises corresponding genomic sequences as indicated in section F) of Table 1. Importantly, the term additionally comprises any known or yet unknown isoform (either as mRNA molecule or transcript or in the form of a polypeptide or protein), splice variant or corresponding derivative which can be derived from said genomic sequence. Corresponding information on the isoforms or splice variants would either be known by the person skilled in the art or could be retrieved with the help of suitable techniques, software tools etc. from databases or information depositories. The presence, size, form and/or identity of isoforms or splice variants may additionally be detected, determined and/or calculated with suitable tools known to the person skilled in the art. Furthermore, the size, identity, location etc. of intron and/or exon sequences and/or boundaries within said genomic sequences would also be known to the person skilled in the art. It is envisaged by the present invention that correspondingly identified intron and exon boundaries may be respected or used during the course of marker detection etc. as described herein.
A "marker" or "tumor marker" according to the present invention may also comprises nucleotide sequences showing a high degree of homology to a marker molecule as indicated in Table 1, in particular to the nucleotide sequence indicated in section D) of Table 1. Furthermore, a "marker" or "tumor marker" according to the present invention may comprise amino acid sequences or protein sequences showing a high degree of homology to a marker molecule as indicated in Table 1, in particular to the amino acid sequence indicated in section E) of Table 1. Nucleic acid sequences according to the present invention may be, for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the sequence as set forth in section D) of Table 1 , amino acid sequences may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section E) of Table 1, nucleic acid sequences encoding amino acid sequences may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%), 96%o, 97%), 98%o or 99%> identical to the sequence as set forth in section E) of Table, or amino acid sequences may be encoded by nucleic acid sequences being at least 75%>, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section D) of Table 1. Further, any variants, mutants and functional domains of the nucleic acid molecules, proteins and polypeptides as mentioned above may be
encompassed. The term "tumor marker gene" or "marker gene" as used herein thus relates to the gene encoding the tumor marker mentioned in Table 1. Preferably, the term relates to a gene expressing a tumor marker protein as indicated in Table 1, e.g. specific exon
combinations derivable from the indicated genomic sequence information of Table or as set forth in the sequences of section D) of Table 1. The term also relates to DNA molecules derived from mRNA transcripts encoding a tumor marker as indicated in Table 1 , preferably cDNA molecules.
A "gene", "genetic unit" or a "nucleotide sequence" is a nucleic acid sequence which may be transcribed under certain physiological or biochemical conditions. The transcribed nucleic acid may further (but must not necessarily) be translated under certain physiological or biochemical conditions into a polypeptide, e.g. when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence may be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
The terms "protein" or "polypeptide" are used herein to designate a produced or naturally occurring polypeptide or a recombinant polypeptide corresponding to the tumor marker as mentioned in Table 1. The term "protein" according to the present invention is to be seen as being interchangeably with the term "polypeptide". The polypeptides or proteins may be encoded by any of the above mentioned nucleic acid molecules. The polypeptides or proteins may further be glycosylated or may be non-glycosylated or may otherwise by modified. In addition, polypeptides or proteins may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. The term "neoplastic" as used in the context of the present invention refers to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, as well as any of a number of characteristic structural and/or molecular features. A "neoplastic cell" is accordingly understood as a cell having specific structural properties, lacking differentiation and in many instances, being capable of invasion and metastasis. Thus the term "neoplastic", "neoplastic disease", "neoplastic disease state" etc. refers to all diseases, disorders, clinical manifestations and/or symptoms characterized by uncontrolled, abnormal cellular proliferation, be it benign or malign in nature, that is, these terms refer to all benign hyperplasia, benign growths, benign lesions, benign tumors and the like as well as to all pre-malignant growths, swellings and lesions, malignant tumors, cancers, malignant cancers, metastases, metastasing cancers and the like.
The term "neoplastic" further also relates in the context of the present invention to a neoplastic disease state as defined herein below. The term "non-neoplastic" relates in the context of the present invention to a condition in which neither benign nor malign proliferation can be detected. Suitable means for said detection are known in the art.
The term "progression of a neoplastic disease" as used herein relates to a switch between different stages of development or genesis of a neoplastic disease and principally refers to a situation in which the neoplastic disease becomes worse and/or spreads in the body. Explicitly encompassed in this term is also the transition of healthy tissue and/or cells into a benign tumor, the transition of a benign tumor into a malignant
lesion/growth/hyperplasia or cancer due to uncontrolled, abnormal proliferation of cells and featuring any of a number of characteristic structural and/or molecular features, as well as the transition between stages or forms of malignant tumors or cancers, e.g. of prostate cancer.
Any changes that are associated with a worsening of the disease, i.e. be it the nature of the transformation (healthy to benign tumor/lesion/growth/hyperplasia, benign tumor/lesion/growth/hyperplasia to malignant tumor or cancer), aggressiveness of the cancer, the localization of the tumor and or /cancerous cells, the occurrence of metastases, presentation of additional or more profound clinical symptoms, recurrence of a tumor after treatment, decreased survival rate, are typically translated into a progression of stages of a given neoplastic disease.
Any worsening of the disease can thus be translated into a switch into the next stage of a given neoplastic disease. Any worsening of a neoplastic disease beyond the stage of a benign tumor, a benign lesion, benign growth, or hyperplasia is understood as a "cancer". Different classifications exist for such switches, e.g. stages 0 and I to IV of the TNM classification, preferably the TNM classification system for prostate cancer as defined herein below, or any other stage or sub-stage of any suitable graduating or scoring system, starting from a healthy condition up to a terminal cancer scenario. Typically such switches are accompanied by a modification of the expression level of the tumor marker or group of tumor markers according to Table 1 , preferably an increase or decrease of the expression level in a test sample in comparison to a previous test sample from the same individual, a test sample from an individual having been diagnosed with a certain type of a neoplastic disease as defined herein above and/or a stage or state of a neoplastic disease as defined herein above, or a value derivable from an information depository on expression data etc.
Progression of a neoplastic disease as defined herein above may further be determined, checked, crosschecked or independently be diagnosed etc. according to the "Gleason score". To determine the Gleason score, typically a grade is assigned to the most common tumor pattern, and a second grade to the next most common tumor pattern. The two grades are added together to get a Gleason score. The Gleason grade is also known as the
Gleason pattern or Gleason sum. The Gleason grade may range from 1 to 5, with 5 having the worst prognosis. The Gleason score typically ranges from 2 to 10, with 10 having the worst prognosis. Methods to apply the Gleason score system, corresponding assessment techniques etc. would be known to the person skilled in the art. The scoring system may be used, for example, in order to verify, check or fine-tune the diagnosis, detection, indication of stages or monitoring according to the present invention.
The term "less progressed stage" as used herein, relates to an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, is/are known or from the same subject at an earlier point in time, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 2 years, 3 years, 4 years, 5 years, 10 years before etc. ("expression level of a less progressed stage"). The term may also refer to an expression level corresponding to the stage of a neoplastic disease as defined herein above or a form of a neoplastic disease, whose disease state or stage is known. The term "disease state" relates to any state or type of cellular or molecular condition between a non-neoplastic or noncancerous cell state and/or healthy condition on the one hand and a terminal cancerous cell state on the other hand. The term, thus, includes benign tumor forms as well as malignant tumor forms. Preferably, the term includes different early proliferation/developmental stages or levels of tumor development in the organism between a non-cancerous cell state and (including) an advanced malignant cancerous cell state. For example, the above mentioned stages may include all stages of the histological grading as per the guidelines of the American Joint Commission on Cancer. As per their standards, one grading possibility is:
GX Grade cannot be assessed
Gl Well differentiated (Low grade)
G2 Moderately differentiated (Intermediate grade)
G3 Poorly differentiated (High grade)
G4 Undifferentiated (High grade)
Further envisaged is the alternative four-tier grading scheme:
Grade 1 Low grade; Well-differentiated
Grade 2 Intermediate grade; Moderately-differentiated
Grade 3 High grade; Poorly-differentiated
Grade 4 Anaplastic; Anaplastic
Also envisaged is the alternative three-tier grading scheme:
Grade 1 Low grade; Well-differentiated
Grade 2 Intermediate grade;
Grade 3 High grade; Poorly-differentiated;
or the two-tier grading scheme
Grade 1 Low grade; Well-differentiated
Grade 2 High grade; Poorly-differentiated.
Such developmental stages may also include all stages of the TNM (Tumor, Node, Metastasis) classification system of malignant tumors as defined by the UICC, e.g. stages 0 and I to IV. The stages may in particular include all TNM stages of prostate cancer as defined herein below. The term also includes stages before TNM stage 0, e.g.
developmental stages in which cancer biomarkers known to the person skilled in the art show a modified expression or expression pattern. In a specific embodiment of the present invention a less progressed stage may be a healthy state. In a further specific embodiment of the present invention a less progressed sate may also be a stage of malignant tumor development, e.g. malignant prostate cancer stages.
Corresponding expression levels or information about the expression level(s) of (a) less progressed stage(s) may be derived, for example, from experimental approaches or from a database of expression patterns or expression levels from previously tested subjects, tissues or cells or from any suitable source of information known to the person skilled in the art. In a specific embodiment of the present invention, the expression level of a less progressed stage can be determined from a reference sample derived from a subject who has been diagnosed to suffer from a certain cancer, and wherein the stage and development has been determined. Correspondingly obtained values and information may also be combined, normalized and statistically processed according to any suitable technique or method known to the person skilled in the art.
The term "more progressed stage" as used herein, relates to the reflection of any changes that are associated with a worsening of the disease as defined herein above, e.g. the aggressiveness of the cancer, the nature of the transformation (benign to malignant), the localization of the tumor and or /cancerous cells, the occurrence of metastases, the presentation of additional or more profound clinical symptoms, recurrence of a tumor after treatment, decreased survival rate, the modification of the expression of bio- or tumor markers, e.g. of tumor marker known to the person skilled in the art like PSAin comparison to a corresponding less progressed stage as defined herein above. Preferably, the term relates to a worsened disease state of a neoplastic disease as defined herein above, e.g. cancer or a malignant tumor in comparison to a less progressed stage as defined herein above. For example, if the less progressed stage is a stage 0 or a healthy state, the more progressed stage may be any higher or more advanced stage, e.g. a stage of any one of stage I, II, III or IV. Preferably, a "more progressed stage" as used herein may be the next worse stage if starting from a less progressed stage as defined herein above. For example, if the less progressed stage is a stage 0 or healthy or a benign tumor, the more progressed stage may be a stage I etc. The "next worse stage" may be reflected by any of the known staging and/or grading systems known to the person skilled in the art. Preferably, the "next worse stage" refers to the staging system provided by the UICC 2002 classification, more preferably the TNM classification for prostate cancer provided herein below. In a specific embodiment of the present invention the staging or grading of a tissue may optionally or additionally be determined, checked, crosschecked or independently be diagnosed by classical staging methods known to the person skilled in the art, e.g. via histological approaches, imaging methods etc.
In a specific embodiment of the present invention a more progressed stage may be determined by a comparison of the expression level of a tumor marker as indicated in Table 1 to a control level or control state of the same tumor marker. The term "control level" (or "control state"), as used herein, relates to an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, e.g. cancerous, non-cancerous, having a tumor, having no tumor, and whose disease stage(s) as defined herein above is/are known. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of the gene(s) of the tumor marker or group of tumor markers according to Table 1 in samples from subjects whose disease state is known. Furthermore, the control level can be derived from a database of expression levels or patterns from previously tested subjects or cells. Moreover, the control level may be multiple control levels whose control levels are determined from multiple reference samples. The control level may accordingly be derived from experimental approaches or from a database of expression levels from previously tested subjects, tissues or cells or from any suitable source of information known to the person skilled in the art. In a specific embodiment of the present invention, the control level can be determined from a reference sample derived from a subject who has been diagnosed to suffer from a certain neoplastic disease as defined herein above, preferably cancer, and wherein the stage and development of the neoplastic disease has been determined, as well as from healthy individuals.
In the context of the present invention, a control level determined from a biological sample that is known not to be neoplastic is called "normal control level". In another embodiment of the present invention, the control level can be from a neoplastic biological sample, e.g. a sample from a subject for which the neoplastic disease as defined herein above, preferably cancer, or prostate cancer, was diagnosed independently, it may be designated as "neoplastic control level". Alternatively, reference samples may comprise material derived from cell lines, e.g. immortalized cancer cell lines, or be derived from tissue xenografts. Preferably, material derived from prostate cancer cell lines or material derived from tissue xenografts with human prostate tissue, in particular with benign and tumor- derived human prostate tissue, may be comprised in a reference sample according to the present invention. Examples of cancer cell lines to be used comprise cells lines PC346P, PC346B, LNCaP, VCaP, DuCaP, PC346C, PC3, DU145, PC346CDD, PC346Flul,
PC346Flu2. Examples of xenografts which may be used comprise PC295, PC310, PC-EW, PC82, PC133, PC135, PC324 and PC374. Preferably an entire panel of cell lines and xenografts may be used, e.g. the human PC346 panel.
Correspondingly obtained values and information may also be combined, normalized and statistically processed according to any suitable technique or method known to the person skilled in the art. By comparing a control level to a measured expression level a modification of the expression may be registered, which may accordingly be used for the determination of the more progressed stage of a neoplastic disease as defined herein above. For such comparison processes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more different control levels may be determined or assessed. Accordingly, the less progressed stage of a neoplastic disease as defined herein above may be determined by assessing the outcome of such comparison process.
In a preferred embodiment of the invention said comparison process comprises the determination of control levels in a sample of an individual. In an even more preferred embodiment of the invention said comparison process comprises the determination of control levels in a sample of an individual afflicted with a neoplastic disease, e.g. cancer. In an even more preferred embodiment of the invention said comparison process comprises the determination of control levels obtained from a sample of an individual afflicted with a neoplastic disease, e.g. cancer, wherein the sample is representative of stages of the histological four-, three-, two-layer grading as per the guidelines of the American Joint Commission on Cancer, as defined herein above.
The comparison processes may further be combined with a comparison with the indications in sections G), K) and/or P) to W) of Table 1. Particularly preferred are control levels determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state is/are known to be prostate tumors, e.g. benign prostate tumors, or prostate cancer, more preferably prostate cancer of stage < T2 (UICC 2002 classification) or of stage >T3 (UICC 2002 classification), or prostate cancer of stage >T3 (UICC 2002 classification) and showing a serum level of PSA of about <10 ng/ml. Also preferred is a control level derived from a healthy tissue or based on expression values from healthy samples or healthy patients, e.g. patients who have independently been analyzed and were found to not suffer from any cancer or tumor, e.g. form prostate tumors or cancer.
The term "expression level" as used herein refers to the amount of any transcript and/or protein derivable from a defined number of cells or a defined tissue portion, preferably to the amount of a transcript and/or protein obtainable in a standard nucleic acid (e.g. RNA) or protein extraction procedure. Suitable extraction methods are known to the person skilled in the art.
The term "modified" or "modified expression level" in the context of the present invention thus denotes a change in the expression level. Expression levels are deemed to be "changed" when the gene expression of the tumor marker or group of tumor markers according to Table 1, e.g. in a sample to be analyzed, differs by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level or the expression level of a less progressed stage, as defined herein above, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level or the expression level of a less progressed stage as defined herein above.
The term "modified" as used throughout the specification relates preferably to a decrease or down-regulation or an increase or up-regulation of the expression level of the tumor marker or group of tumor markers according to Table 1 or a complete inhibition of the expression of the tumor marker or group of tumor markers according to Table 1 if a test sample is compared to a control level or the expression level of a less progressed stage as defined herein above.
In a preferred embodiment of the present invention the expression of the tumor marker(s) or group of tumor markers is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in any of sections P), R), T) and/or V) of Table 1. The term "increased" or
"increased expression level" or "up-regulated expression level" or "increase of expression level" (which may be used synonymously) in the context of the present invention thus denotes a raise in the expression level of the tumor marker or group of tumor markers according to Table 1 between a situation to be analyzed, e.g. a situation derivable from a patient's sample, and a reference point, which could either be a control level derivable from any suitable tumor, e.g. a prostate tumor, or neoplastic disease stage known to the person skilled in the art, e.g. a healthy state, a benign tumor stage or the expression of a less progressed stage as defined herein above. Particularly preferred is an increase or up- regulation between continuous steps in cancer development, e.g. next worse steps as mentioned herein above. Thus, an up-regulation may be present between a healthy tissue (e.g. indicated as pool a2 in Table 1) and a benign tumor tissue (e.g. indicated as pool b2 in Table 1). Alternatively, an up-regulation may be present between a benign tumor tissue (e.g.
indicated as pool b2 in Table 1) and a local cancer showing insignificant disease parameters (e.g. indicated as pool cl in Table 1). Alternatively, an up-regulation may be present between a local cancer showing insignificant disease parameters (e.g. indicated as pool cl in Table 1) and a local cancer showing significant disease parameters (e.g. indicated as pool c2 in Table 1). Alternatively, an up-regulation may be present between a local cancer showing significant disease parameters (e.g. indicated as pool c2 in Table 1) and an advanced cancer disease (e.g. indicated as pool dl in Table 1). Expression levels are deemed to be "increased" when the gene expression of the tumor marker or group of tumor markers according to Table 1, e.g. in a sample to be analyzed, differs by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%), 40%), 50%), or more than 50%> from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level or the expression level of a less progressed stage as defined herein above. In a particularly preferred embodiment of the present invention the tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5 or all tumor markers indicated with a "+" in section P) of Table 1, at least 1, 2, 3, 4 or all tumor markers indicated with a "+" in section R) of Table 1, at least 1, 2, 3 or all tumor markers indicated with a "+" in section T) of Table 1, at least 1, 2, 3, 4 or all tumor markers indicated with a "+" in section V) of Table 1. In a further preferred embodiment of the present invention the tumor marker or group of tumor markers comprises the first 1, 2, 3, 4, 5, 6 tumor markers indicated with a "+" in section P) of Table 1, the first 1, 2, 3, 4, 5 tumor markers indicated with a "+" in section R) of Table 1, the first 1, 2, 3, 4 tumor markers indicated with a "+" in section T) of Table 1, the first 1, 2, 3, 4, 5 tumor markers indicated with a "+" in section V) of Table 1.
In another preferred embodiment of the present invention the expression of the tumor marker(s) or group of tumor markers is decreased (down-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in any of sections Q), S), U) and/or W) of Table 1. The term "reduced" or "reduced expression level" or "down-regulated expression level" or "decrease of expression level" (which may be used synonymously) in the context of the present invention thus denotes a reduction of the expression level of the tumor marker or group of tumor markers according to Table 1 between a situation to be analyzed, e.g. a situation derivable from a patient's sample, and a reference point, which could either be a control level derivable from any suitable prostate tumor or neoplastic disease stage or cancer stage known to the person skilled in the art, e.g. a healthy state, a benign tumor stage or the expression of a less progressed stage as defined herein above. Particularly preferred is a decrease or down-regulation between continuous steps in cancer development, e.g. next worse steps as mentioned herein above. Thus, a down-regulation may be present between a healthy tissue (e.g. indicated as pool a2 in Table 1) and a benign tumor tissue (e.g. indicated as pool b2 in Table 1). Alternatively, a down-regulation may be present between a benign tumor tissue (e.g. indicated as pool b2 in Table 1) and a local cancer showing insignificant disease parameters (e.g. indicated as pool cl in Table 1). Alternatively, a down-regulation may be present between a local cancer showing insignificant disease parameters (e.g. indicated as pool cl in Table 1) and a local cancer showing significant disease parameters (e.g. indicated as pool c2 in Table 1).
Alternatively, a down-regulation may be present between a local cancer showing significant disease parameters (e.g. indicated as pool c2 in Table 1) and an advanced cancer disease (e.g. indicated as pool dl in Table 1). Expression levels are deemed to be "reduced" or "down- regulated" when the gene expression of the tumor marker or group of tumor markers according to Table 1 decreases by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level or the expression level of a less progressed stage as defined herein above. In a particularly preferred embodiment of the present invention the tumor marker or group of tumor markers comprises at least 1 , 2, or all tumor markers indicated with a "+" in section Q) of Table 1, at least 1, 2, 3 or all tumor markers indicated with a "+" in section S) of Table 1, at least 1, 2, 3, 4 or all tumor markers indicated with a "+" in section U) of Table 1, at least 1, 2, 3 or all tumor markers indicated with a "+" in section W) of Table 1. In a further preferred embodiment of the present invention the tumor marker or group of tumor markers comprises the first 1, 2, 3 tumor markers indicated with a "+" in section Q) of Table 1, the first 1, 2, 3, 4 tumor markers indicated with a "+" in section S) of Table 1, the first 1, 2, 3, 4, 5 tumor markers indicated with a "+" in section U) of Table 1, the first 1, 2, 3, 4 tumor markers indicated with a "+" in section W) of Table 1.
In a further embodiment of the present invention the group of tumor marker may comprise markers which are up-regulated and marker which are down-regulated, preferably at least one marker with an increased expression level in the more progressed stage and one marker with a decreased expression level in the more progressed stage. For example, the group of tumor marker may comprise at least one tumor marker indicated with a + in section P), R), T) or V) of Table 1 and at least one tumor marker indicated with a + in section Q), S), U) or W) of Table 1.
In a specific embodiment of the present invention the tumor marker or group of tumor markers may comprises at least 1, 2, 3, 4, 5 or all tumor markers indicated with a "+" in section P) of Table 1 or at least 1, 2, 3, 4 or all tumor markers indicated with a "+" in section R) of Table 1 or at least 1, 2, 3 or all tumor markers indicated with a "+" in section T) of Table 1 or at least 1, 2, 3, 4 or all tumor markers indicated with a "+" in section V) of Table 1 and at least 1, 2, or all tumor markers indicated with a "+" in section Q) of Table 1, or at least 1, 2, 3 or all tumor markers indicated with a "+" in section S) of Table 1, or at least 1, 2, 3, 4 or all tumor markers indicated with a "+" in section U) of Table 1, or at least 1, 2, 3 or all tumor markers indicated with a "+" in section W) of Table 1 , wherein combinations of markers indicated with a "+" from sections P) and Q), R) and S), T) and U) as well as V) and W), respectively, of Table 1 are preferred.
In a further preferred embodiment, the group of tumor markers comprises or consists of AKAP6 and/or CNGB3 and/or KGP2 and/or AKAP1 and/or AKAP9 and/or AKAP13 and/or PDE9A and/or PRKAR1B and/or PDE4C, as indicated in Table 1.
In another embodiment of the present invention the group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the tumor markers of Table 1. The group may further comprise any sub-grouping or combinations of these markers.
In yet another embodiment of the present invention the group of tumor markers comprises those tumor markers which show a p-value of the expression modification of 0.03, 0.04, 0.05, 0.06, 0,07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 016, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.3, 0.4, 0.5, 0.6 or lower as indicated in section K) of Table 1. Preferred are groups of markers which show p-values of the expression of between about 0.03 and about 0.0.4, particularly preferred are groups of markers having a p-value of expression of 0.2 or lower, as indicated in sections L) and/or M) of Table 1. The term "p-value" is a measure of the probability that a variate would assume a value greater than or equal to the observed value strictly by chance and is expressed by the following term: P (z > z0bserved). Thus, in the context of the present invention, the p-value may be seen as a measure of statistical significance.
In another embodiment of the present invention the group of tumor markers comprises least 2 tumor markers corresponding to tumor marker #1 to #2 of Table 1, to tumor marker #2 to #3 of Table 1, to tumor marker #3 to #4 of Table 1, to tumor marker #5 to #6 of Table 1, to tumor marker #6 to #7 of Table 1, to tumor marker #7 to #8 of Table 1 or to tumor marker #8 to #9 of Table 1, or at least 4 tumor markers corresponding to tumor marker #1 to #4 of Table 1, to tumor marker #2 to #5 of Table 1, to tumor marker #3 to #6 of Table 1, to tumor marker #4 to #7 of Table 1, to tumor marker #5 to #8 of Table 1 or to tumor marker #6 to #9 of Table 1. The group of tumor marker according to the present invention may also comprise tumor markers #1 , #3, #5, #7 and #9 of Table 1 , #2, #4, #6, #8, and #9 of Table 1 , #2, #4, #7, #9 and #1 of Table 1, #1, #3, #4, #6 and #8 of Table 1, #5, #4, #9, and # 2 of Table 1, etc.
Additional tumor markers which may be combined with any one or more of the markers of Table 1 , preferably with any combination of these markers or any group of tumor markers or any combination of groups of tumor markers as defined herein above in any suitable manner and thus also form part of the present invention are indicated in the following Table 2:
Table 2:
A) B) C) D) E) F) Marker# Uniprot Function SEQ ID No. SEQ ID No. UniGene Code
Accession Code DNA Protein
1 PPAP PAP 19 20 Hs.388677
2 ACTS MSActin 21 22 Hs.1288
Alpha-methylacyl-CoA
3 AMACR 23 24 Hs.49598 racemase
4 ΑΝΧΑ1 Annexin I 25 26 Hs.287558
5 ΑΝΧΑ7 Annexin VII 27 28 Hs.386741
6 ARPC3 p21-Arc 29 30 Hs.439511
7 ASAHl Acid Ceramidase 31 32 Hs.324808
8 AURKB Aurora B 33 34 Hs.442658
9 BIRC2 cIAP 35 36 Hs.289107
10 BIRC4 XIAP 37 38 Hs.356076
11 CIKS ACT1 39 40 Hs.437508
12 CALD1 L-Caldesmon 41 42 Hs.443811
13 KCC2G CaM Kinase II 43 44 Hs.12436
14 CASP7 Casp7 45 46 Hs.9216
15 CAV1 Caveolin 1 47 48 Hs.74034
16 CAV2 Caveolin 2-20kD 49 50 Hs.139851
17 CBX3 HP1 GAMMA 51 52 Hs.381 189
18 CDC42 Cadc42/RAC 53 54 Hs.355832 CDK2 Cdk2 55 56 Hs.19192
CDK4 Cdk4 57 58 Hs.95577
CDK7 CDK7 59 60 Hs.184298
CARF pl6INK4A 61 62 Hs.421349
RCBT2 RCC1 63 64 Hs.196769
CRYAB alphaB Crystallin 65 66 Hs.408767
CSNK1A1 CK1 67 68 Hs.318381
CSRP1 CRP1 69 70 Hs.108080
DFFA DFF45 71 72 Hs.484782
EDNRA Endothelin 1 Receptor 73 74 Hs.211202
EFS Sin 75 76 Hs.24587
LIMA1 EPLIN-105kD 77 78 Hs.10706
EPS8 Eps8 79 80 Hs.2132
ERCC1 Ercc-l-37kD 81 82 Hs.435981
EZH2 EZH2 83 84 Hs.444082
FLNA ABP280 85 86 Hs.195464
FLOT2 Flotillin-2/ESA 87 88 Hs.18799
GOSR1 GS28 89 90 Hs.124436
GRB7 grb7 91 92 Hs.86859
GELS Gelsolin-88kD 93 94 Hs.446537
TF2B TFIIB 95 96 Hs.258561
HCD2 ERAB 97 98 Hs.171280
HAX1 HAX-1 99 100 Hs.199625
CH60 HSP60 101 102 Hs.79037 IRAKI IRAK 103 104 Hs.182018
ΓΓΑ5 Integrin 5 alpha 105 106 Hs.149609
KLK3 PSA 107 108 Hs.171995
IMA2 Karyopherin alpha 2 109 110 Hs.159557
LHX1 Lim kinase 111 112 Hs.154103
MD2BP MAD2 113 114 Hs.79078
Q6FHX4 Sekl 115 116 Hs.134106
Q499Y8 pan-JNK/SAPKl-45kD 117 118 Hs.25209
MK14 p38 alpha/SAPK2a 119 120 Hs.79107
MAPKAPK3 3PK 121 122 Hs.234521
A2VCR0 EB1 123 124 Hs.408754
MUC18 MCAM 125 126 Hs.51 1397
MCM4 MCM4-92kD 127 128 Hs.460184
MCM6 MCM6 129 130 Hs.4441 18
MKI67 Ki-67-260kD 131 132 Hs.80976
MMP23B MMP23 133 134 Hs.21 1819
MSH2 MSH2 135 136 Hs.440394
Myosin light chain
MYLK 137 138 Hs.386078 kinase(MYLK)
MY06 Myosin VI 139 140 Hs.1 18483
NEXN Nexilin 141 142 Hs.22370
NDKA Nm23 143 144 Hs.l 18638
NSF NSF 145 146 Hs.431279
NUP62 Nucleoporin p62 147 148 Hs.528342
OCLN Occludin 149 150 Hs.171952 OPTO FIP-2-73kD 151 152 Hs.390162
P4HB PDI 153 154 Hs.410578
PAK3 PAK3 155 156 Hs.390616
PCYOX1 Prenylcysteine Lyase 157 158 Hs.278627
P85A PI3 Kinase 159 160 Hs.6241
PP1A PP1 161 162 Hs.183994
MYPT1 MYPT1 163 164 Hs.377908
KAPO PKA RI 165 166 Hs.280342
KAP3 PKA RII beta 167 168 Hs.77439
KPCB PKC beta 169 170 Hs.349845
PRSS8 Prostasin 171 172 Hs.75799
PTRF PTRF 173 174 Hs.437191
RAB11A Rabl l 175 176 Hs.75618
RAB27A RAB27 177 178 Hs.298530
RAB4A Rab4 179 180 Hs.296169
RALA Ral A 181 182 Hs.6906
RBBP4 RBBP 183 184 Hs.16003
ROCK2 ROCK-II/ROK alpha 185 186 Hs.58617
SH2B SH2-B 187 188 Hs.15744
SMAD2 Smad2/3 189 190 Hs.110741
SMCA4 BRG1 191 192 Hs.78202
SMRC2 BAF170 193 194 Hs.236030
SORL LR1 l/SorLA/gp250 195 196 Hs.438159
STK6 Aurora kinase A 197 198 Hs.250822 91 STMN1 Stathmin/Metablastin 199 200 Hs.209983
92 STX12 Syntaxin 13 201 202 Hs.433838
93 TGFB1I1 Hic-5 203 204 Hs.2551 1
94 THRB B Thyroid HNR 205 206 Hs.203213
95 TIMP3 TIMP3 207 208 Hs.245188
96 LAP2A LAP2 209 210 Hs.1 1355
97 TNR19 TROY 211 212 Hs.334174
98 TOP2A TOP02 alpha 213 214 Hs.156346
99 TPD52 TPD52 215 216 Hs.162089
100 TRI25 EFP 217 218 Hs.88914
101 TIF IB KRIP-1 219 220 Hs.433612
102 TYK2 tyk2 221 222 Hs.75516
103 SAE2 Uba2 223 224 Hs.51 1739
104 UBC9 Ubc9 225 226 Hs.302903
105 UHRF1 ICBP90 227 228 Hs.108106
106 XPOl Exportin 229 230 Hs.157367 cAMP-dependent protein
107 KAPCA 231 232 Hs.631630 kinase catalytic SU alpha
In a further aspect the present invention relates to the use of a tumor marker or group of tumor markers as defined herein above as a marker for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed of a neoplastic disease to a more progressed stage of a neoplastic disease, or prognosticating the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease.
The term "diagnosing a cancer disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease" as used herein means that a subject or individual may be considered to be suffering from a more progressed neoplastic disease as defined herein above, e.g. a more progressed prostate cancer, when the expression level of a tumor marker or the group of tumor markers of the present invention is modified, e.g. increased/up-regulated or reduced/down-regulated, compared to the expression level of a less progressed disease state as defined herein above, or compared to a control level as defined herein above. The term "diagnosing" also refers to the conclusion reached through that comparison process. An expression level may be deemed to be modified, when the expression level of a tumor marker or group of tumor markers as defined herein above differs by, for example, between about 1% and 50%, e.g. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% or 40% from the expression level of a less progressed disease state or from a control level as defined herein above, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to such a less progressed disease state or control level. The modification may be an increase or a reduction of said expression level.
In a further embodiment, an additional similarity in the overall gene expression pattern of a group of tumor markers according to the present invention between a sample obtained from a subject and a control sample or a sample corresponding to a less progressed disease state of a neoplastic disease as defined herein above as described herein above, may indicate that the subject is suffering from a more progressed disease stage of the neoplastic disease as defined herein above. In another embodiment of the present invention, the diagnosis may be combined with the elucidation of additional cancer biomarker expression levels, in particular prostate cancer biomarkers. Suitable biomarkers, in particular prostate cancer biomarkers, would be known to the person skilled in the art. For example, the expression of biomarkers like PSA may be tested.
The term "detecting a neoplastic disease associated with a progression from a less progressed stage to a more progressed stage" as used herein means that the presence of a neoplastic disease or disorder as defined herein above in an organism, which is associated with a more progressed stage may be determined or that such a neoplastic disease or disorder as defined herein above may be identified in an organism. The determination or identification of a more progressed neoplastic disease or disorder as defined herein above may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers of the present invention in a sample from a patient or individual to be analyzed and the expression level of a control level as defined herein above, wherein said control level corresponds to the expression level of said more progressed neoplastic disease or disorder as defined herein above. In a preferred embodiment of the present invention a more progressed stage of a neoplastic disease as defined herein above may be detected if the expression level of the tumor marker or group of tumor markers is similar to an expression level of a more progressed stage of the neoplastic disease as defined herein above. The expression level of the more progressed stage of the neoplastic disease as defined herein above may be independently established, e.g. from sample depositories, value databases etc. as mentioned herein above.
The term "graduating a neoplastic disease associated with a progression from a less progressed stage to a more progressed stage" as used herein means that the clinical stage, phase, grade or any other suitable sub-step of features related to the neoplastic disease as defined herein above, such as the progression defined herein above, the transition from a benign to a malignant tumor, the grade of malignancy, the grade of tissue damage to noncancerous tissue, the grade of the extent of tumor growth, the grade of aggressiveness of a tumor, the grade of metastasizing and all other useful and suitable parameters of a cancerous disease or disorder in an organism may be determined in an organism.
In a preferred embodiment the graduating of a more progressed stage of the neoplastic disease as defined herein above may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1 of the present invention in a sample from a patient or individual to be analyzed and a control level as defined herein above, or the expression level of a less progressed stage as defined herein above of the neoplastic disease as defined herein above. In a further preferred embodiment the graduating of a more progressed stage may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1 of the present invention in a sample from a patient or individual to be analyzed and any of the cancer cells, cancer tissues, tumor biopsies, or the cancerous control levels mentioned above. In a further preferred embodiment the graduating of a more progressed stage of the neoplastic disease as defined herein above may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1 of the present invention in a sample from a patient or individual to be analyzed and the above mentioned cancer cells, cancer tissues, tumor biopsies, or the neoplastic control levels being derived from or representative for a less progressed stage of the neoplastic disease as defined herein above. In a further preferred embodiment the graduating of a more progressed stage of the neoplastic disease as defined herein above, preferably cancer, may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1 of the present invention in a sample from a patient or individual to be analyzed and the above mentioned cancer cells, cancer tissues, tumor biopsies, or the neoplastic control levels being derived from or representative for a more progressed stage of the neoplastic disease as defined herein above, preferably cancer.
The term "monitoring a neoplastic disease associated with a progression from a less progressed stage to a more progressed stage" as used herein relates to the
accompaniment of a diagnosed or detected, more progressed neoplastic disease or disorder as defined herein above, e.g. during a treatment procedure or during a certain period of time, typically during 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other period of time. The term "accompaniment" means that states of disease as defined herein above and, in particular, changes of these states of disease may be detected by comparing the expression level of the tumor marker or group of tumor markers of the present invention in a sample to a control level as defined herein above or to the expression level of an established, e.g.
independently established cancer cell or cell line, or to corresponding database values in any type of periodical time segment, e.g. every week, every 2 weeks, every month, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 month, every 1.5 year, every 2, 3, 4, 5, 6, 7, 8,9 or 10 years, during any period of time, e.g. during 2 weeks, 3 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years, respectively. The established, e.g. independently established, cancer cell or cell line giving rise to a control level may be derived from samples corresponding to different stages of neoplastic development, e.g. the stages as mentioned herein above. In a preferred embodiment of the present invention the term relates to the accompaniment of a diagnosed more progressed prostate cancer.
The term "prognosticating a neoplastic disease associated with a progression form a less progressed stage to a more progressed stage" as used herein refers to the prediction of the course or outcome of a diagnosed or detected more progressed stage of the neoplastic disease as defined herein above, e.g. during a certain period of time, during a treatment or after a treatment. The term also refers to a determination of chance of survival or recovery from the disease, as well as to a prediction of the expected survival time of a subject. A prognosis may, specifically, involve establishing the likelihood for survival of a subject during a period of time into the future, such as 6 months, 1 year, 2 years, 3 years, 5 years, 10 years or any other period of time.
The term "progression from a less progressed stage to a more progressed stage" as used herein relates to a switch between different stages of the development or genesis of a neoplastic disease, as defined herein above. Such a progression may be a development in small steps, e.g. from a certain stage to the next, or may alternatively be a development skipping one or more such steps, e.g. from stage I to stage III of the TNM classification. A progression from a less progressed stage to a more progressed stage may be considered as being detected or diagnosed if the expression level of a tumor marker or group of tumor marker according to the present invention is modified, e.g. increased or reduced or both by a value of between 3% to 50%, preferably by a value of 10%>, 15%, 20% or 25% in a test sample in comparison to a previous test sample from the same individual. The modification may be detected over any period of time, preferably over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years, i.e. the value indicated above may be calculated by comparing the expression level of the tumor marker or group of tumor markers at a first point in time and at a second point in time after the above indicated period of time.
In a particularly preferred embodiment of the present invention the term "progression from a less progressed stage to a more progressed stage" relates to a switch from a benign prostate tumor state to a malignant prostate cancer state, as defined herein.
Alternatively, for the comparison test samples from other individuals may be used, e.g. test samples of healthy individuals. Also envisaged is the use of available database information on the expression or the employment of cancer or tumor cell collection samples etc.
In a further embodiment the present invention relates to the diagnosis and detection of a predisposition for developing a more progressed stage of the neoplastic disease as defined herein above. A "predisposition for developing a more progressed stage of the neoplastic disease as defined herein above, preferably cancer" in the context of the present invention is a state of risk of developing a more progressed stage neoplastic disease as defined herein above. Preferably a predisposition for developing a more progressed stage may be present in cases in which the expression level of the tumor marker or group of tumor marker of the present invention as defined herein above is above a normal control level as defined herein above, i.e. a reference expression level derived from tissues or samples of a subject which are evidently healthy. The term "above" in this context relates to an expression level of the tumor marker of group of tumor markers which is increased by about 2% to 20% in comparison to such a control level, preferably increased by about 15%.
Alternatively, a predisposition for developing a more progressed stage of the neoplastic disease as defined herein above, e.g. cancer, in the context of the present invention may be given in situations in which the expression level of the tumor marker or group of tumor markers as defined herein above is above a normal control level and in which further, alternative cancer markers, e.g. PSA, show no modification of expression level or the expression pattern in a less progressed stage of the neoplastic disease as defined herein above. Suitable further cancer markers are known to the person skilled in the art.
Thus, a predisposition for a more progressed stage of the neoplastic disease as defined herein above, e.g. cancer, may be considered as being diagnosed or detected if one of the above depicted situations is observed.
In case a group of tumor markers according to the present invention comprises tumor markers of Table 1 which show an increase of the expression level and, at the same time, other tumor markers of Table 1 which show a reduction of the expression level, the increase and the reduction of the corresponding tumor markers may be calculated
independently, i.e. for each marker or each group of markers with the same tendency
(increase and reduction) separately. The absolute values of such calculations may
subsequently be averaged and used for the calculation of the modification.
The difference between the expression levels of a test biological sample and a control level or the expression level of a less progressed stage of the neoplastic disease as defined herein above, can be normalized to the expression level of one or more control nucleic acids, e.g. housekeeping genes whose expression levels are known not to differ depending on the neoplastic or non-neoplastic state of the cell. Exemplary control genes include inter alia β-actin, glycerinaldehyde 3-phosphate dehydrogenase (GAPDH), 18s R A, ubiquitin C, cytochrome CI, ribosomal protein PI or PBGD. Particularly preferred is the use of GAPDH. In the context of the present invention, the terms "diagnosing" and
"prognosticating" are also intended to encompass predictions and likelihood analyses. Tumor markers or groups of tumor markers may accordingly be used clinically in making decisions concerning treatment modalities, including therapeutic intervention or diagnostic criteria such as a surveillance for the disease. According to the present invention, an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease. Alternatively, the present invention may be used to detect neoplastic cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.
A subject or individual to be diagnosed, monitored or in which a more progressed neoplastic disease as defined herein above, a progression towards such a neoplastic disease as defined herein above, or a predisposition of such a neoplastic disease as defined herein above, is to be detected or prognosticated according to the present invention is an animal, preferably a mammal, more preferably a human being. Also preferred is the use of molecular imaging tools as known to the person skilled in the art, e.g. magnetic resonance imaging (MRI) and/or magnetic photon resonance imaging (MPI) technology in the context of using a tumor marker or group of tumor marker for diagnosing, detecting, monitoring or prognosticating a more progressed neoplastic disease as defined herein above, preferably cancer of the progression towards such a neoplastic disease as defined herein above, preferably cancer. For example, a tumor marker or group of tumor markers according to the present invention may be used as a marker for diagnosing, detecting, monitoring or prognosticating pre-malignant growths, swellings and lesions, malignant tumors, malignant cancers, metastases and the like or the progression towards more progressed states of such neoplastic disease and disorders as defined above, for example malignant, hormone- sensitive prostate cancer and/or the progression towards more progressed cancer states, in approaches like MRI or MPI that allow for online detection of the diagnostic marker within a human subject.
In another aspect the present invention relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand for the expression product(s) or protein(s) of said tumor marker or a group of tumor markers as defined above.
The term "nucleic acid affinity ligand for the expression product of a tumor marker or group of tumor markers" as used herein refers to a nucleic acid molecule being able to specifically bind to a transcript or a DNA molecule derived the nucleic acid molecules of said tumor marker or a group of tumor markers as defined above, preferably to the nucleotide sequences, (DNA sequence(s)) depicted in section D) of Table 1 or to the complementary nucleotide sequences (DNA sequence(s)) of the sequence(s) depicted in section D) of Table 1 or a corresponding RNA molecule. The nucleic acid affinity ligand may also be able to specifically bind to a DNA sequence being at least 75%, 80%>, 85%, 90%>, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section D) of Table 1 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section E) of Table 1 or to any fragments of said sequences.
The term "peptide affinity ligand for the protein of a tumor marker or a group of tumor markers" as used herein refers to a peptide molecule being able to specifically bind to the proteins of the tumor marker or group of tumor markers according to Table 1. The peptide molecule may preferably be able to specifically bind to a protein or polypeptide comprising the amino acid sequence(s) as set forth in section E) of Table 1. The peptide affinity ligand may also be able to specifically bind to a protein or polypeptide comprising an amino acid sequence encoded by a DNA sequence being at least 75%, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section E) of Table 1 or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section E) of Table 1 or to any fragments of said sequences.
The term "peptide" in the context of the affinity ligand of the present invention refers to any type of amino acid sequence comprising more than 2 amino acids, e.g.
polypeptide structures, protein structures or functional derivatives thereof. Furthermore, the peptide may be combined with further chemical moieties or functionalities.
The term "expression product" as used herein refers to a transcript or an mR A molecule of the tumor marker or group of tumor markers according to Table 1 generated by the expression of the corresponding genomic sequence according to Table 1. More preferably, the term relates to a processed transcript of the tumor marker or group of tumor markers according to Table 1 as defined herein above, e.g. via the sequence(s) as set forth in sections D) and/or F) of Table 1. A person skilled in the art would know how to determine the identity, size, length and any other useful parameter of transcripts of tumor markers according to the present invention based on the information provided in said section F) of Table 1.
The term "protein of a tumor marker or group of tumor markers" as used herein also refers to any polypeptide, protein, in particular to the polypeptides or proteins as set forth in section E) of Table 1 or any domain, epitope, oligopeptide, or peptide derivable therefrom.
In a preferred embodiment of the present invention the composition of the present invention may comprise one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid and/or peptide affinity ligands selected from the group consisting of a set of oligonucleotides specific for the expression product of the tumor marker or group of tumor markers according to Table 1 , a probe specific for the expression product of the tumor marker or group of tumor markers according to Table 1 , an aptamer specific for the expression product or for the protein of the tumor marker or group of tumor markers according to Table 1, an antibody specific for the protein the tumor marker or group of tumor markers according to Table 1 and an antibody variant specific for the protein of the tumor marker or group of tumor markers according to Table 1. The composition of the present invention may, for example, comprise a set of oligonucleotides specific for the expression product the tumor marker or group of tumor markers according to Table 1 and/or a probe specific for the expression product of the tumor marker or group of tumor markers according to Table 1.
The term "oligonucleotide specific for the expression product the tumor marker or group of tumor markers according to Table 1 " as used herein refers to a nucleotide sequence which is complementary to the sense- or antisense-strand of the tumor marker or group of tumor markers according to Table 1. Preferably, the oligonucleotide is
complementary to the DNA sequence(s) shown in section D) of Table 1, or to the
complementary DNA sequence of the sequence shown in section D) of Table 1. The oligonucleotide sequence may also be complementary to a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section D) of Table 1 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section E) of Table 1. The oligonucleotide may have any suitable length and sequence known to the person skilled in the art, as derivable from the sequence(s) shown in section D) of Table 1 or its complement. Typically, the oligonucleotide may have a length of between 8 and 60 nucleotides, preferably of between 10 and 35 nucleotides, more preferably a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides. Oligonucleotide sequences specific for the expression product of the tumor marker or group of tumor markers according to Table 1 may be defined with the help of software tools known to the person skilled in the art.
In a further embodiment of the present invention the oligonucleotide sequences may be complementary to genomic sequences localized in (an) exon(s) of the gene(s) encoding for the tumor marker or group of tumor markers according to Table 1.
Corresponding information on genomic loci is indicated in section F) of Table 1. An oligonucleotide usable as a forward primer may be localized at the boundary between exonic and intronic sequences. Such boundary positions may be determined with the help of any suitable tool, based on the information provided in section F) of Table 1.
The term "probe specific for the expression product of the tumor marker or group of tumor markers according to Table 1 " as used herein means a nucleotide sequence which is complementary to the sense- or antisense-strand of the tumor marker or group of tumor markers according to Table 1. Preferably, the probe is complementary to the DNA sequence(s) depicted in section D) of Table 1 or to the complementary DNA sequence of the sequence(s) shown in section D) of Table 1. The probe sequence may also be complementary to a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o or 99% identical to the sequence(s) as set forth in the sequences indicated in section D) of Table 1 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as shown in section E) of Table 1. The probe may have any suitable length and sequence known to the person skilled in the, as derivable from the sequence(s) shown in section D) or its/their complement. Typically, the probe may have a length of between 6 and 300 nucleotides, preferably of between 15 and 60 nucleotides, more preferably a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Probe sequences specific for the expression product of the tumor marker or group of tumor markers according to Table 1 may be defined with the help of software tools known to the person skilled in the art. The composition of the present invention may additionally or alternatively comprise an aptamer specific for the expression product or protein of the tumor marker or group of tumor markers according to Table 1.
The term "aptamer specific for the expression product of the tumor marker or group of tumor markers according to Table 1 " as used herein refers to a short nucleic acid molecule, e.g. RNA, DNA, PNA, CNA, HNA, LNA or ANA or any other suitable nucleic acid format known to the person skilled in the art, being capable of specifically binding to the tumor marker or group of tumor markers according to Table 1, preferably the DNA molecule with (a) sequence(s) as shown in section D) of Table 1. More preferably, the nucleic acid aptamer molecule may specifically bind to a DNA sequence(s) shown in section D) of Table 1 or a double stranded derivative thereof. The nucleic acid aptamer according to the present invention may also bind to an RNA molecule corresponding to the transcript(s) of the tumor marker or group of tumor markers according to Table 1, preferably an RNA molecule corresponding to the DNA sequence(s) as shown in section D) of Table 1. The nucleic acid aptamer may further be capable of specifically binding to a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as shown in section D) of Table 1 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the sequence(s) as shown in section D) of Table 1 or RNA molecules corresponding to these sequences. A nucleic acid aptamer according to the present invention may further be combined with additional moieties, e.g. with interacting portions like biotin or enzymatic functionalities like ribozyme elements.
The term "aptamer specific for the protein of the tumor marker or group of tumor markers according to Table 1 " as used herein refers to (a) short peptide(s) capable of interacting and specifically binding the protein(s) of the tumor marker or group of tumor markers according to Table 1. The peptide aptamer(s) may preferably be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (the) amino acid sequence(s) as shown in section E) of Table 1. The peptide aptamer(s) may also be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (an) amino acid sequence(s) encoded by (a) DNA sequence(s) being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o or 99%) identical to the sequence(s) as shown in section D) of Table 1 or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%>, 85%, 90%>, 91%>, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in shown in section E) of Table 1. Typically, (a) peptide aptamer(s) is/are a variable peptide loop, comprising for example 10 to 20 amino acids. In the context of the present invention the peptide aptamer(s) may preferably be attached at one or both ends to a scaffold structure. The scaffold structure may be any molecule, preferably a protein, which has good solubility properties. Suitable scaffold molecules would be known to the person skilled in the art. A preferred scaffold molecule to be used in the context of the present invention is the bacterial protein thioredoxin-A. The aptamer peptide loop may preferably be inserted within a reducing active site of the scaffold molecule. Alternatively, staphylococcal protein A and domains thereof and derivatives of these domains, such as protein Z or lipocalins may be used as scaffold structures in the context of the present invention.
Peptide aptamers may be generated according to any suitable method known to the person skilled in the art, e.g. via yeast two-hybrid approaches.
In another preferred embodiment of the present invention the composition may comprise, or may additionally comprise, an antibody or a group of antibodies specific for the expression product or protein of a tumor marker or group of tumor markers as defined herein, e.g. specific for one or more of the protein(s) of the tumor marker according to Table 1. Particularly preferred is an antibody which specifically binds to an expression product, protein or peptide comprising the amino acid sequence as indicated in section E) of Table 1 , or a part or fragment of said sequence. . In another aspect of the invention relates to an antibody specific for the tumor marker according to Table 1. Preferably, such an antibody specifically binds to a protein or polypeptide having the amino acid shown in section E) of Table 1 , or any derivative, fragment etc. thereof as defined herein above. Such antibodies are contemplated for any application, use, method, composition, immunoassay, screening method and pharmaceutical compositions as defined in the present application. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i. e. molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e. g., IgG, IgE, IgM, IgD, IgA and IgY), class (e. g., IgGl, IgG2, IgG3, lgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. Preferred epitopes according to the present invention are amino acids 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, 191-200, 201-210 etc. or any other specific stretch of amino acids of a protein of the tumor markers of the present invention, preferably of the sequence as shown in section E) of Table 1. Further comprised are all other suitable epitopes, which can be recognized, determined, described and subsequently be employed according to methods known to the person skilled in the art.
The term "specific for the tumor marker according to Table 1" as used herein refers to the immunospecific detection and binding of an antibody to an antigenic epitope as defined herein above. The term "specifically binding" excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens, in particular with antigens comprising the same antigenic epitope detected by the present antibody.
In a preferred embodiment antibodies of the invention include polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, Fab' fragments, fragments produced by a Fab expression library, F(ab')2, Fv, disulfide linked Fv, minibodies, diabodies, scFv, sc(Fv)2, whole
immunoglobulin molecules small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins, camelized antibodies, VHH containing antibodies, anti- idiotypic (anti-Id) antibodies (including, e. g., anti-Id antibodies to antibodies of the invention) and epitope-binding fragments of any of the above. Most preferably, the antibodies are human antigen-binding antibody fragments of the present invention and include Fab, Fab' and F (ab')2, Fv, single-chain Fvs (scFv), sc(Fv)2, single-chain antibodies, disulfide- linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
The antibodies according to the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e. g., mouse and rat), donkey, monkey, rabbit, goat, guinea pig, camel, horse, or chicken.
The antibodies according to the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. In a particularly preferred embodiment the present invention relates to antibodies that do not bind any other analog, ortholog, or homo log of a polypeptide of the present invention. However, also antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are included in the present invention.
In a specific embodiment the present invention also envisages an antigen and/or an epitope which is detected, recognized or specifically bound by an antibody against a tumor marker protein as defined in Table 1.
Such antigens or epitopes may preferably be derived from a tumor marker or group of tumor marker sequences as depicted in Table 1. The antigen or epitope sequences encompassed by the present invention may, for example, comprise, have, or alternatively consist of amino acids 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, 191- 200, 201-210 etc. or any other specific stretch of amino acids of the N- or C-terminus of a protein or expression product of the tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, preferably of the sequence as shown in section E) of Table 1. Furthermore, the antigen or epitope sequences encompassed by the present invention may, for example, comprise, have, or alternatively consist of amino acids 1-10, 2-11, 3-12, 4- 13, 5-14, 6-15, 7-16, 8-17, 9-18, 10-19, 11-20, 12-21, 13-22, 14-23, 15-24, 16-25, 17-26, 18- 27, 19-28, 20-29, 21-30, 22-31, 23-32, 24-33, 25-34, 26-35, 27-36, 28-37, 29-38, 30-39, 31- 40, 32-41, 33-42, 34-43, 35-44, 36-45, 37-46, 38-47, 39-48, 30-49, 41-50, 42-51, 43-52, 44- 53, 45-54, 46-55, 47-56, 48-57, 49-58, 50-59, 51-60, 52-61, 53-62, 54-63, 55-64, 56-65, 57- 66, 58-67, 59-68, 60-69, 61-70, 62-71, 63-72, 64-73, 65-74, 66-75, 67-76, 68-77, 69-78, 70- 79, 71-80, 72-81, 73-82, 74-83, 75-84, 76-85, 77-86, 78-87, 79-88, 80-89, 81-90, 82-91, 83- 92, 84-93, 85-94, 86-95, 87-96, 88-97, 89-98, 90-99, 91-100, 1-20, 2-21, 3-22, 4-23, 5-24, 6- 25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 30-59, 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72, 54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82, 64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92, 74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100, 1-30, 2-31, 3-32, 4- 33, 5-34, 6-35, 7-36, 8-37, 9-38, 10-39, 11-40, 12-41, 13-42, 14-43, 15-44, 16-45, 17-46, 18- 47, 19-48, 20-49, 21-50, 22-51, 23-52, 24-53, 25-54, 26-55, 27-56, 28-57, 29-58, 30-59, 31- 60, 32-61, 33-62, 34-63, 35-64, 36-65, 37-66, 38-67, 39-68, 30-69, 41-70, 42-71, 43-72, 44- 73, 45-74, 46-75, 47-76, 48-77, 49-78, 50-79, 51-80, 52-81, 53-82, 54-83, 55-84, 56-85, 57- 86, 58-87, 59-88, 60-89, 61-90, 62-91, 63-92, 64-93, 65-94, 66-95, 67-96, 68-97, 69-98, 70- 99 or 71-100 of the N- or C-terminus of a protein or expression product of the tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, preferably of the sequence as shown in section E) of Table 1.
The epitope or antigen according to the present invention may further comprise or consist of additional amino acid stretches or fragments of any length or derived from any position, e.g. N-terminus, C-terminus, middle, specific domain, specified exposed region etc. of the protein or expression product of the tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, preferably of the sequence as shown in section E) of Table 1. Particularly preferred are epitopes or antigens comprised in or comprising fragments or stretches of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 270, 300, 350 or 400 amino acids of the protein or expression product of the tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, preferably of the sequence as shown in section E) of Table 1.
The epitope or antigen of the present invention may further be present on extracellular domains, intracellular domains, sterically exposed domains, surface regions, exposed surface regions, interaction domains etc. of any of the tumor marker or group of tumor markers as defined herein, e.g. as derivable from Table 1, preferably of the sequence as shown in section E) of Table 1. Such domains or regions and their position as well as their amino acid composition would be known to the person skilled in the art or could be deduced with known software or database tools, in particular in view of the information provided in sections E) of Table 1. The person skilled in the art may, for example, use three dimensional modeling tools or structure information associated with the Entre Gene ID in order to identify such domains or regions.
Also envisaged are combinations of two or more epitope sequences, e.g. in the form of a fusion construct or the like. Further envisioned are epitope sequences that bear disease-specific sequence mutations. Such contructs may be used for immunological purposes, e.g. for the preparation of vaccines, the elicitation of antibodies etc. The present inventin thus also encompasses the medical use of correspondingly prepared vaccines, pharmaceutical compositions comprising such vaccines, the vaccines themselves, methods of treatment involving the vaccines etc.
In a further embodiment the present invention encompasses a screening method to identify antigen or epitope sequences bound by an antibody against a tumor marker protein as defined in Table 1. Further envisaged are the epitope sequences and/or antigen sequences derived from a screening procedure.
In a further embodiment the antibodies of the invention include derivatives which are modified, for instance by the covalent attachment of any type of molecule to the antibody such that said covalent attachment does not prevent the antibody from specifically binding to the epitope or from generating an anti-idiotypic response. Typical examples of such modifications are glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Chemical modifications may be carried out by known techniques, including specific chemical cleavage, acetylation, formylation etc. Additionally, the derivative may contain one or more non-classical amino acids.
Antibodies may be produced according to any suitable method known to the person skilled in the art. Monoclonal antibodies of defined specificity may be produced using, for instance, the hybridoma technology developed by Kohler and Milstein (Kohler and Milstein, 1976, Eur. J. Immunol, 6: 511-519).
In a further embodiment of the present invention the antibody or fragment thereof as defined herein above may be biotinylated or labeled. In a particularly preferred embodiment said label is a radioactive label, an enzymatic label, a fluorescent label, a chemiluminescent or a bio luminescent label. Alternatively, antibodies may also be labeled or combined with fluorescent polypeptides, e.g. green fluorescent protein (GFP) as well as derivates thereof known to the person skilled in the art.
Alternatively, a polynucleotide encoding an antibody may be generated from a nucleic acid from a suitable source.
In a further embodiment of the present invention a nucleic acid molecule encoding the antibody or fragment thereof as defined herein above may be used for recombinant antibody expression. Preferably, such expression vectors contain the antibody coding sequences and appropriate transcriptional and translational control signals. The vectors may either comprise coding sequences for the variable heavy chain or the variable light chain or for both. Such vectors may also include the nucleotide sequence encoding the constant regions of the antibody molecule. In a preferred embodiment of the present invention mammalian cells, more preferably Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus may be used as an effective expression system for antibodies.
In another aspect the present invention relates to a cell that produces the antibody or fragment thereof as defined herein above. Such a cell may be a hybridoma cell as defined herein above or a cell which expresses a nucleic acid molecule encoding an antibody according to the present invention. Particularly preferred are cells or cell lines which stably express the antibody molecule.
In addition, the antibodies of the present invention or fragments thereof can be fused to any heterologous polypeptide sequence, preferably to those defined herein above, e.g. in order to facilitate antibody purification or to provide target means for the antibody.
In a specific embodiment of the present invention commercially available antibodies against the tumor marker according to Table 1 , in particular against a protein having an amino acid sequence as indicated in section E) of Table 1 may be comprised in the composition or may be used diagnostically.
An affinity ligand, as described herein above, may be labeled with various markers or may be detected by a secondary affinity ligand, labeled with various markers, to allow detection, visualization and/or quantification. This can be accomplished using any suitable labels, which can be conjugated to the affinity ligand capable of interaction with the expression product(s) of the tumor marker or group of tumor markers according to Table 1 or the corresponding protein(s) or to any secondary affinity ligand, using any suitable technique or methods known to the person skilled in the art. The term "secondary affinity ligand" refers to a molecule which is capable of binding to the affinity ligand as defined herein above (i.e. a "primary affinity ligand" if used in the context of a system with two interacting affinity ligands). The binding interaction is preferably a specific binding.Examples of labels that can be conjugated to a primary and/or secondary affinity ligands include fluorescent dyes or metals (e.g. fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g. rhodopsin), chemiluminescent compounds (e.g. luminal, imidazole) and bio luminescent proteins (e.g. luciferin, luciferase), haptens (e.g. biotin).
In a particularly preferred embodiment an affinity ligand to be used as a probe, in particular a probe specific for the expression product(s) as defined herein above, may be labeled with a fluorescent label like 6-FAM, HEX, TET, ROX, Cy3, Cy5, Texas Red or Rhodamine, and/or at the same time with a quenching label like TAMRA, Dabcyl, Black Hole Quencher, BHQ-1 or BHQ-2. A variety of other useful fluorescents and chromophores are described in Stryer, 1968, Science, 162:526-533. Affinity ligands may also be labeled with enzymes (e.g. horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g. 3H, 14C, 32P, 33P, 35S, 125I, nC, 13N, 150, 18F, 64Cu, 62Cu, 124I, 76Br, 82Rb, 68Ga or 18F) or particles (e.g. gold).
The different types of labels may be conjugated to an affinity ligand using various chemistries, e.g. the amine reaction or the thiol reaction. However, other reactive groups than amines and thiols can also be used, e.g. aldehydes, carboxylic acids and glutamine.
In a further embodiment of the present invention the nucleic acid affinity ligand or peptide affinity ligand of the present invention may be modified to function as a contrast agent, e.g. as an imaging contrast agent. The term "contrast agent" as used herein refers to a molecular compound that is capable of specifically interacting with the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and which can be detected by an apparatus positioned outside the human or animal body.
Preferably, such contrast agents are suitable for use in magnetic resonance imaging (MRI) or magnetic photon imaging (MPI). The term "specifically interacting" refer to the property of a molecular compound to preferentially interact with the tumor marker or group of tumor markers according to Table 1 on the cell surface of cells being present within the human or animal body over other proteins that are expressed by such cells. Preferred contrast agents which may also be designated as contrast agent compositions will be capable of specifically detecting molecules having the nucleotide sequence(s) shown in section D) of Table 1 or the amino acid sequence(s) shown in section E) of Table 1 or derivatives or homologous variants thereof as defined herein above. Preferred contrast agents are aptamers specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 as well as antibodies specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Contrast agents, aside from their property of being capable of specifically recognizing the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 will in addition typically comprise a further molecule which is detectable by the specific detection technology used.
The term "modified to function" as used herein thus refers to any suitable modifications known to the person skilled in the art, which may be necessary in order to allow the use of the contrast agent in molecular imaging methods, in particular in MRI or MPI. For example, if fluorescent spectroscopy is used as a detection means, such molecules may comprise fluorophores as detectable marker molecules that can be excited at a specific wavelength.
Alternatively, a radioactive label, e.g. a radioisotope as described herein above may be employed. With respect to preferred contrast agents in accordance with the invention that are suitable for MPJ, the contrast agents such as the above described antibodies may comprise a marker molecule which is detectable by MPJ. Such detectable labels include e.g. USPIOS and 19Fluor.
In a specific embodiment of the present invention a composition may additionally comprise accessory ingredients like PCR buffers, dNTPs, a polymerase, ions like bivalent cations or monovalent cations, hybridization solutions, secondary affinity ligands like, e.g. secondary antibodies, detection dyes and any other suitable compound or liquid necessary for the performance of a detection based on any of the affinity ligands or contrast agents as defined herein above, which is known to the person skilled in the art.
In another aspect the present invention relates to the use of a nucleic acid or peptide affinity ligand for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, for the preparation of a composition for diagnosing, detecting, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage to a more progressed stage or the progression from a less progressed stage to a more progressed stage or a predisposition for a more progressed stage, as described herein above. The composition is preferably for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed cancer stage to a more progressed cancer stage or the progression from a less progressed cancer stage to a more progressed cancer stage or a predisposition for a more progressed cancer stage in an individual, e.g. a patient or subject to be analyzed or examined.
In a preferred embodiment the present invention relates to the use of a set of oligonucleotides specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or a probe specific for the expression product(s) the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably specific for a nucleic acid sequence having a sequence as indicated in section D) of Table 1 or being complementary to such a sequence, for the preparation of a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage, as described herein above.
In another preferred embodiment the present invention relates to the use of an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably a protein having an amino acid sequence as shown in section E) of Table 1, for the preparation of a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease, as described herein above. The set of oligonucleotides is preferably for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease in an individual, e.g. a patient or subject to be analyzed or examined. In a further specific embodiment the present invention relates to the use of an antibody specific for protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or an antibody variant specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 for the preparation of a composition for diagnosing, detecting, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of said neplastsic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease as described herein above.
In a preferred embodiment of the present invention a composition as defined herein above is a diagnostic composition.
In another aspect the present invention relates to a diagnostic kit for diagnosing, detecting, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease, comprising a set of oligonucleotides specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a probe specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an antibody specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and an antibody variant specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Typically, the diagnostic kit of the present invention contains one or more agents allowing the specific detection of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. The agents or ingredients of a diagnostic kit may, according to the present invention, be comprised in one or more containers or separate entities. The nature of the agents is determined by the method of detection for which the kit is intended. Where detection at the mR A expression level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, i.e. via the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , is intended, the agents to be comprised may be a set of oligonucleotides specific for the expression product(s) of said tumor marker or group of tumor markers and/or a probe specific for the expression product(s) of said tumor marker or group of tumor markers, which may be optionally labeled according to methods known in the art, e.g. with labels described herein above. In addition or alternatively, an aptamer specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be comprised. Where detection at the protein level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 is intended, the agents to be comprised may be antibodies or compounds containing an antigen-binding fragment of an antibody or antibody variants specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, as described herein above. In addition or alternatively an aptamer specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be comprised.
Alternatively, a diagnostic kit may comprise a contrast agent as defined herein above.
The presence of specific proteins may also be detected using other compounds that specifically interact with the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, e.g. specific substrates or ligands. Preferably, a diagnostic kit of the present invention contains detection reagents for expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Such detection reagents comprise, for example, buffer solutions, labels or washing liquids etc. Furthermore, the kit may comprise an amount of a known nucleic acid molecule or protein, which can be used for a calibration of the kit or as an internal control. Typically, a diagnostic kit for the detection of expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may comprise accessory ingredients like a PCR buffers, dNTPs, a polymerase, ions like bivalent cations or monovalent cations, hybridization solutions etc. A diagnostic kit for the detection of protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may also comprise accessory ingredients like secondary affinity ligands, e.g. secondary antibodies, detection dyes and any other suitable compound or liquid necessary for the performance of a protein detection based known to the person skilled in the art. Such ingredients are known to the person skilled in the art and may vary depending on the detection method carried out.
Additionally, the kit may comprise an instruction leaflet and/or may provide information as to the relevance of the obtained results.
In another aspect the present invention relates to a method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprising at least the step of determining the level of a tumor marker or a group of tumor markers as defined above, in a sample.
The term "determining the level of a tumor marker or group of tumor markers" refers to the determination of the presence or amount of expression product(s) of the tumor marker or tumor marker or group of tumor markers as mentioned herein above or according to Table 1, e.g. (a) transcript(s) of the tumor marker or group of tumor markers according to Table 1, and/or the determination of the presence and/or amount of (a) protein(s) of the tumor marker or group of tumor markers according to Table 1.
The term "level of the tumor marker or group of tumor markers according to Table 1 " thus means the presence or amount of (an) expression product(s) of the tumor marker or group of tumor markers according to Table 1, e.g. (a) transcript(s) of the tumor marker or group of tumor markers according to Table 1, and/or the determination of the presence or amount of (a) protein(s) of the tumor marker or group of tumor markers according to Table 1. The determination of the presence or amount of (an) expression product(s) of the tumor marker or group of tumor markers according to Table 1, e.g. (a) transcript(s) of the tumor marker or group of tumor markers according to Table 1 or (a) protein(s) of the tumor marker or group of tumor markers according to Table 1 may be accomplished by any means known in the art.
In a preferred embodiment of the present invention the determination of the presence or amount of the expression products of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, e.g. transcript(s) and/or of protein(s) of the markers mentioned in Table 1 , comprising for instance sequences as depicted in sections D) and E) of Table 1 , is accomplished by the measurement of nucleic acid or protein levels or by the determination of the biological activity of said tumor marker or group of tumor markers. For example, the measurement of the nucleic acid level of the expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be assessed by separation of nucleic acid molecules (e.g. R A or cDNA) obtained from the sample in agarose or polyacrylamide gels, followed by hybridization with tumor marker specific oligonucleotide probes as defined herein above, e.g. oligonucleotide probes comprising fragments of the sequences indicated in section D) of Table 1, or complementary sequences thereof. Alternatively, the expression level may be determined by the labeling of nucleic acid obtained from the sample followed by separation on a sequencing gel. Nucleic acid samples may be placed on the gel such that patient and control or standard nucleic acid are in adjacent lanes. Comparison of expression levels may be accomplished visually or by means of a densitometer. Methods for the detection of mRNA or expression products are known to the person skilled in the art. Typically, Northern blot analysis may be used for such a purpose.
Alternatively, the nucleic acid level of the expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be detected in a DNA array or microarray approach. Typically, sample nucleic acids derived from subjects to be tested are processed and labeled, preferably with a fluorescent label.
Subsequently, such nucleic acid molecules may be used in a hybridization approach with immobilized capture probes corresponding to the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 of the present invention or known biomarker or cancer marker genes. Suitable means for carrying out microarray analyses are known to the person skilled in the art.
In a standard setup a DNA array or microarray comprises immobilized high- density probes to detect a number of genes. The probes on the array are complementary to one or more parts of the sequence of the marker gene, or to the entire coding region of the marker gene. In the present invention, any type of tumor marker associated polynucleotide may be used as probe for the DNA array, as long as the polynucleotide allows for a specific distinction between the tumor marker expression and the expression of other genes.
Typically, cDNAs, PCR products, and oligonucleotides are useful as probes. For example, a fragment comprising 5'- or 3 '-portions of the tumor markers or group of tumor markers as mentioned herein above or according to Table 1, e.g. of the sequences indicated in section D) of Table 1 may be used as a probe. The DNA array or microarray may comprise probes of one or more of the tumor marker of Table 1, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 etc. or all of the tumor markers or any combination of said markers. Furthermore, any type of fragment or sub-portion of any of the markers sequences may be combined with any further fragment or sub-portion of any of the markers sequences of Table 1. In addition to the determination of the expression of tumor markers according to Table 1 also the determination of the expression of other genes, e.g. additional biomarker or cancer marker genes is envisaged by the present invention.
A DNA array- or microarray-based detection method typically comprises the following steps: (1) Isolating mRNA from a sample and optionally converting the mRNA to cDNA, and subsequently labeling this RNA or cDNA. Methods for isolating RNA, converting it into cDNA and for labeling nucleic acids are described in manuals for micro array technology. (2) Hybridizing the nucleic acids from step 1 with probes for the marker genes. The nucleic acids from a sample can be labeled with a dye, such as the fluorescent dyes Cy3 (red) or Cy5 (blue). Generally a control sample is labeled with a different dye. (3) Detecting the hybridization of the nucleic acids from the sample with the probes and determining at least qualitatively, and more particularly quantitatively, the amounts of mRNA in the sample for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or additional marker genes investigated. The difference in the expression level between sample and control can be estimated based on a difference in the signal intensity. These can be measured and analyzed by appropriate software such as, but not limited to the software provided for example by Affymetrix.
There is no limitation on the number of probes corresponding to the marker genes used, which are spotted on a DNA array. Also, a marker gene can be represented by two or more probes, the probes hybridizing to different parts of a gene. Probes are designed for each selected marker gene. Such a probe is typically an oligonucleotide comprising 5-50 nucleotide residues. Longer DNAs can be synthesized by PCR or chemically. Methods for synthesizing such oligonucleotides and applying them on a substrate are well known in the field of micro-arrays. Genes other than the marker genes may be also spotted on the DNA array. For example, a probe for a gene whose expression level is not significantly altered may be spotted on the DNA array to normalize assay results or to compare assay results of multiple arrays or different assays. Alternatively, the nucleic acid level of expression of the tumor marker or group of tumor markers according to Table 1 may be detected in a quantitative RT-PCR approach, preferably in a real-time PCR approach following the reverse transcription of the mRNA transcript(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Typically, as first step, a transcript is reverse transcribed into a cDNA molecule according to any suitable method known to the person skilled in the art. A quantitative or real-time PCR approach may subsequently be carried out based on a first DNA strand obtained as described above. Preferably, Taqman or Molecular Beacon probes as principal FRET -based probes of this type may be used for quantitative PCR detection. In both cases, the probes, preferably probes of the tumor marker or group of tumor markers according to Table 1 as defined herein above, serve as internal probes which are used in conjunction with a pair of opposing primers that flank the target region of interest, preferably a set of oligonucleotides specific for the tumor marker or group of tumor markers according to Table 1 as defined herein above. Upon amplification of a target segment, the probe may selectively bind to the products at an identifying sequence in between the primer sites, thereby causing increases in FRET signaling relative to increases in target frequency.
Preferably, a Taqman probe to be used for a quantitative PCR approach according to the present invention may comprise (a) oligonucleotide(s) derived from the tumor marker or group of tumor markers according to Table 1 as defined above of about 22 to 30 bases that is labeled on both ends with a FRET pair. Typically, the 5' end will have a shorter wavelength fluorophore such as fluorescein (e.g. FAM) and the 3' end is commonly labeled with a longer wavelength fluorescent quencher (e.g. TAMRA) or a non- fluorescent quencher compound (e.g. Black Hole Quencher).
It is preferred that the probes to be used for quantitative PCR, in particular the probes tumor marker or group of tumor markers as mentioned herein above or according to Table 1, have no guanine (G) at the 5' end adjacent to the reporter dye in order to avoid quenching of the reporter fluorescence after the probe is degraded. A Molecular Beacon probe to be used for a quantitative PCR approach according to the present invention preferably uses FRET interactions to detect and quantify a PCR product, with each probe having a 5' fiuorescent-labeled end and a 3' quencher-labeled end. This hairpin or stem-loop configuration of the probe structure comprises preferably a stem with two short self-binding ends and a loop with a long internal target-specific region of about 20 to 30 bases.
Alternative detection mechanisms which may also be employed in the context of the present invention are directed to a probe fabricated with only a loop structure and without a short complementary stem region. An alternative FRET -based approach for quantitative PCR which may also be used in the context of the present invention is based on the use of two hybridization probes that bind to adjacent sites on the target wherein the first probe has a fluorescent donor label at the 3 ' end and the second probe has a fluorescent acceptor label at its 5' end.
The measurement of protein levels of the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or of any fragments, homologues or derivates derived thereof may be carried out via any suitable detection technique known in the art. Preferably, the protein level of tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and derivatives thereof may be determined immunologically, e.g. by using an antibody specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, preferably an antibody as defined herein above. Alternatively, antibody variants or fragments as defined herein above may be used.
The present invention also envisages the use of peptide affinity ligands like aptamers specific for the protein(s) of the tumor marker or group of tumor markers according to Table 1 as defined herein above.
Determination of the protein levels of the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 can be accomplished, for example, by the separation of proteins from a sample on a polyacrylamide gel, followed by identification of the protein(s) of tumor marker or group of tumor markers as mentioned herein above or according to Table 1 using specifically binding antibodies in a Western blot analysis. Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection. In other embodiments, protein samples are analyzed by mass
spectroscopy.
Within the context of the present invention antibodies specific for (a) protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be placed on a support and be immobilized. Proteins derived from samples or tissues to be analyzed may subsequently be mixed with the antibodies. A detection reaction may then be carried out, e.g. with a second affinity ligand as defined herein above, preferably with a specific antibody.
Immunological tests which may be used in the context of the present invention, in particular for the diagnostic purposes of the present invention, include, for example, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassay like RIA (radio-linked immunoassay), ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fiuorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence
immunoassays, electrochemiluminescence immunoassay (ECLIA) and protein A
immunoassays. Such assays are routine and well known to the person skilled in the art.
Furthermore, the binding affinity of an antibody to an antigen and the off-rate of an antibody- antigen interaction may be determined by competitive binding assays.
In this context, the binding affinity of an antibody to an antigen and the off- rate of an antibody- antigen interaction may be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with a suitable antibody in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off- rates may be determined from the data by any suitable analysis approach, e.g. by a scatchard plot analysis. Competition with a second antibody may also be determined using radioimmunoassays. In this case, the antigen may be incubated with a suitable antibody conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
In addition, aptamers specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, may be used in a method of detecting proteins of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Such aptamers may preferably be labeled in order to allow the detection of a protein- ligand interaction.
The determination of the biological activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be carried out by employing molecular or enzymatic assays specific to the corresponding function or functions of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, e.g. of a tumor marker protein having a sequence as indicted in section E) of Table 1. Suitable techniques would be known to the person skilled in the art. An inhibition of the activity may be carried out by any suitable means known to the person skilled in the art, preferably via the use of suitable antisense nucleotides, siR A molecules or miRNA molecules, more preferably via specifically hybridizing antisense nucleotides, specific siR A or miR A molecules, e.g. as described herein below.
In a further preferred embodiment the biological activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be tested with the help of suitable enzymatic reactions or tests for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , known to the person skilled in the art, or by employing specific inhibitors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. The use of such inhibitors may, for example, be combined with an enzymatic readout system. Typical inhibitors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 to be used comprise antisense molecules, siRNA molecules or miRNA molecules.
The level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may also be detected in methods involving histological or cell- biological procedures. Typically, visual techniques, such as light microscopy or
immunofluoresence microscopy, as well as flow cytometry or luminometry may be used.
The presence of (a) protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 in a cell may, for instance, be detected or determined by removing cells to be tested from samples as defined herein above. Also tissue sections or biopsy samples may be used for these methods. Subsequently, affinity ligands for the tumor marker or group of tumor markers as mentioned herein above or according to
Table 1 may be applied, preferably antibodies or aptamers. Typically, such affinity ligands are labeled, preferably with fluorescent labels as defined herein above. Such a procedure allows for the detection of the tumor marker or group of tumor markers according to Table 1 , for its/their quantification and, in addition, allows to determine the distribution and relative level of the expression thereof.
Such procedures involve the use of visualization methods. Suitable
visualization methods are known to the person skilled in the art. Typical methods to be used comprise fluorometric, luminometric and/or enzymatic techniques. Fluorescence is normally detected and/or quantified by exposing fluorescent labels to light of a specific wavelength and thereafter detecting and/or quantifying the emitted light of a specific wavelength. The presence of a luminescently tagged affinity ligand may be detected and/or quantified by luminescence developed during a chemical reaction. Detection of an enzymatic reaction is due to a color shift in the sample arising from chemical reaction. In a further, preferred embodiment the level of the tumor marker or group of tumor markers according to Table 1 may be determined by suitable molecular imaging techniques, e.g. magnetic resonance imaging (MRI) or magnetic photon imaging (MPI), and/or by using suitable contrast agents, e.g. contrast agents as defined herein above.
In a further preferred embodiment of the present invention a method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, or the progression form a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprises the additional step of comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease.
The expression level of the tumor marker or the group of tumor markers of a less progressed stage of the same neoplastic disease may be an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, is/are known or from the same subject at an earlier point in time or an expression level corresponding to a stage of said neoplastic disease or form of said neoplastic disease, whose disease state or stage is known including different
proliferation/developmental stages of the neoplastic disease as defined herein above, or levels of development of benign hyperplasia, benign growths, benign lesions, benign tumors and the like as well as to all pre-malignant growths, swellings and lesions, malignant tumors, malignant cancers, metastases and the like, in particular such diseases and/or disorders of the prostate in the organism, as described herein above
The term "comparing" as used herein refers to any suitable method of assessing, calculating, evaluating or processing of data. In a preferred embodiment of the present invention the determination of the presence or amount of (an) expression product(s), e.g. (a) transcript(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or of (a) protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, is accomplished by the measurement of nucleic acid or protein levels or by the determination of the biological activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Thus, the expression level(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be determined by a method involving the detection of an mRNA encoded by the genes encoding for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , or the according cDNA sequence, e.g. the sequences indicated in section D) of Table 1, the detection of the protein(s) encoded by the transcript of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or the detection of the biological activity of the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, e.g. of the proteins having the sequences indicated in section E) of Table 1. For example, the measurement of the nucleic acid level of expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be assessed by separation of nucleic acid molecules as described herein above.
In yet another embodiment as a further, additional step a decision on the presence or stage of a more progressed stage of the neoplastic disease as defined herein above, or the progression towards said stage of said neoplastic disease may be based on the results of the comparison step. A more progressed stage of said neoplastic disease may be diagnosed or prognosticated or a progression towards said more progressed stage of said neoplastic disease may be diagnosed or prognosticated in said method according to the corresponding definitions provided herein above in the context of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
In another embodiment of the present invention the above mentioned method is a method of graduating a neoplastic disease, comprising the steps of
(a) determining the level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 in a sample by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers,
(b) comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease; and
(c) deciding on the stage or developmental status of a neoplastic disease based on the results obtained in step (b).
The determination of the level of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be carried out according to steps as defined herein above. Preferably, the determination may be carried out as measurement of a nucleic acid level or protein level according to the herein above described options for such measurements. In one embodiment, steps a), b), c) and/or d) of this method of diagnosis may be performed outside the human or animal body. A more progressed stage of a neoplastic disease may be graduated in said method according to the corresponding definitions provided herein above in the context of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
In another aspect the present invention relates to a method of data acquisition comprising at least the steps of:
(a) testing in an individual for expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1; and
(b) comparing the expression as determined in step (a) to a control level. The testing for expression of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be carried out according to steps as defined herein above. Preferably, the testing may be carried out as measurement of protein levels of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , more preferably according to the herein above described options for such measurements. The term "control level" as used in the context of the method of data acquisition refers to the expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or other suitable markers in a control or the expression level of said marker(s) in a less progressed stage of a neoplastic disease, as defined herein above. The status, nature, amount and condition of the control level may be adjusted according to the necessities.
A comparison of the expression to a control level may be carried out according to any suitable method of assessing, calculating, evaluating or processing of data and particularly aims at the detection of differences between two data sets. A statistical evaluation of the significance of the difference may further be carried out. Suitable statistical methods are known to the person skilled in the art. Obtained data and information may be stored, accumulated or processed by suitable informatics or computer methods or tools known to the person skilled in the art and/or be presented in an appropriate manner in order to allow the practitioner to use the data for one or more subsequent deduction or conclusion steps.
In another embodiment the present invention relates to a method of detecting, diagnosing, monitoring or prognosticating a neoplastic disease as defined herein above associated with a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprising at least the steps of:
(a) testing in at least one sample obtained from at least one individual suspected to suffer from cancer for expression of the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ;
(b) testing in at least one sample obtained from at least one individual suspected to suffer from a neoplastic disease as defined herein above for expression of the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ;
(c) determining the difference in the expression of steps (a) and (b); and
(d) deciding on the presence of a neoplastic disease as defined herein above in particular of a more progressed stage of said neoplastic disease or on the progression of said neoplastic disease, in particular from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease based on the results obtained in step (c).
In one embodiment, steps a), b), c) and/or d) of this method of diagnosis may be performed outside the human or animal body. The testing for expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be carried out according to steps as defined herein above. Preferably the testing may be carried out as measurement of nucleic acid or protein levels of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or by determining the biological activity of said tumor markers, more preferably according to the herein above described options for such measurements. The testing may be carried out in an individual, i.e. in vivo, or outside the individual, i.e. ex vivo or in vitro.
In another aspect the present invention relates to an immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, or for detecting, diagnosing, graduating, monitoring or prognosticating the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprising at least the steps (a) testing in a sample obtained from an individual for the expression of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ;
(b) testing in a control sample for the expression of the same tumor marker or group of tumor markers as in (a);
(c) determining the difference in expression of the tumor marker or group of tumor markers of steps (a) and (b); and
(d) deciding on the presence, stage or risk of recurrence of said neoplastic disease or the progression of said neoplastic disease based on the results obtained in step (c), wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or a group of tumor markers as defined above.
The term "risk of recurrence" as used herein refers to a likelihood or probabilty assessment regarding the chances or the probability that a subject or individual may be afflicted with or may be developing a similar or the same neoplastic disease as defined herein above, preferably cancer, comparable to the one that the subject or individual has been treated for or diagnosed for, based on the expression modification of the tumor marker or a group of tumor markers as defined above. A risk of recurrence may, for example, be present when a tumor marker or group of turmor markers as defined herein above shows a modified expression level, e.g. an increased or decreased expression level in comparison to a less progressed stage, in particular a non-malignant stage, benign tumor stage or a healthy stage, although histological markers, cell shapes etc. or other tumor biomarker, e.g. PSA, show no modification, or a different modification, e.g. an opposite modification or a less pronounced increase or decrease of expression level(s).
The testing for expression of the tumor marker or group of tumor markers according to Table 1 may be carried out according to steps as defined herein above.
Preferably, the testing may be carried out as measurement of protein levels of the tumor marker or group of tumor markers according to Table 1 , more preferably according to the herein above described options for such measurements. As controls or control samples controls as defined herein above may be used.
In a particularly preferred embodiment the testing steps may be based on the use of an antibody specifically binding to a tumor marker according to Table 1 as laid out above, e.g. (a) commercially available antibody/antibodies against (a) protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. A neoplastic disease as defined herein above in particular a more progressed cancer stage, may be diagnosed or prognosticated or a progression of a neoplastic disease as defined herein above, in particular from a less progressed to a more progressed stage of said neoplastic disease, may be diagnosed or prognosticated in said immunoassay according to the corresponding definitions provided herein above in the context of the tumor marker or group of tumor markers according to Table 1 as marker(s) for said neoplastic disease.
In another aspect the present invention relates to a method of identifying an individual for eligibility for a neoplastic disease therapy comprising:
(a) testing in a sample obtained from an individual for the expression of an up-regulated tumor marker or group of tumor markers as defined above, or as indicated in section P), R), T) or V) of Table 1;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said up-regulated tumor marker or group of tumor markers as defined above;
(c) classifying the levels of expression of step (a) relative to levels of step
(b); and
(d) identifying the individual as eligible to receive a neoplastic disease therapy where the individual's sample is classified as having an increased level of expression of an up-regulated tumor marker or group of tumor markers as defined above.
In another aspect the present invention relates to a method of identifying an individual for eligibility for a neoplastic disease therapy comprising:
(a) testing in a sample obtained from an individual for the expression of a down-regulated tumor marker or group of tumor markers as defined above, or as indicated in section Q), S), U) or W) of Table 1;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said down-regulated tumor marker or group of tumor markers as defined above;
(c) classifying the levels of expression of step (a) relative to levels of step
(b); and
(d) identifying the individual as eligible to receive a neoplastic disease therapy where the individual's sample is classified as having a decreased level of expression of a down-regulated tumor marker or group of tumor markers as defined above.
The level of a tumor marker or group of tumor markers may be determined on the nucleic acid, protein or activity level as described herein above. Preferred is the determination of the amount of tumor marker protein and/or transcript(s). In addition the level of a reference gene in a sample may be determined. The term "reference gene" as used herein refers to any suitable gene, e.g. to any steadily expressed and continuously detectable gene, gene product, expression product, protein, peptide or protein variant in the organism of choice. The term also includes gene products such as expressed proteins, peptides, polypeptides, as well as modified variants thereof. The invention hence also includes reference proteins derived from a reference gene. Also encompassed are all kinds of transcripts derivable from the reference gene as well as modifications thereof or secondary parameters linked thereto. Alternatively or additionally, other reference parameters may also be used for reference purposes, e.g. metabolic concentrations, cell sizes etc.
Testing for the expression of a reference gene may be carried out in the same sample used for the determination of the tumor marker or group of tumor markers of the invention. If the testing is carried out in the same sample, a single detection or a multiplex detection approach may be performed.
For the performance of the multiplex detection the concentration of primers and/or probe oligonucleotides may be modified. Furthermore, the concentration and presence of further ingredients like buffers, ions etc. may be modified, e.g. increased or decreased in comparison to manufacturers' indications.
Alternatively, the testing for the expression of a reference gene may be carried out in a different sample, preferably a control sample. Preferably, such a "control sample" may be a control sample from the same individual as the test sample, or a control sample derived from a different source or individual. The control sample may further be either a sample derived from the same tissue, preferably prostate tissue, or be derived from a different tissue type. Examples of preferred alternative tissue types are stromal prostate tissue, bladder epithelial tissue and urethra epithelial tissue. Furthermore, the testing of the test sample for the expression of a reference gene and the testing of control sample for the expression of a tumor marker or group of tumor markers may be combined.
In a further embodiment the control sample may also be tested for the expression of the reference gene. In case more than one sample was tested for the expression of a reference gene, the obtained expression results may be compared and/or averaged or normalized according to any suitable statistical method known to the person skilled in the art.
The term "classifying the levels of expression of step (a) relative to levels of step (b)" as used herein means that the expression in a test sample for a tumor maker or group of tumor markers according to the invention and the expression in a control sample for a tumor maker or group of tumor markers as defined herein are compared, e.g. after normalization against a suitable normalization references. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the control sample, an increased expression in comparison to the control sample, or an reduced expression in comparison to the control sample. The term further means that the expression in a test sample for a tumor marker and the expression in the same test sample for a reference gene are compared, e.g. after normalization against a further gene as normalization reference. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the reference gene, an increased expression in comparison to the reference gene, or an reduced expression in comparison to the reference gene.
According to the classification of the expression results an individual may be considered to be eligible for a neoplastic disease therapy when the expression level of an up- regulated tumor marker as defined herein above, or as indicated in section P), R), T) or V) of Table 1, or the expression level of a group of tumor markers as defined herein above is increased. The expression level is deemed to be "increased" when the tumor marker gene expression, or the expression of the group of tumor marker genes in the test sample is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding tumor marker gene expression, or to the expression of the corresponding group of tumor markers in a control sample, or elevated at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the tumor marker expression, or the expression of the group of tumor markers in a control sample; or when the tumor marker or group of tumor marker gene expression is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the expression of a reference gene in a control sample, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more elevated in comparison to the expression of a reference gene. In a specific embodiment, the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes. Alternatively, instead of the gene expression level the amount of protein present, or the amount of detecting antibodies present may be measured. For these alternative determination procedures the above indicated parameters also indicate an increase of expression.
According to the classification of the expression results an individual may also be considered to be eligible for a neoplastic disease therapy when the expression level of a down-regulated tumor marker as defined herein above, or as indicated in section Q), S), U) or W) of Table 1, or the expression level of a group of tumor markers as defined herein above is decreased. The expression level is deemed to be "decreased" when the tumor marker gene expression, or the expression of the group of tumor marker genes in the test sample is lowered by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding tumor marker gene expression, or to the expression of the corresponding group of tumor markers in a control sample, or lowered at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the tumor marker expression, or to the expression of the group of tumor markers in a control sample; or when the tumor marker or group of tumor marker gene expression is lowered by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the expression of a reference gene in a control sample, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more lowered in comparison to the expression of a reference gene. In a specific embodiment, the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes. Alternatively, instead of the gene expression level the amount of protein present, or the amount of detecting antibodies present may be measured. For these alternative determination procedures the above indicated parameters also indicate a decrease of expression.
In another aspect the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:
(a) testing in a sample obtained from an individual for the expression of an up-regulated tumor marker or group of tumor markers as defined above or as indicated in section P), R), T) or V) of Table 1;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said up-regulated tumor marker or group of tumor markers as defined above;
(c) determining the difference in expression of said up-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said up-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b); and
(d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an increased level of expression of an up-regulated tumor marker or group of tumor markers as defined above. In yet another aspect the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:
(a) testing in a sample obtained from an individual for the expression of a down-regulated tumor marker or group of tumor markers as defined above or as indicated in section Q), S), U) or W) of Table 1 ;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said down-regulated tumor marker or group of tumor markers as defined above;
(c) determining the difference in expression of said down-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said down-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b); and
(d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an decreased level of expression of an down-regulated tumor marker or group of tumor markers as defined above.
The testing of the expression of a tumor marker or group of tumor markers may preferably be carried out via the determination of the amount of tumor marker protein or the determination of the tumor marker protein/expression product activity level. Preferred is the determination of the amount of tumor marker proteins or peptides with the help of specific antibodies binding said tumor marker protein or expression product. Alternatively, the immunoassay may be carried out with any other suitable agent or be combined with the determination of other entities. For example, the assay may be combined with the detection of the presence or amount of nucleic acids, or enzymatic testing methods as described herein. In addition the level of a reference gene as described herein above in a sample may be determined. Testing for the expression of a reference gene may be carried out in the same sample used for the determination of the tumor marker or group of tumor markers. If the testing is carried out in the same sample, a single detection or a parallel or multiplex detection approach may be performed. Preferably, for a parallel or multiplex detection differently labeled primary or secondary antibodies may be used.
Alternatively, the testing for the expression of a reference gene may be carried out in a different sample, preferably a control sample. Preferably, such a control sample may be a control sample from the same individual as the test sample, or a control sample derived from a different source or individual. The control sample may further be either a sample derived from the same tissue, preferably prostate tissue, or be derived from a different tissue type. Examples of preferred alternative tissue types are stromal prostate tissue, bladder epithelial tissue and urethra epithelial tissue. Furthermore, the testing of the test sample for the expression of a reference gene and the testing of control sample for the expression of a tumor marker or group of tumor markers may be combined.
In a further embodiment the control sample may also be tested for the expression of the reference gene. In case more than one sample was tested for the expression of a reference gene, the obtained expression results may be compared and/or averaged or normalized according to any suitable statistical method known to the person skilled in the art.
The term "determining the difference in expression of a tumor marker or a group of tumor markers of step (a) and the expression of a tumor marker or group of tumor markers and/or the reference gene in step (b)" as used herein means that the expression in a test sample for the tumor marker or group of tumor markers and the expression in a control sample for the tumor marker or group of tumor markers are compared, e.g. after
normalization against a suitable normalization references. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the control sample, an increased expression in comparison to the control sample, or an reduced expression in comparison to the control sample. The term further means that alternatively or additionally the expression in a test sample for a tumor marker or group of tumor markers and the expression in the same test sample for a reference gene are compared, e.g. after normalization against a further gene as normalization reference. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the reference gene, or a difference in the expression. The difference may be either an increased expression in comparison to the reference gene, or an reduced expression in comparison to the reference gene.
The term "stratifying an individual or cohort of individuals to neoplastic disease therapy" as used herein means that an individual is identified as pertaining to a group of similar individuals, whose optimal therapy form is a neoplastic disease therapy, preferably a therapy against an earliy prostate cancer form in accordance with the outcome of the expression test as described herein above, in particular in accordance with encountered difference in the tumor marker or group of tumor marker expression level and a reference gene or the tumor marker or group of tumor marker expression level in different samples.
According to the determination of the expression difference an individual may be identified as pertaining to a group of similar individuals whose optimal therapy form is a neoplastic disease therapy when the expression levels of the up-regulated tumor marker or group of tumor markers as defined herein above, or as indicated in section P), R), T) or V) of Table 1 , are increased. The expression level is deemed to be "increased" when the gene expression of the up-regulated tumor marker or group of tumor marker as defined herein above, or as indicated in section P), R), T) or V) of Table 1, is elevated in the test sample by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to said tumor marker or group of tumor marker expression in a control sample, or at least elevated 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to said tumor marker or group of tumor marker expression in a control sample; or when the tumor marker or group of tumor marker gene expression is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the expression of a reference gene in a control sample, or is elevated at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more elevated in comparison to the expression of a reference gene. In a specific embodiment, the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes.
According to the determination of the expression difference an individual may also be identified as pertaining to a group of similar individuals whose optimal therapy form is a neoplastic disease therapy when the expression levels of the down-regulated tumor marker or group of tumor marker as defined herein above, or as indicated in section Q), S), U) or W) of Table 1, are decreased. The expression level is deemed to be "decreased" when the gene expression of the down-regulated tumor marker or group of tumor markers as defined herein above, or as indicated in section Q), S), U) or W) of Table 1, is lowered in the test sample by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to said tumor marker or group of tumor marker expression in a control sample, or at least lowered 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to said tumor marker or group of tumor marker expression in a control sample; or when the tumor marker or group of tumor marker gene expression is lowered by, for example, 5%>, 6%>, 7%>, 8%>, 9%>, 10%>, 15%>, 20%>, 25%o, 30%), 40%), 50%), or more than 50%> in comparison to the expression of a reference gene in a control sample, or is lowered at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more lowered in comparison to the expression of a reference gene. In a specific embodiment, the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes.
An individual being considered to be eligible for a neoplastic disease therapy or being stratified to a neoplastic disease therapy as described herein above may receive any suitable therapeutic treatment for this disease form known to the person skilled the art. In particular, the term "neoplastic disease therapy" as used herein refers to any suitable neoplastic disease therapy, preferably prostate cancer related therapy known to the person skilled in the art, and preferably includes surgical castration by removal of the testes as the main organ of male sex hormone production, chemical castration by e.g., suppression of generation of androgens or by inhibition of the androgen receptor activity, cytotoxic, chemotherapy, targeted therapy (e.g. targeting cellular proteins with chemical molecules to suppress or stimulate their activity), radiation therapy (External Beam Radiation Therapy, Brachytherapy), Cryotherapy, focal therapies like HIFU ablation (High Frequency
Ultrasound ablation), or thermal ablation or any type of combination therapy of at least two of the above mentioned treatment forms either in direct combination, or used in a subsequent form.
Typically, an individual considered to be eligible for neoplastic disease therapy due to an increased expression of a tumor marker or group of tumor marker of the present invention may be deemed to be suffering from a neoplastic disease or be prone to develop a neoplastic disease in the future, e.g. within the next 1 to 24 months. A
correspondingly identified or stratified individual may be treated with a pharmaceutical composition according to the present invention, e.g. as defined herein below. In a further embodiment a correspondingly identified individual may be treated with a pharmaceutical composition according to the present invention in combination with an additional cancer therapy. The term "additional cancer therapy" refers to any types of cancer therapy known to the person skilled in the art. Preferred are cancer therapy forms known for prostate cancer. The term includes, for example, all suitable forms of chemotherapy, radiation therapy, surgery, antibody therapies etc.
Alternatively, a correspondingly identified or stratified individual may also be treated solely with one or more cancer therapies such as a chemotherapy, radiation therapy, surgery, antibody therapies etc. Preferred are cancer therapies typically used for prostate cancer.
In a further embodiment of the present invention the classification method for eligibility or the immunoassay for stratification as described herein above may also be used for monitoring the treatment of an individual, e.g. an individual being classified as suffering from a neoplastic disease. The monitoring process may be carried out as expression determination or protein detection over a prolonged period of time, e.g. during or after treatment sessions, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months, or 1, 2, 3 or more years. The determination steps may be carried out in suitable intervals, e.g. every week, 2 weeks, 3 weeks, every month, 2 months, 3 months, 6 months, 12 months etc.
In a further embodiment of the present invention any treatment scheme as mentioned herein may be adjusted, e.g. enforced or attenuated, or altered in any suitable manner in correspondence with the results of the monitoring process.
In a particularly preferred embodiment of the present invention the reference gene is a housekeeping gene. In human organisms, examples of "housekeeping genes" include inter alia β-actin, glycerinaldehyde 3-phosphate dehydrogenase (GAPDH), porphobilinogen deanimase (PBGD), and ribosomal protein PI . Apart from these genes any other suitable gene may be used as a house-keeping gene, as long as the gene shows an expression or transcription on a steady, non-modified level, in particular during different stages of neoplastic disease development, more preferably during different stages of prostate cancer development. Expression data of a house-keeping gene may be obtained from one or more samples of the same individual or from more individuals, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 1000, 5.000, 10.000 or more. Expression data may also be obtained from databases or from data collections available to the person skilled in the art.
Accordingly normalization and/or comparison with GAPDH or PBGD may preferably be used for the methods of identifying or the immunoassays for discriminating or stratifying individuals. Corresponding determination steps may either be carried out in separate reactions, or, particularly preferred in multiplex reactions. For the performance of the multiplex detection the concentration of primers and/or probe oligonucleotides may be modified. Furthermore, the concentration and presence of further ingredients like buffers, ions etc. may be modified, e.g. increased or decreased in comparison to manufacturers' indications.
In a further embodiment of the present invention the method of identifying an individual for eligibility for a neoplastic disease therapy based on the expression of a tumor marker or group of tumor markers or the immunoassay for stratifying an individual or cohort of individuals as described herein above may further be combined with one or more similar identification methods, based on the expression of one or more different biomarkers.
Preferred is the determination of the level of prostate specific antigen (PSA), more preferably in blood. Thus, if the level of PSA in blood is encountered to be of a range of about 2 to 5 or more ng/ml, preferably of about 2.2 to 4.8 ng/ml or more, 2.4 to 4.4 ng/ml or more, 2.6 to 4.2 ng/ml ore more or 2.8 to 4.0 ng/ml or more, more preferably of about 2.5 to 4 ng/ml or more, an individual may be considered to be suffering from a neoplastic disease, or be likely to develop a neoplastic disease in the near future, i.e. within the next 1, 2, 3, 4, 5, 6, 12, 14, 48 months.The testing for expression of a tumor marker or group of tumor markers may be carried out according to steps as defined herein above.
The detection or measurement of the tumor marker, or of the group or combination of markers as defined herein, i.e. derivable from Table 1, may further be used to monitor the potential re-currence of a neplastic disease, in partiuclar a neoplastic disease of the prostate, after a primary treatment, e.g. surgery, or radiotheary. The tumor marker detection or measurement may be performed every 3, or every 6, or every 12 months after the patient has been treated. If the expression level of the tumor marker, or group of tumor markers as defined herein is going to pass a threshold as defined herein, e.g. as derivable form Table 1 or Table 7, by 5%, 10%, 15%, 20%, 25%, or 30% or more, further
investigations or treatment may be started, be used in for the inhibition of tr
In a preferred embodiment of the present invention the diagnosing, detecting, monitoring or prognosticating as mentioned above is to be carried out on a sample obtained from an individual.
The term "sample obtained from an individual" as used herein relates to any biological material obtained via suitable methods known to the person skilled in the art from an individual, as laid out above. The sample used in the context of the present invention should preferably be collected in a clinically acceptable manner, more preferably in a way that nucleic acids (in particular R A) or proteins are preserved.
The biological samples may include body tissues and/or fluids, such as blood, or blood components like serum or plasma, sweat, sputum or saliva, semen and urine, as well as feces or stool samples. Furthermore, the biological sample may contain a cell extract derived from or a cell population including an epithelial cell, preferably a neoplastic, e.g. cancerous epithelial cell or an epithelial cell derived from tissue suspected to be neoplastic, e.g. cancerous. The sample used in the context of the present invention should preferably be collected in a clinically acceptable manner, more preferably in a way that nucleic acids (in particular RNA) or proteins are preserved.
Alternatively, the biological sample may contain a cell population derived from a glandular tissue, e.g. the sample may be derived from the prostate of a male individual. Additionally, cells may be purified from obtained body tissues and fluids if necessary, and then used as the biological sample. Samples, in particular after initial processing, may be pooled. However, also non-pooled samples may be used.
In a specific embodiment of the present invention the content of a biological sample may also be submitted to an enrichment step. For instance, a sample may be contacted with ligands specific for the cell membrane or organelles of certain cell types, e.g. prostate cells, functionalized for example with magnetic particles. The material concentrated by the magnetic particles may subsequently be used for detection and analysis steps as described herein above or below.
In a specific embodiment of the invention, biopsy or resections samples may be obtained and/or used. Such samples may comprise cells or cell lysates. Furthermore, cells, e.g. tumor cells, may be enriched via filtration processes of fluid or liquid samples, e.g.
blood, urine, sweat etc. Such filtration processes may also be combined with enrichment steps based on ligand specific interactions as described herein above.
In a particularly preferred embodiment of the present invention a sample may be a tissue sample, a urine sample, a biopsy sample, a urine sediment sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a semen sample, or a sample comprising circulating tumor cells.
A subject or individual to be diagnosed, monitored or in which a neoplastic disease as defined herein above, a progression of said neoplastic disease or predisposition for said neoplastic disease is to be detected or prognosticated according to the present invention is an animal, preferably a mammal, more preferably a human being.
In a specific embodiment of the present invention the obtaining step of a sample may be included as a first or additional step in any of the herein mentioned methods, uses or approaches.
In another aspect the present invention relates to a pharmaceutical composition relates to a pharmaceutical composition comprising at least one element selected from the group of: (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (e) a miR A specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (g) a siR A specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (h) an aptamer specific for the expression product of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (i) a small molecule or peptidomimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; and j) an antibody specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an antibody variant specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
The term "a compound directly inhibiting the activity of a tumor marker" as used herein refers to a compound which is capable of decreasing the activity of a tumor marker according to Table 1. Such a compound may be any direct interactor of the tumor marker according to Table 1, which has negative influence on the catalytic activity of the tumor marker or group of tumor markers according to Table 1. Such a compound may preferably be an antagonist of the catalytic activity of the tumor marker according to Table 1 , e.g. of a tumor marker protein having a sequence as indicated in section E) of Table 1.
The term "a compound indirectly inhibiting the activity of a tumor marker " as used herein refers to a compound which is capable of decreasing the activity of the tumor marker or group of tumor markers according to Table 1 by an interaction with a direct interactor of the tumor marker according to Table 1 ("indirect interactor") or via an indirect working pathway not involving an interaction with of the tumor marker according to Table 1. Such a compound may be any direct interactor of an interactor of the tumor marker according to Table 1, e.g. of a tumor marker protein having a sequence as indicated in section E) of Table 1. The effect conveyed by the direct interactor of an interactor of the tumor marker according to Table 1 may be either negative if the interactor of the tumor marker or group of tumor markers according to Table 1 itself has a negative effect on the activity of the tumor marker according to Table 1 , or negative, if the interactor of the tumor marker according to Table 1 has a positive effect on the activity of said tumor marker.
Alternatively, such negatively working indirect integrators may provoke a modification of the binding behavior of directly binding proteins, leading to a decreased activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Typically negatively working indirect interactors may have an inhibitory effect on activators of the tumor marker according to Table 1. Examples of such interactors are enzymatic activities degrading activators of the tumor marker according to Table 1, or proteins capable of binding and quenching activators of the tumor marker according to Table 1. Alternatively, such interactors may positively modulate activities leading to a degradation of the tumor marker according to Table 1, e.g. proteinases. Further examples and their implementation would be known to the person skilled in the art.
Alternatively, an indirect inhibition of the activity of the tumor marker according to Table 1 may be conveyed by compounds deactivating, interfering or disrupting the expression of the endogenous gene(s) of said tumor markers. Examples of such compounds are specific interactors of transcription factors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 that inhibit and/or preclude binding of transcription factors and the basal transcription machinery to the promoters of the tumor marker according to Table 1, specific destabilizing activities of the mR A(s) of the tumor marker according to Table 1 or factors inhibiting the splicing factors specific for the tumor marker according to Table 1. Further examples and their implementation would be known to the person skilled in the art.
The "nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker" as used herein refers to any nucleic acid capable of expressing a mutant form of a naturally occurring protein or polypeptide of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Thus the term refers to a nucleic acid encoding (a) variant(s) of the tumor marker according to Table 1, which comprises an antimorphic modification, in particular which adversely affects the tumor marker according to Table 1 of the invention. Typically, such a behavior may occur if the antimorphic variant can interact with the tumor marker according to Table 1 but blocks some aspect of its function. Preferably, such variants may comprise or lack specific domains of the tumor marker according to Table 1, e.g. one or more protein-protein interacting or dimerization domains, complex assembly domains, one or more membrane-associated domains etc. This is particularly of importance in a protein that functions as a dimer or multimer. If, for example, one part of that protein complex is mutant in some functional aspect of the multimer but is still able to form the multimer it may have a dominant effect on the other wildtype portions of the complex, and a negative effect if the mutation prevents the complex from carrying out its normal function. Thus, especially in the case of tumor markers of the present invention which homomultimerize, such a dominant-negative form can specifically block the action of the wild-type tumor-marker from which it was derived. Tests to identify dominant negative variants include appropriate genetic screenings, for instance readout-systems based on the expression of nucleic acids comprising the nucleotide sequence as indicated in section D) of Table 1 and/or functional assays of the proteins and or polypeptides of the invention comprising the amino acid sequence as indicated in section E) of Table 1 or derivatives thereof.
The term "miR A specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 " refers to a short single-stranded R A molecule of typically 18-27 nucleotides in length, which regulate gene expression of one or more of the tumor marker according to Table 1. miRNAs are encoded by genes from whose DNA they are transcribed but are not translated into a protein. In a natural context miRNAs are first transcribed as primary transcripts or pri-miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the
Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). After integration into an active RISC complex, miRNAs may base pair with their complementary mRNA molecules and inhibit translation or may induce mRNA degradation by the catalytically active members of the RISC complex, e.g. argonaute proteins. Mature miRNA molecules are typically at least partially
complementary to mRNA molecules corresponding to the expression product of the present invention, and fully or partially down-regulate gene expression. Preferably, miRNAs according to the present invention may be 100% complementary to their target sequences. Alternatively, they may have 1, 2 or 3 mismatches, e.g. at the terminal residues or in the central portion of the molecule. miRNA molecules according to the present invention may have a length of between about 18 to 27 nucleotides, e.g. 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides. Preferred are 21 to 23 mers. miRNAs having 100% complementarity may preferably be used for the degradation of nucleic acids according to the present invention, whereas miRNAs showing less than 100% complementarity may preferably be used for the blocking of translational processes.
The term "antisense molecule of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 " refers to nucleic acids corresponding to the sequences indicated in section D) of Table 1 or the complementary strand thereof.
Preferably, the antisense molecule of the invention comprises a sequence complementary to at least a portion of a tumor marker expression product according to the present invention. While antisense molecules complementary to the coding region sequence of tumor marker expression products may be used, those complementary to the transcribed and untranslated region are preferred. Generally, antisense technology can be used to control, i.e. reduce or abolish gene expression through antisense DNA or RNA, or through triple-helix formation. In one embodiment, an antisense molecule may be generated internally by the organism, for example intracellularly by transcription from an exogenous sequence. A vector or a portion thereof may be transcribed, producing an antisense nucleic acid of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense molecule. Corresponding vectors can be constructed by recombinant DNA technology methods known to the person skilled in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells, e.g. vectors as defined herein above.
In another embodiment, the antisense molecule may be separately administered. As an example, the 5' coding portion of a nucleic acid according to the present invention, e.g. of the sequence indicated in section D) of Table 1 may be used to design an antisense RNA or DNA oligonucleotide of from about 6 to 50 nucleotides in length.
Preferably, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides in length.
The antisense nucleic acids of the invention typically comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion of an RNA transcript" as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex or triplex formation in the case of double stranded antisense nucleic acids. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex or triplex. A person skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. Preferably antisense molecules complementary to the 5' end of the transcript, e.g., the 5' untranslated sequence up to and including the AUG initiation codon may be used in for the inhibition of translation. In a further preferred embodiment, sequences
complementary to the 3' untranslated sequences of mR As may also be used.
An antisense molecule according to the present invention may be DNA or
R A or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. An antisense molecule, preferably an antisense olignucleotide or any further antisense nucleic acid molecule according to the present invention or a siR A molecule according to the present invention or any other ncR A molecule according to the present invention as defined herein above can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The molecule may include other appended groups such as peptides (e. g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane or the blood- brain barrier hybridization triggered cleavage agents or intercalating agents. The molecule may accordingly be conjugated to another molecule, e. g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
The antisense molecule or antisense oligonucleotide, miR A- or siRNA molecule, may comprise at least one modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethyl-aminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methyl guanine, 3 -methyl cytosine, 5-methylcytosine, N6-adenine, 7- methyl guanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-
N6isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, and 2,6-diaminopurine. The molecule may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2- fluoroarabinose, xylulose, and hexose. In another embodiment, the molecule comprises alternatively or additionally at least one modified phosphate backbone, e.g. a
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In another embodiment, the antisense molecule, e.g. the antisense oligonucleotide may be an alpha-anomeric oligonucleotide, i.e. an oligonucleotide which forms specific double-stranded hybrids with complementary RNA in which the strands run parallel to each other.
The term "siRNA specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 " refers to a particular type of antisense- molecules, namely small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway to negatively regulate gene expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. These siRNA molecules can vary in length and may be between about 18-28 nucleotides in length, e.g. have a length of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides. Preferably, the molecule has a length of 21, 22 or 23 nucleotides. The siRNA molecule according to the present invention may contain varying degrees of complementarity to their target mRNA, preferably in the antisense strand. siRNAs may have unpaired overhanging bases on the 5' or 3' end of the sense strand and/or the antisense strand. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region. Preferably the siRNA may be double-stranded wherein the double-stranded siRNA molecule comprises a first and a second strand, each strand of the siRNA molecule is about 18 to about 23 nucleotides in length, the first strand of the siRNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA via RNA interference, and the second strand of said siRNA molecule comprises nucleotide sequence that is complementary to the first strand.
Methods for designing suitable siRNAs directed to a given target nucleic acid are known to person skilled in the art. Furthermore, antagonistic siRNA molecules may be obtained according to methods of identifying antagonists as described herein.
The term "aptamer specific for the expression product or specific for the protein of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 " as used herein refers to (a) short peptide(s) capable of interacting and specifically binding the protein(s) of the tumor marker according to Table 1. The peptide aptamer(s) may preferably be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (the) amino acid sequence(s) as indicated in section E) of Table 1. The peptide aptamer(s) may also be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (an) amino acid sequence(s) encoded by (a) DNA sequence(s) being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as indicated in section D) of Table 1 , or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as indicated in section E) of Table 1. Typically, (a) peptide aptamer(s) is/are a variable peptide loop, comprising for example, 10 to 20 amino acids. In the context of the present invention the peptide aptamer(s) may preferably be attached at one or both ends to a scaffold structure. The scaffold structure may be any molecule, preferably a protein, which has good solubility properties. Suitable scaffold molecules would be known to the person skilled in the art. A preferred scaffold molecule to be used in the context of the present invention is the bacterial protein thioredoxin-A. The aptamer peptide loop may preferably be inserted within a reducing active site of the scaffold molecule. Alternatively, staphylococcal protein A and domains thereof and derivatives of these domains, such as protein Z or lipocalins may be used as scaffold structures in the context of the present invention. Peptide aptamers may be generated according to any suitable method known to the person skilled in the art, e.g. via yeast two-hybrid approaches.
In a preferred embodiment the above mentioned peptide aptamer is capable to bind to a protein or polypeptide of the invention corresponding to the sequences indicated in section E) of Table 1 and to reduce the biological activity and/or the enzymatic activity of these/this protein(s) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or by at least 98% or 99% when compared to a control level obtained from an untreated sample.
A "small molecule capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 " as used herein refers to a small organic compound that is preferably biologically active, i.e. a biomolecule, but is preferably not a polymer. Such an organic compound may have any suitable form or chemical property. The compound may be a natural compound, e.g. a secondary metabolite or an artificial compound, which has been designed and generated de novo. In an embodiment of the present invention a small molecule is capable of blocking the interaction between the tumor marker protein(s) and its interactor(s). Methods and techniques for the identification and preparation of small molecules as well as assays for the testing of small molecules are known to the person skilled in the art.
The term "peptidomimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 " in the context of the present invention refers to a small protein-like chain designed to mimic a peptide and capable of binding (a) protein(s) of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Such a peptidomimetic may arise from a modification of an existing peptide, e.g. a peptide or peptide aptamer as defined herein above, in order to alter the molecule's properties. A peptidomimetic may arise from a modification which changes the molecule's stability or binding capability. These modifications typically involve changes to the peptide that will not occur naturally. For example, a peptidomimetic according to the present invention may have altered peptide backbones or may comprise non- natural amino acids. Methods and techniques for the preparation of peptidomimetics as well as assays for the testing of peptidomimetics are known to the person skilled in the art.
A pharmaceutical composition according to the present invention may also comprise an antibody specific for the protein(s) of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an antibody variant specific for said protein, e.g. an antibody or antibody variant as defined herein above.
In a preferred embodiment such an antibody or antibody fragment may be capable of inhibiting the biological activity and/or enzymatic activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
The skilled person would also be aware of the possibility to target and destroy neoplastic and/or cancer cells and tissue by virtue of conjugated antibodies specific for the tumor markers. Thus, in a specific embodiment of the present invention the antibody or fragment thereof as defined herein above may be conjugated to a therapeutic or cytotoxic agent. The term "therapeutic agent" refers to any compound, drug, small molecule or medicament, which is able to confer a therapeutic effect to a cell, a tissue or the entire organism. Examples of such agents are known to the person skilled in the art. The term "cytotoxic agent" refers to any compound, drug, small molecule which is able to confer a toxic effect to a cell or a tissue. Such agents may, for example, comprise compounds which activate endogenous cytotoxic effector systems, as well as radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. The term may also include radioisotopes known in the art, additional antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, R Ase, alpha toxin, ricin, abrin,
Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. The term also refers to cytotoxic produgs. By
"cytotoxic prodrug" is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the invention include glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,
daunorubicin, and phenoxyacetamide derivatives of doxorubicin.
In a preferred embodiment of the present invention the pharmaceutical composition may comprise, or may additionally comprise, an antibody or a group of antibodies specific for the expression product or protein of a tumor marker or group of tumor markers as defined herein, e.g. specific for one or more of the protein(s) of the tumor marker according to Table 1. Particularly preferred is an antibody which specifically binds to an expression product, protein or peptide comprising the amino acid sequence as indicated in section E) of Table 1, or a fragment of said amino acid sequence, e.g. a peptide of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids length.
In a another aspect the present invention relates to a pharmaceutical composition for use in, or for the treatment or prevention of a neoplastic disease, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of a tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of: (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (e) a miRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (g) a siRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (h) an aptamer specific for the expression product of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (i) a small molecule or peptido mimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; and (j) an antibody specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an antibody variant specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Such pharmaceutical composition preferably comprises elements as defined herein above. Preferably, such a pharmaceutical composition may comprise elements or ingredients as defined herein above according to the up-regulation indication provided in any of sections P), R), T) or V) of Table 1. Thus, a pharmaceutical composition may preferably comprise any of the above mentioned elements (a) to j) specifically for tumor markers which are indicated with a "+" in any of sections P), R), T) or V) of Table 1.
In another aspect of the present invention relates to a pharmaceutical comprising at least one element selected from the group of: (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and (e) a miRNA inhibitor specific for a miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
The term "a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 " as used herein refers to a compound which is capable of increasing the activity of one or more of the tumor marker according to Table 1. Such a compound may be any direct interactor of the tumor marker according to Table 1 , which has positive influence on the catalytic activity of the tumor marker according to Table 1. Such a compound may preferably be an agonist of the catalytic activity of the tumor marker according to Table 1.
The term "a compound indirectly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 " as used herein refers to a compound which is capable of increasing the activity of the tumor marker according to Table 1 by an interaction with a direct interactor of the tumor marker according to Table 1 ("indirect interactor") or via an indirect working pathway not involving an interaction with of the tumor marker according to Table 1. Such a compound may be any direct interactor of an interactor of the tumor marker according to Table 1, e.g. of the protein(s) of a tumor marker or group of tumor markers according to section E) of Table 1. The effect conveyed by the direct interactor of an interactor of the tumor marker according to Table 1 may be either positive if the interactor of the tumor marker according to Table 1 itself has a positive effect on the activity of the tumor marker according to Table 1, or negative, if the interactor of the tumor marker according to Table 1 has a negative effect on the activity of the tumor marker according to Table 1.
Alternatively, such positively working indirect integrators may provoke a modification of the binding behavior of directly binding proteins, leading to an increased activity of the tumor marker according to Table 1. Typically negatively working indirect interactors may have an inhibitory effect on inhibitors of the tumor marker according to Table 1. Examples of such interactors are enzymatic activities degrading inhibitors of the tumor marker according to Table 1, or proteins capable of binding and quenching inhibitors of the tumor marker according to Table 1. Alternatively, such interactors may inhibit activities leading to a degradation of the tumor marker according to Table 1, e.g. proteinase inhibitors. Further examples and their implementation would be known to the person skilled in the art.
Alternatively, an indirect stimulation of the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be conveyed by compounds activating, protecting or sustaining the expression of the endogenous gene(s) of the tumor marker according to Table 1. Examples of such compounds are specific transcription factors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, specific stabilizing activities of the mR A(s) of the the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or splice factors specific for the the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Further examples and their implementation would be known to the person skilled in the art.
The "protein of a tumor marker" comprised in the pharmaceutical composition may be a protein of the tumor marker according to Table 1 as defined herein above. In particular, it may be a protein being encoded by splice variant of the tumor marker according to Table 1. More preferably it may have the amino acid sequence as set forth in section E) of Table 1. The "protein of a tumor marker " as used in this context also comprises amino acid sequences being at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%) or 99% identical to the sequences as set forth in section E) of Table 1 and amino acid sequences being encoded by nucleotide sequences being at least 60%>, 70%>, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as indicated in section D) of Table 1. In a further embodiment of the invention the homologous variants of the tumor marker according to Table 1 may additionally or alternatively have a similar or identical localization pattern as the tumor marker according to Table 1 within a cell or within a tissue type.
The term "biologically active equivalent of a tumor marker" as used herein refers to a protein of the tumor marker according to Table 1 which is capable of performing all or a majority of the individual functions of the tumor marker according to Table 1. In a further embodiment of the invention the biologically active equivalents of the tumor marker according to Table 1 may additionally or alternatively have a similar or identical localization pattern as the tumor marker according to Table 1 within a cell or within a tissue type.
Biologically active equivalents of the tumor marker according to Table 1 may also comprise variants of the tumor marker according to Table 1 as defined herein above.
The proteins of the tumor marker according to Table 1 or biologically active equivalents of the tumor marker according to Table 1 according to the present invention may be produced recombinantly by any suitable method known to the person skilled in the art. The present invention, thus, also encompasses methods for the production of the tumor marker proteins according to Table 1 or biologically active equivalents of the tumor markers according to Table 1.
Accordingly, the present invention contemplates vectors containing the polynucleotides encoding the tumor markers according to Table 1 or biologically active equivalents of the tumor markers according to Table 1 as defined herein above, host cells, and the production of the tumor markers according to Table 1 or biologically active equivalents of the tumor markers according to Table 1 by recombinant techniques.
A suitable vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. The vectors may preferably comprise one or more of the nucleotide sequences indicated in section D) of Table 1.
Polynucleotides encoding the tumor markers according to Table 1 or biologically active equivalents of the tumor markers according to Table 1 may be joined to a vector or carrier containing a selectable marker for propagation in a host. A corresponding polynucleotide insert may be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, or the PSA promoter. Other suitable promoters are known to the person skilled in the art. The expression constructs may further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
The polypeptides or proteins may be glycosylated or may be non-glycosylated or may otherwise by modified. In addition, polypeptides or proteins may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Furthermore, the polypeptide, protein or peptide may be modified by acetylation, pegylation, hesylation, formylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, specific chemical cleavage, proteolytic cleavage, a linkage to a cellular ligand or other protein or hapylation, i.e. a fusion with a glycine-rich homo-amino- acid polymer (HAP), etc. Such modifications may be carried out by suitable techniques known to the person skilled in the art. Additionally, the polypeptide, peptide or variant may contain one or more non-classical amino acids.
In addition, the tumor marker proteins according to Table 1 or biologically active equivalents of the tumor markers according to Table 1 of the invention can be chemically synthesized using techniques known in the art, e.g. by using a peptide synthesizer.
The "nucleic acid encoding and expressing the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 " comprised in the pharmaceutical composition as defined herein above refers to any suitable carrier element, e.g. as described herein above, comprising an expressable gene of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Preferably, such a carrier element may comprise the sequence(s) as indicated in section E) of Table 1. Such a carrier element may also comprises nucleotide sequences showing a high degree of homology to the tumor markers according to Table 1, e.g. nucleic acid sequences being at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as indicated in section D) of Table 1 or nucleic acid sequences encoding amino acid sequences being at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%o or 99% identical to the sequence(s) as indicated in section E) of Table 1. Alternatively, the carrier may comprise the genomic sequence of the tumor marker according to Table 1. Furthermore, biologically active equivalents of the tumor markers according to Table 1 as defined herein above may be comprised in a carrier of the present invention.
The polynucleotide encoding the tumor marker according to Table 1 may preferably be joined to a vector containing a selectable marker for propagation in a human cell. In a preferred embodiment the polynucleotide insert may be operatively linked to a PSA promoter.
In one embodiment of the present invention nucleic acids encoding and expressing the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 may be provided via living therapeutics. The term "living therapeutic" means that the tumor markers according to Table 1 or biologically active equivalents of the tumor markers according to Table 1 as defined herein above are expressed in any suitable live carrier. Accordingly, the present invention relates to corresponding polynucleotides which are suitable for expression in a living cell. The present invention also relates to vectors containing such polynucleotides, appropriate host cells, and the production of polypeptides by recombinant techniques in said host cells.
The term "live carrier" relates to any appropriate living host cell or virus known to the person skilled in the art. Representative examples of appropriate hosts include, but are not limited to, bacterial cells such as Escherichia coli or Lactobacillus, fungal cells, such as yeast cells, protozoa, insect cells, or animal cells. Preferably, the term relates to attenuated bacteria, attenuated fungal cells or attenuated protozoa. Representative examples of appropriate viruses include viruses of the group of adenoviruses, retrovirues or lentiviruses, preferably attenuated viruses of the group of adenoviruses, retroviruses or lentiviruses. In a preferred embodiment, probiotic bacterial cells, in particular probiotic Escherichia coli or Lactobacillus cells may be used. More preferably, cells of Escherichia coli Nissle 1973 and even more preferably cells of Lactobacillus casei or Lactobacillus zeae 393 may be used.
The "miRNA inhibitor specific for miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 " comprised in the pharmaceutical composition as defined herein above refers to a nucleic acid molecule encoding a nucleic acid sequence complementary to a miRNA or microRNA molecule of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. The term "complementary" as used herein refers to a perfect complementary between the miRNA inhibitor nucleic acid (sense molecule) and the miRNA (antisense molecule) without any mismatch, as well as situations in which the nucleic acid contains any base mismatches and/or additional or missing nucleotides in comparison to the miRNA molecule. In other embodiments, the two molecules comprise one or more base mismatches or differ in their total numbers of nucleotides (due to additions or deletions). In further embodiments, the "complementary" miRNA inhibitor nucleic acid molecule comprises at least ten contiguous nucleotides showing perfect complementarity with a sequence comprised in the miRNA molecule.
Typically miRNA inhibitor nucleic acid molecules are naturally occurring DNA- or RNA-molecules or synthetic nucleic acid molecules comprising in their sequence one or more modified nucleotides which may be of the same type or of one or more different types.
It is, for example, envisaged by the present invention that such a miRNA inhibitor nucleic acid molecule comprises at least one ribonucleotide backbone unit and at least one deoxyribonucleotide backbone unit. Furthermore, the miRNA inhibitor nucleic acid molecule may contain one or more modifications of the RNA backbone into 2'-0-methyl group or 2'-0-methoxyethyl group (also referred to as "2'-0-methylation"), which prevented nuclease degradation in the culture media and, importantly, also prevented endonucleolytic cleavage by the RNA-induced silencing complex nuclease, leading to irreversible inhibition of the miRNA. Another possible modification, which is functionally equivalent to 2'-0- methylation, involves locked nucleic acids (LNAs) representing nucleic acid analogs containing one or more LNA nucleotide monomers, as defined herein above.
Another class of silencers of miRNA expression to be used in the context of the present invention comprises chemically engineered oligonucleotides named "antagomirs", which represent single-stranded RNA molecules conjugated to cholesterol. The molecules may comprise between 19 and 25 nucleotides. Preferably, the molecule comprises 20, 21, 22, 23 or 24 nucleotides. More preferably, the molecule comprises 23 nucleotides (further details may be derived from Krutzfeldt et al, 2005, Nature, 438: 685-689).
In another embodiment of the present invention miRNA inhibitors as defined herein above may be provided in the form of expression vectors to be introduced into tissue or cells. Alternatively, such vectors may also be introduced in living therapeutics as defined herein above.
Typically, RNAs may be produced from transgenes provided in the form of trans fection or transient expression vectors or carriers. For instance, competitive miRNA inhibitors may be provided as transcripts expressed from strong promoters, containing more than one, preferably multiple, tandem binding sites to a microRNA of interest. In a specific embodiment of the present invention a demethylation agent may be comprised in the pharmaceutical composition according to the present invention. The term "demethylation agent" as used herein refers to an agent capable of demethylating chromatine structures, preferably promoter regions, more preferably the promoter(s) of the tumor marker according to Table 1. Examples of demethylation agents to be used in the context of the present invention are 5-aza-2'-deoxycytidine and 5-azacytidine, which reactivate genes inappropriately silenced by structural chromatin changes that involve DNA methylation and which can reverse these changes and, therefore, restore principal cellular pathways. This typically results in gene re-expression and reversion of some aspects of the transformed state. 5-azacytidine and 5-aza-2'-deoxycytidine typically inactivate DNA cytosine C5- methyltransferases through the formation of stable complexes between the 5-aza-2'- deoxycytidine residues in DNA and the enzyme, thereby mimicking a stable transition state intermediate when bound to the methyltransferase enzyme.
A further agent, which may be comprised in a pharmaceutical composition according to the present invention, either per se or in combination with 5-aza-2'- deoxycytidine and/or 5-azacytidine, is trichostatin A (TSA).
In a another aspect the present invention relates to a pharmaceutical composition for use in, or for the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, wherein said neoplastic disease implies the decreased (down-regulated) expression of a tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of: (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and (e) a miRNA inhibitor specific for a miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Such pharmaceutical composition preferably comprises elements as defined herein above. Preferably, such a pharmaceutical composition may comprise elements or ingredients as defined herein above according to the down-regulation indication provided in any of sections Q), S), U) or W) of Table 1. Thus, a pharmaceutical composition may preferably comprise any of the above mentioned elements (a) to (e) specifically for tumor markers which are indicated with a "+" in any of sections Q), S), U) or W) of Table 1.
In a preferred embodiment of the invention a pharmaceutical composition according to the present invention, e.g. as defined herein above, may further comprise additional compounds being active against neoplastic and/or cancer cells. Furthermore, in another embodiment of the invention the pharmaceutical composition may further comprise hormone-inhibitors, preferably anti-androgens or androgen antagonists like spironolactone, cyproterone acetate, flutamide, nilutamide, bicalutamide, ketoconazole, finasteride or dutasteride.
The skilled person is aware of the fact that also a situation in which a given tumor marker of the invention is up-regulated in a neoplastic disease as defined herein above, e.g. in cancer while in parallel another tumor marker of the invention is down-regulated. It is thus an aim of the present invention to provide also pharmaceutical compositions wherein the pharmaceutical composition contains any combination of such elements as laid out above, e.g. the compounds, proteins, dominant negative proteins, nucleic acids, miR As, siRNAs, antisense RNAs, aptamers, antibodies, peptidomimetics and small molecules, and wherein the composition contains said elements being capable of down-regulating at least one tumor marker according to Table 1 and up-regulating at least one other tumor marker according to Table 1. It is preferred to avoid conflicting and/or opposite functionalities or overlapping functional spectra of the elements of the pharmaceutical compositions as defined herein above, e.g. if the tumor markers have similar functionalities. Due to the different identity of the tumor marker, in such situations the use of highly specific elements like antibodies, siRNAs etc. is envisaged.
In another preferred embodiment of the invention the pharmaceutical relates to the pharmaceutical compositions as laid out in the present description for the treatment of a neoplastic disease as defined herein above, preferably cancer, more preferably prostate cancer.
In further specific embodiment of the present invention the above described pharmaceutical compositions is for the manufacture of a medicament for the treatment of a neoplastic disease, preferably cancer, more preferably prostate cancer.
In a most preferred embodiment of the present invention the pharmaceutical compositions and medicaments of the present invention are capable of reducing the tumor volume of a given tumor, e.g. a prostate carcinoma, by at least 5%, 10%, 20%, 30%>, 40%>, 50%, 60%, 70%, 80%, 90%, or at least 95% when compared to an untreated control.
In a further embodiment the present invention also envisages screening procedures and methods for the identification of an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, a compound directly stimulating or modulating the activity of the tumor marker according to Table 1 , an agonist of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 enzymatic activity, a miR A inhibitor specific for miR A(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, an antagomir, a demethylation agent specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , or a peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. Such screening procedures may comprise the steps of (a) producing cells which express the tumor marker or group of tumor markers according to Table 1 as a polypeptide either as secreted protein or on the cell membrane or as intracellular component, (b) contacting the polypeptide produced in step (a) with a test sample potentially containing an interacting molecule, e.g. an aptamer specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, an agonist of enzymatic activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, and (c) indentifying an interacting molecule by observing binding and/or inhibition or modulation of the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
Alternatively, such screening procedures may comprise the steps of (a) contacting a test sample potentially containing a directly or indirectly interacting molecule, e.g. an aptamer specific for the transcript(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a miRNA inhibitor specific for miRNA(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , an antagomir, a demethylation agent specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 with one or more cells which express the tumor marker according to Table 1 as (a) transcript(s), (b) detecting the expression level of said sequence; and (c) indentifying an interacting molecule by observing binding or a modulation or reduction of the expression level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
The present invention also encompasses an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to
Table 1, an agonist of the enzymatic activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , a miR A inhibitor specific for miRNA(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , an antagomir, a demethylation agent specific for of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and a
peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , obtainable or obtained by a screening procedure or method as described herein above.
In a further aspect the present invention relates to a pharmaceutical composition as defined herein above for the treatment or prevention of a neoplastic disease, preferably cancer.
Further, in yet another aspect, the present invention relates to the use of (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (e) a miRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (g) a siRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (h) an aptamer specific for the expression product of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (i) a small molecule or peptidomimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; and/or (j) an antibody specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an antibody variant specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 for the preparation of a pharmaceutical composition for the treatment or prevention of a neoplastic disease as defined herein above, e.g. cancer, preferably for the treatment or prevention of a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease.
Further, in yet another aspect, the present invention relates to the use of (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and (e) a miR A inhibitor specific for a miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 for the preparation of a pharmaceutical composition for the neoplastic disease as defined herein above, preferably cancer, preferably for the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease.
In another aspect the present invention relates to a method of treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, in particular the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, comprising the administration of (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (e) a miRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (g) a siRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (h) an aptamer specific for the expression product of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (i) a small molecule or peptidomimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; and/or (j) an antibody specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an antibody variant specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 to an individual, in particular to an individual suffering from a neoplastic disease as defined herein above, preferably cancer or being prognosticated to develop said neoplastic disease.
In another aspect the present invention relates to a method of treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, in particular the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, comprising the administration of (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and (e) a miRNA inhibitor specific for a miR A of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 to an individual, in particular to an individual suffering from a neoplastic disease as defined herein above, preferably cancer or being prognosticated to develop said a neoplastic disease.
A pharmaceutical composition according to the present invention may be administered to a patient, subject or individual with the help of various delivery systems known to the person skilled in the art, e.g., via encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction may be topical, enteral or parenteral and may include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, inhalational, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e. g., oral mucosa, rectal and intestinal mucosa, etc.) or by inhalation and may be administered together with other biologically active agents. Administration can be systemic or local. A preferred method of local administration is by direct injection.
In another embodiment the pharmaceutical composition may be delivered directly to internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the site of interest. The pharmaceutical composition may also be administered to disease sites at the time of surgical intervention. In yet another embodiment, the composition can be delivered in a controlled release system.
Preferably the pharmaceutical composition is in a form, which is suitable for oral, local or systemic administration. In a preferred embodiment the pharmaceutical composition is administered locally, orally or systemically.
In a further embodiment the pharmaceutical composition comprises a therapeutically effective amount of the ingredients of the pharmaceutical composition of the present invention as defined herein above and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such a carrier is pharmaceutically acceptable, i.e. is non-toxic to a recipient at the dosage and concentration employed.
Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
The pharmaceutical composition of the invention can be formulated as neutral or salt forms.
Preferably, the pharmaceutical composition may be administered directly or in combination with any suitable adjuvant known to the person skilled in the art. The composition of the present invention can be administered to an animal, preferably to a mammal. "Mammal" as used herein is intended to have the same meaning as commonly understood by one of ordinary skill in the art. Particularly, "mammal" encompasses human beings.
The term "administered" means administration of a therapeutically effective dose of the aforementioned composition. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered, preferably this effect is induction and enhancement of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
The concentration of the active ingredients or compounds of a pharmaceutical composition according to the present invention may be further adjusted to the intended dosage regimen, the intended usage duration, the exact amount and ratio of all ingredients of the composition and further factors and parameter known to the person skilled in the art.
The active agents or compounds according to the present invention may be administered alone or in combination with other treatments. In a preferred embodiment the pharmaceutical composition of the present invention may be administered in combination with an anti-hormone treatment, e.g. an anti-androgen treatment.
The pharmaceutical composition of the present invention can also comprise any suitable preservative known to the person skilled in the art.
Furthermore, the preparations according to the invention may also comprise compounds, which have an antioxidative, free-radical scavenger, antierythematous, antiinflammatory or antiallergic action, in order to supplement or enhance their action. In another preferred embodiment of the present invention active components of the pharmaceutical composition as defined herein above may be fused to a suitable carrier protein, e.g. to Ig Fc receptor proteins or polymeric Ig receptors. Preferably the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or biologically active equivalents thereof as defined herein above may be provided as fusion proteins. The fusion partner may be provided at the N- or C-terminus.
If the pharmaceutical composition according to the present invention is to be administered in the form of a live cell or living therapeutic as defined herein above, transformed and prepared cells may be administered to a patient in any suitable form known to the person skilled in the art. Preferably living therapeutics may be administered in the form of a composition comprising a microorganism, e.g. a Lactobacillus as described above, in an amount between 10^ to 101 cells, preferably 10^ to 10^ cells.
In a further preferred embodiment of the present invention the ratio between two or more ingredients in the pharmaceutical composition or medicament may be suitably adjusted according to the skilled person's knowledge.
Suitable assays may optionally be employed to help identify optimal ratios and/or dosage ranges for ingredients of pharmaceutical compositions of the present invention. The precise dose and the ratio between the ingredients of the pharmaceutical composition as defined herein above to be employed in the formulation will, inter alia, depend on the route of administration, and the exact type of disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses or ingredient ratios may be extrapolated from dose-response curves derived from in vitro or (animal) model test systems.
A typical dose can be, for example, in the range of 0.001 to 1000 μg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
In another aspect the present invention relates to a medical kit for the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising at least one element selected from the group consisting of (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (e) a miR A specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (g) a siR A specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (h) an aptamer specific for the expression product of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; (i) a small molecule or peptido mimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1; and (j) an antibody specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and/or an antibody variant specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; or at least one element selected from the group consisting of (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 and (e) a miRNA inhibitor specific for a miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1.
A medical kit that can be used in the context of the administration of the pharmaceutical composition as defined herein above. In particular, a kit according to the present invention may be used for the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease.
The ingredients of a medical kit may, according to the present invention, be comprised in one or more containers or separate entities. They may preferably be formulated as pharmaceutical compositions or medicaments, more preferably they may be formulated as has been described herein above in the context of the pharmaceutical compositions of the present invention, e.g. they may comprise suitable pharmaceutical carriers etc. Particularly preferred are formulations for topical administration as mentioned herein above in the context of pharmaceutical compositions of the invention. The medical kit according to the present invention may optionally also comprise a documentation which indicates the use or employment of the medical kit and its components. Preferably, instructions comprised in the medical kit of the present invention may comprise recommended treatment options, dosage regimens etc. The medical kit may also comprise an instruction leaflet and/or may provide additional information on the use, dosage etc.
The medical kit of the present invention may be administered to a patient according to any suitable dosage regimen known to the person skilled in the art. The medical kit or kit components may preferably be given once a week, more preferably 2 times, 3 times, 4 times, 5 times or 6 times a week and most preferably daily and or 2 times a day or more often, unless otherwise indicated. During progression of the treatment the dosages may be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., several times a day. In a preferred case a response to the treatment may be monitored using herein described methods and further methods known to those skilled in the art and dosages may accordingly be optimized, e.g., in time, amount and/or composition. Progress can be monitored by periodic assessment. It is also envisaged that the medical kit is employed in co-therapy approaches, i.e. in co-administration with other medicaments or drugs, for example antibiotics, antiviral medicaments or IgG or IgA immunoglobulins, anticancer medicaments and, preferably, anti-hormone medicaments, more preferably anti- androgens as mentioned herein above.
In another aspect the present invention relates to a vaccine comprising the expression product or protein, or any fragment thereof, of an up-regulated tumor marker or group of tumor markers as defined herein, or as indicated in section P), R), T) or V) of Table 1, or in sections F), G), H) or I) of Table 3.
Preferably, the vaccine may comprise a protein or antigen, having, comprising or consisting of an amino acid sequence as defined in section E) of Table 3, or any fragment, variant, derivative or modified form thereof, having a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 300, 350 or 400 amino acids or the entire length or any other suitable length. Table 3:
The vaccine may preferably comprise a nucleic acid molecule encoding a tumor marker of the present invention or a group of tumor markers as defined above, e.g. a nucleic acid molecule comprising a nucleotide sequence as indicated in section D) of Table 3, and/or a vector comprising said nucleic acid molecule, a host cell comprising said vector, an antibody as defined herein, or a CTL specific for an antigen as defined herein. Thus, a vaccine according to the present invention may, for example, comprise polypeptides or proteins of varying length comprising the or comprised in the amino acids as indicated in section E) of Table 3, or a nucleotide sequence encoding such a polypeptide, an expression vectors capable of expressing the polypeptide or comprising said nucleic acid or fragments thereof, e.g. DNA plasmid vectors, viral vectors etc., host cells expressing such a
polypeptide, preferably host cells expressing the polypeptide at the surface of the cell, or secrete the polypeptide. These components or ingredients may be present either separately or in combination or in any sub-grouping or sub-combination of the mentioned items. For example, one, two, three or more different tumor marker antigens or proteins of the present invention may be present either separately or in combination.
In a specific aspect the present invention relates to a vaccine for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of a tumor marker selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAPl 3), High affinity cGMP-specific 35-cyclic
phosphodiesterase 9A (PDE9A), cAMP-dependent protein kinase type I-beta regulatory subunit (PRKARIB), and cAMP-specific 35-cyclic phosphodiesterase 4C (PDE4C) as indicated in sections F), G), H) or I) of Table 3, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 3, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) of Table 3, or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) of Table 3, or any fragment thereof.
The expression product, polypeptide or antigen comprised in the vaccine may comprise one epitope or various epitopes, e.g. a MHC I and/or a MHC II epitope or two or more copies of each or combinations thereof. Corresponding, i.e. encoding nucleic acid molecules may be provided, preferably in the form of DNA or RNA molecules, e.g. DNA vectors or expression vectors. Such vectors may preferably be DNA plasmids or viral vectors. Vectors, in particular viral vectors may be capable of replication or replication- impaired or non-replicating. The term "non-replicating" or "replication-impaired" as used herein means not capable of replication to any significant extent in the majority of normal mammalian cells or normal human cells. Viruses which are non-replicating or replication- impaired may have become so naturally (i.e. they may be isolated as such from nature) or artificially e.g. by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication. Suitable viral vectors for use in a vaccine according to the present invention include non-replicating adenoviruses such as El deletion mutants, vectors based on herpes virus and Venezuelan equine encephalitis virus (VEE). Suitable bacterial vectors include recombinant BCG and recombinant Salmonella and Salmonella transformed with plasmid DNA. Alternative suitable non- viral vectors include lipid-tailed peptides known as lipopeptides, peptides fused to carrier proteins such as KLH either as fusion proteins or by chemical linkage.
In a preferred embodiment a vaccinia virus vector such as MVA or NYVAC may be used. Most preferred is the vaccinia strain modified virus ankara (MVA) or a strain derived therefrom. MVA is a replication impaired vaccinia strain with a good safety record. In most cell types and normal human tissues, MVA does not replicate; limited replication of MVA is observed in a few transformed cell types such as BHK21 cells. Alternatives to vaccinia vectors include pox virus vectors, e.g. avipox vectors such as fowl pox or canarypox vectors. Particularly suitable as an avipox vector is a strain of canarypox known as ALVAC, and strains derived therefrom.
The term "CTL" as used herein means "cytotoxic T lymphocyte" and refers in particular to a CD4+ T lymphocyte, a CD8+ T lymphocyte or a natural killer cell. Preferably, the term refers to a CD8+ T lymphocyte or a natural killer cell. A CTL may be genertad according to any suitable approach for the generation of a CTL, which specifically detects an antigen according to the present invention, preferably a protein having or comprised in a sequence as indicted in section E) of Table 3. Subsequently, such a CTL may perform a cytotoxic reaction, e.g. via the release of the cytotoxins perforin and granulysin, or induce an appoptotic cell reaction, e.g. via the interaction with cell-surface molecules, e.g. Fas proteins expressed on a target cell. The making of CTLs typically comprises the presentation of antigens, preferably of MHC I specific antigenic peptides, to T cells, preferably to CD8+ T cells. Preferably, the antigen or antigenic peptide may be presented by a dendritic cell (DC), more preferably by a MHC I molecule present on a dendritic cell.
Positively reacting T lymphocytes may subsequently be selected, enriched and/or expanded according to suitable methods known to the person skilled in the art. T cells to be used for the production of CTLs according to the present invention may be derived from lymphoid tissue, preferably they may be obtained from a peripheral blood mononuclear cell (PBMC) cell fraction. Typically, PBMCs may be extracted from whole blood using ficoll. Alternatively, PBMC may be extracted from whole blood using a hypotonic lysis. Any other suitable method known to the person skilled in the art may also be used.
PBMCs to be used may be derived from blood obtained from blood donors. Alternatively, autologous PBMCs may be used. For the extraction of autologous cells any suitable method known to the person skilled in the art may be used. In a specific embodiment the present invention relates to such CTLs, as well as to a method of making CTLs specific for the antigen or tumor marker expression product or protein, or group of tumor marker expression products or proteins of the present invention, e.g. comprising the amino acid sequence as indicated in section E) of Table 3, which comprises the step of stimulating autologous T cells in vitro with dendritic cells loaded with a peptide derived from the antigen or a fragment thereof. This procedure may be carried out according to any suitable procedure known in the art.
Typically, the following procedure may be used: non-adherent PBMCs (preferably in an amount of 2 x 106 mL) may be co-cultered in suitable medium, e.g. in human serum, preferably in Aim-V medium supplemented with 10% pooled human serum, with mature dendritic cells preincubated with peptides derived from the antigen of the present invention. The co-cultivation may be carried out according to suitable parameters, e.g. for 7- 10 days. The preincubation with the peptides may also be carried out according to any suitable parameters known to the person skilled in the art. Preferably, the peptide may be used in a concentration of 50μg/ml. Subsequently, T cells may be collected and preferably restimulated with dendritic cells loaded with peptides derived from the antigen. The restimulation may preferably be carried out once in a week. Furthermore, the medium may be supplemented with additional factors, e.g. with IL-2, IL-7 and/or IL-15.
After a repetition of the stimulation cycle, preferably by 3 to 5 times T-cell lines may be established by limiting-dilutions. Subsequently, T-cell line clones may be expanded in T-cell medium comprising IL-2, IL-7 and/or IL-15, e.g. during 2 weeks. The CTLs may additionally be tested for their biological activity according to known methods, e.g. a cytotoxicity test.
Accordingly obtained CTLs may be stored or further expanded or be used for the preparation of medicaments of pharmaceutical compositions. Any suitable deviation from this protocol based on the knowledge of the skilled person is also envisaged by the present invention.
In a further preferred embodiment an epitope to be comprised in a vaccine may be of varying length. It may be a B-cell, T-cell, MHC I specific, or MHC II specific epitope. It may preferably have a length of about 8 to 10 amino acids in the case of a MHC I specific epitope e.g. 8, 9 or 10 amino acids, it may preferably have a length of about 13 to 17 amino acids in the case of a MHC II epitope, e.g. 13, 14, 15, 16 or 17 amino acids.
The epitope or antigen may be capable of eliciting B-cell or T-cell immune responses. The epitope or antigen may alternatively be capable of eliciting CTL or cytotoxic reactions. For a T-helper cell response, the presentation by MHC II molecules may be required. For T-cell response, the presentation by MHC I molecules may be required.
Antigens, peptides or epitopes as defined herein, may also stimulate NK-cells (natural killer cells) that are effective tumor killing cells and may be used for such an approach. Peptides or epitopes according to the present invention, e.g. as defined herein, may also stimulate dendritic cells for enhancing antigenic stimulation of lymphocytes and may be used for such an approach. The activation of cells, e.g. T-cells, NK cells or dendritic cells, may be tested with suitable tests known to the person skilled in the art. For the detection of T-cell activation an ELISPOT assay as known to the person skilled in the art may be used.
The epitopes of the present invention, e.g. fragments of proteins or peptides comprising or comprised in an amino acid sequence as indicated in section E) of Table 3, may be present in a peptide, polypeptide, protein, polyprotein or particle comprising one, two or more epitopes, or as a recombinant string of epitopes or in the context of the native target antigen, or in the form of a mixture or combination of the mentioned entities.
The term "polyprotein" refers to two or more proteins which may be the same, or preferably different, linked together. Particularly preferred in this embodiment is a recombinant proteinaceous particle such as a Ty virus-like particle (VLP).
The term "epitope string" as used herein refers to a juxtaposition or combination of one or more epitopes, e.g. of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more epitopes. Such a string may comprise solely MHC I specific epitopes or MHC II specific epitopes or a combination of both. The epitopes in a string of epitopes or of multiple epitopes may be linked together without intervening sequences so that unnecessary nucleic acid and/or amino acid material is avoided. Alternatively, the epitopes in a string of epitopes or of multiple epitopes may be linked together with intervening sequences. Such intervening sequences may have a length of 1 to 10 amino acids, preferably 2 to 5 amino acids. They may comprise amino acids without influence on the overall structure of the string, e.g. glycine.
In a further embodiment of the present invention also antigens or fragments of antigens may be presented in a string like manner, e.g. linked together. These strings may or may not have intervening sequences as mentioned above.
In addition the string of epitopes or antigens or multiple epitopes may include one or more epitopes recognised by T helper cells, to augment the immune response generated by the epitope or antigen string. Particularly suitable T helper cell epitopes are ones, which are active in individuals of different HLA types, for example T helper epitopes from tetanus (against which most individuals will already be primed). Particularly preferred is a combination of three T helper epitopes and an epitope according to the present invention. Additionally or alternatively, the epitope string may also include one or more B cell epitopes for stimulating B cell responses and antibody production. Suitable T helper and B cell epitopes are known to the person skilled in the art.
In a particularly preferred embodiment the present invention relates to a vaccine as defined herein, or a CTL as defined herein, for the treatment or prevention of a neoplastic disease, preferably for the treatment or prevention of a cancer disease, more preferably of a cancer disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastsic or cancer disease.
If a vaccine according to the present invention is for the treatment or prevention of a neoplastic or cancer disease, it may preferably comprise, in addition to antigens or epitopes as mentioned above, additional antigens or epitopes derived from or representing known tumor associated antigens (TAAs). Furthermore the string of epitopes or multiple epitopes of a corresponding vaccine may include one or more epitopes derived from or representing a known tumor associated antigen (TAA). Examples of such as TAAs which may be included in vaccines of the present invention are MAGE antigen, a SSX antigen family member, NY-ESO-1, Melan-A/MART-1, gplOO, tyrosinase, tyrosinase-related protein 1 (TRP1), TRP2, CEA, PSA, Her2/neu, p53, MUC1, PRAME, sarcosin (N-methylglycin), CA-125 (Carbophydrate antigen- 125) or survivin. The sequence and identity of these and further suitable TAAs would be known to the person skilled in the art or can be derived from a suitable textbook.
In yet another preferred embodiment the present invention envisages a method of inducing an immune response in an individual comprising administering to said subject a therapeutically effective amount of an antigen, vector, epitope etc. as mentioned above. The term "immune response" refers to a therapeutic immune response beneficial for the subject or individual. Such an immune response may be a passive or active immunization, or it may be a short-term or long-term immunoprotection. Preferably the immune response is an immune response against a neoplastic disease, e.g. a cancer disease. The term "immune response against a neoplastic disease" means that a neoplastsic cell or tissue, e.g. a cancerous cell or tissue, may be attacked by components of the immune system, e.g. by CTLs or antibodies, that such a cell or tissue may be reduced in its size or modified in its structure by the mentioned entities or that such cell or tissue may be eliminated by the mentioned entities.
In a further, particularly preferred embodiment the present invention also envisages a method of identifying an individual for eligibility for neoplastic disease therapy or treatment or a corresponding immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease as defined above, wherein the detection of an increased level of expression of a tumor marker or group of tumor markers as described herein above, leads to, or is used for the preparation of a vaccine or of a method of vaccination comprising an antigen or epitope linked to the detected, increased tumor marker or group of tumor markers of the present invention. The present invention thus also encompasses a personalized vaccine or method for vaccination based on the outcome of a method of identifying an individual for eligibility for a neoplastic disease therapy, or a corresponding immunoassay for stratifying an individual or cohort of individuals with such a disease. The term "linked to" as used herein means that the tumor marker or group of tumor markers encountered to be increased according to parameters as defined herein may be used in the form of a protein sequences, e.g. comprising or comprised in the amino acid sequence as indicated in sections E) of Table 3, or in the form of a nucleotide sequence, e.g. comprising or comprised in the nucleic acid sequence as indicated in section D) of Table 3. Such antigens or nucleic acid molecules may be provided in different suitable forms, e.g. as full length sequence, or in the form of fragments, in combination with one or more tumor marker s as defined herein, or in combination with known markers or TAA as described above.
A vaccine according to the present invention or vaccine compounds or ingredients as defined herein above may be given, e.g. in the course of a method for immunization or method of treatment of the present invention, once or more than one time, e.g. 2, 3, 4, 5, 6 or more times, preferably 2, 3 or 4 times according to any suitable vaccination scheme known in the art.
In case a vaccine or vaccine ingredient are given more than once a prime and boost administration may be pursued, e.g. a "prime" administration is followed by one or more "boosts" to achieve the desired effects. The same composition or vaccine ingredient can be administered as the prime and as the one or more boosts. Alternatively, different compositions or vaccine ingredients can be used for priming and boosting. Furthermore, priming and boosting compositions or vaccines may comprise different combinations of vaccine ingredients, e.g. first a combination of a DNA plasmid and peptide or polypeptide, followed a DNA plasmid or vice versa etc.
Furthermore, prime and boost compositions may be different in terms of vectors to be used. In a preferred example, the priming composition may be a viral vector and the boosting composition may also be a viral vector, however derived from a different virus. Alternatively, a prime composition may comprise a DNA or plasmid vector and the boost composition may comprise a viral vector, or vice versa. Further preferred are prime boost schemes in which at least one of the vectors is replication-impaired or non-replicating.
A preferred carrier for a vaccine is a molecule that does not itself induce the production of antibodies harmful to the individual receiving the vaccine. Suitable carriers are typically large, slowly metabolized macro molecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Furthermore, the antigen of the present invention, e.g. an antigen comprising or comprised in a tumor marker protein or expression product as defined in Section E) of Table 3 may be conjugated to carrier elements such as a bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc.
The vaccine used according to the invention may also be provided in frozen, freeze-dried or lyophilized form, which may be thawed, or reconstituted, respectively, when needed.
In a further embodiment of the present invention the vaccine may comprise or be mixed with at least one suitable adjuvant. Adjuvants which are preferred for vaccines comprise 1018 ISS, aluminium salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, Amplivax, AS 15, BCG, CP-870893, CpG7909, CyaA, dSLIM, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V,
Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, SAF, RAS, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL1 72, Virosomes and other Virus-like particles, YF-17DBCG, Aquila's QS21 stimulon, Detox Quil, Superfos, Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant, pertussis toxin (PT), E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S 109, and PT-K9/G129, MF59, or saponin adjuvants.
Adjuvants may be combined in any suitable form and amount with the pharmaceutical composition, kit or vaccine of the present invention. The use of an adjuvant may be adjusted to the concrete purpose of the treatment. Such a use may vary depending on the target cell or tissue, the administration way, treatment scheme etc.
A vaccine or immunological formulation may contain the immunogenic active substance at any suitable concentration, preferably at low concentrations, such as in an immunogenic amount ranging from 0.01 μg to 10 mg. Depending on the nature of the antigen, epitope, vector, vehicle, antibody etc. or on the presence of additional agents such a carriers or adjuvants, a suitable immunogenic dose may be chosen, e.g. in the range of from 0.01 μg to 750 μg, preferably 100 μg to 500 μg. In a further embodiment the vaccine according to the present invention may be provided in the form of a depot vaccine which is to be delivered to the organism over an extended period of time may. In such a case, the amount of ingredients may be higher such as from at least 1 mg to up to more than 10 mg.
A vaccine usually may be provided, for example, in ready-to-use syringes having a volume of from 0.01 to 1 ml, preferably 0.1 to 0.75 ml, of the concentrated solution, or suspension, respectively.
Vaccines of the present invention may be administered to a subject or individual by any suitable method, preferably via injection using either a conventional syringe or a gene gun, such as the Accell® gene delivery system. Delivery of DNA into cells of the epidermis is particularly preferred as this mode of administration provides access to skin-associated lymphoid cells and provides for a transient presence of DNA in the recipient. Both, nucleic acids and/or peptides and/or antibodies can be injected either subcutaneously, epidermally, intradermally, intramucosally such as nasally, rectally and vaginally, intraperitoneally, intravenously, orally or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, needle-less injection,
transcutaneous and transdermal applications. If solids are employed as auxiliary agents for the vaccine formulation, e.g. an adsorbate or a suspended mixture of vaccine ingredient with the auxiliary agent is administered. In special embodiments, the vaccine is administered as a solution, or liquid vaccine, respectively, in an aqueous solvent.
As mentioned above, tumor markers as described herein may be over- expressed or down-regulated upon progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease. Accordingly, pharmaceutically active agents that down-regulate or increase expression of such tumor markers can be used to specifically treat such neoplatic diseses, including neoplastic diseases of the prostate.
Pharmaceutical compositions comprising such active agents have been described in detail above. However, the knowledge of over-expression or down-regulation of tumor markers can also be used to screen molecular libraries of potentially pharmaceutically active agents for identifying pharmaceutically active agents that may be used to specifically treat or prevent the neoplatic diseases escribed herein such as neoplatic diseases of the prostate.
The present invention in one embodiment thus relates to the use of a tumor marker or a group of tumor markers as described herein for identifying pharmaceutically active agents useful in the treatment or prevention of a neoplastic disease. In a preferred embodiment the present invention relates to the use of a tumor marker or a group of tumor markers as described herein for identifying pharmaceutically active agents useful in the treatment or prevention of a neoplastic diease of the prostate. In a more preferred
embodiment the present invention relates to the use of a tumor marker or a group of tumor markers as described herein for identifying pharmaceutically active agents useful in the treatment or prevention of prostate cancer.
The present invention thus e.g. also relates to a method for identifying pharmaceutically active agents which can be used to treat or prevent progression from a less progressed stage of a neoplastic disease or cancer stage to a more progressed stage of a neoplastic disease or cancer stage comprising at least the steps of:
a) providing a cell;
b) determining expression of a tumor marker or a group of tumor markers as described herein in said cell;
c) subjecting said cell to at least one potentially pharmaceutically active agent;
d) determining expression of a tumor marker or a group of tumor markers as described herein in said cell which has been subjected to at least one potentially pharmaceutically active agent;
e) comparing the expression level of step b) and d);
f) classifying the at least one potentially pharmaceutically active agent as an agent that either increases or decreases expression of a tumor marker or a group of tumor markers as described herein.
In step a) one may use a cell obtained from a healthy human being of from a patient suffering from a disease such as a cancer. One may also use a cultured cell line. In a preferred embodiment, these cells and cell lines are outside the human body
In step c) one may subject such cells and cell lines to libraries of potentially pharmaceutically active agents. Such libraries may be small molecule libraries, aptamer libraries, antibody libraries, siR A libraries, peptide libraries and the like.
Determining expression levels in steps b) and d) may be undertaken as is common in the art.
The potentially pharmaceutically active agents can then be classified as an agent that either increases or decreases expression of a tumor marker or a group of tumor markers as described herein. Such agents may be used directly to treat or prevent progression from a less progressed cancer stage to a more progressed cancer stage or they may be used as hits or lead compounds for further drug development. The accordingly encountered active agents are also envisaged as part of the present invention.
An especially preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein the neoplastic disease as defined herein above is a neoplastic disease of the prostate. In a further specific embodiment of the present invention said neoplastic disease of the prostate is a benign prostate tumor, a malignant prostate tumor or prostate cancer. A "malignant prostate tumor " is understood to encompass prostate cancer and vice versa.
The terms "neoplastic disease of the prostate", "benign prostate tumor" and "prostate cancer" refer to different stages and developmental transitions starting form a healthy prostate tissue and/or cells over benign tumor/lesion/growth/hyperplasia, to cancers of the prostate gland. A "neoplastic disease", "prostate tumor" or "prostate cancer" as used herein thus denotes a developmental stage of prostate tissue which can be classified according to the TNM classification by the International Union Against Cancer (UICC). Preferably, the terms relates to a classification of prostate tissue pursuing the following T - Primary Prostate Tumor classification schedule:
TX. Primary tumor cannot be assessed
TO. No evidence of primary tumor
Tl . Clinically inapparent tumor not palpable or visible by imaging
Tla. Tumor incidental histological finding in 5% or less of tissue resected
Tib. Tumor incidental histological finding in more than 5% of tissue resected
Tic. Tumor identified by needle biopsy (e.g., because of elevated PSA) T2. Tumor confined within prostate (Tumor found in one or both lobes by needle biopsy, but not palpable or visible by imaging, is classified as Tic)
T2a. Tumor involves one half of one lobe or less
T2b. Tumor involves more than half of one lobe, but not both lobes
T2c. Tumor involves both lobes
T3. Tumor extends through the prostatic capsule (Invasion into the prostatic apex, or into (but not beyond) the prostate capsule, is not classified as T3, but as T2)
T3a. Extracapsular extension (unilateral or bilateral)
T3b. Tumor invades seminal vesicle(s) T4. Tumor is fixed or invades adjacent structures other than seminal vesicles: bladder neck, external sphincter, rectum, levator muscles, or pelvic wall Nl . Tumor invades regional lymph node(s)
Mia. Tumor invades non-regional lymph node(s)
Mlb. Tumor invades bone(s)
Mlc. Tumor invades other site(s)
G Histopathological Grading
GX. Grade cannot be assessed
Gl . Well differentiated (slight anaplasia) (Gleason 2-4)
G2. Moderately differentiated (moderate anaplasia) (Gleason 5-6)
G3-4. Poorly differentiated/undifferentiated (marked anaplasia) (Gleason 7-
10),
wherein T categories are physical examination, imaging, endoscopy, biopsy, and biochemical tests , N categories are physical examination and imaging tests and M categories are physical examination, imaging, skeletal studies, and biochemical tests, and wherein stages I to IV of prostate cancer correspond to the following scheme:
Stage I: Tla; N0; M0; Gl
Stage II: Tla; NO; MO; G2, 3-4, or
Tib. c; NO; MO; any G, or
T1. T2; N0; MO; any G
Stage III T3; NO; MO; any G
Stage IV T4; NO; MO; any G, or
Any T; Nl; MO; any G, or
Any T; Any N; Ml; any G.
Thus, the term "benign prostate tumor" as used herein particularly refers to a prostate tumor which lacks all three of the malignant properties of a cancer, i.e. does not grow in an unlimited, aggressive manner, does not invade surrounding tissues, and does not metastasize. Typically, a benign prostate tumor implies a mild and non-progressive prostate neoplastic or swelling disease lacking the invasive properties of a cancer. Furthermore, benign prostate tumors are typically encapsulated, and thus inhibited in their ability to behave in a malignant manner. A benign tumor or a healthy condition may be determined by any suitable, independent molecular, histological or physiological method known to the person skilled in the art. In contrast thereto the term "prostate cancer" as used herein relates to a cancer of the prostate gland in the male reproductive system, which occurs when cells of the prostate mutate and begin to multiply out of control. Typically, prostate cancer is linked to an elevated level of prostate-specific antigen (PSA). In one embodiment of the present invention the term "prostate cancer" relates to a cancer showing PSA levels above 4.0. In another embodiment the term relates to cancer showing PSA levels above 2.0. The term "PSA level" refers to the concentration of PSA in the blood in ng/ml. A "prostate cancer" as used herein may further have one of the following grade of Gleason score: Grade 1 (the cancerous prostate closely resembles normal prostate tissue. The glands are small, well- formed, and closely packed), Grade 2 (the tissue still has well- formed glands, but they are larger and have more tissue between them), Grade 3 (the tissue still has recognizable glands, but the cells are darker. At high magnification some of these cells have left the glands and are beginning to invade the surrounding tissue), Grade 4 (the tissue has few recognizable glands. Many cells are invading the surrounding tissue), Grade 5 the tissue does not have recognizable glands. There are often just sheets of cells throughout the surrounding tissue). The grading typically follows the Gleason grading as established by the ASCP.
Typically, prostate cancer is further linked to an elevated level of prostate- specific antigen (PSA). In one embodiment of the present invention the term "prostate cancer" relates to a cancer showing PSA levels above 4.0. In another embodiment the term relates to cancer showing PSA levels above 2.0. The term "PSA level" refers to the concentration of PSA in the blood in ng/ml.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of the neoplastic disease is a healthy prostate tissue. The term "healthy prostate tissue" as used herein refers to a tissue which shows none of the above mentioned characteristics of benign or maligant tumors, in particular of benign or malignant prostate tumorand represents physiologically normally active gland structures.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of the neoplastic disease is a healthy prostate tissue and said more progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above. Particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section P) of Table 1. Further, particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section Q) of Table 1.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of the neoplastic disease is a local prostate cancer with insignificant disease parameters The term "insignificant disease parameters", as used herein, refers to a clinical tumor stage < T2 and/or a Prostate Cancer Volume < 0.5 ml and/or a Gleason Score
< 6, preferably to a clinical tumor stage < T2 and a Gleason Score < 6 and/or a Prostate Cancer Volume < 0.5 ml, more preferably to a clinical tumor stage < T2 and a Gleason Score
< 6 and a Prostate Cancer Volume < 0.5 ml.
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is of stage > T2 (UICC 2002 classification) as defined herein above. In specific embodiments of the present invention the more progressed stage of prostate cancer may imply a stage T2, T2a, T2b or T2c, preferably T2, or higher (UICC 2002 classification) as defined herein above.
A further preferred embodiment of the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc, wherein said less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above, and said more progressed stage of the neoplastic disease is a prostate cancer of stage < T2 (UICC 2002 classification) as defined herein above. Particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section R) of Table 1. Further, particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section S) of Table 1.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is a local prostate cancer with insignificant disease parameters.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is a local prostate cancer with significant disease parameters. The term "significant disease parameters", as used herein, refers to a clinical tumor stage > T2 and/or a Gleason Score > 6 and/or a Prostate Cancer Volume > 0.5 ml, alternatively to a clinical tumor stage > T2 and a Gleason Score > 6 and/or a Prostate Cancer Volume > 0.5 ml, alternatively also to a clinical tumor stage > T2 and a Prostate Cancer Volume > 0.5 ml and a Gleason Score > 6.
In a preferred embodiment, the term "insignificant disease parameters" may refer to a clinical tumor stage < T2 and a Gleason Score < 6 and a Prostate Cancer Volume < 0.5 ml and the term "significant disease parameters" refers to a clinical tumor stage > T2 or a Gleason Score > 6 or a Prostate Cancer Volume > 0.5 ml. A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is of stage > T2 (UICC 2002 classification) as defined herein above.
In specific embodiments of the present invention the more progressed stage of prostate cancer may imply a stage T2, T2a, T2b or T2c, preferably T2, or higher (UICC 2002 classification) as defined herein above.
A further preferred embodiment of the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc, wherein said less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above, and said more progressed stage of the neoplastic disease is a prostate cancer of stage < T2 (UICC 2002 classification) as defined herein above. Particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section R) of Table 1. Further, particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section S) of Table 1.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is a local prostate cancer with insignificant disease parameters.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is a local prostate cancer with significant disease parameters. The term "significant disease parameters", as used herein, refers to a clinical tumor stage > T2 and/or a Gleason Score > 6 and/or a Prostate Cancer Volume > 0.5 ml, alternatively to a clinical tumor stage > T2 and a Gleason Score > 6 and/or a Prostate Cancer Volume > 0.5 ml, alternatively also to a clinical tumor stage > T2 and a Prostate Cancer Volume > 0.5 ml and a Gleason Score > 6.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is a local prostate cancer with insignificant disease parameters, as defined herein above and wherein said more progressed stage of prostate cancer is a local prostate cancer with significant disease parameters, as defined herein above.
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer has a Gleason score <6.
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is of stage < T2 (UICC 2002 classification).
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is of stage < T2 (UICC 2002 classification) and has a Gleason score < 6.
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer has a tumor volume of between about <0.05 and about 3.0ml, e.g. <0.1 ml, <0.2ml, <0.3 ml, <0.4 ml, <0.5ml, <0.6 ml, <0.7 ml, <0.8ml, <0.9 ml, <1.0 ml, <1.1 ml, <1.2ml, <1.3 ml, <1.4 ml, <1.5ml, <1.6 ml, <1.7 ml, <1.8ml, <1.9 ml or <2.0 ml. A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer can be any one of the clinically classifiable stages, grades, and/or sub-steps as defined herein above, wherein said less progressed stage of prostate cancer has a tumor volume of between about <0.05 ml and about 3.0 ml, e.g. <0.1 ml, <0.2 ml, <0.3 ml, <0.4 ml, <0.5ml, <0.6 ml, <0.7 ml, <0.8ml, <0.9 ml, <1.0 ml, <1.1 ml, <1.2ml, <1.3 ml, <1.4 ml, <1.5ml, <1.6 ml, <1.7 ml, <1.8ml, <1.9 ml or <2.0 ml.
The term "Prostate Cancer Volume" as used herein refers to a calculated or measured volume of a prostate tumor or prostate swelling, which may be based on the assessment of the extension of the tumor/s welling in three dimensions and a subsequent determination of the volume. For example, such an assessment may employ suitable morphometric reconstruction techniques, as known to the person skilled in the art. Preferably, such a measurement follows any suitable method of cancer volumetric calculations known to the person skilled in the art, for example guidance as provided in D'Amico et al., Urology, 1997 Mar;49(3):385-91.
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is of stage < T2 (UICC 2002 classification) and has a Gleason score < 6, and has a tumor volume of <0.5 ml.
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer has a Gleason score >6.
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is of stage > T2 (UICC 2002 classification) as defined herein above and/or a prostate cancer with a Gleason Score >7. A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is of stage > T2 (UICC 2002 classification) as defined herein above and/or a prostate cancer with a Gleason Score >6.
In specific embodiments of the present invention the less progressed stage of prostate cancer may imply a Gleason score of 1, 2, 3, 4, 5, or 6, preferably 6. In further specific embodiments of the present invention the less progressed stage of prostate cancer may imply a stage of Tl, Tla, Tib, Tlc,T2, T2, T2a, T2b or T2c, preferably T2 (UICC 2002 classification).
In further specific embodiments of the present invention the less progressed stage of prostate cancer may imply a Gleason score of 1, 2, 3, 4, 5, or 6, preferably 6 and a stage of Tl, Tla, Tib, Tlc,T2, T2, T2a, T2b or T2c, preferably T2 (UICC 2002
classification).
In further specific embodiments of the present invention the more progressed stage of prostate cancer may imply a Gleason score of 7, 8, 9 or 10, preferably 7. In further specific embodiments of the present invention the more progressed stage of prostate cancer may imply a stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification).
In specific embodiments of the present invention the more progressed stage of prostate cancer may imply a Gleason score of 7, 8, 9 or 10, preferably 7 and a stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification).
An even more preferred embodiment of the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc, wherein said less progressed stage of prostate cancer is of stage < T2 (UICC 2002 classification) as defined herein above and has a Gleason score <6, and said more progressed stage of prostate cancer is of stage >T2 (UICC 2002 classification), as defined herein above and has a Gleason score >7. Particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section T) of Table 1. Further, particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section U) of Table 1. In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is a local prostate cancer with significant disease parameters, as defined herein above.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is an advanced prostate cancer disease with a level of PSA of about <10 ng/ml.
In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned
compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is a local prostate cancer with significant disease parameters and wherein said more progressed stage of prostate cancer is an advanced prostate cancer disease with a level of PSA of about <10 ng/ml, e.g. 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 9.5 ng/ml or any other value between about 0.5 up to 9.9999 ng/ml.
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is of stage > T2 (UICC 2002 classification).
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer shows a level of PSA of about <10 ng/ml, e.g. 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 9.5 ng/ml or any other value between about 0.5 up to 9.9999 ng/ml. A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer shows a level of PSA of about <10 ng/ml, e.g. 1 ng/ml, 2 ng/ml, 2.5 ng/ml, 3 ng/ml, 3.5 ng/ml, 4 ng/ml, 4.5 ng/ml, 5 ng/ml, 5.5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 9.5 ng/ml or any other value between about 0.5 up to 9.9999 ng/ml.
A further particularly preferred embodiment relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer can be any one of the clinically classifiable stages, grades, and/or sub-steps as defined herein above, wherein said more progressed stage of prostate cancer shows a serum level of PSA of >2 ng/ml, > 2.5 ng/ml, >3 ng/ml,> 3.5 ng/ml, >4 ng/ml, >4.5 ng/ml,> 5 ng/ml, >5.5 ng/ml, >6 ng/ml, >7 ng/ml, >8 ng/ml, >9 ng/ml, >9.5 ng/ml.
A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is of stage >T3 (UICC 2002 classification) as defined herein above.
In specific embodiments of the present invention the less progressed stage of prostate cancer may imply a stage of T2, T2a, T2b or T2c, preferably T2a,but not T3 or higher pre-treatment.
In further specific embodiments of the present invention the more progressed stage of prostate cancer may imply a stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification).
In specific embodiments of the present invention the more progressed stage of prostate cancer may imply a level of PSA of about <10 ng/ml, e.g. 1 ng/ml, 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 9.5 ng/ml or any other value between about 0.5 up to 9.9999 ng/ml, and a stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification). An even more preferred embodiment of the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc, wherein said less progressed stage of prostate cancer is of stage > T2 but not T3 or higher pre-treatment (UICC 2002 classification) as defined herein above, and said more progressed stage of prostate cancer is of stage > T3 (UICC 2002 classification) as defined herein above, with serum PSA <10 ng/ml as defined herein above. Particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section V) of Table 1. Further, particularly preferred in this context is the use, detection, employment etc. of tumor markers indicated with a "+" in section W) of Table 1.
The following examples and figures are provided for illustrative purposes. It is thus understood that the example and figures are not to be construed as limiting. The skilled person in the art will clearly be able to envisage further modifications of the principles laid out herein.
EXAMPLES
Example 1 -Preparation of samples
Clinical human plasma samples
The clinical samples were human plasma drawn and prepared under standardized conditions. Prepared plasma samples were stored in aliquots at -80°C until use.
The characteristics of the clinical samples were chosen such that they would support as much as possible the identification of diagnostic and prognostic biomarkers.
Four groups of unselected men between ages 50-70 had been selected: 1) controls (healthy), 2) men with a benign prostate disease, 3) men with localised prostate cancers, and 4) men with advanced prostate cancers. Group 1 men were selected based on very low PSA values (<0.5 ng/ml), presumably healthy (i.e., no cancer). Group 2 men were selected based on PSA levels >3 ng/ml, but negative biopsy ((i.e., presumably men with a benign prostate disease). Group 3 men were selected based on PSA levels >3 ng/ml and cancer positive biopsy and pre-treatment stages T2 or less. This group was further divided into group 3A -> men with very small cancers (cancer volume <0.5 ml), clinical stage <=T2 (i.e., presumably indolent prostate cancer), vs. group 3B -> men with bigger cancers (cancer volume >0.5 ml), clinical stage >T2 (i.e., presumably significant prostate cancer). Group 4 men were selected on signs of clinical advanced cancer disease (clinical stage >T3).
The Figures 2 to 5 summarize the characteristics of clinical and diagnostic parameters of the plasma samples
Example 2 - Pooling and quantifying samples
Pooling strategy of plasma samples
Since the proteomics standard approach has multiplexing capabilities up to 8 samples and consists of many fractionation steps, the pooling strategy was based on this. Eight pools were randomly constructed from the available samples:
Pool 1 (pool name: al) 15 individual samples, patient group 1 (healthy, controls)
Pool 2 (pool name: a2) 15 individual samples, patient group 1 (healthy, controls)
Pool 3 (pool name: bl) 15 individual samples, patient group 2 (benign prostate disease)
Pool 4 (pool name: b2) 15 individual samples, patient group 2 (benign prostate disease)
Pool 5 (pool name: cl) 15 individual samples, patient group 3A (local prostate disease, insignificant disease)
Pool 6 (pool name: c2): 15 individual samples, patient group 3B (local prostate disease, significant disease)
Pool 7 (pool name: dl): 15 individual samples, patient group 4 (advanced disease, PSA <10 ng/ml)
Pool 8 (pool name: d2): 15 individual samples, patient group 4 (advanced disease, PSA >10 ng/ml).
A corresponding list of clinical samples is provided in Fig. 1.
Pooling and quantifying samples
15 samples of pool 1 were selected following the list of the clinical samples (Cambridge samples). Samples were retrieved from the -80°C freezer, thawed quickly and put on ice. 2 μΐ of sample were diluted with 98 μΐ of Milli-Q (dilution factor 50). The sample dilution was mixed and centrifuged. 300 μΐ of Bradford Reagent was pipetted into a new vial. 10 μΐ of the sample dilution were added to the 300 μΐ of Bradford reagent. After20 minutes the samples were measured. The protein concentration was approximately 1.5 mg/ml, so samples needed to be diluted 70 times. The samples were pooled by adding 68 μΐ of each of the 15 samples to one tube. The samples were mixed and spun (end volume was 1020 μΐ) and this procedure was repeated for pools 2-8. Finally, samples were stored at -80°C.
Bradford Analysis
A 9000 μg/ml BSA Stock Solution was obtained by dissolving 10.87 mg of
BSA (Bovine Serum Albumin) in 1.20777 ml H20.
The following standards were prepared from this BSA stock solution:
Conc.BSA Dilution Amount of the BSA Amount of Total Volume g/ml) factor Stock Solution (μΐ) H20 (μΐ) (μΐ)
100 90 10 890 900
250 36 25 875 900
500 18 50 850 900
750 12 75 825 900
1000 9 100 800 900
1500 6 150 750 900
10 μΐ of Standard were mixed with 300 μΐ of Bradford Reagent.
The standards were measured after 20 minutes of incubation.
Concentration BSA Absorbance at 595 nm rc = 0.9972
^g/ml)
0 0.000
100 0.057
250 0.154
500 0.306
750 0.501
1000 0.640
1500 0.914 All samples from all pools were measured individually.
Example 3 - Proteomics Workflows
A) One-dimensional Immunodepletion, four-dimensional LC-MALDI MS workflow General protocol for immunodepletion applied to clinical samples
Four 0.22 μιη spin filters (Agilent Technologies) were washed with 100 μΐ water followed by centrifugation for 10 minutes at 10,000 rpm and 10°C. The samples from pools and human serum 4522 (Sigma) were thawed and kept on ice. The Human Serum was diluted 4 times with buffer A (Agilent Technologies) by mixing 400 μΐ HS with 1200 μΐ Buffer A. Pool 1 and Pool 2 were diluted four times with buffer A (Agilent) by mixing 120 μΐ Pool sample with 360 μΐ Buffer A. The HS and Pool 1 and 2 samples were filtered over a 0.22 μιη Spin filter at 10000 rpm and 4°C for 60 minutes. The samples were depleted using LC-3 Agilent HU-14 column. All unbound fractions (UF) and bound fractions (BF) were collected and stored at -80°C.
Desalting of samples with 5 filters
200 ml of a 100 mM TEAB solution (5 ml 1M TEAB were diluted with 45 ml Milli-Q) were prepared. All UF samples were thawed (from -80°C to 4°C). 16 sample tubes of 15 ml were prepared and 4,5 ml 100 mM TEAB were added to each tube. The 16 sample tubes were then placed on ice. 4.5 ml sample were added to each tube. 2 tubes per pool were used. The tubes were then placed back on ice. The samples were filtrated over the 5K Spin Concentrators 4ml tubes (Agilent Technologies). Again 2 tubes per pool were used.
First step: 4 ml sample per tube. Centrifugation for 15 minutes with 4000 rpm at 4°C. 3 ml sample were added to the filter tube. Centrifugation for 12 minutes with 4000 rpm at 4°C. 2 ml sample were added to the filter tube. Centrifugation for 15 minutes with 4000 rpm at 4°C. 3 ml of 100 mM TEAB were added to the filter tube. Centrifugation for 20 minutes with 4000 rpm at 4°C. 3 ml 100 mM of TEAB were added to the filter tube.
Centrifugation for 20 minutes with 4000 rpm at 4°C. 3 ml of 100 mM TEAB were added to the filter tube. Centrifugation for 25 minutes with 4000 rpm at 4°C. Centrifugation for another 10 minutes with 4000 rpm at 4°C. The residue from the filter was pipetted to a new tube and the filter washed with TEAB. All UF samples and the filtrate samples were transferred to a -80°C freezer.
Reducing sample volumes with 5 filters to 150 μΐ.
All UF Pool samples were thawed (from -80°C to 4°C), mixed and centrifuged. 8 Millipore Biomax-5K-NMWL Membrane 0.5 ml filters were used in the following procedure. 500 μΐ sample were pipetted in the filter. Centrifugation for 10 minutes at 10000 rpm and 4°C. 300 μΐ sample were added in the filter. Centrifugation for 10 minutes at 10000 rpm and 4°C. 350 μΐ sample were added in the filter (last part of the sample).
Centrifugation for 15 minutes at 10000 rpm and 4°C. Centrifugation for 3 minutes at 10000 rpm and 4°C. Centrifugation samples 1, 2 and 4 for 2 minutes at 10000 rpm and 4°C. The residue (± 50 μΐ) was pipetted from the filter to a new tube and the filter washed with 2 x 50μ1 TEAB. All UF samples and all filtrate samples were stored at -80°C. The protein concentrations were determined using the Bradford assay as described earlier. For each sample 625 μg protein was needed. The volumes were adjusted such that this amount of protein was taken into analysis.
Denaturation / Alkylation / Digestion.
625 μg of each pool sample was taken into analysis and all samples were adjusted to 8 M urea by adding solid urea (± 0.48 mg urea per 1 μΐ sample). The sample volume needed was pipetted to the amount of ureum. The samples were mixed and spun. Note: due to the ureum the volume increases. The pH of each sample needed to be checked (needs to be around 8). A 500 mM TCEP solution (reducing reagent) was prepared. 2.5 μΐ of the 500 mM TCEP solution were added to each sample tube. The samples were mixed and centrifuged. The solutions were mixed for 1 hour at room temperature. A 200 mM IAA solution was prepared. 37 mg IAA were weighed for 1 ml. 20 μΐ of 200 mM IAA were added to each sample tube. The samples were mixed and spun and then left for 30 minutes at room temperature in the dark. 10 tubes of 5 μg Lys-C (Roche) were prepared and the Lys-C of every tube was dissolved in 50 μΐ H20. In total: 50 μg Lys-C in 500 μΐ H20. 62.5 μΐ of this Lys-C solution were added to each sample tube. Ratio Lys-C : Protein = 1 : 100. The samples were mixed and spun and then kept for 4 hours at 37°C in the dark.
For the trypsin digestion ureum concentration was reduced from 8 M to 2 M by diluting samples 4 times. 15 tubes of 20 μg trypsin were prepared. The trypsin of every tube was dissolved in 20 μΐ 100 mM TEAB. In total: 300 μg trypsin in 300 μΐ TEAB. 25 μΐ of this trypsin solution were added to each sample tube. Ratio Trypsin: Protein = 1 : 25. The samples were mixed and spun (overnight at 37°C).
Protocol for iTRAQ-8-plex labeling
Samples were dried using a speedvac. The samples were dissolved in 300 μΐ 1M TEAB (total sample volume app. 500 μΐ). Samples were sonicated for 5 minutes in an ultrasonic bath to dissolve all proteins. The iTRAQ-8-plex Kit (25 Units) was thawed. This kit contains 8 labels and every label consists in 25 units. 1 Unit is intended for 50 μg proteins. There is one label for each sample. Half of the label was used since samples contain 625 μg total protein.
The samples contain (in total) 500 μΐ 1 M TEAB and urea in water. The percentage organic phase in the sample must be greater than 70%. Therefore the sample must be diluted with IPA. An end volume of 1666 μΐ is necessary to reach 70% organic phase, starting from 500 μΐ. 1666 μΐ - 500 μΐ = 1166 μΐ IPA that had be added to the sample. Each label is dissolved in app. 558 μΐ IPA. The half of that was used (279 μΐ), so 887 μΐ pure IPA was added to the label. 100 μΐ IPA was used to wash all tubes, so there is only 787 μΐ IPA added to the label.
Sample pool 1 was pipetted into one 2 ml tube. Both tubes were rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 787 μΐ IPA were pipetted in a separate tube. 279 μΐ of label 113 were mixed with the 787 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
Sample pool 2 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 787 μΐ IPA were pipetted in a separate tube. 279 μΐ of label 114 were mixed with the 787 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
Sample pool 3 was pipetted into one 2 ml tube. Both tubes were rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 787 μΐ IPA were pipetted in a separate tube. 279 μΐ of label 115 were mixed with the 787 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
Sample pool 4 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 792 μΐ IPA were pipetted in a separate tube. 274 μΐ of label 116 were mixed with the 792 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
Sample pool 5 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 810 μΐ IPA were pipetted in a separate tube. 256 μΐ of label 117 were mixed with the 810 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
Sample pool 6 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 740 μΐ IPA were pipetted in a separate tube. 326 μΐ of label 118 were mixed with the 740 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
Sample pool 7 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 790 μΐ IPA were pipetted in a separate tube. 276 μΐ of label 119 were mixed with the 790 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
Sample pool 8 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 823 μΐ IPA were pipetted in a separate tube. 243 μΐ of label 121 were mixed with the 823 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.
The samples were incubated for 2 hours at room temperature. After two hours samples were placed in a -80°C freezer.
B) Isoelectric focusing of peptides
SCX-Clean up Analysis
To remove unbound iTRAQ-labels a SCX-clean up analysis was done before performing the IEF of peptides.
SCX-Method: SCX PEPT CLEAN.M
Column: PolyLC in.
PolySulfoethyl A
200 x 2.1 mm, 5 μιη, 200 A
Buffers: Buffer A: 25 % ACN in H20, pH 2,5 (Formic acid, app. 2 ml per liter).
Buffer B: Buffer A + 1 M KC1 (74.5 gram in 1 liter).
Gradient program time (min). % B flow (ml/min) Max. Pressure (bar)
0 2.5 0.15 100 10 2.5 0.15 100
11 75 0.15 100
30 75 0.15 100
31 2.5 0.15 100
51 2.5 0.15 100
75 % Buffer B means 0.75 M KC1.
Samples were dissolved in 50 μΐ Buffer A. Volume was app. 140 μΐ. 100 μΐ Buffer A were added to reach a volume of 240 μΐ. 240 μΐ were injected. 1 -minute fractions were collected. SCX-fractions 20 through 32 (from 20 minutes to 33 minutes) were collected in one tube for IEF. Samples were stored in a -80°C freezer.
RPLC-Clean up
To remove the salts (introduced in the SCX clean-up step) a normal RPLC clean-up analysis needed to be done.
RPLC-Method: RP CLEAN 03.M
Column: Agilent P.N. 993967-902
Zorbax Eclipse XDB-C18
150 x 4.6 mm, 5 μιη
Buffers: Buffer A: H20 + 0.1 % TFA.
Buffer B: ACN + 0.1 % TFA.
Gradient: time (min). % B flow (ml/min) Pressure
0.00 5 1.00 200
7.00 5 1.00 200
7.10 75 1.00 200
15.00 75 1.00 200
15.10 5 1.00 200
22.00 5 1.00 200
SCX samples were thawed and speedvacced. Subsequently, samples were dissolved in buffer A., mixed and spun. The samples were sonicated for 5 minutes in an ultrasonic bath to dissolve all peptides. The samples were mixed and spun. 300 μΐ were injected. The fractions were collected in a well plate. An example of a chromatogram is shown in Figure 8. All samples were placed in a -80°C freezer.
Samples were dried separately in the speedvac. The samples were placed back into the -80°C freezer. Preparation of the Peptide Offgel stock solution 1.25X (for PEPTIDE IEF). 3 ml glycerol solution (50%) were added to an empty tube. 300 μΐ of the OFFGEL Ampholyte Buffer were added (pH 3-10), (refrigerator tube 1ml). Milli-Q was added to a total volume of 25 ml. This Peptide O.S.S. was separated into 7 tubes of 2, 88 ml and 9 tubes of 0.48 ml and then stored at -20°C for max. 4 months.
120 μΐ H20 were added to one 480 μΐ Peptide Offgel Sample Solution (POSS) tube. This is the Peptide IPG Strip Rehydration Solution. 720 μΐ H20 were added to one 2880 μΐ POSS. tube. This is the Peptide Offgel Sample Solution.
The 3100 OFFGEL fractionator was prepared (24 strip, pH 3 - 10). The general protocol of the 3100 OFFGEL fractionator was followed. The Peptide Offgel stock solution was used (for PEPTIDE IEF).
The eight iTRAQ labeled samples were thawed. Each sample was dissolved in
300 μΐ POSS. Samples were sonicated for 5 minutes in an Ultrasonic bath to dissolve all peptides. All samples were mixed together in one sample tube (volume increases). All tubes were rinsed with 400 μΐ POSS (3600 μΐ - (8 * 300 μΐ) - increases volume). The sample was mixed well. 150 μΐ of the POSS were pipetted in each IEF fraction. 10 μΐ was used for directly testing the labeling. This 10 μΐ sample was diluted 4 times (30 μΐ Buffer A (H20 + 0.1 % TFA) is added) and analyzed by LC-MALDI. IEF of peptides was started using method OG24PE00 (Offgel 24 strip for peptides; 120 kVh, 8000 Volts, 50 μΑ, 200 mW, 100 hours).
All fractions were collected and placed in the -80°C freezer. To increase recovery the IEF was loaded for a second time with a new Peptide Offgel Stock Solution
(150 μΐ per fraction). IEF was started again and run it for app. 2 hours.
Start conditions: 8.50h, 50 μΑ, 100 V.
End conditions: 11.05h, 50 μΑ, 300 V.
Fractions collected together with the first fractions.
The fractions were dried in the Speed Vac and placed in the -80°C freezer.
High pH RP analysis.
As a second dimension separation reversed phase at high pH had to be performed:
RPLC-Method: RP PEPT HIGH PH JB.M
Column: Agilent P.N. 763973-902
Zorbax 300 Extend - CI 8
150 x 4.6 mm, 3.5 μιη
Buffers: Stocksolution = 20 mM NH4FA, pH 10 (lOx solution) 12.5 gram NH4OH (28%-solution) + 900 ml H20 + 1.62 ml FA (99%- solution).
the pH was adjusted to 10,5 and the volume adjusted to 1 liter.
Buffer A: Stock solution was diluted lOx with H20.
Buffer B: Stock solution was diluted lOx with ACN.
Gradient: time (min). % B flow (ml/min) Pres
0.00 5 0.70 200
3.00 5 0.70 200
30.00 60 0.70 200
32.00 60 0.70 200
33.00 90 0.70 200
38.00 90 0.70 200
38.10 5 0.70 200
45.00 5 0.70 200
Fraction Collector: time (min). Trigg er Mode Timeslices
0.00 Off
1.00 Time-based 1.00
45.00 Off
Fraction Delay Volume = 77 μΐ
Above location = 7 mm
Column by Column
Collection Mode = Discrete fractions
Tray = "eppendorf 2.0 ml Tube Rack"
Plate 1, A1-A9, C1-C9, E1-E9 and Plate 3, A1-A9 were used
High pH Column and buffers were installed on LC 3.
Buffer A was purged for 5 minutes (5 ml/min).
Buffer B was purged for 5 minutes (5 ml/min).
The system was purged with column and start conditions (0.7 ml/min and 5% buffer B). Pressure = 106 bar.
The IEF fractions were dried in the speedvac for 16 hours
IEF 1 = fraction 1 and 2 together. 100 μΐ of buffer A were added to fraction 1. The solution was mixed and spun. The sample was sonicated for 5 minutes in an ultrasonic bath to dissolve all peptides. The solution was mixed and spun. Fraction 1 was pipetted to fraction 2. The tube of fraction 1 was rinsed with 50 μΐ of buffer A. This wash fraction 1 was pipetted to fraction 2. Fraction 2 was mixed and spun. The sample was sonicated for 5 minutes in an ultrasonic bath to dissolve all peptides. The solution was mixed and spun. Fraction 2 was pipetted in the well plate of the autosampler l-A-5 (= 180 μΐ). The tube of fraction 2 was rinsed with 70 μΐ of buffer A. This wash fraction 2 was also pipetted in the Well Plate of the Autosampler l-A-5 (= 250 μΐ). The complete sample was injected. High pH fractions 1-35 were stored in the -80°C freezer. The other samples were treated in the same way as IEF 1 :
IEF 2 = fraction 3 and 4 together. l-A-6, 250 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
IEF 3 = fraction 5 and 6 together. l-A-7, 250 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
IEF 4 = fraction 7 and 8 together. l-A-8, 250 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
IEF 5 = fraction 9 and 10 together. l-A-9, 250 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
IEF 6 = fraction 11 and 12 together. l-A-10, 250 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
IEF 7 = fraction 13 and 14 together. 1-A-l 1, 250 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
IEF 8 = fraction 15 and 16 together. 1-A-l, 250 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
IEF 9 = fraction 17 and 18 together. l-A-2, 250 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
IEF 10 = fraction 19 and 20 together. l-A-3, 250 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
IEF 11 = fraction 21 and 22 together. l-A-4, 325 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
IEF 12 = fraction 23 and 24 together. l-A-5, 350 μΐ
The High pH fractions 1-35 were stored in the -80°C freezer.
After analysis the column was washed by injection of 175 μΐ of buffer A.
Pooling strategy high pH RP fractions
Since reversed phase at high and low pH are not very orthogonal a pooling strategy had to be developed to assure that peptides will still elute in the complete separation window in the final separation technique of this multi-dimensional workflow; reversed phase at low pH:
The fractions 1 through 34 of IEF 1 were thawed. Volume was reduced by using the speedvac.
The following fractions were combined:
IEF 1 A: fractions 2 - 8- 14- 20- 26- 32
IEF 1 B: fractions 3 - 9- 15 - 21 - 27- 33
IEF 1 C: fractions 4 - 10 - 16 -22 -28 -34
IEF 1 D: fractions 5 - 11 - 17 -23 -29
IEF 1 E: fractions 6 - 12 -18- 24- -30
IEF 1 F: fractions 7 - 13 - 19 -25 -31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50)
The samples were dried with the speedvac.
The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 2 were thawed. Volume was reduced by using the speedvac.
The fractions were combined as follows:
IEF 2 A: fractions 2 - 8- 14- 20- 26- 32
IEF 2 B: fractions 3 - 9- 15 - 21 - 27- 33
IEF 2 C: fractions 4 - 10 - 16 -22 -28 -34
IEF 2 D: fractions 5 - 11 - 17 -23 -29
IEF 2 E: fractions 6 - 12 -18- -24- -30
IEF 2 F: fractions 7 - 13 - 19 -25 -31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50)
The samples were dried with the speedvac.
The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 3 were thawed. Volume was reduced by using the speedvac.
The fractions were combined as follows:
IEF 3 A: fractions 2 - 8 - 14-20-26- 32
IEF 3 B: fractions 3-9-15-21-27-33
IEF 3 C: fractions 4 - 10- 16-22-28 - 34
IEF 3 D: fractions 5 - 11 - 17 - 23 - 29
IEF 3 E: fractions 6 - 12 - 18 - 24 - 30 IEF 3 F: fractions 7 - 13 - 19 - 25 - 31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50)
The samples were dried with the speedvac.
The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 4 were thawed. Volume was reduced by using the speedvac.
The fractions were combined as follows:
IEF 4 A: fractions 2 - 8- 14- 20- 26- 32
IEF 4 B: fractions 3 - 9- 15 - 21 - 27- 33
IEF 4 C: fractions 4 - 10 - 16 -22 -28 -34
IEF 4 D: fractions 5 - 11 - 17 -23 -29
IEF 4 E: fractions 6 - 12 -18- -24- -30
IEF 4 F: fractions 7 - 13 - 19 -25 -31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50)
The samples were dried with the speedvac.
The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 5 were thawed. Volume was reduced by using the speedvac.
The fractions were combined as follows:
IEF 5 A: fractions 2 - 8- 14- 20- 26- 32
IEF 5 B: fractions 3 - 9- 15 - 21 - 27- 33
IEF 5 C: fractions 4 - 10 - 16 -22 -28 -34
IEF 5 D: fractions 5 - 11 - 17 -23 -29
IEF 5 E: fractions 6 - 12 -18- -24 -30
IEF 5 F: fractions 7 - 13 - 19 -25 -31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50)
The samples were dried with the speedvac.
The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 6 were thawed. Volume was reduced by using the speedvac.
The fractions were combined as follows:
IEF 6 A: fractions 2 - 8 - 14-20-26- 32
IEF 6 B: fractions 3 - 9 - 15 - 21 - 27 - 33
IEF 6 C: fractions 4- 10- 16-22-28 -34 IEF 6 D: fractions 5 - 11 - 17 - 23 - 29
IEF6E: fractions 6 - 12 -18 - 24 - 30
IEF 6 F: fractions 7 - 13 - 19 - 25 - 31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50)
The samples were dried with the Speed Vac.
The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 7 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:
IEF 7 A: fractions 2 -8- 14- 20- 26- 32
IEF 7 B: fractions 3 -9- 15 - 21 - 27- 33
IEF 7 C: fractions 4 - 10 - 16 -22 -28 -34
IEF 7 D: fractions 5 - 11 - 17 -23 -29
IEF 7 E: fractions 6 - 12 -18- ■24 -30
IEF 7 F: fractions 7 - 13 - 19 -25 -31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50)
The samples were dried with the speedvac.
The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 8 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:
IEF 8 A: fractions 2 -8- 14- 20- 26- 32
IEF 8 B: fractions 3 -9- 15 - 21 - 27- 33
IEF 8 C: fractions 4 - 10 - 16 -22 -28 -34
IEF 8 D: fractions 5 - 11 - 17 -23 -29
IEF 8 E: fractions 6 - 12 -18- -24 -30
IEF 8 F: fractions 7 - 13 - 19 -25 -31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50)
The samples were dried with the speedvac. The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 9 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:
IEF 9 A: fractions 2 -8- 14- 20- 26- 32
IEF 9 B: fractions 3 -9- 15- 21 - 27- 33
IEF 9 C: fractions 4 - 10 - 16 -22 -28 -34
IEF 9 D: fractions 5 - 11 - 17 -23 -29
IEF 9 E: fractions 6 - 12 -18 -24 -30 IEF 9 F: fractions 7 - 13 - 19 - 25 - 31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50). The samples were dried with the speedvac. The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 10 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:
IEF 10 A: fractions 2 -8- 14- 20- 26- 32
IEF 10 B: fractions 3 -9- 15 - 21 - 27- 33
IEF 10 C: fractions 4 - 10 - 16 -22 -28 -34
IEF 10 D: fractions 5 - 11 - 17 -23 -29
IEF 10 E: fractions 6 - 12 -18 -24 -30
IEF 10 F: fractions 7 - 13 - 19 -25 -31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50). The samples were dried with the speedvac. The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 11 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:
IEF 11 A: fractions 2 - 8- 14- 20- 26- 32
IEF 11 B: fractions 3 - 9- 15 - 21 - 27- 33
IEF 11 C: fractions 4 - 10 - 16 -22 -28 -34
IEF 11 D: fractions 5 - 11 - 17 -23 -29
IEF 11 E: fractions 6 - 12 -18- -24 -30
IEF 11 F: fractions 7 - 13 - 19 -25 -31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50). The samples were dried with the speedvac. The dry samples were placed in the -80°C freezer.
The fractions 1 through 34 of IEF 12 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:
IEF 12 A: fractions 2 -8- 14- 20- 26- 32
IEF 12 B: fractions 3 -9- 15 - 21 - 27- 33
IEF 12 C: fractions 4 - 10 - 16 -22 -28 -34
IEF 12 D: fractions 5 - 11 - 17 -23 -29
IEF 12 E: fractions 6 - 12 -18- -24 -30
IEF 12 F: fractions 7 - 13 - 19 -25 -31
Every tube was rinsed with 200 μΐ ACN/H20 (50/50). The samples were dried with the speedvac. The dry samples were placed in the -80°C freezer. Strong cation exchange chromatography
As a third dimension of peptide separation, strong cation exchange (SCX) chromatography was used:
RPLC-Method: SCX_pept_1911.M
Column: PolyLC inc.
PolySulfoethyl A
200 x 2.1 mm, 5 μπι, 200 A
Buffers: Buffer A: 25 % ACN in H20, pH 2,5 (Formic acid, app. 2 ml per liter).
Buffer B: Buffer A + 0.5 M KC1 (37.25 gram in 1 liter).
Gradient: time (min). % B flow (ml/min) Pressure
0.00 5 0.15 200
5.00 5 0.15 200
40.00 90 0.15 200
45.00 90 0.15 200
45.10 5 0.15 200
55.00 5 0.15 200
Fraction Collector: time (min). Trigger Mode Fractions (min.)
0.00 Off
1.00 Time-based 1.00
62.00 Off
Fraction Delay Volume = 77 μΐ
Above location = 7 mm
Column by Column
Collection Mode = Discrete fractions
Tray = "Agilent 96"
Samples were thawed and dissolved in 300 μΐ buffer A, mixed and spun. Samples were sonicated for 5 minutes in an ultrasonic bath to dissolve all peptides, were mix and spun. 300 μΐ of sample were injected onto the SCX column. The fractions were combined in such a way that based on the UV signal 8 new fractions were generated that contain equal amounts of peptide. The same was done for the other 72 samples.
LC-spotter
The final separation step was nano-reversed phase at low pH in combination with spotting the effluent on MALDI plates for off-line mass analyses using MALDI-ToF mass spectrometry. Samples were thawed, dissolved in 70 μΐ Buffer A (H20 + 0.1% TFA), mixed and spun. Samples were sonicated for 5 minutes in an ultrasonic bath to fully dissolve all peptides, then mixed and spun. 40 μΐ of these samples were injected and the remainders stored in the -80°C freezer.
MALDI Methods:
MALDI-MS was performed using a 4800 instrument from Applied
Biosystems. MS and MS/MS of the most intense peaks was done in positive ion mode.
Peak extraction and database searching
Centroid peaks were extracted from the raw data using TS2Mascot 1.0.0. Resulting *.mgf files were searched using Mascot 2.2 or Mascot Deamon (for automated searching). Alternatively, the raw data can directly be searched using Protein Pilot 2.0.
B) Two-dimensional Immunodepletion, two-dimensional LC-MALDI MS workflow
The first depletion step removes fourteen highly abundant human plasma proteins: albumin, IgG, IgA, IgM, fibrinogen, transferrin, alpha- 1 -antitrypsin, alpha-2- macroglobulin, haptoglobin, apo A-I, apo A-II, alpha- 1 acid glycoprotein, complement C3, and apo B. The subsequent depletion step utilizes a SuperMix column (for details on the immunodepletion procedure applied here, see Genway Inc. San Diego, CA) to deplete the next 80 most abundant proteins. Proteins were extracted from the depleted plasma by reversed-phase capture. The extracted proteins were subjected to reduction, alkylation (of cysteine residues) and digestion with trypsin.
The resulting peptide pools were labeled with the primary amine specific iTRAQ reagents; combined into eight-plex iTRAQ mixes which contains six primary samples labeled with the iTRAQ reagents that yield marker fragment ions at m/z 114, 115, 116, 118, 119, and 121. Each iTRAQ mix was analyzed by two-dimensional LC-MS/MS (SCX chromatography followed by RP-HPLC; for details of methods see above under (Proteomics Workflow (A)).
Data processing
All raw spectra were stored as binary objects in an Oracle database, and were viewable using the GPS explorer software from ABI, as delivered with the instrument. For identification and quantification of the peptides' spectra, and for assembly of the peptides into proteins, two different software packages were used. Both packages use peaklists from the peak-picking and de-isotoping software that comes with the instrument.
The first package used was Protein Pilot 2.0 from ABI. This software readily delivers protein and peptide reports in a tabular form for further analysis. It was found that on large datasets (100.000+ spectra), this program shows a remarkable sensitivity. The second package was Mascot, ref M2, in conjunction with an Integra database system and custom Java scripts, all from Matrix Science, London, UK. In this case, the data pipeline is as follows. First, the peaklists are extracted from the instrument database for each jobrun (typically an LC-run with 381 spots), using TS2Mascot (Matrix Science). The resulting *.mgf files (typically each containing a few 100's of MS2 spectra) are imported into the Integra database. The names (*) of the *.mgf files were chosen to indicate which plate and fraction the LC-run is from. In the mgf file, the header above each separate peaklist indicates the spot number. Hence, for each MS-2 spectrum, the exact position on any of the MALDI plates can be retrieved, together with all peak intensities, the resulting peptide assignments by Mascot, the Mascot score and expectation value. These values were extracted from the
Integra database using a custom java script. By matching of the spotname and the exact (to 0.001 Da) precursor mass, the strength and signal to noise of the precursor peak in the MS-1 spectrum could be retrieved from the instrument database and included in the resulting 'Mascot MS-2 report'. This report is more complete than the PP report as it has one line for each MS-2 spectrum, and because the peak strengths in MS-1 and MS-2 are available. This was used to correct the ITRAQ 8-plex labels for contributions from immonium-ions with masses of 112 and 120 Da. For matches below the homology score, Mascot reports only the best matching peptide sequence, but not the protein it has taken the sequence from.
Therefore, the protein accessions and names were retrieved using the DB-toolkit software. After defining a cut-off value equal to 1 for the maximum acceptable expectation value, the average ITRAQ ratio's per unique peptide were calculated. Average ITRAQ ratio's for the proteins were calculated by averaging over all peptides from the same protein. All protein identifications were made using human sequences from SwissProt 56.0.
Two datasets were processed. These were on the same 8 pooled samples, using the same type of mass spectrometer but different sample depletion and fractionation, as described. Another difference was in the labeling of the pools, as indicated below. Table 4:
In the protein ITRAQ profiles, the pool names were used. In the supporting peptide reports, the ITRAQ labels were used to identify the samples and table Ml can be used to refer to the correct sample by taking the provenance of the data into account.
Table 5, below, lists some of the parameters of the two sets, using Mascot as described.
Data quality
Before starting the data-analysis, the data were inspected in several ways. First of all, inventors expected that for most spectra, the ITRAQ peaks are nearly equal, as most spectra come from peptides of highly abundant proteins. These will most likely not be influenced by the presence of disease, but rather reflect normal physiology. A typical raw spectrum in the ITRAQ region is shown in Figs. 7 and 8. The ITRAQ peaks were nearly always detectable, as shown in Fig. 9, and ratio's were close to 1. It could be confirmed that this was the typical behavior by plotting the ITRAQ peak strengths for the 8 ITRAQ labels against the average over all 8 peaks for each spectrum. To get an idea of the biological and technical variation, the log2 of the ratio of ITRAQ peak strength were plotted over average peak strength for two pairs of samples from clinically equivalent groups, al and a2, and bl and b2. A normal distribution was found, also for samples cl, c2, dl and d2. This again confirmed that markers for disease are rare, as expected.
Also data reproducibility was checked, by going back to some spots and taking the spectrum again. It is clear that reproducibility is good, and the differences measured between equivalent samples such as al and a2 must reflect biological variation or technical variation due to sample preparation such as digestion or labeling.
Two software packages were used for protein identification, as it is known that this can increase the coverage of the proteome under study. This was also found in the present study, see Table 6 below. It was checked that the peptide quantitation by contrast was nearly identical for the two software packages, small differences can be attributed to differences in isotope corrections and peak selections.
Table 6: Number of unique protein accessions (SwissProt 56.0); in total, 2350 distinct proteins were identified.
Protein identification with tandem mass spectrometry is essentially a two-step process. In the first step, the software determines the peptide sequence that best explains a spectrum / peaklist. In the next step, peptides are assembled into proteins. ITRAQ protein ratio's are obtained by averaging the peptide ratio's. Thus, the data that go into the analysis are tables with on each line a protein accession, protein name and the relative (PP) or absolute (Mascot) ITRAQ peak strengths. Depending on the software, there are additional columns; for an assessment of the strength of the protein identification, in the Mascot protein report, the number of supporting spectra and for the PP protein report the 'unused' parameter are used. For each protein identification, the supporting peptide assignments and spectral data are available in a peptide report. Protein and peptide reports for the combined sets A&B are attached. Example 4 - Data analysis and scoring Data analysis
The data analysis started with either absolute intensities or intensity ratios for each and each sample. First, some preprocessing on these data is performed:
1. In case of absolute intensities, each sample's measurements were first scaled such that they yield the same total intensity.
2. Each protein's intensities/ratios were divided by the average over the eight samples, such that (new) ratios with a mean value of one for each protein are obtained.
3. A log2 transformation of the ratios resulting from the previous step is then used.
On these log-ratio data, three techniques to identify interesting proteins were applied:
correlation-based, t-tests, and a scoring method.
Correlation-based analysis
In the correlation-based analysis, inventors first computed the Pearson correlation for each protein's log-ratio profile with a reference profile. This was only performed for proteins that had no missing ITRAQ values, and the following three reference profiles were used:
To find proteins with a gradually increasing/decreasing profile over the samples, inventors used a reference profile 0, 0, 1, 1, 1.9, 2.1, 2.9, 3.1 for samples al, a2, bl, b2, cl, c2, dl, d2, respectively, i.e. a step from each group to the next one, and a small differentiation between cl and c2, and between dl and d2.
1. To find diagnostic proteins, a reference profile 0, 0, 0, 0, 1, 1, 1, 1, was used i.e. there is a step between sample b2 (benign) and cl (local).
2. To find prognostic proteins, a reference profile 0, 0, 0, 0, 0, 1, 1, 1, was used i.e. there is a step between sample cl (local insignificant) and c2 (local significant).
The closer a protein's Pearson correlation is to -1 or to +1, the better.
However, Pearson correlation alone is not enough; it is also important that the log-ratio values over a protein's eight measurements shows enough variation (otherwise, differences might not be reproducibly measurable). Therefore, also the standard deviation of each protein's profile was computed. Next, for each protein these two figures were combine into one criterion by taking the square of its Pearson correlation, and multiplying that with the protein's standard deviation. The higher this criterion is, the better.
In order to assess the significance of the findings, random label-permutation experiments were performed, i.e. the reference profile was shuffled, and the above analysis was performed again (actually, all possible shuffling of the reference profiles can be checked, as for the three reference profiles above there are only 100, 80, 70 and 56 unique
permutations, respectively). Then, inventors were able to compare the observed number of proteins with a criterion value above a certain threshold on the original data, with the expected number of such proteins obtained on randomized data, to get an idea what the fraction of such proteins is that one gets by chance (i.e. we estimate a false discovery rate).
T-tests
In the second analysis, inventors aimed at finding interesting steps in the proteins' profiles by means of t-tests. Inventors focused on identifying profiles that have different values for the first four samples (al - b2) as compared to the next three (cl - dl), and for the first five (al - cl) compared to the next two (c2 - dl). The reason to omit sample d2 is that it can already be distinguished by its exceptional high PSA scores.
For the t-test, only proteins that had all ITRAQ values for the first seven samples were considered. The p-values for the respective t-tests were computed, and corrected for multiple testing by a method controlling the false discovery rate.
Scoring method
The third method applied is a heuristic scoring method, which allows for missing values. Inventors applied it at first to the intensity data, and interpret a missing value as a very low value. Again, inventors checked for the first four samples (al - b2) as compared to the next three (cl - dl), and for the first five (al - cl) compared to the next two (c2 - dl). For each protein, the heuristic compares each measurement in the first group to each measurement in the second group (i.e. 12 comparisons for al - b2 vs. cl - dl and 10 comparisons for al - cl vs. c2 - dl). A score is given to each of these comparisons:
+4 if the second intensity is over 4 times the first one
+3 if the second intensity is over 2 times the first one
-4 if the first intensity is over 4 times the second one
-3 if the first intensity is over 2 times the second one If one of the two values is missing, then a score of +/-4 is given if the other intensity is large enough (more than 500), and +/-3 if it is smaller. If both values are missing, or in other cases, a score of 0 is given to this pair of intensities.
Next, all 12 or 10 scores, respectively, are added, and the sums are divided by the maximum attainable score to get a result in the range [-1, +1]. Proteins with a score close to -1 or +1 are now considered as interesting.
The scoring heuristic can also be applied on ratio data, although in that case the intensity information cannot be taken into account in the comparison to a missing value.
Results from the above experiments and test are depicted in Table 7. Table 7 shows proteins that might be useful as tumor markers, in particular prostate cancer biomarkers to improve the differential diagnosis of benign vs. malignant prostate disease as well as providing an individual prognosis of prostate cancer progression.
Table 7:
In some embodiments, the present invention relates to the following items:
Item 1 : A tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8 or all tumor markers selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAPl 3), High affinity cGMP-specific 35-cyclic phosphodiesterase 9A
(PDE9A),cAMP-dependent protein kinase type I-beta regulatory subunit (PRKARIB), and cAMP-specific 35-cyclic phosphodiesterase 4C (PDE4C), indicated in Table 1.
Item 2: The group of tumor marker of item 1, wherein said group consists of AKAP6 and/or CNGB3 and/or KGP2 and/or AKAPl and/or AKAP9 and/or AKAPl 3 and/or PDE9A and/or PRKARIB and/or PDE4C. Item 3 : The tumor marker or group of tumor markers of item 1 or 2, wherein the expression of the marker(s) is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section P), R), T) or V) of Table 1. Item 4: The tumor marker or group of tumor markers of item 1 or 2, wherein the expression of the marker(s) is decreased (down-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section Q), S), U) or W) of Table 1. Item 5: The group of tumor markers of any one of items 1 to 4, wherein the p-value of the expression modification is 0.2 or lower, as indicated in section L) or M) of Table 1.
Item 6: A composition for diagnosing, detecting, graduating, monitoring or
prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand for the expression product(s) or protein(s) of a tumor marker or a group of tumor markers as defined in any one of items 1 to 5.
Item 7: A method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the step of determining the level of a tumor marker or a group of tumor markers as defined in any one of items 1 to 5 in a sample.
Item 8: The method of item 7, wherein the determining step is accomplished by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers.
Item 9: The method of item 8, wherein said method is a method of graduating a neoplastic disease, comprising the steps of
(a) determining the level of a tumor marker or a group of tumor markers as defined in any one of items 1 to 5 in a sample by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers,
(b) comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease; and
(c) deciding on the stage or developmental status of a neoplastic disease based on the results obtained in step (b).
Item 10: Use of a tumor marker or a group of tumor markers as defined in any one of items 1 to 5 as a marker for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease.
Item 11 : An immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or for detecting, diagnosing, monitoring or prognosticating the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the steps
(a) testing in a sample obtained from an individual for the expression of a tumor marker or a group of tumor markers according to any one of items 1 to 5;
(b) testing in a control sample for the expression of the same tumor marker or group of tumor markers as in (a);
(c) determining the difference in expression of the tumor marker or group of tumor markers of steps (a) and (b); and
(d) deciding on the presence, stage or risk of recurrence of a neoplastic disease or the progression of the neoplastic disease based on the results obtained in step (c), wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or a group of tumor markers as defined in any one of items 1 to 5.
Item 12: A pharmaceutical composition for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of a tumor marker or group of tumor markers as defined in item 3, comprising at least one element selected from the group of:
(a) a compound directly inhibiting the activity of a tumor marker as defined in item 3, preferably an antagonist of said tumor marker enzymatic activity;
(b) a compound indirectly inhibiting the activity of a tumor marker as defined in item 3;
(c) a dominant negative form of a protein of a tumor marker as defined in item 3 or a biologically active equivalent thereof;
(d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker as defined in item 3;
(e) a miR A specific for a tumor marker as defined in item 3; (f) an antisense molecule of a tumor marker as defined in item 3;
(g) a siR A specific for a tumor marker as defined in item 3;
(h) an aptamer specific for the expression product of a tumor marker as defined in item 2 or for the protein of a tumor marker as defined in item 3;
(i) a small molecule or peptido mimetic capable of specifically binding to the protein of a tumor marker as defined in item 3; and
(j) an antibody specific for the protein of a tumor marker as defined in item 3 and/or an antibody variant specific for the protein of a tumor marker as defined in item 3.
Item 13: A pharmaceutical composition for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the decreased (down-regulated) expression of a tumor marker or group of tumor markers as defined in item 4, comprising at least one element selected from the group of:
(a) a compound directly stimulating or modulating the activity of a tumor marker as defined in item 4, preferably an agonist of said tumor marker enzymatic activity;
(b) a compound indirectly stimulating or modulating the activity of a tumor marker as defined in item 4;
(c) a protein of a tumor marker as defined in item 4 or a biologically active equivalent thereof;
(d) a nucleic acid encoding and expressing a protein of a tumor marker as defined in item 4; and
(e) a miR A inhibitor specific for a miRNA of a tumor marker as defined in item 4.
Item 14: Use of a tumor marker or a group of tumor markers of any one of items 1 to 5 for identifying pharmaceutically active agents useful in the treatment or prevention of cancer, preferably in the treatment or prevention of prostate cancer.
Item 15: The tumor marker or group of tumor markers of any one of items 1 to 5, the composition of item 6, the method of any one of items 7 to 9, the use of item 10 or 14, the immunoassay of item 11, or the pharmaceutical composition of item 12 or 13, wherein said neoplastic disease is prostate cancer. Item 16: The tumor marker or group of tumor markers of any one of items 1 to 5, the composition of item 6, the method of any one of items 7 to 9, the use of item 10 or 14, the immunoassay of item 11, or the pharmaceutical composition of item 12 or 13, wherein said less progressed stage is a healthy prostate tissue, and said more progressed stage is a benign prostate tumor.
Item 17: The group of tumor markers, composition, method, use, immunoassay or pharmaceutical composition of item 15, wherein said less progressed stage is a benign prostate tumor, and said more progressed stage is a < T2 (UICC 2002 classification) prostate cancer.
Item 18: The group of tumor markers, composition, method, use, immunoassay or pharmaceutical composition of item 15, wherein said less progressed stage is a < T2 (UICC 2002 classification) prostate cancer with a Gleason score < 6, and said more progressed stage is a > T2 (UICC 2002 classification) prostate cancer and/or a prostate cancer with a Gleason score > 6.
Item 19: The group of tumor markers, composition, method, use, immunoassay or pharmaceutical composition of item 15, wherein said less progressed stage is a >T2 (UICC 2002 classification) prostate cancer but not T3 or higher pre-treatment, and said more progressed stage is a > T3 (UICC 2002 classification) prostate cancer with serum PSA < 10 ng/ml.

Claims

CLAIMS:
1. A tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8 or all tumor markers selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAP13), High affinity cGMP-specific 35-cyclic phosphodiesterase 9A
(PDE9A),cAMP-dependent protein kinase type I-beta regulatory subunit (PRKAR1B), and cAMP-specific 35-cyclic phosphodiesterase 4C (PDE4C), indicated in Table 1.
2. The group of tumor marker of claim 1, wherein said group consists of AKAP6 and/or CNGB3 and/or KGP2 and/or AKAPl and/or AKAP9 and/or AKAPl 3 and/or PDE9A and/or PRKAR1B and/or PDE4C.
3. The tumor marker or group of tumor markers of claim 1 or 2, wherein the expression of the marker(s) is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section P), R), T) or V) of Table 1.
4. The tumor marker or group of tumor markers of claim 1 or 2, wherein the expression of the marker(s) is decreased (down-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section Q), S), U) or W) of Table 1.
5. The group of tumor markers of any one of claims 1 to 4, wherein the p-value of the expression modification is 0.2 or lower, as indicated in section L) or M) of Table 1.
6. A composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand for the expression product(s) or protein(s) of a tumor marker or a group of tumor markers as defined in any one of claims 1 to 5.
7. A method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the step of determining the level of a tumor marker or a group of tumor markers as defined in any one of claims 1 to 5 in a sample.
8. The method of claim 7, wherein the determining step is accomplished by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers.
9. The method of claim 8, wherein said method is a method of graduating a neoplastic disease, comprising the steps of
(a) determining the level of a tumor marker or a group of tumor markers as defined in any one of claims 1 to 5 in a sample by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers,
(b) comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease; and
(c) deciding on the stage or developmental status of a neoplastic disease based on the results obtained in step (b).
10. Use of a tumor marker or a group of tumor markers as defined in any one of claims 1 to 5 as a marker for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease.
11. An immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or for detecting, diagnosing, monitoring or prognosticating the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the steps
(a) testing in a sample obtained from an individual for the expression of a tumor marker or a group of tumor markers according to any one of claims 1 to 5;
(b) testing in a control sample for the expression of the same tumor marker or group of tumor markers as in (a);
(c) determining the difference in expression of the tumor marker or group of tumor markers of steps (a) and (b); and
(d) deciding on the presence, stage or risk of recurrence of a neoplastic disease or the progression of the neoplastic disease based on the results obtained in step (c), wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or a group of tumor markers as defined in any one of claims 1 to 5.
12. A method of identifying an individual for eligibility for a neoplastic disease therapy comprising:
(a) testing in a sample obtained from an individual for the expression of an up-regulated tumor marker or group of tumor markers as defined in claim 3;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of a tumor marker or a group of tumor markers as defined in claim 3;
(c) classifying the levels of expression of step (a) relative to levels of step
(b); and (d) identifying the individual as eligible to receive a neoplastic disease therapy where the individual's sample is classified as having an increased level of expression of a tumor marker or a group of tumor markers as defined in claim 3.
13. A method of identifying an individual for eligibility for a neoplastic disease therapy comprising:
(a) testing in a sample obtained from an individual for the expression of a down-regulated tumor marker or group of tumor markers as defined in claim 4;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of a tumor marker or a group of tumor markers as defined in claim 4;
(c) classifying the levels of expression of step (a) relative to levels of step
(b); and
(d) identifying the individual as eligible to receive a neoplastic disease therapy where the individual's sample is classified as having a decreased level of expression of a tumor marker or a group of tumor markers as defined in claim 4.
14. An immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:
(a) testing in a sample obtained from an individual for the expression of an up-regulated tumor marker or group of tumor markers as defined in claim 3;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of a tumor marker or a group of tumor markers as defined in claim 3;
(c) determining the difference in expression of a tumor marker or a group of tumor markers as defined in claim 3 of steps (a) and the expression of a tumor marker or a group of tumor markers as defined in claim 3 and/or the reference gene in step (b); and
(d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an increased level of expression of a tumor marker or a group of tumor markers as defined in claim 3.
15. An immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:
(a) testing in a sample obtained from an individual for the expression of a down-regulated tumor marker or group of tumor markers as defined in claim 4;
(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of a tumor marker or a group of tumor markers as defined in claim 4;
(c) determining the difference in expression of a tumor marker or a group of tumor markers as defined in claim 4 of steps (a) and the expression of a tumor marker or a group of tumor markers as defined in claim 4 and/or the reference gene in step (b); and
(d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has a decreased level of expression of a tumor marker or a group of tumor markers as defined in claim 4.
16. The method of any one of claims 7 to 9, or 12 to 13, or the immunoassay of any one of claims 11, 14 or 15, wherein said method or immunoassay comprises the additional step of determining the level of prostate specific antigen (PSA).
17. A pharmaceutical composition for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of a tumor marker or group of tumor markers as defined in claim 3, comprising at least one element selected from the group of:
(a) a compound directly inhibiting the activity of a tumor marker as defined in claim 3, preferably an antagonist of said tumor marker enzymatic activity;
(b) a compound indirectly inhibiting the activity of a tumor marker as defined in claim 3;
(c) a dominant negative form of a protein of a tumor marker as defined in claim 3 or a biologically active equivalent thereof;
(d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker as defined in claim 3;
(e) a miR A specific for a tumor marker as defined in claim 3; (f) an antisense molecule of a tumor marker as defined in claim 3;
(g) a siR A specific for a tumor marker as defined in claim 3;
(h) an aptamer specific for the expression product of a tumor marker as defined in claim 2 or for the protein of a tumor marker as defined in claim 3;
(i) a small molecule or peptido mimetic capable of specifically binding to the protein of a tumor marker as defined in claim 3; and
(j) an antibody specific for the protein of a tumor marker as defined in claim 3 and/or an antibody variant specific for the protein of a tumor marker as defined in claim 3.
18. A pharmaceutical composition for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the decreased (down-regulated) expression of a tumor marker or group of tumor markers as defined in claim 4, comprising at least one element selected from the group of:
(a) a compound directly stimulating or modulating the activity of a tumor marker as defined in claim 4, preferably an agonist of said tumor marker enzymatic activity;
(b) a compound indirectly stimulating or modulating the activity of a tumor marker as defined in claim 4;
(c) a protein of a tumor marker as defined in claim 4 or a biologically active equivalent thereof;
(d) a nucleic acid encoding and expressing a protein of a tumor marker as defined in claim 4; and
(e) a miR A inhibitor specific for a miRNA of a tumor marker as defined in claim 4.
19. A vaccine for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (up- regulated) expression of a tumor marker selected from a group of cAMP -regulatory pathway members comprising A-kinase anchor protein 6 (AKAP6), cyclic nucleotide-gated cation channel beta-3 (CNBG3), cGMP-dependent protein kinase 2 (KGP2), A-kinase anchor protein 1 (AKAPl), A-kinase anchor protein 9 (AKAP9), A-kinase anchor protein 13 (AKAP13), High affinity cGMP-specific 35-cyclic phosphodiesterase 9A (PDE9A), cAMP- dependent protein kinase type I-beta regulatory subunit (PRKAR1B), and cAMP-specific 35- cyclic phosphodiesterase 4C (PDE4C) as indicated in sections F), G), H) or I) of Table 3, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 3, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) of Table 3, or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) of Table 3, or any fragment thereof.
20. Use of a tumor marker or a group of tumor markers of any one of claims 1 to 5 for identifying pharmaceutically active agents useful in the treatment or prevention of a neoplastic disease, preferably in the treatment or prevention of prostate cancer.
21. The tumor marker or group of tumor markers of any one of claims 1 to 5, the composition of claim 6, the method of any one of claims 7 to 9, 12, 13 or 16, the use of claim 10 or 20, the immunoassay of any one of claims 11 , 14, 15 or 16, the pharmaceutical composition of claim 17 or 18, or the vaccine of claim 19, wherein said neoplastic disease is prostate cancer.
22. The tumor marker or group of tumor markers of any one of claims 1 to 5, the composition of claim 6, the method of any one of claims 7 to 9, 12, 13 or 16, the use of claim 10 or 20, the immunoassay of any one of claims 11, 14, 15 or 16, the pharmaceutical composition of claim 17 or 18, or the vaccine of claim 19, wherein said less progressed stage is a healthy prostate tissue, and said more progressed stage is a benign prostate tumor.
23. The group of tumor markers, composition, method, use, immunoassay, pharmaceutical composition or vaccine of claim 21, wherein said less progressed stage is a benign prostate tumor, and said more progressed stage is a < T2 (UICC 2002 classification) prostate cancer.
24. The group of tumor markers, composition, method, use, immunoassay, pharmaceutical composition or vaccine of claim 21, wherein said less progressed stage is a < T2 (UICC 2002 classification) prostate cancer with a Gleason score < 6, and said more progressed stage is a > T2 (UICC 2002 classification) prostate cancer and/or a prostate cancer with a Gleason score > 6.
25. The group of tumor markers, composition, method, use, immunoassay, pharmaceutical composition or vaccine of claim 21, wherein said less progressed stage is a >T2 (UICC 2002 classification) prostate cancer but not T3 or higher pre-treatment, and said more progressed stage is a > T3 (UICC 2002 classification) prostate cancer with serum PSA < 10 ng/ml.
EP10760034A 2009-09-03 2010-09-02 NOVEL TUMOR MARKERS SELECTED FROM A GROUP OF cAMP-REGULATORY PATHWAY MEMBERS Withdrawn EP2473853A1 (en)

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