WO2011073896A1 - Nouveaux marqueurs tumoraux - Google Patents

Nouveaux marqueurs tumoraux Download PDF

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Publication number
WO2011073896A1
WO2011073896A1 PCT/IB2010/055796 IB2010055796W WO2011073896A1 WO 2011073896 A1 WO2011073896 A1 WO 2011073896A1 IB 2010055796 W IB2010055796 W IB 2010055796W WO 2011073896 A1 WO2011073896 A1 WO 2011073896A1
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WIPO (PCT)
Prior art keywords
marker
molecular
tumor
group
tumor markers
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PCT/IB2010/055796
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English (en)
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WO2011073896A8 (fr
Inventor
Ralf Hoffmann
Hugo Matthieu Visser
Tim Hulsen
Leonie Waanders
Edwin Peter Romijn
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Koninklijke Philips Electronics N.V.
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Priority claimed from EP09179125A external-priority patent/EP2295977A1/fr
Application filed by Koninklijke Philips Electronics N.V. filed Critical Koninklijke Philips Electronics N.V.
Priority to PCT/IB2010/055805 priority Critical patent/WO2011073905A1/fr
Priority to PCT/IB2010/055803 priority patent/WO2011073903A1/fr
Priority to PCT/IB2010/055801 priority patent/WO2011073901A1/fr
Publication of WO2011073896A1 publication Critical patent/WO2011073896A1/fr
Publication of WO2011073896A8 publication Critical patent/WO2011073896A8/fr

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    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • 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 cancer disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor markers is modified when comparing the expression in the less progressed stage and in the more progressed stage.
  • the present invention further relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating a cancer 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 cancer 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 cancer disease therapy, as well as to a pharmaceutical composition based on the inhibition of the expression of said tumor markers.
  • Cancer is a class of diseases in which a group of cells display uncontrolled growth, invasion and sometimes metastasis. These three malignant properties of cancers differentiate them from benign tumors, which are self-limited and do not invade or metastasize.
  • 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.
  • phosphodiesterase PDE7 has been shown to be linked to chronic lymphocytic leukemia. Yet, for many cancer types or cancer progression forms there is no adequate marker molecule available.
  • Prostate cancer for example, is traditionally diagnosed via the serum level of prostate-specific antigen (PSA).
  • PSA prostate-specific antigen
  • PSA is not prostate cancer-specific and can be raised due to other circumstances, leading to a large number of false-positives (cancer is not found in around 70% of men with raised PSA levels who undergo biopsy).
  • cancer is not found in around 70% of men with raised PSA levels who undergo biopsy.
  • the present invention addresses this need and provides means and methods which allow the diagnosis and detection of cancer, in particular prostate cancer.
  • novel tumor markers from samples of cancer patients.
  • tissue or bodily fluid samples in particular serum samples, of patients representing different progression stages of cancer novel tumor markers could be identified which were shown to be differentially expressed, depending on the stage of progression of cancer.
  • the identification was achieved via the detection of autoantibodies derived from bodily fluid samples on an array of human proteins.
  • the novel molecular tumor markers are significantly up-regulated when comparing samples from patients having a less progressed stage of cancer to samples from patients having a more progressed stage of the disease.
  • These molecular markers are in certain embodiments of the present invention complemented by additional diagnostic factors such as a patient's age or the outcome of a patient's digital rectal examination. These markers are accordingly considered as markers for cancer prediction and a decision tool for the stratification of certain cancer surveillance regimes, as well as for the prognosis and monitoring of cancer progression. Diagnostic methods and uses based on the tumor markers of the present invention can thus
  • 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 cancer 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 by increase (i.e. increased or up-regulated) 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 one molecular tumor marker selected from Table 1, Table 2 or Table 3.
  • said group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or all molecular tumor markers of Table 1 and/or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or all of the molecular tumor markers of Table 2, and/or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or all of the molecular tumor markers of Table 3.
  • the p-value of the expression modification of said group of tumor markers is 0.006 or lower.
  • the group of tumor markers comprises at least 2 molecular tumor markers corresponding to tumor marker #1 to #2 of Table 1, or at least 3 molecular tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 5 molecular tumor markers corresponding to tumor marker #1 to #5 of Table 1, or at least 9 molecular tumor markers corresponding to tumor marker #1 to #9 of Table 1, or at least 12 molecular tumor markers corresponding to tumor marker #1 to #12 of Table 1, or at least 15 molecular tumor markers corresponding to tumor marker #1 to #15 of Table 1, or at least 17 molecular tumor markers corresponding to tumor marker #1 to #17 of Table 1, and/or at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #113 of Table 2 and/or at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #50 of Table 3.
  • the group of tumor markers as defined above additionally comprises protein specific antigen (PSA) as molecular marker.
  • PSA protein specific antigen
  • said PSA is total PSA, or free PSA, or complexed PSA and/or the ratio of free and total PSA and/or the ratio of complexed and total PSA.
  • the group of tumor markers as defined above comprises a patient's age and/or the outcome of a patient's digital rectal examination (DRE) as additional diagnostic factor.
  • DRE digital rectal examination
  • the specificity for detection of the progression of a cancer disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above is at least 40% and the sensitivity for detection of the progression of a cancer disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above is at least 90%.
  • the specificity for detection of the progression of a cancer disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above is at least 30%> and the sensitivity for detection of the progression of a cancer disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above is at least 95%.
  • the group of tumor markers comprises at least one marker combination of selected from the combinations shown in Table 4 (i.e. of combinations 1 to 244, as indicated in column A of Table 4) or shown in Table 5 (i.e. of combinations 1 to 171, as indicated in column A of Table 5).
  • the group of tumor markers comprises at least one of the following combinations of molecular markers and/or of additional diagnostic factors:
  • the present invention relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating a cancer disease 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, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand for the expression product(s) or protein(s) of said molecular tumor marker or group of tumor markers as defined above.
  • said peptide affinity ligand is an antibody.
  • nucleic acid affinity ligand or peptide affinity ligand is modified to function as an imaging contrast agent.
  • the present invention relates to a method for detecting, diagnosing, monitoring or prognosticating a cancer disease 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 comprising at least the step of determining the level of a molecular tumor marker or 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 molecular 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 molecular tumor marker or the group of tumor markers as defined above in one or more samples of a less progressed stage of the same cancer.
  • said method is a method of graduating cancer, comprising the steps of:
  • control level is the expression level of the molecular tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same cancer
  • step (c) deciding on the stage or developmental status of cancer 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 cancer disease 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.
  • the present invention relates to an immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, or for detecting, diagnosing, monitoring or prognosticating the progression from a less progressed cancer stage to a more progressed cancer stage comprising at least the steps of
  • step (d) deciding on the presence, stage or risk of recurrence of cancer or the progression of cancer based on the results obtained in step (c),
  • testing steps are based on the use of an antibody specifically binding (a) protein(s) of a molecular 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 cancer 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 cancer disease comprising:
  • step (c) determining the difference in expression of a molecular tumor marker or a group of tumor markers as defined above of steps (a) and the expression of a molecular tumor marker or a 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 cancer disease therapy based on the results obtained in step (c), where the individual's sample has an increased level of expression of a molecular tumor marker or a group of tumor markers as defined above.
  • said immunoassay or said methods as defined above comprise the additional step of determining the level of prostate specific antigen (PSA) and/or of determining the patient's age; and/or of determining the outcome of the patient's DRE.
  • PSA prostate specific antigen
  • said PSA is total PSA, or free PSA, or complexed PSA and/or the ratio of free and total PSA and/or the ratio of complexed and total PSA.
  • the present invention relates to a pharmaceutical composition for the treatment or prevention of a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, wherein said cancer disease implies the increased (up-regulated) expression of a molecular tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of:
  • said antibody as described in the context of a composition for diagnosing, detecting, graduating, monitoring or prognosticating a cancer disease as mentioned herein above, or in the context of a pharmaceutical composition as mentioned herein above is an antibody against the expression product or protein or a fragment thereof, of a molecular tumor marker or group of tumor markers as mentioned above.
  • said antibody is an autoantibody against a the expression product or protein or a fragment thereof of a molecular tumor marker or group of tumor markers as defined above.
  • said autoantibody is an autoantibody against the expression product or protein of a molecular tumor marker or group of tumor markers as defined above, wherein said expression product or protein comprises an amino acid sequence as indicated in section G) of Table 1, Table 2 or Table 3, or any fragment thereof.
  • the present invention relates to a vaccine for the treatment or prevention of a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 1 , Table 2 or Table 3 or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) or G) of Table 1 , Table 2 or 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) or G) of Table 1 , Table 2 or Table 3 or any fragment thereof.
  • An especially preferred embodiment of the present invention relates to the above mentioned tumor marker or group of tumor markers, the above mentioned
  • compositions for the above mentioned methods, the abovementioned use, the above mentioned immunoassay, or the above mentioned pharmaceutical compositions or vaccine, wherein said cancer or cancer disease is prostate cancer.
  • said less progressed stage of prostate cancer 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, or the above defined pharmaceutical compositions is of stage ⁇ T2 (UICC 2002 classification), Gleason score ⁇ 6, and said more progressed stage of prostate cancer is of stage >T2 (UICC 2002 classification), Gleason score >6.
  • Fig. 1 depicts a list of clinical samples (and corresponding clinical groups) used for the experiments described in the present application.
  • the group no in the following Figures refer to the group numbers in Fig. 1.
  • 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 100 ng/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) separation of peptides.
  • Fig. 7 (A-N) gives an illustrative overview over the AUC under a ROC of tumor markers according to the present invention for discrimination between clinical insignificant from significant prostate cancer.
  • Fig. 8 (A-Y) gives an illustrative overview over the expression values of tumor markers according to the present invention over the five clinical groups studied (see Fig 1 for details).
  • Fig. 9 gives an overview over the ProtoArray® Immune Response Biomarker Profiling assay controls.
  • On the left side a negative control image is shown, in which the array was incubated with buffer followed by the detection reagent.
  • the inset shows the pattern of control proteins including Alexa Fluor®647-conjugated anti-mouse antibody (boxes in the lower row), a gradient of human IgG (boxes in the upper row), and a mouse anti-biotin antibody (boxes in the middle row).
  • the murine antibody is recognized by the Alexa Fluor®647-conjugated anti-human antibody used for detection.
  • Fig. 10 depicts the process of selection of antigens for the testing of the antigens on targeted arrays for marker verification purposes.
  • tumor markers as depicted in Table 1 , Table 2 or Table 3 differentially expressed, depending on the stage of progression of cancer when testing samples of patients.
  • tumor markers and tumor marker combinations are statistically significantly up-regulated when comparing samples from a less progressed stage to a more progressed stage of a cancer 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 %.
  • 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 cancer 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 by increase (up-regulated) 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 one molecular tumor marker selected from the following Table 1 : .
  • the present invention further relates to a tumor marker or group of tumor markers associated with the progression of a cancer 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 by increase (up-regulated) 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 one molecular tumor marker selected from the following Table 2:
  • the present invention further relates to a tumor marker or group of tumor markers associated with the progression of a cancer 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 by increase (up-regulated) 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 one molecular tumor marker selected from the following Table 3.
  • Table 3 partially overlaps with Table 1. Identical markers are indicated by the same SEQ ID NO.
  • molecular marker or “molecular tumor marker”, as used herein, relates to a gene, genetic unit, antigen or sequence (a nucleotide sequence or amino acid or protein sequence) as defined in Table 1, Table 2 or Table 3, whose expression level is modified, i.e. increased, in a cancerous cell, or in a cancerous tissue or in any type of sample comprising cancerous cells or 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, antibodies, antigens, homologues and/or derivatives thereof indicated as marker# 1 to # 17 in Table 1, marker #1 to #113 in Table 2, or marker #1 to #200 in Table 3, having the nucleotide or amino acid sequences of SEQ ID NOs: 1 to 957, respectively, wherein the nucleotide sequences, corresponding amino acid sequences, or derivatives or fragments thereof as used for the underlying immune response experiments are comprised as indicated in Table 1 , 2 or 3, or as derivable from the Examples.
  • the term also comprises corresponding genomic sequences which can be obtained from any suitable database or information depositories, e.g. the Genbank database.
  • the term additionally comprises any known or yet unknown isoform (either as mR A 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.
  • 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.
  • molecular marker or “molecular tumor marker” may additionally comprise molecules specifically binding to the expression products of said marker genes.
  • binding molecules are antibodies which specifically bind to proteins expressed by said tumor marker genes.
  • the present invention thus envisages as molecular tumor marker also antibodies which specifically bind to proteins comprising the amino acid sequence as indicated in section E) of Table 1, 2 or 3, more preferably to proteins comprising the amino acid sequence as indicated in section G) of Table 1, 2 or 3, or to epitopes derived from the amino acid sequences indicated in sections E) or G) of Table 1, 2 or 3.
  • a “molecular marker” or “molecular 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, Table 2 or Table 3, in particular to the nucleotide sequence indicated in section D) of Table 1, Table 2 or Table 3. Furthermore, a “molecular marker” or “molecular 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, Table 2 or Table 3, in particular to the amino acid sequence indicated in section E) or section G) of Table 1, Table 2 or Table 3.
  • 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
  • Table 2 or Table 3 amino acid sequences may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o or 99%> identical to the sequence as set forth in section E) or section G) of Table 1
  • Table 2 or Table 3 nucleic acid sequences encoding 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) or section G) of Table 1
  • Table 2 or Table 3 or amino acid sequences may be encoded by nucleic acid sequences being at least 75%>, 80%
  • molecular tumor marker gene or “molecular marker gene” as used herein thus relates to the gene encoding the tumor marker mentioned in Table 1 , Table 2 or Table 3.
  • the term relates to a gene expressing a tumor marker protein as indicated in Table 1, Table 2 or Table 3, e.g. specific exon combinations derivable from the indicated genomic sequence information of Table 1, Table 2 or Table 3 or as set forth in the sequences of section D) of Table 1, Table 2 or Table 3.
  • the term also relates to DNA molecules derived from mRNA transcripts encoding a molecular tumor marker as indicated in Table 1, Table 2 or Table 3, 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 molecular tumor marker as mentioned in Table 1, Table 2 or Table 3.
  • 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 abovementioned 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.
  • the term "marker” or “tumor marker”, as used herein, relates to a marker comprising a molecular tumor marker as defined herein above, as well as one or more additional diagnostic factors.
  • diagnostic factor means factors known to be of significance in the identification, progression, development or diagnosis of a cancer disease. Such a factor may be, for example, a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE). Further suitable diagnostic factors would be known to the skilled person and may also be considered.
  • diagnostic factors may be determined, assessed and rated according to suitable rating schemes. It is particularly preferred to convert the diagnostic factor into numerical data according to schemes and procedures known in the art. Further details may be derived from qualified publications or textbooks, e.g. from Kranse et al, The Prostate, 68,1674-80 (2008); Roobol et al, Eur Urol, 57(l),79-85 (2009); Roobol et al, Eur Urol, 182(5), 2112-20 (2009).
  • cancer or “cancer disease” 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 “cancerous cell” is accordingly understood as a cell having specific structural properties, lacking differentiation and in many instances, being capable of invasion and metastasis.
  • cancerous relates in the context of the present invention to a cancerous disease state as defined herein above.
  • non-cancerous relates in the context of the present invention to a condition in which neither benign nor malign
  • Suitable means for said detection are known in the art.
  • progression of cancer relates to a switch between different stages of cancer development and principally refers to a situation in which the cancer disease becomes worse and/or spreads in the body. Any changes that are associated with a worsening of the disease, i.e. be it 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, 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 cancer disease.
  • any worsening of the disease can thus be translated into a switch into the next stage of a given cancer disease, 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 staging or scoring system, starting from a healthy condition up to a terminal cancer scenario.
  • 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 staging or scoring system, starting from a healthy condition up to a terminal cancer scenario.
  • Such switches are accompanied by a modification of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, 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 cancer type and/or tumor or cancer stage or state, or a value derivable from an information depository on expression data etc.
  • Progression of cancer 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 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.
  • 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 a cancer stage or cancer form, whose disease state or stage is known.
  • disease state relates to any state or type of cellular or molecular condition between a non-cancerous cell state and/or healthy and (including) a terminal cancerous cell state.
  • the term thus, includes benign tumor forms as well as malignant tumor forms.
  • the term includes different cancerous proliferation/developmental stages or levels of tumor development in the organism between (and excluding) a non-cancerous cell state and
  • these 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 benign tumor
  • cancers e.g. malignant prostate cancer.
  • 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 PSA or PCA3 in comparison to a corresponding less progressed stage as defined herein above.
  • bio- or tumor markers e.g. of tumor marker known to the person skilled in the art like PSA or PCA3 in comparison to a corresponding less progressed stage as defined herein above.
  • the term relates to a worsened disease state of a tumor or cancer in comparison to a less progressed stage as defined herein above.
  • 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. For example, if the less progressed stage is a stage I, the more progressed stage may be a stage II.
  • 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 molecular tumor marker as indicated in Table 1 , Table 2 or Table 3 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 molecular tumor marker or group of tumor markers according to Table 1 , Table 2 or Table 3 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.
  • the control level 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 of the cancer or tumor disease has been determined, as well as from healthy individuals.
  • control level determined from a biological sample that is known not to be cancerous is called "normal control level".
  • the control level can be from a cancerous biological sample, e.g. a sample from a subject for which cancer, in prostate cancer was diagnosed independently, it may be designated as "cancerous 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.
  • xenografts which may be used comprise PC295, PC310, PC-EW, PC82, PC133, PC135, PC324 and PC374.
  • 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 cancer disease.
  • 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 cancer disease may be determined by a 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.
  • the comparison processes may further be combined with a comparison with the indications in sections H), and I) of Table 1, Table 2 or Table 3, wherein section H) indicates the averaged detected amount or expression of the marker in the less progressed stage, and section I) indicates the averaged detected amount or expression of the marker in the more progressed stage.
  • 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 cancer, more preferably prostate cancer of stage ⁇ T2 (UICC 2002 classification), and Gleason score ⁇ 6.
  • control levels related to prostate cancer of stage >T2 (UICC 2002 classification), or prostate cancer of stage >T2 (UICC 2002 classification) and Gleason score >6 may be used.
  • 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. The amount may also be determined indirectly via the binding of an antibody, e.g. as the amount of bound antibody etc.
  • 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 molecular tumor marker or group of tumor markers according to Table 1, Table 2 or Table 3, 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 an increase or up-regulation of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, Table 2 or Table 3 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 molecular 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, for example, in sections I) (more progressed stage) and H) (less progressed stage) of Table 1, Table 2 or Table 3.
  • the expression of the molecular 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 section I) vs. section H) of Table 1, Table 2 or Table 3.
  • 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 molecular tumor marker or group of tumor markers according to Table 1 , Table 2 or Table 3 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 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.
  • Expression levels are deemed to be "increased" when the gene expression of the molecular tumor marker or group of tumor markers according to Table 1, Table 2 or Table 3, 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.
  • the tumor marker or group of tumor markers comprises the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 molecular tumor markers of Table 1.
  • the tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
  • the tumor marker or group of tumor markers comprises the first 1, 2, 3, 4, 6, 7, 8, 9, 11, 1,2, 13, 14, 16, 17, 18, 19, 21, 22, 23, 24, 26, 27, 28, 29, 31, 32, 33, 34, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 51, 52, 53, 55, 56, 57, 58, 59, 61, 62, 63, 64, 66, 67, 68, 69, 71, 72, 73, 74, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113 molecular tumor markers of Table 2.
  • the tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 6, 7, 8, 9, 11, 1,2, 13, 14, 16, 17, 18, 19, 21, 22, 23, 24, 26, 27, 28, 29, 31, 32, 33, 34, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 51, 52, 53, 55, 56, 57, 58, 59, 61, 62, 63, 64, 66, 67, 68, 69, 71, 72, 73, 74, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or all molecular tumor markers indicated in Table 2.
  • the tumor marker or group of tumor markers comprises the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • the tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 30, 32, 35, 37, 40, 42, 45, 46, 47, 48, 49, 50, 55, 65, 70 or all molecular tumor markers indicated in Table 3.
  • the group of tumor markers comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195 or all of the molecular tumor markers of Table 3.
  • a group of tumor markers comprising at least 1, 2, 3, 4, 6, 7, 8, 9, 11, 1,2, 13, 14, 16, 17, 18, 19, 21, 22, 23, 24, 26, 27, 28, 29, 31, 32, 33, 34, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 51, 52, 53, 55, 56, 57, 58, 59, 61, 62, 63, 64, 66, 67, 68, 69, 71, 72, 73, 74, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,
  • the group may further comprise any sub-grouping or combinations of these markers.
  • the group of tumor markers may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or all molecular tumor markers of Table 1 and/or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110 or all of the molecular tumor markers of Table 2, and/or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or all of the molecular tumor markers of Table 3.
  • the group of tumor markers comprises those tumor markers which show a p-value of the expression modification of 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.006, 0.0007, 0.0008, 0.0009, 0.001, 0.0011, 0.0012, 0.0013, 0.0014, 0.005, 0.06, 0.007, 0.008, 0.009, 0.01, 0.012, 0.013, 0.0135, 0.0139, or lower as indicated in section H) of Table 1, Table 2 or Table 3.
  • the term "p-value" is a measure of the probability that a variant 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 b se rved). 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 at least 2 molecular tumor markers corresponding to tumor marker #1 to #2 of Table 1, or at least 3 molecular tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 5 molecular tumor markers corresponding to tumor marker #1 to #5 of Table 1, or at least 9 molecular tumor markers corresponding to tumor marker #1 to #9 of Table 1, or at least 12 molecular tumor markers corresponding to tumor marker #1 to #12 of Table 1, or at least 15 molecular tumor markers corresponding to tumor marker #1 to #15 of Table 1, or at least 17 molecular tumor markers corresponding to tumor marker #1 to #17 of Table 1.
  • the group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker#l to #30, #30 to #60, #60 to #90, #90 to #113, #1 to #60, #60 to #113 or #1 to #113 of Table 2.
  • the group of tumor marker comprises at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #50 of Table 3.
  • the group of tumor markers comprises at least 2 molecular tumor markers corresponding to tumor marker #1 to #2 of Table 1, or at least 3 molecular tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 5 molecular tumor markers corresponding to tumor marker #1 to #5 of Table 1, or at least 9 molecular tumor markers corresponding to tumor marker #1 to #9 of Table 1, or at least 12 molecular tumor markers corresponding to tumor marker #1 to #12 of Table 1, or at least 15 molecular tumor markers corresponding to tumor marker #1 to #15 of Table 1, or at least 17 molecular tumor markers corresponding to tumor marker #1 to #17 of Table 1; and at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #113 of Table 2.
  • the group of tumor markers comprises at least 2 molecular tumor markers corresponding to tumor marker #1 to #2 of Table 1, or at least 3 molecular tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 5 molecular tumor markers corresponding to tumor marker #1 to #5 of Table 1, or at least 9 molecular tumor markers corresponding to tumor marker #1 to #9 of Table 1, or at least 12 molecular tumor markers corresponding to tumor marker #1 to #12 of Table 1, or at least 15 molecular tumor markers corresponding to tumor marker #1 to #15 of Table 1, or at least 17 molecular tumor markers corresponding to tumor marker #1 to #17 of Table 1; and at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #50 of Table 3.
  • the group of tumor markers comprises at least 2 molecular tumor markers corresponding to tumor marker #1 to #2 of Table 1, or at least 3 molecular tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 5 molecular tumor markers corresponding to tumor marker #1 to #5 of Table 1, or at least 9 molecular tumor markers corresponding to tumor marker #1 to #9 of Table 1, or at least 12 molecular tumor markers corresponding to tumor marker #1 to #12 of Table 1, or at least 15 molecular tumor markers corresponding to tumor marker #1 to #15 of Table 1, or at least 17 molecular tumor markers corresponding to tumor marker #1 to #17 of Table 1; and at least 1,
  • the group of tumor markers comprises at least 5 tumor markers corresponding to tumor marker #1 to #5 of Table 3, at least 10 tumor markers corresponding to tumor marker #1 to #10 of Table 3, at least 15 tumor markers corresponding to tumor marker #1 to #15 of Table 3, at least 20 tumor markers corresponding to tumor marker #1 to #20 of Table 3, at least 25 tumor markers corresponding to tumor marker #1 to #25 of Table 3, at least 30 tumor markers corresponding to tumor marker #1 to #30 of Table 3, at least 35 tumor markers corresponding to tumor marker #1 to #35 of Table 3, at least 40 tumor markers corresponding to tumor marker #1 to #40 of Table
  • the group of tumor markers may comprise tumor markers #1 to #55, tumor markers #1 to #60, tumor markers #1 to #65, tumor markers #1 to #70, tumor markers #1 to #75, tumor markers #1 to #80, tumor markers #1 to #85, tumor markers #1 to #90, tumor markers #1 to #95, tumor markers #1 to #100, tumor markers #1 to #105, tumor markers #1 to #110, tumor markers #1 to #115, tumor markers #1 to #120, tumor markers #1 to #125, tumor markers #1 to #130, tumor markers #1 to #135, tumor markers #1 to #140, tumor markers #1 to #145, tumor markers #1 to #150, tumor markers #1 to #155, tumor markers #1 to #160, tumor markers #1 to #165, tumor markers #1 to #170, tumor markers #1 to #175, tumor markers #1 to #180, tumor markers #1 to #185, tumor markers #1 to #190, tumor markers #1 to #195, tumor markers #1 to #200 of
  • the group of tumor marker according to the present invention may also comprise tumor markers #1, #3, #5, #7 and #9 of Table 3, #2, #4, #6, #8, and #10 of Table 3, #3, #5, #7, #9 and #11 of Table 3, #4, #6, #8, #10, and #12 of Table 3, #5, #7, #9, #11 and #13 of Table 3, #6, #8, #10, #12 and #14 of Table 3, #7, #9, #11, #13 and #15 of Table 3, #8, #10, #12, #14and #16 of Table 3, #9, #11, #13, #15 and #17 of Table 3, #10, #12, #14, #16 and #18 of Table 3, #11, #13, #15, #17 and #19 of Table 3, #12, #14, #16, #18 and #20 of Table 3, #13, #15, #17, #19 and #21 of Table 3 etc.
  • the present invention relates to a group of tumor markers of Table 1, 2 or 3, which comprise EGFR (Endothelian derived Growth Factor Receptor), or EGFR variants, mutants or iso forms.
  • tumor marker #11 of Table 3 i.e. the EGFR L858R mutant form.
  • tumor marker #18 of Table 3 i.e. the EGFR L861Q mutant form.
  • a group of tumor markers comprising marker #11 and/or #18 of Table 3, i.e. the EGFR L858R mutant form and/or the EGFR L861Q mutant form.
  • This group may also comprise one or more of any of the other tumor markers mentioned in Table 3.
  • it may comprises at least 5, 10, 15 more of any of the tumor markers of Table 1, or it may comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more of any of the tumor markers of Table 2, or it may comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more of any of the tumor markers of Table 3.
  • it may comprises at least 5 tumor markers corresponding to tumor marker #1 to #5 of Table 1, Table 2 or Table 3, at least 10 tumor markers corresponding to tumor marker #1 to #10 of Table 1, Table 2 or Table 3, at least 15 tumor markers corresponding to tumor marker #1 to #15 of Table 1, Table 2 or Table 3, at least 20 tumor markers corresponding to tumor marker #1 to #20 of Table 2 or Table 3, at least 25 tumor markers corresponding to tumor marker #1 to #25 of Table 2 or Table 3, at least 30 tumor markers corresponding to tumor marker #1 to #30 of Table 2 or Table 3, at least 35 tumor markers corresponding to tumor marker #1 to #35 of Table 2 or Table 3, at least 40 tumor markers corresponding to tumor marker #1 to #40 of Table 2 or Table 3, at least 45 tumor markers corresponding to tumor marker #1 to #45 of Table 2 or Table 3 or at least 50 tumor markers corresponding to tumor marker #1 to #50 of Table 2 or Table 3.
  • said group of EGFR mutant related tumor markers may comprise tumor markers #1 to #55 of Table 2 or Table 3, tumor markers #1 to #60 of Table 2 or Table 3, tumor markers #1 to #65 of Table 2 or Table 3, tumor markers #1 to #70 of Table 2 or Table 3, tumor markers #1 to #75 of Table 2 or Table 3, tumor markers #1 to #80 of Table 2 or Table 3, tumor markers #1 to #85 of Table 2 or Table 3, tumor markers #1 to #90 of Table 2 or Table 3, tumor markers #1 to #95 of Table 2 or Table 3, tumor markers #1 to #100 of Table 2 or Table 3, tumor markers #1 to #105 of Table 2 or Table 3, tumor markers #1 to #110 of Table 2 or Table 3, tumor markers #1 to #115 of Table 2 or Table 3, or tumor markers #1 to #120, tumor markers #1 to #125, tumor markers #1 to #130, tumor markers #1 to #135, tumor markers #1 to #140, tumor markers #1 to #145, tumor markers #1 to #150, tumor markers #1 to #155, tumor markers #1 to #160, tumor markers #1 to #165, tumor markers #1 to #170, tumor markers #1 to #175,
  • the group of EGFR mutant related tumor marker according to the present invention may also comprise tumor markers #1, #3, #5, #7 and #9 of Table 3, #2, #4, #6, #8, and #10 of Table 3, #3, #5, #7, #9 and #13 of Table 3, #4, #6, #8, #10, and #14 of Table 3, #5, #7, #9, #13 and #15 of Table 3, #6, #8, #10, and #14 of Table 3, #7, #9, #13, #15 and #17 of Table 3, #8, #10, #14and #16 of Table 3, #9, #13, #15, #17 and #19 of Table 3, #10, #14, #16 and #20 of Table 3, #13, #15, #17, #19 and #21 of Table 3, #14, #16, #20 and #22 of Table 3, #13, #15, #17, #19 and #21 of Table 3 etc.
  • At least molecular marker # 1 of Table 1 HZ
  • optionally at least one additional marker of Table 1, Table 2 or Table 3 and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or
  • DRE digital rectal examination
  • PSA PSA as further molecular marker
  • POPDC2 at least molecular marker # 4 of Table 1
  • additional diagnostic factors such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or
  • At least molecular marker # 6 of Table 1 SOD2
  • additional marker of Table 1, Table 2 or Table 3 optionally at least one additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or
  • At least molecular marker # 7 of Table 1 (PDCD5), optionally at least one additional marker of Table 1, Table 2 or Table 3, and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or
  • At least molecular marker # 8 of Table 1 (RNF41), optionally at least one additional marker of Table 1, Table 2 or Table 3, and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or
  • (xii) at least molecular marker # 12 of Table 1 (GLOl), optionally at least one additional marker of Table 1, Table 2 or Table 3, and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or
  • (xiii) at least molecular marker # 13 of Table 1 (C9orf97), optionally at least one additional marker of Table 1, Table 2 or Table 3, and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or
  • (xiv) at least molecular marker # 14 of Table 1 (CAP2), optionally at least one additional marker of Table 1, Table 2 or Table 3, and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or
  • (xv) at least molecular marker # 15 of Table 1 (SNF1LK2), optionally at least one additional marker of Table 1, Table 2 or Table 3, and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or
  • xvi at least molecular marker # 16 of Table 1 (TRIM63), optionally at least one additional marker of Table 1, Table 2 or Table 3, and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or
  • any of the above described tumor marker groupings may comprise one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE).
  • additional diagnostic factors such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE).
  • DRE digital rectal examination
  • any of the above described tumor marker groups may comprise a patient's age. In another preferred embodiment of the present invention any of the above described tumor marker groups may the outcome of a patient's digital rectal examination (DRE) as additional diagnostic factor. In yet another preferred embodiment of the present invention any of the above described tumor marker groups may comprise a patient's age and the outcome of a patient's digital rectal examination (DRE) additional diagnostic factor.
  • DRE digital rectal examination
  • patient's age refers to the number of years of a patient's life. These numbers may be used as numerical data within a combination of molecular markers, e.g. expression data obtained from molecular tumor markers as defined herein above. According to suitable procedures this numerical data may be weighted and assessed according to the type or stage of disease. In a specific embodiment of the present invention the high number of years of a patient's life may have a positive effect on the overall significance of a diagnostic/prognostic statement associated with a group of tumor markers as defined herein.
  • DRE digital rectal examination
  • the term "digital rectal examination” or “DRE” as used herein refers to an digital inspection of a patient's rectum whereby lumps, enlargements, or areas of hardness that might indicate cancer, can be identified.
  • the outcome of a patient's digital rectal examination (DRE) may be classified according to suitable numerical schemes for DRE.
  • a numerical scheme of DRE results may comprise the following values:
  • any of the above described tumor marker groupings or groups may additionally comprise PSA as a further molecular marker.
  • PSA refers to the gene, transcript, or expression product, e.g. protein, of human prostate specific antigen. Corresponding sequences would be known to the person skilled in the art and can be obtained from public data repositories such as Genbank or the EMBL database. The present invention thus envisages the assessment of PSA at different levels, e.g.
  • PSA at the level of mR A or the level of protein.
  • condition of PSA may be determined, e.g. whether PSA protein is free circulating in the blood/serum, whether it is complexed to other factors or proteins, how much PSA is present totally etc.
  • presence of PSA gene or transcript variants or mutants may be determined and is correspondingly considered as further molecular marker within the context of the present invention.
  • PSA may be total PSA protein, free PSA protein, complexed PSA protein and/or the ratio of free and total PSA protein or the ratio of complexed and total PSA protein, benign PSA, nicked PSA, proPSA, truncated forms of proPSA in particular -2proPSA or any ratios and/or combinations of these values.
  • the determination of PSA may be carried out according to any suitable methods known to the skilled person, e.g. via PCR tests, via antibody tests, e.g. by using monoclonal antibodies, via immune sandwich assays, ELISA, or RIA.
  • An example of a suitable test is the elecsys and cobas immunoassay.
  • the determined amount of PSA may be compared to suitable controls e.g. from healthy subjections, earlier taken samples, etc. or to values known form the prior art, or to database entries etc.
  • the group of tumor markers comprises at least one marker combination shown in the following Table 4 (e.g. of combinations 1 to 244, as indicated in column A of Table 4; the identity of the markers and or their sequences may be derived from Table 1, Table 2 or Table 3, where the corresponding SEQ ID NOs. are indicated; alternatively, public database entries as indicated in column C of Table 4 may be used in order to obtain suitable marker sequences):
  • the combinations provided in Table 4 may comprise further molecular markers or additional diagnostic factors, e.g. one or more of the molecular markers of Table 1, 2 or 3.
  • the present invention also envisages at least one marker combination derivable from the following Table 5 (e.g. of combinations 1 to 171, as indicated in column A of Table 5; the identity of the markers and or their sequences may be derived from Table 1, Table 2 or Table 3, where the corresponding SEQ ID NOs. are indicated; alternatively, public database entries as indicated in column C of Table 5 may be used in order to obtain suitable marker sequences):
  • Table 5 e.g. of combinations 1 to 171, as indicated in column A of Table 5; the identity of the markers and or their sequences may be derived from Table 1, Table 2 or Table 3, where the corresponding SEQ ID NOs. are indicated; alternatively, public database entries as indicated in column C of Table 5 may be used in order to obtain suitable marker sequences:
  • the combinations provided in Table 5 may comprise further molecular markers or additional diagnostic factors, e.g. one or more of the molecular markers of Table 1, 2 or 3.
  • the specificity for detection of the progression of a cancer disease from a less progressed stage to a more progressed stage of a group of tumor markers as defined above may be at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39%, preferably at least 40%, more preferably at least 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 % with a sensitivity for detection of the progression of a cancer disease from a less progressed stage to a more progressed stage of a group of tumor markers as defined above of at least 85%>, preferably 90%>.
  • markers of Table 1 may be associated with any grouping of markers of Table 1 , Table 2 and/or Table 3 as well as the above mentioned additional diagnostic factors. It is preferred that the sensitivity and specificity values as indicated above may be associated with combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more markers, more preferably with combinations of 3 or 5 markers.
  • the specificity for detection of the progression of a cancer disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above may be at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 %>, preferably at least 30%>, more preferably at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46 % with a sensitivity for detection of the progression of a cancer disease from a less progressed stage to a more progressed stage of a group of tumor markers as defined above of at least 92%>, preferably 95%>.
  • sensitivity and specificity values may be associated with combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more markers, more preferably with combinations of 3 or 5 markers.
  • the specificity and sensitivity of a group or combination of markers according to the present invention may be determined according to suitable procedures known to the person skilled in the art. Preferably, the specificity and sensitivity may be determined as explained in Example 8. The methodology and the corresponding results are also envisaged as further embodiments of the present invention.
  • the specificity and sensitivity may be a specificity and/or sensitivity as indicated in sections D) or E) of Table 4 or 5, or of corresponding Tables in the Examples.
  • the group or combination of tumor markers may, thus, have any of the sensitivities or specificities as indicated in sections D) or E) of Table 4 or 5, or of corresponding Tables in the Examples.
  • the group of tumor markers may comprise at least one of the following combinations of elements, i.e. molecular markers and/or of additional diagnostic factors:
  • 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, monitoring or prognosticating a cancer disease 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.
  • diagnosis a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage means that a subject or individual may be considered to be suffering from a more progressed cancer stage, 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 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.
  • additional diagnostic factors such as a patient's age, the outcome of DRE, or the determination of PSA may also be considered and/or combined with the determined expression levels and the corresponding results.
  • diagnosis 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 molecular tumor marker or group of tumor markers as defined herein above differs by, for example, between about 1% and 100%, e.g. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more 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 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 cancer disease state as described herein above, may indicate that the subject is suffering from a more progressed cancer disease stage.
  • 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 PCA3 may be tested.
  • detecting a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage means that the presence of a cancer disease or disorder in an organism, which is associated with a more progressed cancer stage may be determined or that such a disease or disorder may be identified in an organism.
  • the determination or identification of a more progressed cancer disease or disorder may be accomplished by a comparison of the expression level of the molecular 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 cancer disease or disorder.
  • the determination of additional diagnostic factors may also be considered and/or combined with the determined expression levels and the corresponding results.
  • a more progressed cancer stage may be detected if the expression level the molecular tumor marker or group of tumor markers is similar to an expression level of a more progressed cancer stage.
  • the expression level of the more progressed cancer stage may be independently established, e.g. from sample depositories, value databases etc. as mentioned herein above.
  • grade a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage means that the clinical stage, phase, grade or any other suitable sub-step of cancer disease related features such as the transition from a benign to a malignant tumor, the grade of malignancy, the grade of tissue damage to non-cancerous 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. Furthermore, in certain embodiments of the invention, the determination of additional diagnostic factors, such as a patient's age, the outcome of DRE, or the
  • determination of PSA may also be considered and/or combined with the determined values and the corresponding results.
  • the graduating of a more progressed cancer stage may be accomplished by a comparison of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, Table 2 or Table 3 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 cancer stage as defined herein above.
  • the graduating of a more progressed cancer stage may be accomplished by a comparison of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, Table 2 or Table 3 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 cancer stage may be accomplished by a comparison of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, Table 2 or Table 3 of the present invention in a sample from a patient or individual to be analyzed and the above cancer cells, cancer tissues, tumor biopsies, or the cancerous control levels being derived from or representative for a less progressed stage of the cancer.
  • the graduating of a more progressed cancer stage may be accomplished by a comparison of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, Table 2 or Table 3 of the present invention in a sample from a patient or individual to be analyzed and the above cancer cells, cancer tissues, tumor biopsies, or the cancerous control levels being derived from or representative for a more progressed stage of the cancer.
  • the determination of the graduating cancer may be accomplished by a comparison of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, Table 2 or Table 3 of the present invention in a sample from a patient or individual to be analyzed and the above cancer cells, cancer tissues, tumor biopsies, or the cancerous control levels being derived from or representative for an identical stage of the cancer.
  • the term "monitoring a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage” as used herein relates to the accompaniment of a diagnosed or detected, more progressed cancer disease or disorder, 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 molecular 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 cancer development, e.g. the stages as mentioned herein above. In a preferred embodiment of the present invention the term relates to the
  • prognosticating a cancer disease associated with a progression form a less progressed cancer stage to a more progressed cancer stage refers to the prediction of the course or outcome of a diagnosed or detected more progressed cancer stage, 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 cancer stage to a more progressed cancer stage relates to a switch between different stages of cancer development.
  • 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 cancer stage to a more progressed cancer stage may be considered as being detected or diagnosed if the expression level of a molecular 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 or other diagnostic values 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 cancer stage to a more progressed cancer stage relates to a switch from a healthy state or a benign prostate tumor state to a malignant prostate cancer state.
  • test samples from other individuals may be used, e.g. test samples of healthy individuals.
  • test samples of healthy individuals are also envisaged.
  • the present invention relates to the diagnosis and detection of a predisposition for developing a more progressed cancer stage.
  • predisposition for developing a more progressed cancer stage in the context of the present invention is a state of risk of developing a more progressed cancer stage.
  • a predisposition for developing a more progressed cancer stage may be present in cases in which the expression level of the molecular tumor marker or group of tumor markers of the present invention as defined herein above is above a normal control level as defined herein above, e.g. a reference expression level derived from tissues or samples of a subject which are evidently healthy, or other reference diagnostic values.
  • the term "above” in this context relates to an expression level of the molecular 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 cancer stage in the context of the present invention may be given in situations in which the expression level of the molecular 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 cancer stage.
  • further cancer markers are known to the person skilled in the art.
  • a predisposition for a more progressed cancer stage may be considered as being diagnosed or detected if one of the above depicted situations is observed.
  • control nucleic acids e.g. housekeeping genes whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell.
  • control genes include inter alia ⁇ -actin, glycerinaldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein PI, 18s R A, ubiquitin C, cytochrom C-l or PBGD.
  • 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 results 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 cancerous 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 cancer, a progression towards such cancer or a predisposition to such cancer is to be detected or prognosticated according to the present invention is an animal, preferably a mammal, more preferably a human being.
  • 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 malignant, hormone-sensitive prostate cancer or the progression towards more progressed cancer states in approaches like MPJ 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 a cancer disease 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, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand for the expression product(s) or protein(s) of said molecular tumor marker or a group of tumor markers as defined above.
  • nucleic acid affinity ligand for the expression product of a molecular 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 molecular 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, 2 or 3, or to the complementary nucleotide sequences (DNA sequence(s)) of the sequence(s) depicted in section D) of Table 1, 2 or 3, 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, 2 or 3 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) or section G) of Table 1, 2 or 3 or to any fragments of said sequences.
  • peptide affinity ligand for the protein of a molecular tumor marker or a group of tumor markers refers to a peptide molecule being able to specifically bind to the proteins of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • 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) or section G) of Table 1 , 2 or 3.
  • the peptide affinity ligand may also be able to
  • 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 molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3, generated by the expression of the corresponding genomic sequence according to Table 1 , 2 or 3. More preferably, the term relates to a processed transcript of the molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 as defined herein above, e.g. via the sequence(s) as set forth in sections D) and/or F) of Table 1 , 2 or 3. 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 , 2 or 3.
  • protein of a molecular 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) or section G) of Table 1 , 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3, a probe specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1 , 2 or 31 , an aptamer specific for the expression product or for the protein of the molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3, an antibody specific for the protein the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 and an antibody variant specific for the protein of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • nucleic acid and/or peptide affinity ligands selected from the group consisting of a set of oligonucleotides
  • composition of the present invention may, for example, comprise a set of oligonucleotides specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 and/or a probe specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • oligonucleotide specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 refers to a nucleotide sequence which is complementary to the sense- or antisense-strand of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • the oligonucleotide is complementary to the DNA sequence(s) shown in section D) of Table 1, 2 or 3, or to the complementary DNA sequence of the sequence shown in section D) of Table 1, 2 or 3.
  • 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, 2 or 3 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) or section G) of Table 1, 2 or 3.
  • 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, 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 may be defined with the help of software tools known to the person skilled in the art.
  • the oligonucleotide sequences may be complementary to genomic sequences localized in (an) exon(s) of the gene(s) encoding for the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • Corresponding information on genomic loci can be derived from any suitable database or information depositary, e.g. by using the data provided in section F) of Table 1, 2 or 3.
  • 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, which can be derived from any suitable database or information depositary, e.g. by using the data provided in section F) of Table 1, 2 or 3.
  • probe specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 means a nucleotide sequence which is complementary to the sense- or antisense-strand of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • the probe is complementary to the DNA sequence(s) depicted in section D) of Table 1, 2 or 3 or to the complementary DNA sequence of the sequence(s) shown in section D) of Table 1, 2 or 3.
  • 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% or 99% identical to the sequence(s) as set forth in the sequences indicated in section D) of Table 1, 2 or 3 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) or section G) of Table 1, 2 or 3.
  • 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 molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • aptamer specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, preferably the DNA molecule with (a) sequence(s) as shown in section D) of Table 1, 2 or 3. More preferably, the nucleic acid aptamer molecule may specifically bind to a DNA sequence(s) shown in section D) of Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, preferably an RNA molecule corresponding to the DNA sequence(s) as shown in section D) of Table 1, 2 or 3.
  • 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, 2 or 3 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 , 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 refers to (a) short peptide(s) capable of interacting and specifically binding the protein(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • 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) or section G) of Table 1, 2 or 3.
  • 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 shown in section D) of Table 1, 2 or 3 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) or section G) of Table 1, 2 or 3.
  • (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 molecular tumor marker or group of tumor markers as defined herein, e.g. specific for one or more of the protein(s) of the molecular tumor marker according to Table 1, 2 or 3.
  • 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, 2 or 3 or a part or fragment of said sequence.
  • an antibody which specifically binds to an expression product, protein, or peptide comprising the amino acid sequence indicated in section G) of Table 1, 2 or 3, or a part or fragment of said sequence.
  • such an antibody may be an autoantibody, e.g. an autoantibody against the expression product or protein of a molecular tumor marker or group of tumor markers as defined herein above, more preferably an autoantibody against the expression product or protein of a molecular tumor marker or group of tumor markers wherein said expression product or protein comprises an amino acid sequence as indicated in section G) of Table 1, 2 or 3, or comprises 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 invention thus relates to an antibody specific for one or more of the molecular tumor markers according to Table 1, 2 or 3.
  • an antibody specifically binds to a protein or polypeptide having or comprising the amino acid shown in section E) or section G) of Table 1, 2 or 3, 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.
  • 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) or section G) of Table 1, 2 or 3.
  • antibodies binding to epitopes or antigens comprised in or comprising the amino acid sequence as mentioned in section G) of Table 1, 2 or 3, or parts of said sequence, e.g. fragments 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 or 100 amino acids.
  • 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 molecular tumor marker according to Table 1, 2 or 3" 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 nonspecific 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 autoantibodies, preferably human autoantibodies, 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, 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.
  • autoantibodies preferably human autoantibodies, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, Fab' fragments, fragments produced by a
  • 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. In a particularly preferred embodiment the present invention relates to antibodies that do not bind any other analog, ortholog, or homolog 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.
  • autoantibody refers to an antibody generated by the immune system that is directed against one or more of the individual's own proteins.
  • Autoantibodies are normally generated by the immune system against the body's normal constituents or proteins, for example in reaction to the overexpression of said proteins or sudden appeance of said proteins after cellular modifications or transitions, e.g. during the development of a cancer disease or cancer.
  • autoantibodies are normally generated by the immune system against the body's normal constituents or proteins, for example in reaction to the overexpression of said proteins or sudden appeance of said proteins after cellular modifications or transitions, e.g. during the development of a cancer disease or cancer.
  • autoantibodies are envisaged which typically detect a molecular tumor marker protein as defined in Table 1, 2 or 3.
  • the invention thus envisages antibodies, in particular human autoantibodies against the molecular tumor marker proteins as defined in Table 1, 2 or 3.
  • the present invention also envisages an antigen and/or an epitope which is detected, recognized or specifically bound by an antibody against a molecular tumor marker protein as defined in Table 1, 2 or 3, more preferably bound by a human autoantibody against a molecular tumor marker protein as defined in Table 1, 2 or 3.
  • the present invention encompasses a screening method to identify antigen or epitope sequences bound by an antibody, in particular a human autoantibody against a molecular tumor marker protein as defined in Table 1, 2 or 3. Further envisaged are the epitope sequences and/or antigen sequences derived from a screening procedure.
  • the present invention also envisages antigen or epitope sequences which are bound, recognized, specifically recognized or detected by an autoantibody as defined herein.
  • antigens or epitopes may preferably be derived from a molecular tumor marker or group of tumor marker sequences as depicted in Table 1, 2 or 3.
  • 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 molecular tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2 or 3, preferably of the sequence as shown in section E) or section G) of Table 1, 2 or 3.
  • 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,
  • 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 molecular tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2 or 3, preferably of the sequence as shown in section E) or section G) of Table 1, 2 or 3.
  • 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 molecular tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2 or 3, preferably of the sequence as shown in section E) or section G) of Table 1 , 2 or 3
  • 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 molecular tumor marker or group of tumor markers as defined herein, e.g. as derivable from Table 1, 2 or 31, preferably of the sequence as shown in section E) or section G) of Table 1, 2 or 3.
  • 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), F) or G) of Table 1, 2 or 3.
  • 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 invention 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 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). 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.
  • 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 molecular tumor marker according to Table 1, 2 or 3 in particular against a protein having an amino acid sequence as indicated in section E) or section G) of Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 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.
  • second 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
  • 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.
  • 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.
  • 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 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).
  • contrast agent compositions will be capable of specifically detecting molecules having the nucleotide sequence(s) shown in section D) of Table 1, 2 or 3 or the amino acid sequence(s) shown in section E) or section G) of Table 1, 2 or 3or derivatives or homologous variants thereof as defined herein above.
  • contrast agents are aptamers specific for the expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 as well as antibodies specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • Contrast agents aside from their property of being capable of specifically recognizing the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 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 MRI.
  • 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, for the preparation of a composition for diagnosing, detecting, monitoring or prognosticating cancer disease 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, as described herein above.
  • the composition is preferably for diagnosing, detecting, monitoring or prognosticating cancer disease 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or a probe specific for the expression product(s) the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably specific for a nucleic acid sequence having a sequence as indicated in section D) of Table 1, 2 or 3 or being complementary to such a sequence, for the preparation of a composition for diagnosing, detecting, monitoring or prognosticating cancer disease 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, as described herein above.
  • the present invention relates to the use of an ap tamer specific for the expression product(s) or protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably a protein having an amino acid sequence as shown in section E) or section G) of Table 1, 2 or 3, for the preparation of a composition for diagnosing, detecting, monitoring or
  • the set of oligonucleotides is preferably for diagnosing, detecting, monitoring or prognosticating cancer disease 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 an antibody specific for protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or an antibody variant specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 for the preparation of a composition for diagnosing, detecting, monitoring or prognosticating cancer disease 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 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 cancer disease 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, comprising a set of oligonucleotides specific for the expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, a probe specific for the expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and/or an aptamer specific for the expression product(s) or protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or an antibody specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1
  • the diagnostic kit of the present invention contains one or more agents allowing the specific detection of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • 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 mRNA expression level of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be comprised.
  • the agents to be comprised may be antibodies (including autoantibodies) or compounds containing an antigen-binding fragment of an antibody or antibody variants specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, as described herein above.
  • an aptamer specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be comprised.
  • a diagnostic kit may comprise a contrast agent as defined herein above.
  • a kit according to the present invention may be a companion diagnostics test kit.
  • a diagnostic kit of the present invention contains detection reagents for expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 or the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 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 cancer disease 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 comprising at least the step of determining the level of a molecular tumor marker or a group of tumor markers as defined above, in a sample.
  • determining the level of a molecular tumor marker or group of tumor markers refers to the determination of the presence or amount of expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, e.g. (a) transcript(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, and/or the determination of the presence and/or amount of (a) protein(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • level of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 thus means the presence or amount of (an) expression product(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, e.g. (a) transcript(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, and/or the determination of the presence or amount of (a) protein(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • transcript(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 or (a) protein(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 may be accomplished by any means known in the art.
  • the determination of the presence or amount of the expression products of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, e.g. transcript(s) and/or of protein(s) of the markers mentioned in Table 1, 2 or 3, comprising for instance sequences as depicted in sections D), E) or G) of Table 1, 2 or 3, is accomplished by the measurement of nucleic acid or protein levels or by the determination of the biological activity of said molecular tumor marker or group of tumor markers.
  • the measurement of the nucleic acid level of the expression of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 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 molecular 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, 2 or 3, 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.
  • the nucleic acid level of the expression of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 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.
  • such nucleic acid molecules may be used in a hybridization approach with immobilized capture probes corresponding to the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 of the present invention or known bio marker 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 molecular 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 molecular 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 molecular tumor markers or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, e.g. of the sequences indicated in section D) of Table 1, 2 or 3 may be used as a probe.
  • the DNA array or microarray may comprise probes of one or more of the molecular tumor marker of Table 1, 2 or 3, 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, 2 or 3.
  • the determination of the expression of molecular tumor markers according to Table 1, 2 or 3 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 m NA 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • 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 molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, 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.
  • G guanine
  • Molecular Beacon probe to be used for a quantitative PCR approach preferably uses FRET interactions to detect and quantify a PCR product, with each probe having a 5' fluorescent-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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and derivatives thereof may be determined immunologically, e.g. by using an antibody specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an antibody as defined herein above.
  • the presence and/or amount of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be determined with an antibody specifically binding to or detecting a protein or antigen or epitope thereof of the proteins as indicated in section G) of Table 1, 2 or 3, or any fragment thereof, or any epitope comprised therein, as defined herein above.
  • an antibody specifically binding to or detecting a protein or antigen or epitope thereof of the proteins as indicated in section G) of Table 1, 2 or 3, or any fragment thereof, or any epitope comprised therein, as defined herein above Further preferred are autoantibodies according to the present invention. Alternatively, antibody variants or fragments as defined herein above may be used.
  • the present invention also envisages the use of peptide affinity ligands like ap tamers specific for the protein(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 as defined herein above.
  • Determination of the protein levels of the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 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 spectroscopy.
  • antibodies specific for (a) protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 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.
  • a molecular tumor marker expression product or protein or the expression product or protein of a group of molecular tumor marker preferably as derivable from Table 1, 2 or 3 (e.g. as defined in sections E) and/or section G)), which are or can specifically be bound by an autoantibody or a group of autoantibodies as mentioned herein above may be placed on a support and/or be immobilized.
  • the present invention in particular envisages an array of peptide sequences, protein sequences or expression products comprising one or more of the molecular tumor markers or groups of tumor marker as defined herein above.
  • the array may be provided in any suitable form known to the person skilled in the art and may comprise one or more suitable control elements, proteins or antibodies as would be known to the person skilled in the art.
  • the bound elements in the array may further be of any suitable length, comprising peptides/proteins of e.g. 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8 amino acids length or any other suitable length.
  • one or more antibodies, in particular autoantibodies present in a sample or tissue to be analyzed may be mixed with the presented peptides, proteins, epitopes etc., e.g. the proteins, peptides etc. immobilized on an array.
  • a detection reaction may then be carried out, e.g. with a second affinity ligand as defined herein above, preferably with a specific antibody.
  • the present invention also encompasses an autoantibody bound or detected in such an approach.
  • 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, fluorescent 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may be used in a method of detecting proteins of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be carried out by employing molecular or enzymatic assays specific to the corresponding function or functions of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, e.g. of a molecular tumor marker protein having a sequence as indicted in section E) or section G) of Table 1, 2 or 3. 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 miR A molecules, more preferably via specifically hybridizing antisense nucleotides, specific siRNA or miRNA molecules, e.g. as described herein below.
  • the biological activity of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be tested with the help of suitable enzymatic reactions or tests for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, known to the person skilled in the art, or by employing specific inhibitors of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • the use of such inhibitors may, for example, be combined with an enzymatic readout system.
  • Typical inhibitors of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 to be used comprise antisense molecules, siR A molecules or miR A molecules.
  • the level of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 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.
  • the presence of (a) protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 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.
  • affinity ligands for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be applied, preferably antibodies or aptamers.
  • affinity ligands are labeled, preferably with fluorescent labels as defined herein above.
  • 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.
  • the level of the molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 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 cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, or the progression form a less progressed cancer stage to a more progressed cancer stage 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 molecular tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same cancer.
  • the expression level of the molecular tumor marker or the group of tumor markers of a less progressed stage of the same cancer 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 cancer stage or cancer form, whose disease state or stage is known including different cancerous proliferation/developmental stages or levels of tumor development in the organism , as described herein above.
  • 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or of (a) protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, is accomplished by the measurement of nucleic acid or protein levels or by the determination of the biological activity of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • the expression level(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may be determined by a method involving the detection of an mR A encoded by the genes encoding for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, or the according cDNA sequence, e.g.
  • the measurement of the nucleic acid level of expression of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may be assessed by separation of nucleic acid molecules as described herein above.
  • a decision on the presence or stage of a more progressed cancer stage or the progression towards said stage may be based on the results of the comparison step.
  • a more progressed cancer stage may be diagnosed or prognosticated or a progression towards said more progressed cancer stage may be diagnosed or prognosticated in said method according to the corresponding definitions provided herein above in the context of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • the above mentioned method is a method of graduating cancer, comprising the steps of
  • control level is the expression level of the molecular tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same cancer
  • step (c) deciding on the stage or developmental status of cancer based on the results obtained in step (b).
  • the determination of the level of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 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 cancer stage may be graduated in said method according to the corresponding definitions provided herein above in the context of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • the present invention relates to a method of data acquisition comprising at least the steps of:
  • step (b) comparing the expression as determined in step (a) to a control level.
  • the testing for expression of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may be carried out according to steps as defined herein above.
  • the testing may be carried out as measurement of protein levels of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, more preferably according to the herein above described options for such measurements.
  • control level refers to the expression of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or other suitable markers in a control or the expression level of said marker(s) in a less progressed cancer stage, 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 cancer disease associated with a more progressed cancer stage or the progression from a less progressed cancer stage to a more progressed cancer stage comprising at least the steps of:
  • step (c) determining the difference in the expression of steps (a) and (b); and (d) deciding on the presence of cancer, in particular of a more progressed cancer stage or on the progression of cancer, in particular from a less progressed cancer stage to a more progressed cancer stage 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 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 cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, or for detecting, diagnosing, monitoring or prognosticating the progression from a less progressed cancer stage to a more progressed cancer stage comprising at least the steps
  • step (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 cancer or the progression of cancer based on the results obtained in step (c) ,
  • testing steps are based on the use of an antibody specifically binding (a) protein(s) of a molecular 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 molecular tumor marker or a group of tumor markers as defined above.
  • a risk of recurrence may, for example, be present when a molecular 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 molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 may be carried out according to steps as defined herein above.
  • the testing may be carried out as measurement of protein levels of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, more preferably according to the herein above described options for such measurements.
  • controls or control samples controls as defined herein above may be used.
  • testing steps may be based on the use of an antibody specifically binding to a molecular tumor marker according to Table 1, 2 or 3 as laid out above, e.g. (a) commercially available antibody/antibodies against (a) protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • a cancer in particular a more progressed cancer stage, may be diagnosed or prognosticated or a progression of cancer, in particular form a less progressed to a more progressed cancer stage, may be diagnosed or prognosticated in said immunoassay according to the corresponding definitions provided herein above in the context of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 as cancer marker(s).
  • the present invention relates to a method of identifying an individual for eligibility for a cancer disease therapy comprising: (a) testing in a sample obtained from an individual for the expression of a molecular tumor marker or a group of tumor markers as defined above;
  • step (c) classifying the levels of expression of step (a) relative to levels of step
  • the level of a molecular 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 molecular tumor marker protein and/or transcript(s). In addition the level of a reference gene in a sample may be determined.
  • reference gene 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 molecular 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 molecular 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 maker 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, 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 cancer disease therapy when the expression level of a molecular tumor marker as defined herein above or in Table 1, 2 or 3, or the expression level of a groups of tumor markers as defined herein above is increased.
  • the expression level is deemed to be "increased" when the molecular 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 molecular tumor marker gene expression, or 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 molecular tumor marker expression, or the expression of the group of tumor markers in a control sample; or when the molecular tumor marker or group of tumor marker gene expression is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%>, 25%o, 30%o, 40%o, 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
  • the gene expression level 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.
  • the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a cancer disease comprising:
  • step (c) determining the difference in expression of a molecular tumor marker or group of tumor markers as defined above of step (a) and the expression of a molecular 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 cancer disease therapy based on the results obtained in step (c), where the individual's sample has an increased level of a molecular tumor marker or group of tumor marker expression.
  • the testing of the expression of a molecular tumor marker or group of tumor markers may preferably be carried out via the determination of the amount of molecular tumor marker protein or the determination of the molecular tumor marker protein/expression product activity level. Preferred is the determination of the amount of molecular tumor marker proteins or peptides with the help of specific antibodies binding said tumor marker protein or expression product, e.g. an autoantibody as defined herein.
  • 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 molecular 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 molecular 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 molecular tumor marker or a group of tumor markers of step (a) and the expression of a molecular 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 molecular tumor marker or group of tumor markers and the expression in a control sample for the molecular 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.
  • test sample for a molecular 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 a cancer 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 cancer disease therapy, preferably a therapy against an early prostate cancer, or malignant 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 molecular tumor marker or group of tumor marker expression level and a reference gene or the molecular 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 cancer disease therapy when the molecular tumor marker or group of tumor marker expression levels are increased.
  • the expression level is deemed to be "increased" when the molecular tumor marker or group of tumor marker gene expression 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 molecular 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
  • the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes.
  • cancer disease therapy refers to any suitable cancer 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,
  • 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 cancer disease therapy due to an increased expression of a molecular tumor marker or group of tumor marker of the present invention may be deemed to be suffering from a cancer disease or be prone to develop a cancer 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.
  • 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 cancer 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
  • the classification method for eligibility or the immunoassay for stratification as described herein above more specifically be used for the detection of or in the context of aberrant expression of EGFR (Endothelian derived Growth Factor Receptor). Particulary preferred is the detection, measurement or determination of the presence and/or expression level of tumor marker #11 and/or #18 and/or of a group of tumor markers comprising tumor marker #11 and/or #18 of Table 3 or as defined herein, even more preferably of an EGFR L858R mutant form and/or an EGFR L861Q mutant form.
  • EGFR Endothelian derived Growth Factor Receptor
  • a specific cancer disease treatment may be initiated, or an ongoing cancer disease treatment may be modified in order to reflect the result.
  • tumors are treated with anti-EGFR treatments like small molecule inhibitors of EGFR tyrosine kinase activity, e.g., gefitinib, erlotinib, or by the use of anti-EGFR antibodies like cetuximab or panitumumab.
  • the reference gene is a housekeeping gene or a phosphodiesterase gene.
  • housekeeping genes include inter alia ⁇ -actin, glycerinaldehyde 3 -phosphate
  • 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 cancer 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.
  • 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.
  • the method of identifying an individual for eligibility for a cancer disease therapy based on the expression of a molecular 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 bio markers.
  • the above mentioned method for detecting, diagnosing, monitoring or prognosticating a cancer disease, or for identifying an individual for eligibility for a cancer disease therapy, or the above mentioned immunoassay for detecting, diagnosing, monitoring or prognosticating a cancer disease or for stratifying an individual or cohort of individuals with a cancer disease comprises the additional step of determing the level of PSA, and/or of determining the patient's age; and/or of determining the outcome of the patient's DRE as defined herein above.
  • the determination of the level of PSA may include the analysis of the PSA gene, transcript, or expression product, e.g. protein, of human prostate specific antigen. Furthermore, the condition of PSA may be determined, e.g. whether PSA protein is free circulating in the blood/serum, whether it is complexed to other factors or proteins, how much PSA is present totally etc. Furthermore, the presence of PSA gene or transcript variants or mutants may be determined.
  • the amount of total PSA protein, of free PSA protein, of complexed PSA protein and/or the ratio of the amount of free and total PSA protein or the ratio of the amount of complexed and total PSA protein, and/or the amount or presence of benign PSA, nicked PSA, proPSA, of truncated forms of proPSA in particular -2proPSA or any ratios and/or combinations of these values may be determined.
  • the determination of PSA may be carried out according to any suitable methods known to the skilled person, e.g. via PCR tests, via antibody tests, e.g. by using monoclonal antibodies , via immune sandwich assays, ELISA, or RIA.
  • a suitable test is the elecsys and cobas immunoassay.
  • the determined amount of PSA may be compared to suitable controls e.g. from healthy subjections, earlier taken samples, etc. or to values known form the prior art, or to database entries etc.
  • the determination of the level of PSA in blood is encountered to be in a range of about 2 to 5 ng/ml or more, 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 cancer disease, or be likely to develop a neoplasstic 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 molecular tumor marker or group of tumor markers may be carried out according to steps as defined herein above. These values may further be combined with the expression level of any of the molecular tumor markers or groups of tumor marker according to the present invention, or with additional diagnostic factors as defined herein.
  • the measurement of a marker, or of a group or combination of molecular markers as defined above, i.e. from Table 1, 2 or 3, in a patient serum sample may be used to identify those patients which have a cancer disease, in particular a prostate cancer disease, that is of indolent, or of insignificant nature.
  • the measurement of a tumor marker, or of a group or combination of tumor markers, e.g. those as defined in Table 1, 2 or 3, may accordingly be used to identify those patients which have a cancer disease, in particular a prostate cancer disease, that is of indolent, or of insignificant nature. This may preferably be done on a blood sample.
  • the detection of the molecular marker or group of markers as defined above which may be carried out as mentioned herein above, wherein the detected expression level is below a given threshold as defined herein, e.g. in Table 1, 2 or 3 or derivable from the Examples, indicates a >95%, >90%, >85%, >80%, >75%, >70%, >65%, or >60%, probability that the identified cancer disease, in particular prostate cancer disease is of indolent, or of insignificant nature.
  • the measurement of a molecular marker, or of a group or combination of markers as defined above, i.e. from Table 1, 2 or 3, in a patient serum sample may be used to identify those patients which have a cancer disease, in particular a prostate cancer disease, that is of significant nature whereas an indolent, insignificant cancer disease, in particular prostate cancer disease would stay undetected.
  • the measurement of a molecular tumor marker, or of a group or combination of tumor markers, e.g. those as defined in Table 1, 2 or 3, may accordingly be used to identify a cancer disease, in particular a prostate cancer disease that has a very high probability to progress over time and therefore requires radical intervention. This may preferably be done on a blood sample.
  • the detection of the marker or group of markers as defined above which may be carried out as mentioned herein above, wherein the detected expression level is above a given threshold as defined herein, e.g. in Table 1, 2 or 3 or derivable from the Examples, indicates a >95%, >90%, >85%, >80%, >75%, >70%, >65%, or >60% probability that the identified cancer disease, in particular prostate cancer disease is a significant disease and is going to progress in the future.
  • the detection or measurement of the molecular tumor marker, or of the group or combination of markers as defined herein, i.e. derivable from Table 1, 2 or 3, may further be used to monitor the progression of a potential cancer disease, in particular prostate in case the initial measurement of the markers, or combination of markers indiciated the absence of a significant disease.
  • the tumor marker detection or measurement including the determination of additonal diagnostic factors such as DRE, may be performed every 3, or every 6, or every 12 months after the patient has been moved into an active surveillance regime. If the expression level of the molecular tumor marker, or group of tumor markers as defined herein is going to pass a threshold as defined herein, e.g. in Table 1, 2 or 3or derivable from the Examples, by 5%, 10%, 15%, 20%>, 25%, or 30%> or more, further investigations or treatment may be started.
  • 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 RNA) 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 cancerous epithelial cell or an epithelial cell derived from tissue suspected to be 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. Additionally, cells may be purified from obtained body tissues and fluids if necessary, and then used as the biological sample.
  • 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 cancer, a progression of cancer or predisposition for cancer 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an antagonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (e) a miR A specific for a molecular tumor marker or group of tumor markers as mentioned herein above
  • a compound directly inhibiting the activity of a molecular tumor marker refers to a compound which is capable of decreasing the activity of a molecular tumor marker according to Table 1, 2 or 3.
  • a compound may be any direct interactor of the molecular tumor marker according to Table 1, 2 or 3, which has negative influence on the catalytic activity of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.
  • Such a compound may preferably be an antagonist of the catalytic activity of the molecular tumor marker according to Table 1, 2 or 3, e.g. of a molecular tumor marker protein having a sequence as indicated in section E) or section G) of Table 1, 2 or 3.
  • a compound indirectly inhibiting the activity of a molecular tumor marker refers to a compound which is capable of decreasing the activity of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 by an interaction with a direct interactor of the molecular tumor marker according to Table 1, 2 or 3 ("indirect interactor") or via an indirect working pathway not involving an interaction with of the molecular tumor marker according to Table 1, 2 or 3.
  • Such a compound may be any direct interactor of an interactor of the molecular tumor marker according to Table 1, 2 or 3, e.g. of a molecular tumor marker protein having a sequence as indicated in section E) or section G) of Table 1, 2 or 3.
  • the effect conveyed by the direct interactor of an interactor of the molecular tumor marker according to Table 1, 2 or 3 may be either negative if the interactor of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 itself has a negative effect on the activity of the molecular tumor marker according to Table 1, 2 or 3, or negative, if the interactor of the t molecular umor marker according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • negatively working indirect interactors may have an inhibitory effect on activators of the molecular tumor marker according to Table 1, 2 or 3. Examples of such interactors are enzymatic activities degrading activators of the molecular tumor marker according to Table 1, 2 or 3, or proteins capable of binding and quenching activators of the molecular tumor marker according to Table 1, 2 or 3.
  • such interactors may positively modulate activities leading to a degradation of the molecular tumor marker according to Table 1, 2 or 3, e.g. proteinases.
  • an indirect inhibition of the activity of the molecular tumor marker according to Table 1 , 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 that inhibit and/or preclude binding of transcription factors and the basal transcription machinery to the promoters of the molecular tumor marker according to Table 1, 2 or 3, specific destabilizing activities of the mR A(s) of the molecular tumor marker according to Table 1, 2 or 3 or factors inhibiting the splicing factors specific for the molecular tumor marker according to Table 1, 2 or 3. 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 molecular tumor marker refers to any nucleic acid capable of expressing a mutant form of a naturally occurring protein or polypeptide of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3.
  • the term refers to a nucleic acid encoding (a) variant(s) of the molecular tumor marker according to Table 1, 2 or 3, which comprises an antimorphic modification, in particular which adversely affects the molecular tumor marker according to Table 1, 2 or 3 of the invention.
  • such a behavior may occur if the antimorphic variant can interact with the molecular tumor marker according to Table 1, 2 or 3 but blocks some aspect of its function.
  • such variants may comprise or lack specific domains of the molecular tumor marker according to Table 1, 2 or 3, 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.
  • 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, 2 or 3 and/or functional assays of the proteins and or polypeptides of the invention comprising the amino acid sequence as indicated in section E) or section G) of Table 1, 2 or 3 or derivatives thereof.
  • RNA specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 refers to a short single- stranded RNA molecule of typically 18-27 nucleotides in length, which regulate gene expression of one or more of the molecular tumor marker according to Table 1, 2 or 3.
  • miRNAs are encoded by genes from whose DNA they are transcribed but are not translated into a protein.
  • 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).
  • RISC RNA-induced silencing complex
  • miRNAs 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%
  • 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 refers to nucleic acids corresponding to the sequences indicated in section D) of Table 1, 2 or 3 or the complementary strand thereof.
  • the antisense molecule of the invention comprises a sequence complementary to at least a portion of a molecular tumor marker expression product according to the present invention. While antisense molecules complementary to the coding region sequence of molecular tumor marker expression products may be used, those complementary to the transcribed and untranslated region are preferred.
  • antisense technology can be used to control, i.e. reduce or abolish gene expression through antisense DNA or RNA, or through triple-helix formation.
  • 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, 2 or 3 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 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 RNA 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 siRNA molecule according to the present invention or any other ncRNA 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, miRNA- 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-methoxyamin
  • 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 R A in which the strands run parallel to each other.
  • siRNA specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • 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.
  • 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.
  • 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 refers to (a) short peptide(s) capable of interacting and specifically binding the protein(s) of the molecular tumor marker according to Table 1, 2 or 3.
  • 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) or section G) of Table 1, 2 or 3.
  • 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 indicated in section D) of Table 1, 2 or 3, 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) or section G) of Table 1, 2 or 3.
  • (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) or section G) of Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3" 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.
  • a small molecule is capable of blocking the interaction between the molecular tumor marker protein(s) and its interactor(s).
  • peptidomimetic capable of specifically binding to the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 refers to a small protein- like chain designed to mimic a peptide and capable of binding (a) protein(s) of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • 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.
  • peptidomimetic 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • the skilled person would also be aware of the possibility to target and destroy cancer cells and tissue by virtue of conjugated antibodies specific for the tumor markers.
  • 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.
  • 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.
  • the present invention relates to a pharmaceutical composition for use in, or for the treatment or prevention of a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, wherein said cancer disease implies the increased (up-regulated) expression of a molecular 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an antagonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative
  • Such pharmaceutical composition preferably comprises elements as defined herein above.
  • a pharmaceutical comprising at least one element selected from the group of: (a) a compound directly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an agonist of said molecular tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and (e) a miR A inhibitor specific for a miR A of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • a compound directly stimulating or modulating the activity of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 refers to a compound which is capable of increasing the activity of one or more of the molecular tumor marker according to Table 1, 2 or 3.
  • a compound may be any direct interactor of the molecular tumor marker according to Table 1, 2 or 3, which has positive influence on the catalytic activity of the molecular tumor marker according to Table 1, 2 or 3.
  • Such a compound may preferably be an agonist of the catalytic activity of the molecular tumor marker according to Table 1, 2 or 3.
  • a compound indirectly stimulating or modulating the activity of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 refers to a compound which is capable of increasing the activity of the molecular tumor marker according to Table 1, 2 or 3by an interaction with a direct interactor of the molecular tumor marker according to Table 1, 2 or 3 ("indirect interactor") or via an indirect working pathway not involving an interaction with of the molecular tumor marker according to Table 1, 2 or 3.
  • Such a compound may be any direct interactor of an interactor of the molecular tumor marker according to Table 1, 2 or 3, e.g. of the protein(s) of a molecular tumor marker or group of tumor markers according to section E) or section G) of Table 1, 2 or 3.
  • the effect conveyed by the direct interactor of an interactor of the molecular tumor marker according to Table 1, 2 or 3 may be either positive if the interactor of the molecular tumor marker according to Table 1, 2 or 3 itself has a positive effect on the activity of the molecular tumor marker according to Table 1, 2 or 3, or negative, if the interactor of the molecular tumor marker according to Table 1, 2 or 3 has a negative effect on the activity of the molecular tumor marker according to Table 1, 2 or 3.
  • such positively working indirect integrators may provoke a modification of the binding behavior of directly binding proteins, leading to an increased activity of the molecular tumor marker according to Table 1, 2 or 3.
  • negatively working indirect interactors may have an inhibitory effect on inhibitors of the molecular tumor marker according to Table 1, 2 or 3.
  • interactors are enzymatic activities degrading inhibitors of the molecular tumor marker according to Table 1, 2 or 3, or proteins capable of binding and quenching inhibitors of the molecular tumor marker according to Table 1, 2 or 3.
  • such interactors may inhibit activities leading to a degradation of the molecular tumor marker according to Table 1, 2 or 3, 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be conveyed by compounds activating, protecting or sustaining the expression of the endogenous gene(s) of the molecular tumor marker according to Table 1, 2 or 3.
  • compounds are specific transcription factors of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, specific stabilizing activities of the mR A(s) of the the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or splice factors specific for the the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • Further examples and their implementation would be known to the person skilled in the art.
  • the "protein of a molecular tumor marker” comprised in the pharmaceutical composition may be a protein of the molecular tumor marker according to Table 1, 2 or 3 as defined herein above.
  • it may be a protein being encoded by splice variant of the molecular tumor marker according to Table 1, 2 or 3. More preferably it may have the amino acid sequence as set forth in section E) or section G) of Table 1, 2 or 3.
  • the "protein of a molecular tumor marker" also comprises amino acid sequences being at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences as set forth in section E) or section G) of Table 1, 2 or 3 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, 2 or 3.
  • homologous variants of the molecular tumor marker according to Table 1, 2 or 3 may additionally or alternatively have a similar or identical localization pattern as the molecular tumor marker according to Table 1, 2 or 3 within a cell or within a tissue type.
  • biologically active equivalent of a molecular tumor marker refers to a protein of the molecular tumor marker according to Table 1, 2 or 3 which is capable of performing all or a majority of the individual functions of the molecular tumor marker according to Table 1, 2 or 3.
  • the biologically active equivalents of the molecular tumor marker according to Table 1, 2 or 3 may additionally or alternatively have a similar or identical localization pattern as the molecular tumor marker according to Table 1, 2 or 3 within a cell or within a tissue type.
  • Biologically active equivalents of the molecular tumor marker according to Table 1, 2 or 3 may also comprise variants of the molecular tumor marker according to Table 1, 2 or 3 as defined herein above.
  • the proteins of the molecular tumor marker according to Table 1, 2 or 3 or biologically active equivalents of the molecular tumor marker according to Table 1, 2 or 3 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 molecular tumor marker proteins according to Table 1, 2 or 3 or biologically active equivalents of the molecular tumor markers according to Table 1, 2 or 3.
  • the present invention contemplates vectors containing the polynucleotides encoding the molecular tumor markers according to Table 1, 2 or 3 or biologically active equivalents of the molecular tumor markers according to Table 1, 2 or 3 as defined herein above, host cells, and the production of the molecular tumor markers according to Table 1, 2 or 3 or biologically active equivalents of the molecular tumor markers according to Table 1, 2 or 3 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, 2 or 3.
  • Polynucleotides encoding the molecular tumor markers according to Table 1, 2 or 3 or biologically active equivalents of the molecular tumor markers according to Table 1, 2 or 3 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.
  • 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 U
  • 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.
  • 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.
  • molecular tumor marker proteins according to Table 1, 2 or 3 or biologically active equivalents of the molecular tumor markers according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3" 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • a carrier element may comprise the sequence(s) as indicated in section E) or section G) of Table 1, 2 or 3.
  • Such a carrier element may also comprises nucleotide sequences showing a high degree of homology to the molecular tumor markers according to Table 1, 2 or 3, 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, 2 or 3 or nucleic acid sequences encoding amino 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 E) or section G) of Table 1, 2 or 3.
  • the carrier may comprise the genomic sequence of the molecular tumor marker according to Table 1, 2 or 3.
  • biologically active equivalents of the molecular tumor markers according to Table 1 , 2 or 3 as defined herein above may be comprised in a carrier of the present invention.
  • the polynucleotide encoding the molecular tumor marker according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may be provided via living therapeutics.
  • living therapeutic means that the molecular tumor markers according to Table 1, 2 or 3 or biologically active equivalents of the molecular tumor markers according to Table 1, 2 or 3 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, retroviruses 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3" 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • 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.
  • miR A 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.
  • R As may be produced from transgenes provided in the form of tranfection 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 molecular tumor marker according to Table 1, 2 or 3.
  • 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).
  • TSA trichostatin A
  • the present invention relates to a pharmaceutical composition for use in, or for the treatment or prevention of a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, wherein said cancer 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an agonist of said molecular tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a
  • a pharmaceutical composition according to the present invention may further comprise additional compounds being active against 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 cancer disease 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, miRNAs, 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 , 2 or 3 and up-regulating at least one other tumor marker according to Table 1, 2 or 3.
  • the pharmaceutical composition contains any combination of such elements as laid out above, e.g. the compounds, proteins, dominant negative proteins, nucleic acids, miRNAs, siRNAs, antisense RNAs, aptamers, antibodies, peptidomimetics and small molecules, and wherein the composition contains said elements being capable of down-regulating
  • 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 cancer, particularly prostate cancer.
  • the above described pharmaceutical compositions is for the manufacture of a medicament for the treatment of cancer, particularly prostate cancer.
  • the pharmaceutical compositions and medicaments of the present invention are capable of reducing the tumor volume of a given 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, a compound directly stimulating or modulating the activity of the molecular tumor marker according to Table 1, 2 or 3, an agonist of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 enzymatic activity, a miR A inhibitor specific for miR A(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, an antagomir, a demethylation agent specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3, or a peptidomimetic specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • Such screening procedures may comprise the steps of (a) producing cells which express the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 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.
  • 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, a miR A inhibitor specific for miR A(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, an antagomir, a demethylation agent specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, a peptidomimetic specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 with one or more cells which express the molecular tumor marker according to Table 1, 2 or 3 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
  • the present invention also encompasses an aptamer specific for the expression product(s) or protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, a compound directly stimulating or modulating the activity of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, an agonist of the enzymatic activity of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, a miRNA inhibitor specific for miRNA(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3, an antagomir, a demethylation agent specific for of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and a peptidomimetic specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, 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 use in, or for the treatment or prevention of cancer.
  • the present invention relates to the use of (a) a compound directly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an antagonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (e) a miR A specific for a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (f) an antisense molecule of
  • the present invention relates to the use of (a) a compound directly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an agonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and (e) a miRNA inhibitor specific for a miRNA of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 for the preparation of a pharmaceutical composition for the treatment or
  • the present invention relates to a method of treatment or prevention of cancer, in particular the treatment or prevention of a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, comprising the administration of (a) a compound directly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an antagonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (e) a mim
  • the present invention relates to a method of treatment or prevention of cancer, in particular the treatment or prevention of a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, comprising the administration of (a) a compound directly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an agonist of said molecular tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and (e) a miRNA inhibitor specific for
  • 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.
  • 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.
  • 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 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 1012 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.
  • 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 cancer, comprising at least one element selected from the group consisting of (a) a compound directly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an antagonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (e) a miR A specific for a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table
  • peptidomimetic capable of specifically binding to the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; and (j) an antibody specific for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and/or an antibody variant specific for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; or at least one element selected from the group consisting of (a) a compound directly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3, preferably an agonist of said molecular tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table
  • 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 cancer disease, preferably a cancer disease associated with a progression from a less progressed stage of said cancer disease to a more progressed stage of said cancer 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 a molecular tumor marker or group of tumor markers as defined herein.
  • the vaccine may comprise a protein or antigen, having, comprising or consisting of an amino acid sequence as defined in section E) of Table 1, 2 or 3, more preferably as defined in section G) of Table 1, 2 or 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.
  • the vaccine may comprise a nucleic acid molecule encoding a molecular 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 1, 2 or 3,and/or a vector comprising said nucleic acid molecule, a host cell comprising said vector, an antibody as defined herein, in particular an autoantibody according to the present invention, or a CTL specific for an antigen as defined herein.
  • a nucleic acid molecule encoding a molecular 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 1, 2 or 3,and/or a vector comprising said nucleic acid molecule, a host cell comprising said vector, an antibody as defined herein, in particular an autoantibody according to the present invention, or a CTL specific for an anti
  • a vaccine according to the present invention may, for example, comprise polypeptides or proteins of varying length comprising the amino acids sequence of SEQ ID NOs: 201 to 600, a nucleotide sequence encoding such a polypeptide, e.g. the nucleotide sequences of SEQ ID NOs: 1 to 200, or of amino acid sequences or nucleotide sequences as defined in Table 2, 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.
  • 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
  • compositions 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 molecular 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 cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 1, 2 or 3, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) or G) of Table 1, 2 or 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) or G) of Table 1, 2 or 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 sections E) or G) of Table 1, 2 or 3. Subsequently, such a CTL may perform a cytotoxic 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
  • 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.
  • 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.
  • For the extraction of autologous cells 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 molecular tumor marker expression product or protein, or group of molecular tumor marker expression products or proteins of the present invention, e.g. comprising the amino acid sequence as indicated in sections E) or G) of Table 1, 2 or 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. 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.
  • 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 cancer disease, more preferably a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage.
  • a vaccine according to the present invention is for the treatment or prevention of a 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
  • TAA Carbophydrate antigen-125
  • 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.
  • 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 cancer disease.
  • the term "immune response against a cancer disease” means that 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 a cancer disease therapy or treatment or a corresponding immunoassay for stratifying an individual or cohort of individuals with a cancer disease as defined above, wherein the detection of an increased level of expression of a molecular 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 molecular 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 cancer 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 molecular 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) or G) of Table 1, 2 or 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 1, 2, or 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.
  • the present invention relates to a method for screening for immunogenic epitopes of any of the molecular tumor marker or group of tumor marker expression products or proteins, e.g. a protein comprising or comprised in an amino acid sequence as mentioned in sections E) or G) of Table 1, 2 or 3.
  • a method may preferably comprise:
  • Immobilization of an antigen, expression product or protein of the present invention e.g. a protein comprising or comprised in amino acid sequence as mentioned in sections E) or G) of Table 1, 2 or 3; preferably an antigen may be used which is detected or identified according to a method of identifying an individual for eligibility for a cancer disease therapy or treatment or a corresponding immunoassay for stratifying an individual or cohort of individuals with such a disease, or according to any other detection or diagnosis method of the present invention as described herein.
  • the immobilization may be carried out according to any suitable method known to the person skilled in the art and also comprises the employment of pre-manufactured protein arrays etc.
  • an individual's sample preferably an individual's serum, e.g. the sample or serum which was initially tested positive with regard to the antigen of (i), in particular the antibody or autoantibody present in said serum.
  • the incubation may be carried out according to any suitable method or procedure known to the person skilled in the art, e.g. according to parameters known for the interaction between immobilized antigens and antibodies .
  • short protein fragments or peptides as used here means peptides or fragments of a length of about 10 to 30 amino acids, preferably of about 15 to 25 amino acids, more preferably of about 15 to 20 amino acids of the initially immobilized antigen, expression product or protein, e.g. a protein comprising or comprised in amino acid sequence as mentioned in sections E) or G) of Table 1, 2 or 3.
  • the peptides or protein fragments may additionally be modified, varied or derivatized according to suitable methods known in the art.
  • impede refers to a complete or almost complete abolition of the interaction between the immobilized antigen and serum ingredients in comparison to an interaction reaction between the immobilized antigen and serum ingredients in the presence of a control peptide which has no sequence or structure similarity with the antigen and/or which is not able to influence said interaction.
  • Peptides or protein fragments and their corresponding amino acid sequences, as well as derived or modified variants thereof, which are selected or identified according to the above described method, are also encompassed in a particularly preferred embodiment of the present invention.
  • Such peptides or protein fragments may preferably be used for any of the diagnostic or prognostic methods or assays according to the present invention. They may for example be synthetically produced, immobilized on substrates as described herein above, employed in detection procedures etc. Furthermore, such peptides or protein fragments may preferably be used for vaccination methods as described herein above, or be comprised in a vaccine according to the present invention. More than one such fragment may be provided in the form of a string of proteins as described above.
  • the present invention thus also refers to a personalized vaccine comprising antigenic compounds or protein fragments identified as being detected by an individual's autoantibody. Such vaccine may preferably be used for the treatment of a cancer disease.
  • 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 molecular tumor marker protein or expression product as defined in Section E) or G) of Table 1, 2, or 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 IS S, aluminium salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, Amplivax, AS 15, BCG, CP-870893, CpG7909, CyaA, dSLIM, IC30,
  • 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.
  • the present invention relates to a tumor marker or group of tumor markers, a composition, a methods, a use, an immunoassay, or a pharmaceutical composition as mentioned above, wherein said cancer disease, is any cancer with aberrant expression of EGFR (Endothelian derived Growth Factor Receptor). More preferably, said cancer disease is a cancer disease which is linked to an EGFR mutation, variation, isoform or any other type of modification, even more preferably a cancer disease which is linked to an EGFR L858R mutant form and/or an EGFR L861Q mutant form. An example of such a cancer disease is non- small cell lung cancer.
  • EGFR Endothelian derived Growth Factor Receptor
  • An especially preferred embodiment the present invention relates to any of the abovementioned tumor marker or group of tumor markers, the abovementioned
  • compositions for the abovementioned methods, the abovementioned use, the abovementioned immunoassay, kits or the abovementioned pharmaceutical compositions etc., wherein said cancer disease or cancer is prostate cancer.
  • prostate cancer 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.
  • a "prostate cancer” as used herein denotes a prostate cancer which can be classified according to the TNM classification by the International Union against Cancer (UICC) into stages I to IV.
  • UICC International Union against Cancer
  • the term relates to the classification of prostate cancer pursuing the following T - Primary Prostate Tumor classification schedule:
  • 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)
  • T3b Tumor invades seminal vesicle(s)
  • 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)
  • G2 Moderately differentiated (moderate anaplasia) (Gleason 5-6) G3-4. Poorly differentiated/undifferentiated (marked anaplasia) (Gleason 7- 10),
  • 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
  • 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 abovementioned
  • insignificant disease parameters refers to a clinical tumor stage ⁇ T2 and a Gleason Score ⁇ 6 and a Prostate Cancer Volume ⁇ 0.5 ml.
  • 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.
  • the present invention relates to any of the abovementioned tumor marker or group of tumor markers, the above mentioned
  • compositions wherein said more progressed stage of prostate cancer is a local prostate cancer with significant disease parameters.
  • significant disease parameters refers to a clinical tumor stage > T2 or a Gleason Score > 6 or a Prostate Cancer Volume > 0.5 ml, alternatively to a clinical tumor stage > T2 and/or a Gleason Score > 6 and 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.
  • the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the abovementioned
  • compositions wherein said less progressed stage of prostate cancer is a local prostate cancer with insignificant disease parameters and wherein said more progressed stage of prostate cancer is a local prostate cancer with significant disease parameters.
  • a further, particularly preferred embodiment the present invention relates to any of the abovementioned tumor marker or group of tumor markers, the abovementioned compositions, the abovementioned methods, the abovementioned use, the abovementioned immunoassay, kits or the abovementioned 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 abovementioned tumor marker or group of tumor markers, the abovementioned compositions, the abovementioned methods, the abovementioned use, the abovementioned immunoassay, kits or the abovementioned 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 abovementioned 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 abovementioned use, the abovementioned immunoassay, kits or the abovementioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer has 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 abovementioned compositions, the above mentioned methods, the abovementioned 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 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 Tic or 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 Tic or T2 (UICC 2002 classification).
  • 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 T2, T2a, T2b, T2c, preferably T2 (UICC 2002 classification).
  • the more progressed stage of prostate cancer may imply a Gleason score of 5 or 6, preferably 6 and a stage of T3, T3a, T3b, T3c, preferably T3 (UICC 2002 classification).
  • the most 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 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/or has a Gleason score >6.
  • Another most 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 abovementioned use, the abovementioned 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 has a tumor volume ⁇ 0.5 ml and said more progressed stage of prostate cancer is of stage >T2 (UICC 2002 classification), as defined herein above and/or has a Gleason score >6 and/or has a tumor volume of > 0.5 ml.
  • HIRP Host Immune Response Profiling
  • Host Immune Response Profiling is a technology that allows the identification of host immune reactions against body proteins that are changed in its characteristics due the development, or the progression, or the treatment of a disease condition.
  • the change in characteristics may mean a change in expression of a protein, the change in
  • the immune system of the host is able to detect such changes via an innate or humoral immune reaction against any sort of non-normal body proteins. In that sense the host immune system picks up any change in e.g. cellular proteins that occur while a disease may develop and is therefore able to potentially pick up this process way before the disease is going to manifest in the form of clinical symptoms. Further, as the progression of a disease is often associated with increased proliferation of cells (particularly in any type of tumor disease) this technology is especially suited to detect early signs of progression of disease thereby providing significant value for the prognosis of the future development of a given disease.
  • the technology works by the immobilization of body proteins that were previously expressed in a vertebrate cellular expression system, like insect cells, or in mammalian cells. After expression the proteins are purified, spotted and immobilized onto a solid support (e.g., glass slides, 96-well Microtiter plates), and subsequently incubated with human serum of plasma of a disease patient, or of a normal/healthy control.
  • a solid support e.g., glass slides, 96-well Microtiter plates
  • the binding of antibodies in the blood sample to the immobilized proteins is detected be a secondary fluorescence-labeled antibody which is directed against, and specific for the constant part of human IgG.
  • Such antibodies are typically called auto-antibodies as they react against body- owned proteins.
  • This detection step ensures that only auto-antibodies that are present in patient/control blood against body proteins will be detected.
  • the reaction patterns against patient samples can then be compared to corresponding patterns of control samples to select those that are able to discriminate between different sample groups (e.g., healthy vs. disease).
  • the strength of this technique is the blood-based detection of events that are typically closely linked to changes on the cellular level as a disease starts to develop or starts to progress.
  • Auto-antibodies may typically be developed by the host against cellular proteins derived from the diseased tissue, and which are secreted at small levels into the circulation during apoptosis or necrosis of cells, or that are unnaturally secreted into the circulation as full or fragmented proteins.
  • 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 biopsies (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- and post-treatment stages T2 or less, and Gleason scores 6 or less.
  • Group 4 men were selected based on PSA levels >3 ng/ml and cancer positive biopsy and pre-treatment stages T2 or less, post-treatment stages >T2, Gleason scores >7.
  • Group 5 men were selected on signs of clinical advanced cancer disease (clinical stage >T3).
  • Samples were retrieved from the -80°C freezer, thawed quickly and put on ice. 2 ⁇ 1 of sample were diluted with 98 ⁇ 1 of Milli-Q (dilution factor 50). The sample dilution was mixed and centrifuged. 300 ⁇ 1 of Bradford Reagent was pipetted into a new vial. 10 ⁇ 1 of the sample dilution were added to the 300 ⁇ 1 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 ⁇ 1 of each of the 15 samples to one tube. The samples were mixed and spun (end volume was 1020 ⁇ 1) and this procedure was repeated for pools 2-8. Finally, samples were stored at -80°C.
  • BSA Stock Solution was obtained by dissolving 10.87 mg of BSA (Bovine Serum Albumin) in 1.20777 ml H 2 0.
  • the standards were measured after 20 minutes of incubation.
  • FIG. 2 shows a representative negative control image displaying the regular pattern of control proteins spotted in one subarray. The purpose of these control proteins is to provide reference points for data acquisition and analysis.
  • the Alexa Fluor® conjugated antibody (boxed in white) allows proper alignment of the spotfinding software for data acquisition.
  • the anti- biotin antibody (boxed in green) serves as a control for another ProtoArray® application, but is recognized by the anti-human IgG antibody used for detection in this assay.
  • ProtoArray® subarray contains a gradient of human IgG (boxed in red), which serves as a control for proper performance of the detection reagent.
  • Anti- human IgG antibody is also spotted as a gradient in every subarray. This antibody binds to IgG present in the serum sample and serves as a control for proper assay performance. Assay Performance
  • the maximum group average background signal observed across the five groups was 437 relative fluorescence units (RFU) in group 2, with the cumulative average background across all samples equal to 419. Maximal signals were observed at >65,000 RFU for all of the samples, indicating a dynamic range of greater than 2 logs.
  • Table 7 shows the mean signals for the spots corresponding to the highest concentration of anti-human IgG on each array.
  • Arrays profiled with samples 46, 60, 61, 62, 84, 108, and 109 had mean signal intensities that were greater than two standard deviations below the mean for all arrays (highlighted dark gray). The low signals may be the result of lower IgG levels or differences in immunoglobulin binding proteins in the corresponding samples. To ensure the use of the highest quality data set, those samples highlighted dark gray in Table 7 were excluded from the analysis and candidate autoantigen tables in this report.
  • Example 4 Data analysis and scoring
  • the array data were analyzed as follows. The raw results (fluorescence from duplex, neighboring spots) were converted to semi-ready data by averaging the intensities of the two spots. The resulting file contained 8302 rows for the different spotted proteins and 113 columns for the samples that had passed the quality control (described before) . As a final step, all arrays were normalized to have the same median intensity. We found this an essential step as the median intensities of the arrays varied by more than a factor of two in either direction before normalization.
  • the proteins were ranked according to their p-value from a one-sided Wilcoxon test comparing groups 3 and 4 (signal in 4 greater than in 3).
  • groups 3 and 4 signal in 4 greater than in 3
  • the full and dashed line indicate the median and the mean normalized intensity per patient group, respectively.
  • the top-left number indicates the rank of the protein according to the Wilcoxon test, with number one the protein with the lowest P-value.
  • the PSA values measured clinically for each patient were added as a separate row to the data as a benchmark, also PSA (gene name KLK3) could be detected, which appears with rank 42.
  • the measurement of a marker, or a combination of markers from Table 1, 2, 3 in a patient serum sample is used to select patients for active surveillance.
  • the measurement of the marker, or the combinations of markers above a given threshold hereby indicates a >95%, >90%, >85%, >80%, >75%, >70%, >65%, or >60% chance that the identified prostate cancer is indolent, insignificant disease and is not going to progress over time.
  • the measurement of the marker, or a combination of markers from Table 1, 2, 3 is further used to monitor the potential progression of the prostate cancer.
  • the marker(s) measurement is performed every 3, or every 6, or every 12 months after the patient has been moved into an avtive surveillance regime.
  • Example 6 Identification of men with significant, progressive prostate cancer for stratification towards radical treatment
  • the measurement of a marker, or a combination of markers from Table 1, 2, 3 in a serum sample of a patient before a biopsy is perfromed is used to identify prostate cancers that have a very high probability to progress over time and therefore require radical intervention.
  • the measurement of the marker, or the combinations of markers above a given threshold hereby indicates a >95%, >90%, >85%, >80%, >75%, >70%, >65%, or >60% chance that the identified prostate cancer is significant disease and is going to progress in the future.
  • the measurement of the marker, or the combinations of markers below a given threshold indicates a >95%, >90%, >85%, >80%, >75%, >70%, >65%, or >60% chance that in case a prostate cancer is present it is insignificant disease and is not going to progress in the future.
  • the measurement of the marker, or a combination of markers from Table 1, 2, 3 in a serum sample of a patient is further used to monitor the progression of a potential prostate cancer in case the initial measurement of the markers, or combination of markers indiciated the absence of significant disease.
  • the marker(s) measurement is performed every 3, or every 6, or every 12 months after the patient has been moved into an avtive surveillance regime. If the marker, or marker combination measurement is going to pass a given threshold by 5%, 10%, 15%, 20%>, 25%, or 30% further investigations or treatment will be started.
  • This workflow provides a very effective way of prostate cancer patient management towards treatment of selectively those patients that need intervention to save their life.
  • Example 7 Selection of antigens for the testing of such antigens on targeted arrays for marker verification purposes
  • markers 213 antigens were selected from the initial discovery that was performed on 120 Prostate Cancer (PCa) patient derived blood samples. The process is outlined in Figure 10.
  • the 120 blood samples were coming from 5 clinically defined patient groups ranging from normal/healthy patients over patients with a benign prostate disease to finally those patients with significant and advanced prostate cancer:
  • PCa indolent/non-aggressive vs. significant/aggressive Prostate Cancer
  • Table 8 Numbers of indolent vs. aggressive PCa spread of four independent data sets.
  • Samples in each data set were selected such that the diagnostic specificity of PSA at a diagnostic sensitivity was corresponding to a historical value demonstrated on larger sample collections of ca 20%.
  • antigens were analyzed on their power to discriminate between indolent and aggressive prostate cancer patients.
  • the clinical characteristics of the patients are:
  • the diagnostic specificity was calculated at a diagnostic sensitivity of 90%>. Subsequently, individual antigens were selected based on their overall performance on all four analyzed data sets. Only markers were selected with a minimum specificity of >20% on at least 3 of the 4 data sets to continue with.
  • the diagnostic sensitivity is defined as the probability of the identification of a disease condition; the sensitivity is expressed as a percentage as it also indicates how many disease cases out 100 is a test procedure able to correctly identify.
  • the value 100-sensitivity is defined as the False-Negative Rate (FNR) as this number indicates the percentage of disease cases that are missed by a diagnostic test or procedure. For instance, a diagnostic sensitivity of a test procedure of 90% indicates that that 90%> of disease cases out of a tested population can be correctly identified by the test procedure as having the disease condition.
  • the diagnostic specificity is defined as the probability of the identification of a non-disease condition (vs.
  • a disease condition by a diagnostic test/procedure
  • the specificity is expressed as a percentage as it also indicates how many non-disease cases out 100 is a test procedure able to correctly identify.
  • the value 100-sensitivity is defined as the False-Negative Rate (FPR) as this number indicates the percentage of non-disease cases that are incorrectly classified as having the disease.
  • FPR False-Negative Rate
  • a diagnostic specificity of a test procedure of 50% indicates that that 50%> of non-disease cases out of a tested population can be correctly identified by the test procedure as not having the disease condition.
  • Tables 9 and 10 provide an overview over the data points of DATA set 1, DATA set 2 (both in Table 9), and DATA set 3 and DATA set 4 (both in Table 10), respectively, measured, the derived p-values and the derived specificity at 90%> sensitivity of all 213 candidates.
  • Table 12 Selected marker candidates including PSA, Patient Age, & DRE to generate 5- antigen signatures.
  • the PSA values were included for each patient, the patient age, as well as the DRE (Digital Rectal Examination) as additional parameters.
  • the PSA value typically a value from 1-30 representing the concentration of this protein in patients per Milliliter blood
  • the DRE result is typically represented by a single digit number from 1-5 each representing different pathophysiological conditions of the prostate.
  • the DRE numeric values were scaled according to PSA and patient age by a factor of 10 or 100. In consequence, 20 marker candidates were included to build 3 antigen signatures, and 13 marker candidates to build 5 antigen signatures for the purpose to discriminate between indolent and aggressive Prostate Cancer.
  • the identified marker/antigen combinations are able to identify aggressive Prostate Cancer at a very high diagnostic sensitivity of >90%, and at the same time discriminate between indolent and aggressive Prostate Cancer with specificity of >40% (3 marker/antigen signatures), or a specificity of >45% (5 marker/antigen signatures). Further, if the diagnostic sensitivity level is increased to >95%> with very low aggressive cases missed the specificity is still at a minimal level of >30% (3 marker/antigen signatures), or a specificity of >35% (5
  • the max specificity which can be achieved by using PSA, Patient Age or DRE is 23% (at 90% sensitivity), or 11% (at 95% sensitivity).
  • the identified marker/antigen combinations are able to achieve a max specificity of 55% (at 90%) sensitivity), and 46%> (at 95%> sensitivity) respectively.
  • the diagnostic sensitivity is increased to 95% the diagnostic specificity can even be increased by a factor of 4 over the currently used gold standard PSA (11% vs. 46% for a 5-marker/antigen signature).
  • Example 9 Marker verification and identification of 5-parameter signatures to discriminate indolent from aggressive prostate cancer with high sensitivity for patients with PSA range o f 4-10 ng/ml
  • PCa indolent/non-aggressive vs. significant/aggressive Prostate Cancer
  • Table 17 Numbers of indolent vs. aggressive PCa spread of four independent data sets (DATA l to DATA 4). Please note that for the further processing of the data only those patient samples with PSA 4-10 ng/ml were selected.
  • the patient samples were derived from patient cohorts with the following characteristics: i) Control, very low PSA (GROUP J)
  • the diagnostic sensitivity is defined as the probability of the identification of a disease condition; the sensitivity is expressed as a percentage as it also indicates how many disease cases out 100 is a test procedure able to correctly identify.
  • the value 100-sensitivity is defined as the False-Negative Rate (FNR) as this number indicates the percentage of disease cases that are missed by a diagnostic test or procedure. For instance, a diagnostic sensitivity of a test procedure of 90% indicates that that 90%> of disease cases out of a tested population can be correctly identified by the test procedure as having the disease condition.
  • the diagnostic specificity is defined as the probability of the identification of a non-disease condition (vs.
  • a disease condition by a diagnostic test/procedure
  • the specificity is expressed as a percentage as it also indicates how many non-disease cases out 100 is a test procedure able to correctly identify.
  • the value 100-sensitivity is defined as the False-Negative Rate (FPR) as this number indicates the percentage of non-disease cases that are incorrectly classified as having the disease.
  • FPR False-Negative Rate
  • a diagnostic specificity of a test procedure of 50% indicates that that 50%> of non-disease cases out of a tested population can be correctly identified by the test procedure as not having the disease condition.
  • Tables 9 and 10 above provide an overview over the data points of DATA set 1, DATA set 2 (both in Table 9), and DATA set 3 and DATA set 4 (both in Table 10), respectively, measured, the derived p-values and the derived specificity at 90%> sensitivity of all 213 candidates.
  • Table 19b 5- Marker/ Antigen Combinations with a minimal diagnostic specificity of >40% at a diagnostic sensitivity level of >90%.
  • Tables 19a to 19c numerous combinations have been defined that perform significantly better compared to PSA, Patient Age, or DRE alone (see Tables above), which can be used to discriminate between indolent and aggressive Prostate Cancer based on the measurement of such antigens in a patient blood sample as described here.
  • the identified marker/antigen combinations are able to identify aggressive Prostate Cancer at a very high diagnostic sensitivity of >90%, and at the same time discriminate between indolent and aggressive Prostate Cancer with specificity of >40%.
  • the max specificity which can be achieved by using PSA, Patient Age or DRE is 23% (at 90%> sensitivity) (see Tables Example 2).
  • the identified marker/antigen combinations are able to achieve a max specificity of 56%> (at 90%> sensitivity). Such it is possible by using the disclosed marker combinations to increase the diagnostic specificity at 90% diagnostic sensitivity by a factor of >2 over the currently used gold standard PSA (23% vs. 56% for a 5-marker/antigen signature).
  • the present invention relates to the following items:
  • Item 1 A tumor marker or group of tumor markers associated with the progression of a cancer 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 by increase (up- regulated) 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 one tumor marker selected from
  • Item 2 The group of tumor markers of item 1, wherein said group comprises at least
  • Item 3 The group of tumor markers of item 1 or 2, wherein the p-value of the expression modification is 0.006 or lower.
  • Item 4 The group of tumor markers of any one of items 1 to 3, wherein the group comprises at least 5 tumor markers corresponding to tumor marker #1 to #5 of Table 3, at least 10 tumor markers corresponding to tumor marker #1 to #10 of Table 3, at least 15 tumor markers corresponding to tumor marker #1 to #15 of Table 3, at least 20 tumor markers corresponding to tumor marker #1 to #20 of Table 3, at least 25 tumor markers corresponding to tumor marker #1 to #25 of Table 3, at least 30 tumor markers corresponding to tumor marker #1 to #30 of Table 3, at least 35 tumor markers corresponding to tumor marker #1 to #35 of Table 3, at least 40 tumor markers corresponding to tumor marker #1 to #40 of Table 3, at least 45 tumor markers corresponding to tumor marker #1 to #45 of Table 3 or at least 50 tumor markers corresponding to tumor marker #1 to #50 of Table 3.
  • Item 5 A composition for diagnosing, detecting, graduating, monitoring or prognosticating a cancer disease 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, 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 4.
  • Item 6 The composition of item 5, wherein said nucleic acid affinity ligand or peptide affinity ligand is modified to function as an imaging contrast agent.
  • Item 7 A method for detecting, diagnosing, monitoring or prognosticating a cancer disease 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, 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 4 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 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 cancer.
  • Item 10 The method of item 9, wherein 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 cancer disease
  • step (c) deciding on the stage or developmental status of cancer based on the results obtained in step (b).
  • Item 11 Use of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4 as a marker for detecting, diagnosing, graduating, monitoring or prognosticating a cancer disease 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.
  • Item 12 An immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, or for detecting, diagnosing, monitoring or prognosticating the progression from a less progressed cancer stage to a more progressed cancer stage comprising at least the steps
  • step (d) deciding on the presence, stage or risk of recurrence of cancer or the progression of cancer based on the results obtained in step (c),
  • 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 4.
  • Item 13 A method of identifying an individual for eligibility for a cancer disease therapy comprising:
  • step (c) classifying the levels of expression of step (a) relative to levels of step
  • step (c) determining the difference in expression of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4 of steps (a) and the expression of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4 and/or the reference gene in step (b);
  • step (d) stratifying an individual or cohort of individuals to a cancer 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 any one of items 1 to 4.
  • Item 15 The method of any one of items 7 to 10 or 13, or the immunoassay of item 12 or 14, wherein said method or immunoassay comprises the additional step of determining the level of prostate specific antigen (PSA).
  • PSA prostate specific antigen
  • Item 16 A pharmaceutical composition for the treatment or prevention of a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage, wherein said cancer disease implies the increased (up-regulated) expression of a tumor marker or group of tumor markers as defined in any one of items 1 to 4, comprising at least one element selected from the group of:
  • Item 17 The composition for diagnosing, detecting, graduating, monitoring or prognosticating of item 5 or the pharmaceutical composition of item 16, wherein said antibody is an autoantibody against the expression product or protein or a fragment thereof, of a tumor marker or group of tumor markers as defined in any one of items 1 to 4.
  • Item 18 The composition or pharmaceutical composition of item 17, wherein said autoantibody is an autoantibody against the expression product or protein of a tumor marker of group of tumor markers as defined in any one of items 1 to 4, wherein said expression product or protein comprises an amino acid sequence as indicated in section G) of Table 3, or any fragment thereof.
  • Item 19 A vaccine for the treatment or prevention of a cancer disease associated with a progression from a less progressed stage of a cancer disease to a more progressed stage of a cancer disease, 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) or G) 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) or G) of Table 3, or any fragment thereof.
  • Item 20 The tumor marker or group of tumor markers of any one of items 1 to 4, the composition of item 5 or 6, 17 or 18, the method of any one of items 7 to 10, 13 or 15, the use of item 11, the immunoassay of item 12, 14 or 15, the pharmaceutical composition of item 16, 17 or 18, or the vaccine of item 19, wherein said cancer disease is prostate cancer.
  • Item 21 The tumor marker or group of tumor markers, composition, method, use, immunoassay, pharmaceutical composition or vaccine of item 20, wherein said less progressed stage of prostate cancer is of stage ⁇ T2 (UICC 2002 classification), and Gleason score ⁇ 6, and said more progressed stage of prostate cancer is of stage >T2 (UICC 2002 classification), and/or Gleason score >6.

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Abstract

La présente invention porte sur un marqueur tumoral ou un groupe de marqueurs tumoraux associés à l'évolution d'une maladie cancéreuse d'un stade moins avancé à un stade plus avancé, l'expression des marqueurs tumoraux étant modifiée lors de la comparaison de l'expression dans le stade moins avancé avec celle dans le stade plus avancé. La présente invention porte en outre sur une composition destinée à diagnostiquer, détecter, évaluer, surveiller ou pronostiquer une maladie cancéreuse associée à une évolution d'un stade de cancer moins avancé à un stade de cancer plus avancé comprenant des ligands d'affinité pour les produits d'expression des marqueurs tumoraux, sur des procédés correspondants, et sur l'utilisation desdits marqueurs tumoraux pour détecter, diagnostiquer, évaluer, surveiller ou pronostiquer une maladie cancéreuse associée à une évolution d'un stade de cancer moins avancé à un stade de cancer plus avancé. La présente invention porte en outre sur un immuno-essai correspondant, sur un procédé d'identification d'une personne pour son éligibilité à une thérapie de maladie cancéreuse, ainsi que sur une composition pharmaceutique basée sur l'inhibition de l'expression desdits marqueurs tumoraux.
PCT/IB2010/055796 2009-12-14 2010-12-14 Nouveaux marqueurs tumoraux WO2011073896A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2807347C2 (ru) * 2018-12-28 2023-11-14 СиДжей ЧЕИЛДЗЕДАНГ КОРПОРЕЙШН Генетический маркер для связанного с helicobacter pylori заболевания

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002090580A1 (fr) * 2001-05-03 2002-11-14 National Cancer Centre Of Singapore Pte Ltd Identification du cancer du foie par detection de l'expression aberrante de proteines se liant au facteur de croissance analogue a l'insuline
WO2006125021A2 (fr) * 2005-05-16 2006-11-23 Dana Farber Cancer Institute, Inc. Methodes de detection du cancer
DE102006056784A1 (de) * 2006-12-01 2008-06-05 Meyer, Helmut E., Prof.Dr. Biomarker für die Diagnose von Pankreaskrebs
WO2009020932A2 (fr) * 2007-08-03 2009-02-12 Biocept, Inc. Hybridation in situ pour détection de marqueurs d'arn et d'adn
WO2009090269A2 (fr) * 2008-01-18 2009-07-23 Katholieke Universiteit Leuven, K.U.Leuven R & D Marqueurs et procédés de diagnostic pour une métastase

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002090580A1 (fr) * 2001-05-03 2002-11-14 National Cancer Centre Of Singapore Pte Ltd Identification du cancer du foie par detection de l'expression aberrante de proteines se liant au facteur de croissance analogue a l'insuline
WO2006125021A2 (fr) * 2005-05-16 2006-11-23 Dana Farber Cancer Institute, Inc. Methodes de detection du cancer
DE102006056784A1 (de) * 2006-12-01 2008-06-05 Meyer, Helmut E., Prof.Dr. Biomarker für die Diagnose von Pankreaskrebs
WO2009020932A2 (fr) * 2007-08-03 2009-02-12 Biocept, Inc. Hybridation in situ pour détection de marqueurs d'arn et d'adn
WO2009090269A2 (fr) * 2008-01-18 2009-07-23 Katholieke Universiteit Leuven, K.U.Leuven R & D Marqueurs et procédés de diagnostic pour une métastase

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KOHLER; MILSTEIN, EUR. J. IMMUNOL., vol. 6, 1976, pages 511 - 519
KRANSE ET AL., THE PROSTATE, vol. 68, 2008, pages 1674 - 80
ROOBOL ET AL., EUR UROL, vol. 182, no. 5, 2009, pages 2112 - 20
ROOBOL ET AL., EUR UROL, vol. 57, no. 1, 2009, pages 79 - 85
TAMRA; DABCYL; BLACK HOLE QUENCHER: "BHQ-1 or BHQ-2. A variety of other useful fluorescents and chromophores are described in Stryer", SCIENCE, vol. 162, 1968, pages 526 - 533

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2807347C2 (ru) * 2018-12-28 2023-11-14 СиДжей ЧЕИЛДЗЕДАНГ КОРПОРЕЙШН Генетический маркер для связанного с helicobacter pylori заболевания

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