WO2011027310A1

WO2011027310A1 - Novel tumor markers

Info

Publication number: WO2011027310A1
Application number: PCT/IB2010/053942
Authority: WO
Inventors: Ralf Hoffmann; Edwin P. Romijn; Hugo M. Visser; Tim Hulsen
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2009-09-03
Filing date: 2010-09-02
Publication date: 2011-03-10
Also published as: WO2011027310A8

Abstract

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 and/or activation of the expression of said tumor markers.

Description

NOVEL TUMOR MARKERS

FIELD OF THE INVENTION

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 and/or activation of the expression of said tumor markers. BACKGROUND OF THE INVENTION

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. Among men, the three most commonly diagnosed cancers are prostate, lung and colorectal cancer in developed countries. Particularly prostate cancer is the most common malignancy in European males. In 2002 in Europe, an estimated 225,000 men were newly diagnosed with prostate cancer and about 83,000 died from this disease.

Several different proteins or peptides have been associated with cancer development. For instance, phosphodiesterase PDE7 has been shown to be linked to chronic lymphocytic leukemia. Yet, for many cancer types or cancer progression forms there is no adequate marker molecule available.

Prostate cancer, for example, is traditionally diagnosed via the serum level of prostate-specific antigen (PSA). However, PSA is not prostate cancer-specific and can be raised due to other circumstances, leading to a large number of false-positives (cancer is not found in around 70% of men with raised PSA levels who undergo biopsy). Furthermore, there will be an unpredictable number of false-negatives who later develop prostate cancer in the presence of a "normal" PSA test.

Therefore, there is a need for the provision of a new and effective, alternative diagnosis perspective for the detection, monitoring and prognostication of cancer, in particular of prostate cancer. There is in particular a need for tumor markers that can increase the diagnostic specificity and differentiate between harmless and aggressive neoplastic diseases. SUMMARY OF THE INVENTION

The present invention addresses this need and provides means and methods which allow the diagnosis and detection of cancer, in particular prostate cancer.

The above objective is accomplished by the identification of novel tumor markers from samples of cancer patients. Using tissue or bodily fluid 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. In particular, it was demonstrated by the present inventors that the novel tumor markers are significantly down-regulated or up-regulated when comparing samples from patients having a less progressed stage of cancer to samples from patients having a more progressed stage of the disease. Therefore, these markers are 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

advantageously be employed for (i) detecting and diagnosing cancer forms, in particular prostate cancer forms, (ii) prognosticating cancer forms, in particular prostate cancer forms, (iii) monitoring of cancer progression towards more progressed cancer stages, in particular of prostate cancer, and (iv) distinguishing between less and more progressed cancer forms, in particular less and more progressed prostate cancer forms ,and most advantageously less progressed prostate cancer forms wherein the prostate cancer is of stage < T2 (UICC 2002 classification) and Gleason score < 6 and more progressed prostate cancer forms wherein the prostate cancer is of stage >T2 (UICC 2002 classification) and Gleason score >7. Finally, pharmaceutical compositions based on these tumor markers will provide novel therapeutic avenues in the treatment of cancer, in particular prostate cancer. The present invention, thus relates in a first aspect to a tumor marker or group of tumor markers associated with the progression of a 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 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 Table 1 or Table 2.

In a preferred embodiment of the present invention the expression of said marker(s) is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section I) of Table 1 or 2.

In another preferred embodiment the expression of the marker(s) is decreased (down-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section J) of Table 1 or 2.

In yet another preferred embodiment of the present invention said group of tumor markers comprises at least one marker with a decreased expression in said more progressed stage and at least one marker with an increased expression in said more progressed stage.

In a further preferred embodiment of the present invention said 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 or all tumor markers of Table 1, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or all of the tumor markers of Table 2.

In another preferred embodiment of the present invention the p-value of the expression modification of said group of tumor markers is 0.0006 or lower.

In a further embodiment of the present invention the group of tumor markers comprises at least 3 tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 6 tumor markers corresponding to tumor marker #1 to #6 of Table 1, or at least 9 tumor markers corresponding to tumor marker #1 to #9 of Table 1, or at least 12 tumor markers corresponding to tumor marker #1 to #12 of Table 1, or at least 15 tumor markers

corresponding to tumor marker #1 to #15 of Table 1, or at least 18 tumor markers

corresponding to tumor marker #1 to #18 of Table 1, or at least 21 tumor markers

corresponding to tumor marker #1 to #21 of Table 1, or at least 25 tumor markers

corresponding to tumor marker #1 to #25 of Table 1 and/or at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 tumor markers selected from tumor marker #1 to #50 of Table 2. In a further embodiment of the present invention the group of tumor markers comprises:

(i) at least marker # 1 of Table 1 (K1C10) and at least one additional marker of Table 1 or Table 2; or

(ii) at least marker # 2 of Table 1 (HYOU1) and at least one additional marker of Table 1 or Table 2; or

(iii) at least marker # 3 of Table 1 (HBA) and at least one additional marker of Table 1 or Table 2; or

(iv) at least marker # 4 of Table 1 (PROC) and at least one additional marker of Table 1 or Table 2; or

(v) at least marker # 5 of Table 1 (ICl) and at least one additional marker of Table 1 or Table 2; or

(vi) at least marker # 6 of Table 1 (VTDB) and at least one additional marker of Table 1 or Table 2; or

(vii) at least marker # 7 of Table 1 (IGHA1) and at least one additional marker of Table 1 or Table 2; or

(viii) at least marker # 8 of Table 1 (PCOC1) and at least one additional marker of Table 1 or Table 2; or

(ix) at least marker # 9 of Table 1 (BST1) and at least one additional marker of Table 1 or Table 2; or

(x) at least marker # 10 of Table 1 (GRK7) and at least one additional marker of Table 1 or Table 2; or

(xi) at least marker # 11 of Table 1 (C07) and at least one additional marker of Table 1 or Table 2; or

(xii) at least marker # 12 of Table 1 (ADIPO) and at least one additional marker of Table 1 or Table 2; or

(xiii) at least marker # 13 of Table 1 (CHL1) and at least one additional marker of Table 1 or Table 2; or

(xiv) at least marker # 14 of Table 1 (TSP1) and at least one additional marker of Table 1 or Table 2; or

(xv) at least marker # 15 of Table 1 (SAA4) and at least one additional marker of Table 1 or Table 2; or

(xvi) at least marker # 16 of Table 1 (CD 14) and at least one additional marker of Table 1 or Table 2; or (xvii) at least marker # 17 of Table 1 (HEMO) and at least one additional marker of Table 1 or Table 2; or

(xviii) at least marker # 18 of Table 1 (HRG) and at least one additional marker of Table 1 or Table 2; or

(^χί^χ) ^at least marker # 19 of Table 1 (MMP2) and at least one additional marker of Table 1 or Table 2; or

(xx) at least marker # 20 of Table 1 (CYTC) and at least one additional marker of Table 1 or Table 2; or

(xxi) at least marker # 21 of Table 1 (CXCL7) and at least one additional marker of Table 1 or Table 2; or

(xxii) at least marker # 22 of Table 1 (FBLN3) and at least one additional marker of Table 1 or Table 2; or

(xxiii) at least marker # 23 of Table 1 (DOPO) and at least one additional marker of Table 1 or Table 2; or

(xxiv) at least marker # 24 of Table 1 (HBB) and at least one additional marker of Table 1 or Table 2; or

(xxv) at least marker # 25 of Table 1 (APOA1) and at least one additional marker of Table 1 or Table 2.

In a further aspect 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 tumor marker or group of tumor markers as defined above.

In a preferred embodiment of the present invention said nucleic acid affinity ligand or peptide affinity ligand is modified to function as an imaging contrast agent.

In yet another aspect 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 tumor marker or group of tumor markers as defined above, in a sample. In a preferred embodiment the determining step of said method is

accomplished by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of the tumor marker or group of tumor markers as defined above.

In another preferred embodiment of the present invention said method comprises the additional step of comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same cancer.

In yet another preferred embodiment of the present said method is a method of graduating cancer, comprising the steps of:

(a) determining the level of a tumor marker or group of tumor markers as defined above in a sample by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers, and

(b) comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same cancer; and

(c) deciding on the stage or developmental status of cancer based on the results obtained in step (b).

In a further aspect the present invention relates to the use of said tumor marker or a group of tumor markers as defined above as a marker for detecting, diagnosing, graduating, monitoring or prognosticating a 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.

In yet another aspect 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

(a) testing in a sample obtained from an individual for the expression of a tumor marker or a group of tumor markers as defined above;

(b) testing in a control sample for the expression of the same tumor marker or group of tumor markers as in (a); (c) determining the difference in expression of the tumor marker or group of tumor markers of steps (a) and (b); and

(d) deciding on the presence, stage or risk of recurrence of cancer or the progression of cancer based on the results obtained in step (c),

wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or group of tumor markers as defined above.

In another aspect the present invention relates to a method of identifying an individual for eligibility for a cancer disease therapy comprising:

(a) testing in a sample obtained from an individual for the expression of an up-regulated tumor marker or group of tumor markers as defined above;

(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said up-regulated tumor marker or group of tumor markers as defined above;

(c) classifying the levels of expression of step (a) relative to levels of step (b); and

(d) identifying the individual as eligible to receive a cancer disease therapy where the individual's sample is classified as having an increased level of expression of an up-regulated tumor marker or group of tumor markers as defined above.

(a) testing in a sample obtained from an individual for the expression of a down-regulated tumor marker or group of tumor markers as defined above;

(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said down-regulated tumor marker or group of tumor markers as defined above;

(c) classifying the levels of expression of step (a) relative to levels of step

(b); and

(d) identifying the individual as eligible to receive a cancer disease therapy where the individual's sample is classified as having a decreased level of expression of a down-regulated tumor marker or group of tumor markers as defined above.

In yet another aspect the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a cancer disease comprising:

(a) testing in a sample obtained from an individual for the expression of an up-regulated tumor marker or group of tumor markers as defined above; (b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said up-regulated tumor marker or group of tumor markers as defined above;

(c) determining the difference in expression of said up-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said up-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b); and

(d) stratifying an individual or cohort of individuals to a cancer disease therapy based on the results obtained in step (c), where the individual's sample has an increased level of expression of an up-regulated tumor marker or group of tumor markers as defined above.

(c) determining the difference in expression of said down-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said down-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b); and

(d) stratifying an individual or cohort of individuals to a cancer disease therapy based on the results obtained in step (c), where the individual's sample has an decreased level of expression of an down-regulated tumor marker or group of tumor markers as defined above.

In a preferred embodiment said immunoassay or said method as mentioned above comprises the additional step of determining the level of prostate specific antigen (PSA).

In an additional aspect the present invention relates to a pharmaceutical composition for the treatment or prevention of a 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 an up-regulated tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of:

(a) a compound directly inhibiting the activity of a tumor marker as defined above, preferably an antagonist of said tumor marker enzymatic activity;

(b) a compound indirectly inhibiting the activity of a tumor marker as defined above;

(c) a dominant negative form of a protein of a tumor marker as defined above or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker as defined above;

(e) a miR A specific for a tumor marker as defined above;

(f) an antisense molecule of a tumor marker as defined above;

(g) a siR A specific for a tumor marker as defined above;

(h) an aptamer specific for the expression product of a tumor marker as defined above or for the protein of a tumor marker as defined above;

(i) a small molecule or peptidomimetic capable of specifically binding to the protein of a tumor marker as defined above; and

(j) an antibody specific for the protein of a tumor marker as defined above and/or an antibody variant specific for the protein of a tumor marker as defined above.

In another aspect the present invention relates to a pharmaceutical composition for the treatment or prevention of a 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 down-regulated tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of:

(a) a compound directly stimulating or modulating the activity of a tumor marker as defined above, preferably an agonist of said tumor marker enzymatic activity;

(b) a compound indirectly stimulating or modulating the activity of a tumor marker as defined above;

(c) a protein of a tumor marker as defined above or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing a protein of a tumor marker as defined above; and (e) a miR A inhibitor specific for a miR A of a tumor marker as defined above.

In another aspect the present invention relates to 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, wherein said cancer disease implies the increased (up-regulated) expression of a tumor marker or gourp of tumor markers as defined above, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 6, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) of Table 6, or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) of Table 6, or any fragment thereof.

In another aspect the present invention relates to the use of a tumor marker or a group of tumo markers as defined above for identifying pharmaceutically active agents useful in the treatment or prevention of a cancer disease.

In a preferred embodiment the present invention relates to the use of a tumor marker or a group of tumo markers as defined above for identifying pharmaceutically active agents useful in the treatment or prevention of prostate cancer.

An especially preferred embodiment of the present invention relates to the above mentioned tumor marker or group of tumor markers, the above mentioned

compositions, the above mentioned methods, the abovementioned use, the above mentioned immunoassay, the above mentioned pharmaceutical compositions, or the above mentioned vaccine, wherein said cancer is prostate cancer .

In a particularly preferred embodiment of the present invention 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, the above defined pharmaceutical compositions or the above mentioned vaccine, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 (A and B) depicts a list of clinical samples used for the experiments described in the present application. Fig. 2 shows the statistical distribution of sample collection dates (A) and age distribution (B) of clinical samples.

Fig. 3 indicates the statistical distribution of PSA values of the clinical

samples with maximally 20ng/ml (A) or maximally 200ng/ml.

Fig. 4 indicates the statistical distribution of prostate volumes (A) or total size of cancer tissue in the biopsy core (B) of the clinical samples. Fig. 5 shows the statistical distribution of total size of non-cancerous tissue in the biopsy core (A) or primary Gleason score groups 3 and 4 (B) and secondary Gleason score groups 3 and 4 (C) of the clinical samples.

Fig. 6 shows a corresponding Bradford standard curve using bovine serum albumin (BSA).

Fig. 7 shows an exemplary strong cation exchange chromatography (SCX)- Chromatogram of sample clean-up to remove unbound iTRAQ-labels. Fig. 8 shows an exemplary chromatogram of a normal RPLC clean-up

analysis to remove salts introduced in the SCX clean-up step.

Fig. 9 depicts an exemplary chromatogram of a high pH RPLC for final separation of peptides.

Fig. 10 gives an illustrative overview over the expression of tumor markers according to the present invention. The transition is indicated by the interruption of the line.

Fig. 11 depicts the steps of MS-based MRM targeted protein quantification using a triple quadrupole mass spectrometer.

Fig. 12 shows a comparison of selected reaction monitoring (SRM), where only one transition is utilized, versus multiple reaction monitoring

(MRM), where six transitions are monitored. SRM is following only one combination of Ql m/z and Q3 m/z. Depicted are two peptides, an endogenous and heavy labeled peptide that differ only in their C- terminal amino acid and six fragments per peptide. Only the C- terminal fragment ion masses (e.g. x-, y"-, and z-ions) are different between the endogenous and heavy labeled version.

Fig. 13 provides a schematic representation of peptide fragmentation as well the difference between selected reaction monitoring (SRM) and multiple reaction monitoring (MRM). Fig. 14 shows an MRM assay design workflow. Assays were designed with synthetic peptides. A full scan MS/MS spectrum was recorded for each target peptide which is the basis of transition selection and retention time determination. Detectability of the endogenous peptide with the validated assay was verified in the target matrix.

Fig. 15 (A-D) gives an illustrative overview over the expression values of tumor markers according to the present invention over the clinical groups studied.

Fig. 16 (A-B) gives an illustrative overview over the AUC under a ROC of tumor markers according to the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

It was shown that tumor markers as depicted in Table 1, Table 2, Table 3 and Table 4 are differentially expressed, depending on the stage of progression of cancer when testing samples of patients. In particular, it was demonstrated by the present inventors that tumor markers are statistically significantly down-regulated or up-regulated when comparing samples from a less progressed stage to a more progressed stage of a cancer disease.

Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.

Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given.

As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise.

In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, and even more preferably ±5 %.

It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of is considered to be a preferred embodiment of the term "comprising of .

If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only. Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. relate to steps of a method or use there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.

It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

As has been set out above, a first aspect of the present invention pertains to a tumor marker or group of tumor markers associated with the progression of a 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 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 the following Table 1.

Table 1:

NB: cl = local prostate cancer, insignificant disease corresponding to a clinical tumor stage < T2 (UICC 2002 classification) and Gleason Score < 6 and Prostate Cancer Volume < 0.5 ml c2 = local prostate cancer, significant disease corresponding to a clinical tumor stage > T2 (UICC 2002 classification); and/or Gleason Score > 6; and/or Prostate Cancer Volume > 0.5 ml

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 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 the following Table 2:

Table 2:

NB: cl = local prostate cancer, insignificant disease corresponding to a clinical tumor stage < T2 (UICC 2002 classification) and Gleason Score < 6 and Prostate Cancer Volume < 0.5 ml c2 = local prostate cancer, significant disease corresponding to a clinical tumor stage > T2 (UICC 2002 classification); and/or Gleason Score > 6; and/or Prostate Cancer Volume > 0.5 m

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 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 the following Table 3, which comprises all markers of Table 2 as well as additional markers not listed in Table 1 or 2:

Table 3:

NB: cl = local prostate cancer, insignificant disease corresponding to a clinical tumor stage < T2 (UICC 2002 classification) and Gleason Score < 6 and Prostate Cancer Volume < 0.5 ml c2 = local prostate cancer, significant disease corresponding to a clinical tumor stage > T2 (UICC 2002 classification); and/or Gleason Score > 6;

and/or Prostate Cancer Volume > 0.5 ml

In a specific embodiment of the present invention 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 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 Table 1 and/or Table 2 and/or Table 3.

The term "marker" or "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. decreased or 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, homologues and/or derivatives thereof, in particular fragments , antibodies binding them, and/or antigens indicated as marker #1 to #25 in Table 1, having the nucleotide or amino acid sequences as indicated in sections D) and E), respectively, wherein the uneven numbers indicate the nucleotide sequences and the following even numbers the corresponding amino acid sequences. The term further refers to the genes, genetic units, sequences, proteins, protein sequences, antibodies, antigens, homologues and/or derivatives thereof indicated as marker #1 to #96 in Table 2, having the nucleotide or amino acid sequences as indicated in sections D) and E), respectively, wherein the uneven numbers indicate the nucleotide sequences and the following even numbers the corresponding amino acid sequences. The term further refers to the genes, genetic units, sequences, proteins, protein sequences, antibodies, antigens, homologues and/or derivatives thereof indicated as marker #1 to #317 in Table 3, having the nucleotide or amino acid sequences as indicated in sections D) and E), respectively, wherein the uneven numbers indicate the nucleotide sequences and the following even numbers the corresponding amino acid sequences. The term also comprises corresponding genomic sequences as indicated in section F) of Tables 1, 2 and 3.

Importantly, 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. Furthermore, the size, identity, location etc. of intron and/or exon sequences and/or boundaries within said genomic sequences would also be known to the person skilled in the art. It is envisaged by the present invention that correspondingly identified intron and exon boundaries may be respected or used during the course of marker detection etc. as described herein.

In a specific embodiment the term "marker" or "tumormarker" also comprises peptides, peptide sequences (amino acid sequences or encoding DNA or R A sequences) or derivatives or homologues thereof, wherein said peptide is derived from, overlapping with, or partially overlapping with a tumor marker sequence as defined in Table 1, 2 or 3. In a particularly preferred embodiment of the present invention said peptides are the peptides depicted in the following Table 4, or derived therefrom:

Table 4:

A) B) C) D) E) F)

Marker Uniprot Marker Function SEQ ID SEQ ID Peptide

# Accession NO. NO. amino (SEQ ID NO indicated in Code nucleotide acid section E))

sequence sequence

Keratin type I cyto skeletal

1 K1C10 851 852 GSLGGGFSSGGFSGGSFSR

10

Hypoxia up-regulated

2 HYOU1

protein 1 853 854 LQDLTLR

3 HBA Hemoglobin subunit alpha 855 856 MFLSFPTTK

Vitamin K-dependent

4 PROC 857 858 TFVLNFIK

protein C

Plasma protease CI

5 IC1 859 860 GVTSVSQIFHSPDLAIR inhibitor

Vitamin D-binding protein

6 VTDB 861 862 HLSLLTTLSNR

precursor

7 IGHA1 Ig alpha- 1 chain C region 863 864 YLTWASR

Procollagen C-

8 PCOC1 865 866 VFDLELHPACR

endopeptidase enhancer 1

9 BST1 ADP-ribosyl cyclase 2 867 868 GFFADYEIPNLQK G protein-coupled receptor

10 GRK7 869 870 AGTNGYMAPEILMEK kinase 7

11 C07 Complement component C7 871 872 NWYTCNEGYSLIGNPVAR

12 ADIPO Adiponectin 873 874 GDIGETGVPGAEGPR

Neural cell adhesion

13 CHL1 875 876 VMTPAVYAPYDVK molecule LI -like protein

14 TSP1 Thrombospondin- 1 877 878 TrVTTLQDSIR

15 SAA4 Serum amyloid A-4 protein 879 880 GPGGVWAAK

Monocyte differentiation

16 CD14

antigen CD 14 881 882 AFPALTSLDLSDNPGLGER

17 HEMO Hemopexin 883 884 NFPSPVDAAFR

18 HRG Histidine-rich glycoprotein 885 886 DSPVLIDFFEDTER

19 MMP2 72 kDa type IV collagenase 887 888 IIGYTPDLDPETVDDAFAR

20 CYTC Cystatin-C 889 890 ALDFAVGEYNK

21 CXCL7 Platelet basic protein 891 892 NIQSLEVIGK

EGF-containing fibulin-like

22 FBLN3 extracellular matrix protein 893 894 DIDECDrVPDACK

1

23 DOPO Dopamine beta-hydroxylase 895 896 VISTLEEPTPQCPTSQGR

24 HBB Hemoglobin subunit beta 897 898 WAGVANALAHK

25 APOA1 Apolipoprotein A-I 899 900 LLDNWDSVTSTFSK

In a specific embodiment the term "marker" or "tumor marker" may additionally comprise molecules specifically binding to the expression products of said marker genes. Preferred examples of said binding molecules are antibodies which specifically bind to proteins expressed by said tumor marker genes. The present invention thus envisages as tumor marker also antibodies which specifically bind to proteins or peptides comprising the amino acid sequence as indicated in section E) of Table 1, 2, 3 or 4, or to epitopes derived from the amino acid sequences indicated in section E) of Table 1, 2, 3 or 4.

A "marker" or "tumor marker" according to the present invention may also comprises nucleotide sequences showing a high degree of homology to a marker molecule as indicated in Table 1, 2, 3 or 4, in particular to the nucleotide sequence indicated in section D) of Table 1, 2, 3 or 4. Furthermore, a "marker" or "tumor marker" according to the present invention may comprise amino acid sequences or protein sequences showing a high degree of homology to a marker molecule as indicated in Table 1, 2, 3 or 4, in particular to the amino acid sequence indicated in section E) of Table 1, 2, 3 or 4.

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, 2, 3 or 4, 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) of Table 1, 2, 3 or 4, 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) of Table, or amino acid sequences may be encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section D) of Table 1, 2, 3 or 4. Further, any variants, mutants and functional domains of the nucleic acid molecules, proteins and polypeptides as mentioned above may be encompassed. The term "tumor marker gene" or "marker gene" as used herein thus relates to the gene encoding the tumor marker mentioned in Table 1, 2 or 3. Preferably, the term relates to a gene expressing a tumor marker protein as indicated in Table 1, 2 or 3, e.g. specific exon combinations derivable from the indicated genomic sequence information of Table or as set forth in the sequences of section D) of Table 1, 2 or 3. The term also relates to DNA molecules derived from mR A transcripts encoding a tumor marker as indicated in Table 1, 2, 3 or 4, preferably cDNA molecules.

A "gene", "genetic unit" or a "nucleotide sequence" is a nucleic acid sequence which may be transcribed under certain physiological or biochemical conditions. The transcribed nucleic acid may further (but must not necessarily) be translated under certain physiological or biochemical conditions into a polypeptide, e.g. when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence may be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.

The terms "protein" or "polypeptide" are used herein to designate a produced or naturally occurring polypeptide or a recombinant polypeptide corresponding to the tumor marker as mentioned in Table 1, 2, 3 or 4. The term "protein" according to the present invention is to be seen as being interchangeably with the term "polypeptide". The

polypeptides or proteins may be encoded by any of the above mentioned nucleic acid molecules. The polypeptides or proteins may further be glycosylated or may be non- glycosylated or may otherwise by modified. In addition, polypeptides or proteins may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. In certain embodiments the term may also relate to fragments of proteins of the present invention or peptides according to the present invention, e.g. peptides comprised in the proteins according to the present invention, or peptides as defined herein.

The term "cancer" 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.

The term "cancerous" relates in the context of the present invention to a cancerous disease state as defined herein above or below. The term "non-cancerous" relates in the context of the present invention to a condition in which neither benign nor malign proliferation can be detected. Suitable means for said detection are known in the art.

The term "progression of cancer" as used herein 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 graduating, staging or scoring system, starting from a healthy condition up to a terminal cancer scenario. Typically such switches are accompanied by a modification of the expression level of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, 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". To determine the Gleason score, typically a grade is assigned to the most common tumor pattern, and a second grade to the next most common tumor pattern. The two grades are added together to get a Gleason score. The Gleason grade is also known as the Gleason pattern or Gleason sum. The Gleason grade may range from 1 to 5, with 5 having the worst prognosis. The Gleason score typically ranges from 2 to 10, with 10 having the worst prognosis. Methods to apply the Gleason score system, corresponding assessment techniques etc. would be known to the person skilled in the art. The scoring system may be used, for example, in order to verify, check or fine-tune the diagnosis, detection, indication of stages or monitoring according to the present invention.

The term "less progressed stage" as used herein, relates to an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, is/are known or from the same subject at an earlier point in time, e.g. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 months, 2 years, 3 years, 4 years, 5 years, 10 years before etc. ("expression level of a less progressed stage"). The term may also refer to an expression level corresponding to a cancer stage or cancer form, whose disease state or stage is known. The term "disease state" relates to any state or type of cellular or molecular condition between a non-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. Preferably, 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

(including) a terminal cancerous cell state. For example, 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:

GX Grade cannot be assessed

Gl Well differentiated (Low grade)

G2 Moderately differentiated (Intermediate grade)

G3 Poorly differentiated (High grade)

G4 Undifferentiated (High grade)

Further envisaged is the alternative four-tier grading scheme:

Grade 1 Low grade; Well-differentiated

Grade 2 Intermediate grade; Moderately-differentiated Grade 3 High grade; Poorly-differentiated

Grade 4 Anaplastic; Anaplastic

Also envisaged is the alternative three-tier grading scheme:

Grade 1 Low grade; Well-differentiated

Grade 2 Intermediate grade;

Grade 3 High grade; Poorly-differentiated;

or the two-tier grading scheme

Grade 1 Low grade; Well-differentiated

Grade 2 High grade; Poorly-differentiated.

Such developmental stages may also include all stages of the TNM (Tumor,

Node, Metastasis) classification system of malignant tumors as defined by the UICC, e.g. stages 0 and I to IV. The stages may in particular include all TNM stages of prostate cancer as define herein below. The term also includes stages before TNM stage 0, e.g.

developmental stages in which cancer biomarkers known to the person skilled in the art show a modified expression or expression pattern. In a specific embodiment of the present invention a less progressed stage may be a healthy state. In a further specific embodiment of the present invention a less progressed sate may also be a stage of benign tumor

development, e.g. benign prostate tumors. Particularly preferred are less progressed stages of malignant 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. In a specific embodiment of the present invention, the expression level of a less progressed stage can be determined from a reference sample derived from a subject who has been diagnosed to suffer from a certain cancer, and wherein the stage and development has been determined. Correspondingly obtained values and information may also be combined, normalized and statistically processed according to any suitable technique or method known to the person skilled in the art.

The term "more progressed stage" as used herein, relates to the reflection of any changes that are associated with a worsening of the disease as defined herein above, e.g. the aggressiveness of the cancer, the nature of the transformation (benign to malignant), the localization of the tumor and or /cancerous cells, the occurrence of metastases, the presentation of additional or more profound clinical symptoms, recurrence of a tumor after treatment, decreased survival rate, the modification of the expression of bio- or tumor markers, e.g. of tumor marker known to the person skilled in the art like PSA or PCA3 in comparison to a corresponding less progressed stage as defined herein above. Preferably, the term relates to a worsened disease state of a tumor or cancer in comparison to a less progressed stage as defined herein above. For example, if the less progressed stage is a stage 0 or healthy state, the more progressed stage may be any higher or more advanced stage, e.g. a stage of any one of stage I, II, III or IV. Preferably, a "more progressed stage" as used herein may be the next worse stage if starting from a less progressed stage as defined herein above. For example, if the less progressed stage is a stage 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. Preferably, the "next worse stage" refers to the staging system provided by the UICC 2002 classification, more preferably the TNM classification for prostate cancer provided herein below. In a specific embodiment of the present invention the staging or grading of a tissue may optionally or additionally be determined, checked, crosschecked or independently be diagnosed by classical staging methods known to the person skilled in the art, e.g. via histological approaches, imaging methods etc.

In a specific embodiment of the present invention a more progressed stage may be determined by a comparison of the expression level of a tumor marker as indicated in Table 1, 2, 3 or 4 to a control level or control state of the same tumor marker. The term

"control level" (or "control state"), as used herein, relates to an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, e.g. cancerous, non-cancerous, having a tumor, having no tumor, and whose disease stage(s) as defined herein above is/are known. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of the gene(s) of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 in samples from subjects whose disease state is known.

Furthermore, the control level can be derived from a database of expression levels or patterns from previously tested subjects or cells. Moreover, the control level may be multiple control levels whose control levels are determined from multiple reference samples. The control level may accordingly be derived from experimental approaches or from a database of expression levels from previously tested subjects, tissues or cells or from any suitable source of information known to the person skilled in the art. In a specific embodiment of the present invention, the control level can be determined from a reference sample derived from a subject who has been diagnosed to suffer from a certain cancer, and wherein the stage and development of the cancer or tumor disease has been determined, as well as from healthy individuals.

In the context of the present invention, a control level determined from a biological sample that is known not to be cancerous is called "normal control level". In another embodiment of the present invention, 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". Alternatively, reference samples may comprise material derived from cell lines, e.g. immortalized cancer cell lines, or be derived from tissue xenografts. Preferably, material derived from prostate cancer cell lines or material derived from tissue xenografts with human prostate tissue, in particular with benign and tumor-derived human prostate tissue, may be comprised in a reference sample according to the present invention. Examples of cancer cell lines to be used comprise cells lines PC346P, PC346B, LNCaP, VCaP, DuCaP, PC346C, PC3, DU145,

PC346CDD, PC346Flul, PC346Flu2. Examples of xenografts which may be used comprise PC295, PC310, PC-EW, PC82, PC133, PC135, PC324 and PC374. Preferably an entire panel of cell lines and xenografts may be used, e.g. the human PC346 panel.

Correspondingly obtained values and information may also be combined, normalized and statistically processed according to any suitable technique or method known to the person skilled in the art. By comparing a control level to a measured expression level a modification of the expression may be registered, which may accordingly be used for the determination of the more progressed stage of a cancer disease. For such comparison processes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more different control levels may be determined or assessed. Accordingly, the less progressed stage of a cancer diseased may be determined by a assessing the outcome of such comparison process.

In a preferred embodiment of the invention said comparison process comprises the determination of control levels in a sample of an individual. In an even more preferred embodiment of the invention said comparison process comprises the determination of control levels in a sample of an individual afflicted with a cancer or neoplastic disease, e.g. prostate cancer. In an even more preferred embodiment of the invention said comparison process comprises the determination of control levels obtained from a sample of an individual afflicted with a cancer or neoplastic disease, e.g. prostate 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 I), J), K) and L) of Table 1 and/or sections G), H), I) and J) of Table 2 or 3. 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), even more preferably prostate cancer of stage >T2 (UICC 2002 classification) and Gleason score >7.

The term "expression level" as used herein refers to the amount of any transcript and/or protein derivable from a defined number of cells or a defined tissue portion, preferably to the amount of a transcript and/or protein obtainable in a standard nucleic acid (e.g. RNA) or protein extraction procedure. Suitable extraction methods are known to the person skilled in the art. The amount may also be determined indirectly via the binding of an antibody, e.g. as the amount of bound antibody etc.

The term "modified" or "modified expression level" in the context of the present invention thus denotes a change in the expression level. Expression levels are deemed to be "changed" when the gene expression of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, e.g. in a sample to be analyzed, differs by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level or the expression level of a less progressed stage, as defined herein above, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level or the expression level of a less progressed stage as defined herein above.

The term "modified" as used throughout the specification relates preferably to a decrease or down-regulation or an increase or up-regulation of the expression level of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 or a complete inhibition of the expression of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 if a test sample is compared to a control level or the expression level of a less progressed stage as defined herein above.

In a preferred embodiment of the present invention the expression of the tumor marker(s) or group of tumor markers is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section I) of Table 1, 2 or 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 tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 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 tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, e.g. in a sample to be analyzed, differs by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level or the expression level of a less progressed stage as defined herein above. In a particularly preferred embodiment of the present invention the tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 5, 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, 50, 55, 65, 70 or all tumor markers indicated with a "+" in section I) of Table 1, 2 or 3. In a further preferred embodiment of the present invention the tumor marker or group of tumor markers comprises the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23tumor markers indicated with a "+" in section I) of Table 1, or the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, 24, 25, 27, 29, 30, 32, 35, 37, 40, 42, 45 tumor markers indicated with a "+" in section I) of Table 2. In a further preferred embodiment of the present invention 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, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or all tumor markers indicated with a "+" in section I) of Table 3. In a further preferred embodiment of the present invention the tumor markers or group of tumor markers comprises the first 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, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90 tumor markers indicated with a "+" in section I) of Table 3.

In another preferred embodiment of the present invention the expression of the tumor marker(s) or group of tumor markers is decreased (down-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section J) of Table 1, 2, 3 or 4. The term "reduced" or "reduced expression level" or "down-regulated expression level" or "decrease of expression level" (which may be used synonymously) in the context of the present invention thus denotes a reduction of the expression level of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 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 "reduced" or "down-regulated" when the gene expression of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 decreases by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level or the expression level of a less progressed stage as defined herein above. In a particularly preferred embodiment of the present invention the tumor marker or group of tumor markers comprises at least 1 or 2 or all tumor markers indicated with a "+" in section J) of Table 1. In a further preferred embodiment of the present invention the tumor markers or group of tumor markers comprises the first 1 , 2 tumor markers indicated with a "+" in section J) of Table 1. In a further particularly preferred embodiment of the present invention 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 or all tumor markers indicated with a "+" in section J) of Table 2. In a further preferred embodiment of the present invention the tumor markers or group of tumor markers comprises the first 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 tumor markers indicated with a "+" in section J) of Table 2. In a further preferred embodiment of the present invention 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, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or all tumor markers indicated with a "+" in section J) of Table 3. In a further preferred embodiment of the present invention the tumor markers or group of tumor markers comprises the first 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, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90 tumor markers indicated with a "+" in section J) of Table 3.

In a further embodiment of the present invention the group of tumor marker may comprise markers which are up-regulated and markers which are down-regulated, preferably at least one marker with an increased expression level in the more progressed stage and one marker with a decreased expression level in the more progressed stage. For example, the group of tumor marker may comprise at least one tumor marker indicated with a + in section I) of Table 1, 2 or 3 and at least one tumor marker indicated with a + in section J) of Table 1, 2 or 3.

In a specific embodiment of the present invention the group of tumor marker may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more tumor markers indicated with a "+" in section I) of Table land at least 1 or 2 tumor markers indicated with a "+" in section J) of Table 1.

In a further specific embodiment of the present invention the group of tumor marker may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more tumor markers indicated with a "+" in section I) of Table land 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 or more tumor markers indicated with a "+" in section J) of Table 2.

In a further specific embodiment of the present invention the group of tumor marker may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more tumor markers indicated with a "+" in section I) of Table land 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, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90 or more tumor markers indicated with a "+" in section J) of Table 3.

In a specific embodiment of the present invention the group of tumor marker may comprise at leastl, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, 24, 25, 27, 29, 30, 32, 35, 37, 40 or more tumor markers indicated with a "+" in section I) of Table 2. and at least 1 or 2 tumor markers indicated with a "+" in section J) of Table 1.

In a specific embodiment of the present invention the group of tumor marker may comprise at leastl, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, 24, 25, 27, 29, 30, 32, 35, 37, 40 or more tumor markers indicated with a "+" in section I) ofTable 2 and 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 or more tumor markers indicated with a "+" in section J) of Table 2.

In a specific embodiment of the present invention the group of tumor marker may comprise 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 or more tumor markers indicated with a "+" in section I) ofTable 2 and 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, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90 or more tumor markers indicated with a "+" in section J) of Table 3. In a further specific embodiment of the present invention the group of tumor marker may comprise 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, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90 or more tumor markers indicated with a "+" in section I) of Table 3 and at least 1 or 2 tumor markers indicated with a "+" in section J) of Table 1.

In a further specific embodiment of the present invention the group of tumor marker may comprise 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, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90 or more tumor markers indicated with a "+" in section I) of Table 3 and 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 or more tumor markers indicated with a "+" in section J) of Table 2.

In a further specific embodiment of the present invention the group of tumor marker may comprise 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, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90 or more tumor markers indicated with a "+" in section I) of Table 3 and 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, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90 or more tumor markers indicated with a "+" in section J) of Table 3.

In another preferred embodiment of the present invention the 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 or all tumor markers of Table 1, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or all of the tumor markers of Table 2. In a further embodiment of the present invention the group of tumor markers 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 or all tumor markers of Table 1, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 170, 180, 200, 220, 250, 300 or all of the tumor markers of Table 3.

In another embodiment of the present invention 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 or all of the tumor markers of Table 2. Also preferred is a group of tumor marker 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, 86, 87, 88, 89 of the tumor markers indicated in Table 2. The group may further comprise any sub-grouping or combinations of these markers.

In yet another embodiment of the present invention the group of tumor markers comprises those tumor markers which show a p-value of the expression modification of 0.00002, 0.00003, 0.00004, 0.00005, 0.00006, 0.00007, 0.00008, 0.00009, 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.02, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.035, 0.04, 0.045 or lower as indicated in section G) of Table 1 or section K) of Table 2. In a further embodiment the groups of markers may show p-values of the expression of between about 0.001 and about 0.025 or have a p-value of expression of 0.022 or lower, as indicated in section G) of Table 1 or section K) of Table 2. Preferred are groups of markers which show p-values of expression of 0.0006 or lower. The term "p-value" is a measure of the probability that a variate would assume a value greater than or equal to the observed value strictly by chance and is expressed by the following term: P (z > z₀bserved). Thus, in the context of the present invention, the p-value may be seen as a measure of statistical significance.

In another embodiment of the present invention the group of tumor markers comprises at least 3 tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 6 tumor markers corresponding to tumor marker #1 to #6 of Table 1, or at least 9 tumor markers corresponding to tumor marker #1 to #9 of Table 1, or at least 12 tumor markers corresponding to tumor marker #1 to #12 of Table 1, or at least 15 tumor markers

corresponding to tumor marker #1 to #25 of Table 1 and/or at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 tumor markers selected from tumor marker #1 to #50 of Table 2.

The group of tumor markers may also comprises tumor markers corresponding to tumor marker #1 to #2 of Table 1, or tumor markers corresponding to tumor marker #1 to #4 of Table 1, or tumor markers corresponding to tumor marker #1 to #5 of Table 1, or tumor markers corresponding to tumor marker #1 to #7 of Table 1, or tumor markers corresponding to tumor marker #1 to #8 of Table 1, or tumor markers corresponding to tumor marker #1 to #10 of Table 1, or tumor markers corresponding to tumor marker #1 to #11 of Table 1, or tumor markers corresponding to tumor marker #1 to #13 of Table 1, or tumor markers corresponding to tumor marker #1 to #14 of Table 1, or tumor markers corresponding to tumor marker #1 to #16 of Table l,or tumor markers corresponding to tumor marker #1 to #17 of Table 1, or tumor markers corresponding to tumor marker #1 to #19 of Table 1, or tumor markers corresponding to tumor marker #1 to #20 of Table 1, or tumor markers corresponding to tumor marker #1 to #22 of Table 1, or tumor markers corresponding to tumor marker #1 to #23 of Table 1, or tumor markers corresponding to tumor marker #1 to #24 of Table 1.

The group of tumor marker according to the present invention may also comprise tumor markers #1, #3, #5, #7 and #9 of Table 1, or #2, #4, #6, #8, and #10 of Table

1, or #3, #5, #7, #9 and #11 of Table 1, or #4, #6, #8, #10, and #12 of Table 1, or #5, #7, #9, #11 and #13 of Table 1, or #6, #8, #10, #12 and #14 of Table 1, or #7, #9, #11, #13 and #15 of Table 1, or #8, #10, #12, #14 and #16 of Table l, or #9, #11, #13, #15 and #17 of Table 1, or #10, #12, #14, #16 and #17 of Table 1, or #11, #13, #15, #17 of Table 1 etc.

In another embodiment of the present invention the group of tumor markers comprises at least 5 tumor markers corresponding to tumor marker #1 to #5 of Table 2, at least 10 tumor markers corresponding to tumor marker #1 to #10 of Table 2, at least 14 tumor markers corresponding to tumor marker #1 to #15 of Table 2, at least 19 tumor markers corresponding to tumor marker #1 to #20 of Table 2, at least 24 tumor markers corresponding to tumor marker #1 to #25 of Table 2, at least 29 tumor markers corresponding to tumor marker #1 to #30 of Table 2, at least 34 tumor markers corresponding to tumor marker #1 to #35 of Table 2, at least 39 tumor markers corresponding to tumor marker #1 to #40 of Table

2, at least 44 tumor markers corresponding to tumor marker #1 to #45 of Table 2 or at least 48 tumor markers corresponding to tumor marker #1 to #50 of Table 2. Furthermore, 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, or tumor markers #1 to #96 of Table 2. The group of tumor marker according to the present invention may also comprise tumor markers #1, #3, #5, #7 and #9 of Table 2, #2, #4, #6, and #8 of Table 2, #3, #5, #7, #9 and #11 of Table 2, #4, #6, #8, and #12 of Table 2, #5, #7, #9, #11 and #13 of Table 2, #6, #8, #12 and #14 of Table 2, #7, #9, #11, #13 and #15 of Table 2, #8, #12, #14 and #16 of Table 2, #9, #11, #13, #15 and #17 of Table 2, #12, #14, #16 and #18 of Table 2, #11, #13, #15, #17 and #19 of Table 2, #12, #14, #16, #18 and #20 of Table 2, #13, #15, #17, #19 and #21 of Table 2 etc.

In a further embodiment the group of tumor marker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315 or all of the tumor markers of Table 3. Further envisaged is a group of tumor marker 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, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 or more of the tumor markers indicated in Table 3. The group may further comprise any sub-grouping or combinations of these markers. The group may also comprise any sub-groupings or combinations with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more markers of Table 1.

In a preferred embodiment of the present invention the group of tumor markers comprises :

(v) at least marker # 5 of Table 1 (IC1) and at least one additional marker of Table 1 or Table 2; or

(viii) at least marker # 8 of Table 1 (PCOC1) and at least one additional marker of Table 1 or Table 2; or (ix) at least marker # 9 of Table 1 (BSTl) and at least one additional marker of Table 1 or Table 2; or

(x) at least marker # 10 of Table GR 7) and at least one additional marker of Table 1 or Table 2; or

(xi) at least marker # 11 of Table C07) and at least one additional marker of Table 1 or Table 2; or

(xii) at least marker # 12 of Table ADIPO) and at least one additional marker of Table 1 or Table 2; or

(xiii) at least marker # 13 of Table CHL1) and at least one additional marker of Table 1 or Table 2; or

(xiv) at least marker # 14 of Table TSPl) and at least one additional marker of Table 1 or Table 2; or

(xv) at least marker # 15 of Table SAA4) and at least one additional marker of Table 1 or Table 2; or

(^xvl) at least marker # 16 of Table CD 14) and at least one additional marker of Table 1 or Table 2; or

(xvii) at least marker # 17 of Table HEMO) and at least one additional marker of Table 1 or Table 2; or

(xviii) at least marker # 18 of Table HRG) and at least one additional marker of Table 1 or Table 2; or

(xix) at least marker # 19 of Table MMP2) and at least one additional marker of Table 1 or Table 2; or

(xx) at least marker # 20 of Table CYTC) and at least one additional marker of Table 1 or Table 2; or

(^xxl) ^at least marker # 21 of Table CXCL7) and at least one additional marker of Table 1 or Table 2; or

(xxii) at least marker # 22 of Table FBLN3) and at least one additional marker of Table 1 or Table 2; or

(xxiii) at least marker # 23 of Table DOPO) and at least one additional marker of Table 1 or Table 2; or

(xxiv) at least marker # 24 of Table HBB) and at least one additional marker of Table 1 or Table 2; or

(xxv) at least marker # 25 of Table APOAl) and at least one additional marker of Table 1 or Table 2. In yet another embodiment of the present invention the group of tumor markers comprises :

(i) at least marker #1 of Table 1 (K1C10) and at least one additional marker of Table 3; or

(ii) at least marker #2 of Table 1 (HYOU1) and at least one additional marker of Table 3; or

(iii) at least marker #3 of Table 1 (HBA) and at least one additional marker of Table 3; or

(iv) at least marker #4 of Table 1 (PROC) and at least one additional marker of Table 3; or

(v) at least marker #5 of Table 1 (IC1) and at least one additional marker of Table 3; or

(vi) at least marker #6 of Table 1 (VTDB) and at least one additional marker of Table 3; or

(vii) at least marker #7 of Table 1 (IGHA1) and at least one additional marker of Table 3; or

(viii) at least marker #8 of Table 1 (PCOC1) and at least one additional marker of Table 3; or

(ix) at least marker #9 of Table 1 (BSTl) and at least one additional marker of Table 3; or

(x) at least marker #10 of Table 1 (GRK7) and at least one additional marker of Table 3; or

(xi) at least marker #11 of Table 1 (C07) and at least one additional marker of Table 3; or

(xii) at least marker #12 of Table 1 (ADIPO) and at least one additional marker of Table 3; or

(xiii) at least marker #13 of Table 1 (CHL1) and at least one additional marker of Table 3; or

(xiv) at least marker #14 of Table 1 (TSP1) and at least one additional marker of Table 3; or

(xv) at least marker #15 of Table 1 (SAA4) and at least one additional marker of Table 3; or

(xvi) at least marker #16 of Table 1 (CD 14) and at least one additional marker of Table 3; or (xvii) at least marker #17 of Table 1 (HEMO) and at least one additional marker of Table 3; or

(xviii) at least marker # 18 of Table 1 (HRG) and at least one additional marker of Table 3; or

(^xrx) ^at least marker # 19 of Table 1 (MMP2) and at least one additional marker of Table 3; or

(xx) at least marker # 20 of Table 1 (CYTC) and at least one additional marker of Table 3; or

(xxi) at least marker # 21 of Table 1 (CXCL7) and at least one additional marker of Table 3; or

(xxii) at least marker # 22 of Table 1 (FBLN3) and at least one additional marker of Table 3; or

(xxiii) at least marker # 23 of Table 1 (DOPO) and at least one additional marker of Table 3; or

(xxiv) at least marker # 24 of Table 1 (HBB) and at least one additional marker of Table 3; or

(xxv) at least marker # 25 of Table 1 (APOA1) and at least one additional marker of Table 3.

In yet another embodiment of the present invention the group of tumor markers comprises:

(i) at least marker #1 of Table 1 (K1C10) and marker #2 of Table 1 (HYOUl) and optionally at least one additional marker of Table 1 or 2; or

(ii) at least marker #1 of Table 1 (K1C10) and marker #2 of Table 1 (HYOUl) and marker #3 of Table 1 (HBA) and optionally at least one additional marker of Table 1 or 2; or

(iii) at least marker #1 of Table 1 (K1C10) and marker #2 of Table 1 (HYOUl) and marker #3 of Table 1 (HBA) and marker #4 of Table 1 (PROC) and optionally at least one additional marker of Table 1 or 2; or

(iv) at least marker #1 of Table 1 (K1C10) and marker #2 of Table 1 (HYOUl) and marker #3 of Table 1 (HBA) and marker #4 of Table 1 (PROC) and marker #5 of Table 1 (IC1) and optionally at least one additional marker of Table 1 or 2; or

(v) at least marker #1 of Table 1 (K1C10) and marker #3 of Table 1 (HBA) and optionally at least one additional marker of Table 1 or 2; or

(vi) at least marker #1 of Table 1 (K1C10) and marker #4 of Table 1 (PROC) and optionally at least one additional marker of Table 1 or 2; or

(vii) at least marker # 1 of Table 1 (Kl C 10) and marker #5 of Table 1 (ICl) and optionally at least one additional marker of Table 1 or 2; or

(viii) at least marker #1 of Table 1 (K1C10) and marker #3 of Table 1 (HBA) and marker #4 of Table 1 (PROC) and optionally at least one additional marker of Table 1 or 2; or

(ix) at least marker #1 of Table 1 (K1C10) and marker #3 of Table 1 (HBA) and marker #4 of Table 1 (PROC) and marker #5 of Table 1 (ICl) and optionally at least one additional marker of Table 1 or 2; or

(x) at least marker #2 of Table 1 (HYOU 1 ) and marker #3 of Table 1

(HBA) and optionally at least one additional marker of Table 1 or 2; or

(xi) at least marker #2 of Table 1 (HYOU1) and marker #3 of Table 1 (HBA) and marker #4 of Table 1 (PROC) and optionally at least one additional marker of Table 1 or 2; or

(xii) at least marker #2 of Table 1 (HYOU1) and marker #3 of Table 1

(HBA) and marker #4 of Table 1 (PROC) and marker #5 of Table 1 (ICl) and optionally at least one additional marker of Table 1 or 2.

Additional tumor markers which may be combined with any one or more of the markers of Table 1, 2, 3 or 4, preferably with any combination of these markers or any group of tumor markers or any combination of groups of tumor markers as defined herein above in any suitable manner and thus also form part of the present invention are indicated in the following Table 5:

Table 5:

A) B) C) D) E) F) Marker# Uniprot Function SEQ ID No. SEQ ID No. UniGene Code

Accession Code DNA Protein

1 PPAP PAP 635 636 Hs.388677

2 ACTS MSActin 637 638 Hs.1288

Alpha-methylacyl-CoA

3 AMACR 639 640 Hs.49598 racemase

4 ANXA1 Annexin I 641 642 Hs.287558

5 ANXA7 Annexin VII 643 644 Hs.386741 ARPC3 p21-Arc 645 646 Hs.439511

ASAH1 Acid Ceramidase 647 648 Hs.324808

AURKB Aurora B 649 650 Hs.442658

BIRC2 cIAP 651 652 Hs.289107

BIRC4 XIAP 653 654 Hs.356076

CIKS ACT1 655 656 Hs.437508

CALD1 L-Caldesmon 657 658 Hs.443811

KCC2G CaM Kinase II 659 660 Hs.12436

CASP7 Casp7 661 662 Hs.9216

CAV1 Caveolin 1 663 664 Hs.74034

CAV2 Caveolin 2-20kD 665 666 Hs.139851

CBX3 HP1 GAMMA 667 668 Hs.381189

CDC42 Cadc42/RAC 669 670 Hs.355832

CDK2 Cdk2 671 672 Hs.19192

CDK4 Cdk4 673 674 Hs.95577

CDK7 CDK7 675 676 Hs.184298

CARF pl6INK4A 677 678 Hs.421349

RCBT2 RCC1 679 680 Hs.196769

CRYAB alphaB Crystallin 681 682 Hs.408767

CSNK1A1 CK1 683 684 Hs.318381

CSRP1 CRPl 685 686 Hs.108080

DFFA DFF45 687 688 Hs.484782

EDNRA Endothelin 1 Receptor 689 690 Hs.211202

EFS Sin 691 692 Hs.24587 LIMA1 EPLIN-105kD 693 694 Hs.10706

EPS8 Eps8 695 696 Hs.2132

ERCC1 Ercc-l-37kD 697 698 Hs.435981

EZH2 EZH2 699 700 Hs.444082

FLNA ABP280 701 702 Hs.195464

FLOT2 Flotillin-2/ESA 703 704 Hs.18799

GOSR1 GS28 705 706 Hs.124436

GRB7 grb7 707 708 Hs.86859

GELS Gelsolin-88kD 709 710 Hs.446537

TF2B TFIIB 711 712 Hs.258561

HCD2 ERAB 713 714 Hs.171280

HAX1 HAX-1 715 716 Hs.199625

CH60 HSP60 717 718 Hs.79037

IRAKI IRAK 719 720 Hs.182018

ΓΓΑ5 Integrin 5 alpha 721 722 Hs.149609

KLK3 PSA 723 724 Hs.171995

IMA2 Karyopherin alpha 2 725 726 Hs.159557

LHX1 Lim kinase 727 728 Hs.154103

MD2BP MAD2 729 730 Hs.79078

Q6FHX4 Sekl 731 732 Hs.134106

Q499Y8 pan-JNK/SAPKl-45kD 733 734 Hs.25209

MK14 p38 alpha/SAPK2a 735 736 Hs.79107

MAPKAPK3 3PK 737 738 Hs.234521

A2VCR0 EB1 739 740 Hs.408754 MUC18 MCAM 741 742 Hs.511397

MCM4 MCM4-92kD 743 744 Hs.460184

MCM6 MCM6 745 746 Hs.444118

MKI67 Ki-67-260kD 747 748 Hs.80976

MMP23B MMP23 749 750 Hs.211819

MSH2 MSH2 751 752 Hs.440394

Myosin light chain

MYLK

kinase(MYLK) 753 754 Hs.386078

MY06 Myosin VI 755 756 Hs.118483

NEXN Nexilin 757 758 Hs.22370

NDKA Nm23 759 760 Hs.l 18638

NSF NSF 761 762 Hs.431279

NUP62 Nucleoporin p62 763 764 Hs.528342

OCLN Occludin 765 766 Hs.171952

OPTO FIP-2-73kD 767 768 Hs.390162

P4HB PDI 769 770 Hs.410578

PAK3 PAK3 771 772 Hs.390616

PCYOX1 Prenylcysteine Lyase 773 774 Hs.278627

P85A PI3 Kinase 775 776 Hs.6241

PP1A PP1 777 778 Hs.l 83994

MYPT1 MYPT1 779 780 Hs.377908

KAPO PKA RI 781 782 Hs.280342

KAP3 PKA RII beta 783 784 Hs.77439

KPCB PKC beta 785 786 Hs.349845

PRSS8 Prostasin 787 788 Hs.75799 78 PTRF PTRF 789 790 Hs.437191

79 RAB11A Rabl l 791 792 Hs.75618

80 RAB27A RAB27 793 794 Hs.298530

81 RAB4A Rab4 795 796 Hs.296169

82 RALA Ral A 797 798 Hs.6906

83 RBBP4 RBBP 799 800 Hs.16003

84 ROCK2 ROCK-II/ROK alpha 801 802 Hs.58617

85 SH2B SH2-B 803 804 Hs.15744

86 SMAD2 Smad2/3 805 806 Hs.110741

87 SMCA4 BRG1 807 808 Hs.78202

88 SMRC2 BAF170 809 810 Hs.236030

89 SORL LR1 l/SorLA/gp250 811 812 Hs.438159

90 STK6 Aurora kinase A 813 814 Hs.250822

91 STMN1 Stathmin Metablastin 815 816 Hs.209983

92 STX12 Syntaxin 13 817 818 Hs.433838

93 TGFB1I1 Hic-5 819 820 Hs.25511

94 THRB B Thyroid HNR 821 822 Hs.203213

95 TIMP3 TIMP3 823 824 Hs.245188

96 LAP2A LAP2 825 826 Hs.11355

97 TNR19 TROY 827 828 Hs.334174

98 TOP2A TOP02 alpha 829 830 Hs.156346

99 TPD52 TPD52 831 832 Hs.162089

100 TRI25 EFP 833 834 Hs.88914

101 TIF IB KRIP-1 835 836 Hs.433612 102 TYK2 tyk2 837 838 Hs.75516

103 SAE2 Uba2 839 840 Hs.511739

104 UBC9 Ubc9 841 842 Hs.302903

105 UHRF1 ICBP90 843 844 Hs.108106

106 XPOl Exportin 845 846 Hs.157367 cAMP-dependent protein

107 KAPCA 847 848 Hs.631630 kinase catalytic SU alpha

In a further aspect the present invention relates to the use of a tumor marker or group of tumor markers as defined herein above as a marker for diagnosing, detecting, 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 term "diagnosing a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage" as used herein 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 or reduced/down-regulated, compared to the expression level of a less progressed disease state as defined herein above, or compared to a control level as defined herein above. The term "diagnosing" also refers to the conclusion reached through that comparison process. An expression level may be deemed to be modified, when the expression level of a tumor marker or group of tumor markers as defined herein above differs by, for example, between about 1% and 50%, e.g. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% or 40%) from the expression level of a less progressed disease state or from a control level as defined herein above, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to such a less progressed disease state or control level. The modification may be an increase or a reduction of said expression level.

In a further embodiment, an additional similarity in the overall gene expression pattern of a group of tumor markers according to the present invention between a sample obtained from a subject and a control sample or a sample corresponding to a less progressed cancer disease state as described herein above, may indicate that the subject is suffering from a more progressed cancer disease stage. In another embodiment of the present invention, the diagnosis may be combined with the elucidation of additional cancer biomarker expression levels, in particular prostate cancer biomarkers. Suitable biomarkers, in particular prostate cancer biomarkers, would be known to the person skilled in the art. For example, the expression of biomarkers like PSA may be tested.

The term "detecting a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage" as used herein 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 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. In a preferred embodiment of the present invention a more progressed cancer stage may be detected if the expression level the 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.

The term "graduating a cancer disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage" as used herein 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.

In a preferred embodiment the graduating of a more progressed cancer stage may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 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. In a further preferred embodiment the graduating of a more progressed cancer stage may be

accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 of the present invention in a sample from a patient or individual to be analyzed and any of the cancer cells, cancer tissues, tumor biopsies, or the cancerous control levels mentioned above. In further preferred embodiment the graduating of a more progressed cancer stage may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1 , 2, 3 or 4 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. In further preferred embodiment the graduating of a more progressed cancer stage may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 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. In a further preferred embodiment the determination of the graduating cancer may be accomplished by a comparison of the expression level of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 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. The term

"accompaniment" means that states of disease as defined herein above and, in particular, changes of these states of disease may be detected by comparing the expression level of the tumor marker or group of tumor markers of the present invention in a sample to a control level as defined herein above or to the expression level of an established, e.g. independently established cancer cell or cell line, or to corresponding database values in any type of periodical time segment, e.g. every week, every 2 weeks, every month, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 month, every 1.5 year, every 2, 3, 4, 5, 6, 7, 8,9 or 10 years, during any period of time, e.g. during 2 weeks, 3 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years, respectively. The established, e.g. independently established, cancer cell or cell line giving rise to a control level may be derived from samples corresponding to different stages of cancer development, e.g. the stages as mentioned herein above. In a preferred embodiment of the present invention the term relates to the

accompaniment of a diagnosed more progressed prostate cancer.

The term "prognosticating a cancer disease associated with a progression form a less progressed cancer stage to a more progressed cancer stage " as used herein 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.

The term "progression from a less progressed cancer stage to a more progressed cancer stage" as used herein relates to a switch between different stages of cancer development. Such a progression may be 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 tumor marker or group of tumor marker according to the present invention is modified, e.g. increased or reduced or both by a value of between 3% to 50%, preferably by a value of 10%, 15%, 20% or 25% in a test sample in comparison to a previous test sample from the same individual. The modification may be detected over any period of time, preferably over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years, i.e. the value indicated above may be calculated by comparing the expression level of the tumor marker or group of tumor markers at a first point in time and at a second point in time after the above indicated period of time.

In a particularly preferred embodiment of the present invention the term

"progression from a less progressed 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. Alternatively, for the comparison test samples from other individuals may be used, e.g. test samples of healthy individuals. Also envisaged is the use of available database information on the expression or the employment of cancer cell collection samples etc.

In a further embodiment the present invention relates to the diagnosis and detection of a predisposition for developing a more progressed cancer stage. A

"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. Preferably a predisposition for developing a more progressed cancer stage may be present in cases in which the expression level of the tumor marker or group of tumor marker of the present invention as defined herein above is above a normal control level as defined herein above, i.e. a reference expression level derived from tissues or samples of a subject which are evidently healthy. The term "above" in this context relates to an expression level of the tumor marker of group of tumor markers which is increased by about 2% to 20% in comparison to such a control level, preferably increased by about 15%.

Alternatively, a predisposition for developing a more progressed cancer stage in the context of the present invention may be given in situations in which the expression level of the tumor marker or group of tumor markers as defined herein above is above a normal control level and in which further, alternative cancer markers, e.g. PSA, show no modification of expression level or the expression pattern in a less progressed cancer stage. Suitable further cancer markers are known to the person skilled in the art.

Thus, 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.

In case a group of tumor markers according to the present invention comprises tumor markers of Table 1, 2, 3 or 4 which show an increase of the expression level and, at the same time, other tumor markers of Table 1, 2, 3 or 4 which show a reduction of the expression level, the increase and the reduction of the corresponding tumor markers may be calculated independently, i.e. for each marker or each group of markers with the same tendency (increase and reduction) separately. The absolute values of such calculations may subsequently be averaged and used for the calculation of the modification.

The difference between the expression levels of a test biological sample and a control level or the expression level of a less progressed cancer stage can be normalized to the expression level of one or more control nucleic acids, e.g. housekeeping genes whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell. Exemplary control genes include inter alia β-actin, glycerinaldehyde 3-phosphate dehydrogenase (GAPDH), 18s R A, ubiquitin C, cytochrome CI, ribosomal protein PI or PBGD. Particularly preferred is the use of GAPDH.

In the context of the present invention, the terms "diagnosing" and

"prognosticating" are also intended to encompass predictions and likelihood analyses. Tumor markers or groups of tumor markers may accordingly be used clinically in making decisions concerning treatment modalities, including therapeutic intervention or diagnostic criteria such as a surveillance for the disease. According to the present invention, an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease. Alternatively, the present invention may be used to detect 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 such cancer is to be detected or prognosticated according to the present invention is an animal, preferably a mammal, more preferably a human being.

Particularly preferred is the use of molecular imaging tools as known to the person skilled in the art, e.g. magnetic resonance imaging (MRI) and/or magnetic photon resonance imaging (MPI) technology in the context of using a tumor marker or group of tumor marker for diagnosing, detecting, monitoring or prognosticating a more progressed cancer of the progression towards such a cancer. For example, a tumor marker or group of tumor markers according to the present invention may be used as a marker for diagnosing, detecting, monitoring or prognosticating 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.

In another aspect 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 tumor marker or a group of tumor markers as defined above.

The term "nucleic acid affinity ligand for the expression product of a tumor marker or group of tumor markers" as used herein refers to a nucleic acid molecule being able to specifically bind to a transcript or a DNA molecule derived the nucleic acid molecules of said tumor marker or a group of tumor markers as defined above, preferably to the nucleotide sequences, (DNA sequence(s)) depicted in section D) of Table 1, 2, 3 or 4 or to the complementary nucleotide sequences (DNA sequence(s)) of the sequence(s) depicted in section D) of Table 1, 2, 3 or 4 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, 3 or 4 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section E) of Table 1 , 2, 3 or 4 or to any fragments of said sequences.

The term "peptide affinity ligand for the protein of a tumor marker or a group of tumor markers" as used herein refers to a peptide molecule being able to specifically bind to the proteins or peptides of the tumor marker or group of tumor markers according to Table

1 , 2, 3 or 4. The peptide molecule may preferably be able to specifically bind to a protein, polypeptide or peptide comprising the amino acid sequence(s) as set forth in section E) of Table 1 , 2, 3 or 4. The peptide affinity ligand may also be able to specifically bind to a protein, polypeptide or peptide comprising an amino acid sequence encoded by a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the sequence as set forth in section E) of Table 1 , 2, 3 or 4 or to a protein, polypeptide or peptide comprising an amino acid sequence being at least 75%, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section E) of Table 1 , 2, 3 or 4 or to any fragments of said sequences.

The term "peptide" in the context of the affinity ligand of the present invention refers to any type of amino acid sequence comprising more than 2 amino acids, e.g.

polypeptide structures, protein structures or functional derivatives thereof. Furthermore, the peptide may be combined with further chemical moieties or functionalities.

The term "expression product" as used herein refers to a transcript or an mRNA molecule of the tumor marker or group of tumor markers according to Table 1 , 2, 3 or 4 generated by the expression of the corresponding genomic sequence according to Table 1 ,

2, 3 or 4. More preferably, the term relates to a processed transcript of the tumor marker or group of tumor markers according to Table 1 , 2, 3 or 4 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.

The term "protein of a tumor marker or group of tumor markers" as used herein also refers to any polypeptide, protein, in particular to the polypeptides or proteins as set forth in section E) of Table 1 , 2, 3 or 4 or any domain, epitope, oligopeptide, or peptide derivable therefrom. A "peptide" in this context is any peptide derived from said protein, or overlapping or partially overlapping therewith. In a preferred embodiment said peptide is a peptide as defined in Table 4. In a preferred embodiment of the present invention the composition of the present invention may comprise one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid and/or peptide affinity ligands selected from the group consisting of a set of oligonucleotides specific for the expression product of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, a probe specific for the expression product of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, an aptamer specific for the expression product or for the protein of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, an antibody specific for the protein the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 and an antibody variant specific for the protein of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4. The composition of the present invention may, for example, comprise a set of oligonucleotides specific for the expression product the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 and/or a probe specific for the expression product of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4.

The term "oligonucleotide specific for the expression product the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4" as used herein refers to a nucleotide sequence which is complementary to the sense- or antisense-strand of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4. Preferably, the

oligonucleotide is complementary to the DNA sequence(s) shown in section D) of Table 1, 2, 3 or 4, or to the complementary DNA sequence of the sequence shown in section D) of Table 1, 2, 3 or 4. 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, 3 or 4 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section E) of

Table 1, 2, 3 or 4. 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, 3 or 4 or its complement. Typically, the oligonucleotide may have a length of between 8 and 60 nucleotides, preferably of between 10 and 35 nucleotides, more preferably a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 nucleotides. Oligonucleotide sequences specific for the expression product of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 may be defined with the help of software tools known to the person skilled in the art. In a further embodiment of the present invention the oligonucleotide sequences may be complementary to genomic sequences localized in (an) exon(s) of the gene(s) encoding for the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4. Corresponding information on genomic loci is indicated in section F) of Table 1, 2, 3 or 4. An oligonucleotide usable as a forward primer may be localized at the boundary between exonic and intronic sequences. Such boundary positions may be determined with the help of any suitable tool, based on the information provided in section F) of Table 1, 2, 3 or 4.

The term "probe specific for the expression product of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4" as used herein means a nucleotide sequence which is complementary to the sense- or antisense-strand of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4. Preferably, the probe is

complementary to the DNA sequence(s) depicted in section D) of Table 1, 2, 3 or 4 or to the complementary DNA sequence of the sequence(s) shown in section D) of Table 1, 2, 3 or 4. 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, 3 or 4 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%>, 85%, 90%>, 91%>, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as shown in section E) of Table 1, 2, 3 or 4. The probe may have any suitable length and sequence known to the person skilled in the, as derivable from the sequence(s) shown in section D) or its/their complement. Typically, the probe may have a length of between 6 and 300 nucleotides, preferably of between 15 and 60 nucleotides, more preferably a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Probe sequences specific for the expression product of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 may be defined with the help of software tools known to the person skilled in the art. The composition of the present invention may additionally or alternatively comprise an aptamer specific for the expression product or protein of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4.

The term "aptamer specific for the expression product of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4" as used herein refers to a short nucleic acid molecule, e.g. RNA, DNA, PNA, CNA, ETNA, LNA or ANA or any other suitable nucleic acid format known to the person skilled in the art, being capable of specifically binding to the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, preferably the DNA molecule with (a) sequence(s) as shown in section D) of Table 1 , 2, 3 or 4. More preferably, the nucleic acid aptamer molecule may specifically bind to a DNA sequence(s) shown in section D) of Table 1 , 2, 3 or 4 or a double stranded derivative thereof. The nucleic acid aptamer according to the present invention may also bind to an RNA molecule corresponding to the transcript(s) of the tumor marker or group of tumor markers according to Table 1 , 2, 3 or 4, preferably an RNA molecule corresponding to the DNA sequence(s) as shown in section D) of Table 1 , 2, 3 or 4. 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, 3 or 4 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, 3 or 4 or RNA molecules corresponding to these sequences. A nucleic acid aptamer according to the present invention may further be combined with additional moieties, e.g. with interacting portions like biotin or enzymatic functionalities like ribozyme elements.

The term "aptamer specific for the protein of the tumor marker or group of tumor markers according to Table 1 , 2, 3 or 4" as used herein refers to (a) short peptide(s) capable of interacting and specifically binding the protein(s) of the tumor marker or group of tumor markers according to Table 1 , 2, 3 or 4. The peptide aptamer(s) may preferably be able to specifically bind to (a) protein(s), polypeptide(s) or peptide(s) comprising (the) amino acid sequence(s) as shown in section E) of Table 1 , 2, 3 or 4. 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, 3 or 4 or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the sequence(s) as set forth in shown in section E) of Table 1 , 2, 3 or 4. Typically, (a) peptide aptamer(s) is/are a variable peptide loop, comprising for example 10 to 20 amino acids. In the context of the present invention the peptide aptamer(s) may preferably be attached at one or both ends to a scaffold structure. The scaffold structure may be any molecule, preferably a protein, which has good solubility properties. Suitable scaffold molecules would be known to the person skilled in the art. A preferred scaffold molecule to be used in the context of the present invention is the bacterial protein thioredoxin-A. The aptamer peptide loop may preferably be inserted within a reducing active site of the scaffold molecule. Alternatively, staphylococcal protein A and domains thereof and derivatives of these domains, such as protein Z or lipocalins may be used as scaffold structures in the context of the present invention.

Peptide aptamers may be generated according to any suitable method known to the person skilled in the art, e.g. via yeast two-hybrid approaches.

In another preferred embodiment of the present invention the composition may comprise, or may additionally comprise an antibody or a group of antibodies specific for the expression product or protein of a tumor marker or group of tumor markers as defined herein, e.g. specific for one or more of the protein(s) of the tumor marker according to Table 1, 2, 3 or 4. Particularly preferred is an antibody which specifically binds to an expression product, protein or peptide comprising the amino acid sequence as indicated in section E) of Table 1 , 2, 3 or 4 or a part or fragment of said sequence. Even more preferred is an antibody which specifically binds to an expression product, protein, or peptide comprising the amino acid sequence indicated in Table 4, or a part or fragment of said sequence.

In another aspect the invention thus relates to an antibody specific for one or more of the tumor markers according to Table 1, 2, 3 or 4. Preferably, such an antibody specifically binds to a protein or polypeptide having or comprising the amino acid shown in section E) of Table 1, 2, 3 or 4, or any derivative, fragment etc. thereof as defined herein above. Such antibodies are contemplated for any application, use, method, composition, immunoassay, screening method and pharmaceutical compositions as defined in the present application. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i. e. molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e. g., IgG, IgE, IgM, IgD, IgA and IgY), class (e. g., IgGl, IgG2, IgG3, lgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. Preferred epitopes according to the present invention are amino acids 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, 191-200, 201-210 etc. or any other specific stretch of amino acids of a protein of the tumor markers of the present invention, preferably of the sequence as shown in section E) of Table 1, 2, 3 or 4. Particularly preferred are antibodies binding to epitopes or antigens comprised in or comprising the amino acid sequence as mentioned in section Table 4, or parts of said sequence, e.g. fragments of 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. Further envisaged are all other suitable epitopes, which can be recognized, determined, described and subsequently be employed according to methods known to the person skilled in the art.

The term "specific for the tumor marker according to Table 1, 2, 3 or 4" as used herein refers to the immunospecific detection and binding of an antibody to an antigenic epitope as defined herein above. The term "specifically binding" excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens, in particular with antigens comprising the same antigenic epitope detected by the present antibody.

In a preferred embodiment antibodies of the invention include polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain

antibodies, Fab fragments, Fab' fragments, fragments produced by a Fab expression library, F(ab')2, Fv, disulfide linked Fv, minibodies, diabodies, scFv, sc(Fv)2, whole

immunoglobulin molecules small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins, camelized antibodies, 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.

Most preferably, the antibodies are human antigen-binding antibody fragments of the present invention and include Fab, Fab' and F (ab')2, Fv, single-chain Fvs (scFv), sc(Fv)2, single-chain antibodies, disulfide- linked Fvs (sdFv) and fragments comprising either a VL or VH domain.

The antibodies according to the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e. g., mouse and rat), donkey, monkey, rabbit, goat, guinea pig, camel, horse, or chicken.

The antibodies according to the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. In a particularly preferred embodiment the present invention relates to antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention. However, also antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are included in the present invention.

In a specific embodiment the present invention also envisages an antigen and/or an epitope which is detected, recognized or specifically bound by an antibody against a tumor marker protein as defined in Table 1, 2, 3 or 4.

Such antigens or epitopes may preferably be derived from a tumor marker or group of tumor marker sequences as depicted in Table 1, 2, 3 or 4. The antigen or epitope sequences encompassed by the present invention may, for example, comprise, have, or alternatively consist of amino acids 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, 191-200, 201-210 etc. or any other specific stretch of amino acids of the N- or C- terminus of a protein or expression product of the tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2, 3 or 4, preferably of the sequence as shown in section E) of Table 1, 2, 3 or 4. Furthermore, the antigen or epitope sequences encompassed by the present invention may, for example, comprise, have, or alternatively consist of amino acids 1-10, 2-11, 3-12, 4-13, 5-14, 6-15, 7-16, 8-17, 9-18, 10-19, 11-20, 12- 21, 13-22, 14-23, 15-24, 16-25, 17-26, 18-27, 19-28, 20-29, 21-30, 22-31, 23-32, 24-33, 25- 34, 26-35, 27-36, 28-37, 29-38, 30-39, 31-40, 32-41, 33-42, 34-43, 35-44, 36-45, 37-46, 38- 47, 39-48, 30-49, 41-50, 42-51, 43-52, 44-53, 45-54, 46-55, 47-56, 48-57, 49-58, 50-59, 51- 60, 52-61, 53-62, 54-63, 55-64, 56-65, 57-66, 58-67, 59-68, 60-69, 61-70, 62-71, 63-72, 64- 73, 65-74, 66-75, 67-76, 68-77, 69-78, 70-79, 71-80, 72-81, 73-82, 74-83, 75-84, 76-85, 77- 86, 78-87, 79-88, 80-89, 81-90, 82-91, 83-92, 84-93, 85-94, 86-95, 87-96, 88-97, 89-98, 90- 99, 91-100, 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 30-59, 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72, 54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82, 64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92, 74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100, 1-30, 2-31, 3-32, 4-33, 5-34, 6-35, 7-36, 8-37, 9-38, 10-39, 11-40, 12- 41, 13-42, 14-43, 15-44, 16-45, 17-46, 18-47, 19-48, 20-49, 21-50, 22-51, 23-52, 24-53, 25- 54, 26-55, 27-56, 28-57, 29-58, 30-59, 31-60, 32-61, 33-62, 34-63, 35-64, 36-65, 37-66, 38- 67, 39-68, 30-69, 41-70, 42-71, 43-72, 44-73, 45-74, 46-75, 47-76, 48-77, 49-78, 50-79, 51- 80, 52-81, 53-82, 54-83, 55-84, 56-85, 57-86, 58-87, 59-88, 60-89, 61-90, 62-91, 63-92, 64- 93, 65-94, 66-95, 67-96, 68-97, 69-98, 70-99 or 71-100 of the N- or C-terminus of a protein or expression product of the tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2, 3 or 4, preferably of the sequence as shown in section E) of Table 1, 2, 3 or 4.

The epitope or antigen according to the present invention may further comprise or consist of additional amino acid stretches or fragments of any length or derived from any position, e.g. N-terminus, C-terminus, middle, specific domain, specified exposed region etc. of the protein or expression product of the tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2, 3 or 4, preferably of the sequence as shown in section E) of Table 1, 2, 3 or 4. Particularly preferred are epitopes or antigens comprised in or comprising fragments or stretches of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 270, 300, 350 or 400 amino acids of the protein or expression product of the tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2, 3 or 4, preferably of the sequence as shown in section E) of Table 1, 2, 3 or 4.

The epitope or antigen of the present invention may further be present on extracellular domains, intracellular domains, sterically exposed domains, surface regions, exposed surface regions, interaction domains etc. of any of the tumor marker or group of tumor markers as defined herein, e.g. as derivable from Table 1, 2, 3 or 4, preferably of the sequence as shown in section E) of Table 1, 2, 3 or 4. Such domains or regions and their position as well as their amino acid composition would be known to the person skilled in the art or could be deduced with known software or database tools, in particular in view of the information provided in sections E) of Table 1, 2, 3 or 4. The person skilled in the art may, for example, use three dimensional modeling tools or structure information associated with the Entre Gene ID in order to identify such domains or regions.

Also envisaged are combinations of two or more epitope sequences, e.g. in the form of a fusion construct or the like. Further envisioned are epitope sequences that bear disease-specific sequence mutations. Such contructs may be used for immunological purposes, e.g. for the preparation of vaccines, the elicitation of antibodies etc. The present inventin thus also encompasses the medical use of correspondingly prepared vaccines, pharmaceutical compositions comprising such vaccines, the vaccines themselves, methods of treatment involving the vaccines etc.

In a further embodiment the present invention encompasses a screening method to identify antigen or epitope sequences bound by an antibody against a tumor marker protein as defined in Table 1, 2, 3 or 4. Further envisaged are the epitope sequences and/or antigen sequences derived from a screening procedure.

In a further embodiment the antibodies of the invention include derivatives which are modified, for instance by the covalent attachment of any type of molecule to the antibody such that said covalent attachment does not prevent the antibody from specifically binding to the epitope or from generating an anti-idiotypic response. Typical examples of such modifications are glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Chemical modifications may be carried out by known techniques, including specific chemical cleavage, acetylation, formylation etc. Additionally, the derivative may contain one or more non-classical amino acids.

Antibodies may be produced according to any suitable method known to the person skilled in the art. Monoclonal antibodies of defined specificity may be produced using, for instance, the hybridoma technology developed by Kohler and Milstein (Kohler and Milstein, 1976, Eur. J. Immunol, 6: 511-519).

In a further embodiment of the present invention the antibody or fragment thereof as defined herein above may be biotinylated or labeled. In a particularly preferred embodiment said label is a radioactive label, an enzymatic label, a fluorescent label, a chemiluminescent or a bio luminescent label. Alternatively, antibodies may also be labeled or combined with fluorescent polypeptides, e.g. green fluorescent protein (GFP) as well as derivates thereof known to the person skilled in the art.

Alternatively, a polynucleotide encoding an antibody may be generated from a nucleic acid from a suitable source.

In a further embodiment of the present invention a nucleic acid molecule encoding the antibody or fragment thereof as defined herein above may be used for recombinant antibody expression. Preferably, such expression vectors contain the antibody coding sequences and appropriate transcriptional and translational control signals. The vectors may either comprise coding sequences for the variable heavy chain or the variable light chain or for both. Such vectors may also include the nucleotide sequence encoding the constant regions of the antibody molecule. In a preferred embodiment of the present invention mammalian cells, more preferably Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus may be used as an effective expression system for antibodies. In another aspect the present invention relates to a cell that produces the antibody or fragment thereof as defined herein above. Such a cell may be a hybridoma cell as defined herein above or a cell which expresses a nucleic acid molecule encoding an antibody according to the present invention. Particularly preferred are cells or cell lines which stably express the antibody molecule.

In addition, the antibodies of the present invention or fragments thereof can be fused to any heterologous polypeptide sequence, preferably to those defined herein above, e.g. in order to facilitate antibody purification or to provide target means for the antibody.

In a specific embodiment of the present invention commercially available antibodies against the tumor marker according to Table 1, 2, 3 or 4, in particular against a protein having an amino acid sequence as indicated in section E) of Table 1, 2, 3 or 4 may be comprised in the composition or may be used diagnostically.

An affinity ligand, as described herein above, may be labeled with various markers or may be detected by a secondary affinity ligand, labeled with various markers, to allow detection, visualization and/or quantification. This can be accomplished using any suitable labels, which can be conjugated to the affinity ligand capable of interaction with the expression product(s) of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 or the corresponding protein(s) or to any secondary affinity ligand, using any suitable technique or methods known to the person skilled in the art.

The term "secondary affinity ligand" refers to a molecule which is capable of binding to the affinity ligand as defined herein above (i.e. a "primary affinity ligand" if used in the context of a system with two interacting affinity ligands). The binding interaction is preferably a specific binding.Examples of labels that can be conjugated to a primary and/or secondary affinity ligands include fluorescent dyes or metals (e.g. fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g. rhodopsin), chemiluminescent compounds (e.g. luminal, imidazole) and bio luminescent proteins (e.g. luciferin, luciferase), haptens (e.g. biotin).

In a particularly preferred embodiment an affinity ligand to be used as a probe, in particular a probe specific for the expression product(s) as defined herein above, may be labeled with a fluorescent label like 6-FAM, HEX, TET, ROX, Cy3, Cy5, Texas Red or Rhodamine, and/or at the same time with a quenching label like TAMRA, Dabcyl, Black Hole Quencher, BHQ-1 or BHQ-2. A variety of other useful fluorescents and chromophores are described in Stryer, 1968, Science, 162:526-533. Affinity ligands may also be labeled with enzymes (e.g. horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g. ³H, ¹⁴C, ³²P, ³³P, ³⁵S, ¹²⁵I, ⁿC, ¹³N, ¹⁵0, ¹⁸F, ⁶⁴Cu, ⁶²Cu, ¹²⁴I, ⁷⁶Br, ⁸²Rb, ⁶⁸Ga or ¹⁸F) or particles (e.g. gold).

The different types of labels may be conjugated to an affinity ligand using various chemistries, e.g. the amine reaction or the thiol reaction. However, other reactive groups than amines and thiols can also be used, e.g. aldehydes, carboxylic acids and glutamine.

In a further preferred embodiment of the present invention the nucleic acid affinity ligand or peptide affinity ligand of the present invention may be modified to function as a contrast agent, e.g. as an imaging contrast agent. The term "contrast agent" as used herein refers to a molecular compound that is capable of specifically interacting with the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4 and which can be detected by an apparatus positioned outside the human or animal body. Preferably, such contrast agents are suitable for use in magnetic resonance imaging (MPJ) or magnetic photon imaging (MPI). The term "specifically interacting" refer to the property of a molecular compound to preferentially interact with the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 on the cell surface of cells being present within the human or animal body over other proteins that are expressed by such cells.

Preferred contrast agents which may also be designated as contrast agent compositions will be capable of specifically detecting molecules having the nucleotide sequence(s) shown in section D) of Table 1, 2, 3 or 4 or the amino acid sequence(s) shown in section E) of Table 1, 2, 3 or 4 or derivatives or homologous variants thereof as defined herein above. Preferred contrast agents are aptamers specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 as well as antibodies specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Contrast agents, aside from their property of being capable of specifically recognizing the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4 will in addition typically comprise a further molecule which is detectable by the specific detection technology used.

The term "modified to function" as used herein thus refers to any suitable modifications known to the person skilled in the art, which may be necessary in order to allow the use of the contrast agent in molecular imaging methods, in particular in MPJ or MPI. For example, if fluorescent spectroscopy is used as a detection means, such molecules may comprise fluorophores as detectable marker molecules that can be excited at a specific wavelength.

Alternatively, a radioactive label, e.g. a radioisotope as described herein above may be employed. With respect to preferred contrast agents in accordance with the invention that are suitable for MRI, the contrast agents such as the above described antibodies may comprise a marker molecule which is detectable by MRI. Such detectable labels include e.g. USPIOS and 19Fluor.

In a specific embodiment of the present invention a composition may additionally comprise accessory ingredients like PCR buffers, dNTPs, a polymerase, ions like bivalent cations or monovalent cations, hybridization solutions, secondary affinity ligands like, e.g. secondary antibodies, detection dyes and any other suitable compound or liquid necessary for the performance of a detection based on any of the affinity ligands or contrast agents as defined herein above, which is known to the person skilled in the art.

In another aspect the present invention relates to the use of a nucleic acid or peptide affinity ligand for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, 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.

In a preferred embodiment the present invention relates to the use of a set of oligonucleotides specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or a probe specific for the expression product(s) the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably specific for a nucleic acid sequence having a sequence as indicated in section D) of Table 1, 2, 3 or 4 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.

In another preferred embodiment the present invention relates to the use of an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably a protein having an amino acid sequence as shown in section E) of Table 1, 2, 3 or 4, 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 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.

In a further preferred embodiment the present invention relates to the use of an antibody specific for protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or an antibody variant specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 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.

In a preferred embodiment of the present invention a composition as defined herein above is a diagnostic composition.

In another aspect the present invention relates to a diagnostic kit for diagnosing, detecting, monitoring or prognosticating 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 tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, a probe specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or an antibody specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and an antibody variant specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Typically, the diagnostic kit of the present invention contains one or more agents allowing the specific detection of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. 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 m NA expression level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, i.e. via the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, is intended, the agents to be comprised may be a set of oligonucleotides specific for the expression product(s) of said tumor marker or group of tumor markers and/or a probe specific for the expression product(s) of said tumor marker or group of tumor markers, which may be optionally labeled according to methods known in the art, e.g. with labels described herein above. In addition or alternatively, an aptamer specific for the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be comprised. Where detection at the protein level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 is intended, the agents to be comprised may be antibodies or compounds containing an antigen-binding fragment of an antibody or antibody variants specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, as described herein above. In addition or alternatively an aptamer specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be comprised.

Alternatively, a diagnostic kit may comprise a contrast agent as defined herein above.

The presence of specific proteins may also be detected using other compounds that specifically interact with the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, e.g. specific substrates or ligands. Preferably, a diagnostic kit of the present invention contains detection reagents for expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Such detection reagents comprise, for example, buffer solutions, labels or washing liquids etc. Furthermore, the kit may comprise an amount of a known nucleic acid molecule or protein, which can be used for a calibration of the kit or as an internal control. Typically, a diagnostic kit for the detection of expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may comprise accessory ingredients like a PCR buffers, dNTPs, a polymerase, ions like bivalent cations or monovalent cations, hybridization solutions etc. A diagnostic kit for the detection of protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may also comprise accessory ingredients like secondary affinity ligands, e.g. secondary antibodies, detection dyes and any other suitable compound or liquid necessary for the performance of a protein detection based known to the person skilled in the art. Such ingredients are known to the person skilled in the art and may vary depending on the detection method carried out.

Additionally, the kit may comprise an instruction leaflet and/or may provide information as to the relevance of the obtained results.

In another aspect the present invention relates to a method for detecting, diagnosing, monitoring or prognosticating a 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 above, in a sample.

The term "determining the level of a tumor marker or group of tumor markers" refers to the determination of the presence or amount of expression product(s) of the tumor marker or tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, e.g. (a) transcript(s) of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, and/or the determination of the presence and/or amount of (a) protein(s) or a peptide or fragment thereof of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4.

The term "level of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4" thus means the presence or amount of (an) expression product(s) of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, e.g. (a) transcript(s) of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, and/or the determination of the presence or amount of (a) protein(s) and/or (a) peptide(s) of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4. The determination of the presence or amount of (an) expression product(s) of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, e.g. (a) transcript(s) of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 or (a) protein(s) and/or (a) peptide(s) of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 may be

accomplished by any means known in the art.

In a preferred embodiment of the present invention the determination of the presence or amount of the expression products of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, e.g. transcript(s) and/or of protein(s) of the markers mentioned in Table 1, 2, 3 or 4, comprising for instance sequences as depicted in sections D) and E) of Table 1, 2, 3 or 4, is accomplished by the measurement of nucleic acid or protein levels or by the determination of the biological activity of said tumor marker or group of tumor markers.

For example, the measurement of the nucleic acid level of the expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be assessed by separation of nucleic acid molecules (e.g. RNA or cDNA) obtained from the sample in agarose or polyacrylamide gels, followed by

hybridization with tumor marker specific oligonucleotide probes as defined herein above, e.g. oligonucleotide probes comprising fragments of the sequences indicated in section D) of Table 1, 2, 3 or 4, or complementary sequences thereof. Alternatively, the expression level may be determined by the labeling of nucleic acid obtained from the sample followed by separation on a sequencing gel. Nucleic acid samples may be placed on the gel such that patient and control or standard nucleic acid are in adjacent lanes. Comparison of expression levels may be accomplished visually or by means of a densitometer. Methods for the detection of mRNA or expression products are known to the person skilled in the art.

Typically, Northern blot analysis may be used for such a purpose.

Alternatively, the nucleic acid level of the expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be detected in a DNA array or microarray approach. Typically, sample nucleic acids derived from subjects to be tested are processed and labeled, preferably with a fluorescent label. Subsequently, such nucleic acid molecules may be used in a hybridization approach with immobilized capture probes corresponding to the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 of the present invention or known biomarker or cancer marker genes. Suitable means for carrying out microarray analyses are known to the person skilled in the art.

In a standard setup a DNA array or microarray comprises immobilized high- density probes to detect a number of genes. The probes on the array are complementary to one or more parts of the sequence of the marker gene, or to the entire coding region of the marker gene. In the present invention, any type of tumor marker associated polynucleotide may be used as probe for the DNA array, as long as the polynucleotide allows for a specific distinction between the tumor marker expression and the expression of other genes.

Typically, cDNAs, PCR products, and oligonucleotides are useful as probes. For example, a fragment comprising 5'- or 3 '-portions of the tumor markers or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, e.g. of the sequences indicated in section D) of Table 1, 2, 3 or 4 may be used as a probe. The DNA array or microarray may comprise probes of one or more of the tumor marker of Table 1, 2, 3 or 4, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 etc. or all of the tumor markers or any combination of said markers. Furthermore, any type of fragment or sub-portion of any of the markers sequences may be combined with any further fragment or sub-portion of any of the markers sequences of Table 1, 2, 3 or 4. In addition to the determination of the expression of tumor markers according to Table 1, 2, 3 or 4 also the determination of the expression of other genes, e.g. additional biomarker or cancer marker genes is envisaged by the present invention.

A DNA array- or microarray-based detection method typically comprises the following steps: (1) Isolating mRNA from a sample and optionally converting the mRNA to cDNA, and subsequently labeling this RNA or cDNA. Methods for isolating RNA, converting it into cDNA and for labeling nucleic acids are described in manuals for micro array technology. (2) Hybridizing the nucleic acids from step 1 with probes for the marker genes. The nucleic acids from a sample can be labeled with a dye, such as the fluorescent dyes Cy3 (red) or Cy5 (blue). Generally a control sample is labeled with a different dye. (3) Detecting the hybridization of the nucleic acids from the sample with the probes and determining at least qualitatively, and more particularly quantitatively, the amounts of mRNA in the sample for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or additional marker genes investigated. The difference in the expression level between sample and control can be estimated based on a difference in the signal intensity. These can be measured and analyzed by appropriate software such as, but not limited to the software provided for example by Affymetrix. There is no limitation on the number of probes corresponding to the marker genes used, which are spotted on a DNA array. Also, a marker gene can be represented by two or more probes, the probes hybridizing to different parts of a gene. Probes are designed for each selected marker gene. Such a probe is typically an oligonucleotide comprising 5-50 nucleotide residues. Longer DNAs can be synthesized by PCR or chemically. Methods for synthesizing such oligonucleotides and applying them on a substrate are well known in the field of micro-arrays. Genes other than the marker genes may be also spotted on the DNA array. For example, a probe for a gene whose expression level is not significantly altered may be spotted on the DNA array to normalize assay results or to compare assay results of multiple arrays or different assays. Alternatively, the nucleic acid level of expression of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 may be detected in a quantitative RT-PCR approach, preferably in a real-time PCR approach following the reverse transcription of the mRNA transcript(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4.

Typically, as first step, a transcript is reverse transcribed into a cDNA molecule according to any suitable method known to the person skilled in the art. A quantitative or real-time PCR approach may subsequently be carried out based on a first DNA strand obtained as described above. Preferably, Taqman or Molecular Beacon probes as principal FRET -based probes of this type may be used for quantitative PCR detection. In both cases, the probes, preferably probes of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 as defined herein above, serve as internal probes which are used in conjunction with a pair of opposing primers that flank the target region of interest, preferably a set of oligonucleotides specific for the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 as defined herein above. Upon amplification of a target segment, the probe may selectively bind to the products at an identifying sequence in between the primer sites, thereby causing increases in FRET signaling relative to increases in target frequency.

Preferably, a Taqman probe to be used for a quantitative PCR approach according to the present invention may comprise (a) oligonucleotide(s) derived from the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 as defined above of about 22 to 30 bases that is labeled on both ends with a FRET pair. Typically, the 5' end will have a shorter wavelength fluorophore such as fluorescein (e.g. FAM) and the 3' end is commonly labeled with a longer wavelength fluorescent quencher (e.g. TAMRA) or a non- fluorescent quencher compound (e.g. Black Hole Quencher). It is preferred that the probes to be used for quantitative PCR, in particular the probes tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, have no guanine (G) at the 5' end adjacent to the reporter dye in order to avoid quenching of the reporter fluorescence after the probe is degraded. A Molecular Beacon probe to be used for a quantitative PCR approach according to the present invention preferably uses FRET interactions to detect and quantify a PCR product, with each probe having a 5' 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 tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or of any fragments, homologues or derivates derived thereof may be carried out via any suitable detection technique known in the art. Preferably, the protein level of tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and derivatives thereof may be determined immunologically, e.g. by using an antibody specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an antibody as defined herein above. In a particularly preferred embodiment of the present invention the presence and/or amount of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4 may be determined with an antibody specifically binding to or detecting a protein or antigen or epitope thereof of the proteins or peptides as indicated in section E) of Table 1, 2, 3 or 4, or any fragment thereof, or any epitope comprised therein, as defined herein above. Alternatively, antibody variants or fragments as defined herein above may be used.

The present invention also envisages the use of peptide affinity ligands like aptamers specific for the protein(s) of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 as defined herein above. Determination of the protein levels of the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 can be accomplished, for example, by the separation of proteins from a sample on a polyacrylamide gel, followed by identification of the protein(s) of tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 using specifically binding antibodies in a Western blot analysis. Alternatively, proteins can be separated by two- dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection. In other embodiments, protein samples are analyzed by mass spectroscopy.

Within the context of the present invention antibodies specific for (a) protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 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.

The present invention further envisages an array of peptide sequences, protein sequences or expression products comprising one or more of the 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.

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, electrochemiluminescence immunoassay (ECLIA) and protein A

immunoassays. Such assays are routine and well known to the person skilled in the art.

Furthermore, the binding affinity of an antibody to an antigen and the off-rate of an antibody- antigen interaction may be determined by competitive binding assays.

In this context, the binding affinity of an antibody to an antigen and the off- rate of an antibody- antigen interaction may be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with a suitable antibody in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off- rates may be determined from the data by any suitable analysis approach, e.g. by a scatchard plot analysis. Competition with a second antibody may also be determined using radioimmunoassays. In this case, the antigen may be incubated with a suitable antibody conjugated to a labeled compound (e.g., ³H or ¹²⁵I) in the presence of increasing amounts of an unlabeled second antibody.

In addition, aptamers specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, may be used in a method of detecting proteins of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Such aptamers may preferably be labeled in order to allow the detection of a protein- ligand interaction.

The determination of the biological activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be carried out by employing molecular or enzymatic assays specific to the corresponding function or functions of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, e.g. of a tumor marker protein having a sequence as indicted in section E) of Table 1, 2, 3 or 4. 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.

In a further preferred embodiment the biological activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be tested with the help of suitable enzymatic reactions or tests for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, known to the person skilled in the art, or by employing specific inhibitors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. The use of such inhibitors may, for example, be combined with an enzymatic readout system. Typical inhibitors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 to be used comprise antisense molecules, siRNA molecules or miR A molecules.

The level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may also be detected in methods involving histological or cell-biological procedures. Typically, visual techniques, such as light microscopy or immunofluoresence microscopy, as well as flow cytometry or luminometry may be used.

The presence of (a) protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 in a cell may, for instance, be detected or determined by removing cells to be tested from samples as defined herein above. Also tissue sections or biopsy samples may be used for these methods. Subsequently, affinity ligands for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be applied, preferably antibodies or aptamers. Typically, such affinity ligands are labeled, preferably with fluorescent labels as defined herein above. Such a procedure allows for the detection of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, for its/their quantification and, in addition, allows determining the distribution and relative level of the expression thereof.

Such procedures involve the use of visualization methods. Suitable

visualization methods are known to the person skilled in the art. Typical methods to be used comprise fluorometric, luminometric and/or enzymatic techniques. Fluorescence is normally detected and/or quantified by exposing fluorescent labels to light of a specific wavelength and thereafter detecting and/or quantifying the emitted light of a specific wavelength. The presence of a luminescently tagged affinity ligand may be detected and/or quantified by luminescence developed during a chemical reaction. Detection of an enzymatic reaction is due to a color shift in the sample arising from chemical reaction.

In a further, preferred embodiment the level of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 may be determined by suitable molecular imaging techniques, e.g. magnetic resonance imaging (MRI) or magnetic photon imaging (MPI), and/or by using suitable contrast agents, e.g. contrast agents as defined herein above. In a further preferred embodiment of the present invention a method for detecting, diagnosing, monitoring or prognosticating a 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 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 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.

The term "comparing" as used herein refers to any suitable method of assessing, calculating, evaluating or processing of data. In a preferred embodiment of the present invention the determination of the presence or amount of (an) expression product(s), e.g. (a) transcript(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or of (a) protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, is

accomplished by the measurement of nucleic acid or protein levels or by the determination of the biological activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Thus, the expression level(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be determined by a method involving the detection of an mR A encoded by the genes encoding for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, or the according cDNA sequence, e.g. the sequences indicated in section D) of Table 1, 2, 3 or 4, the detection of the protein(s) encoded by the transcript of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or the detection of the biological activity of the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, e.g. of the proteins having the sequences indicated in section E) of Table 1, 2, 3 or 4. For example, the measurement of the nucleic acid level of expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be assessed by separation of nucleic acid molecules as described herein above.

In yet another embodiment as a further, additional step a decision on the presence or stage of a more progressed 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 tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4.

In another embodiment of the present invention the above mentioned method is a method of graduating cancer, comprising the steps of

(a) determining the level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 in a sample by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers,

The determination of the level of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 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 tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4.

In another aspect the present invention relates to a method of data acquisition comprising at least the steps of: (a) testing in an individual for expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; and

(b) comparing the expression as determined in step (a) to a control level. The testing for expression of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be carried out according to steps as defined herein above. Preferably, the testing may be carried out as measurement of protein levels of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, more preferably according to the herein above described options for such measurements. The term "control level" as used in the context of the method of data acquisition refers to the expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 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.

In another embodiment 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:

(a) testing in at least one sample obtained from at least one individual suspected to suffer from cancer for expression of the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4;

(b) testing in at least one control sample obtained from at least one individual not suffering from cancer or suffering from a less progressed cancer stage for the expression of the expression product(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4;

(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).

In one embodiment, steps a), b), c) and/or d) of this method of diagnosis may be performed outside the human or animal body. The testing for expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be carried out according to steps as defined herein above. Preferably the testing may be carried out as measurement of nucleic acid or protein levels of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or by determining the biological activity of said tumor markers, more preferably according to the herein above described options for such measurements. The testing may be carried out in an individual, i.e. in vivo, or outside the individual, i.e. ex vivo or in vitro.

In another aspect the present invention relates to an immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a 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

(a) testing in a sample obtained from an individual for the expression of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4;

(b) testing in a control sample for the expression of the same tumor marker or group of tumor markers as in (a);

(c) determining the difference in expression of the tumor marker or group of tumor markers of steps (a) and (b); and

wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or a group of tumor markers as defined above.

The term "risk of recurrence" as used herein refers to a likelihood or probabilty assessment regarding the chances or the probability that a subject or individual may be afflicted with or may be developing a similar or the same cancer or neoplastic disease as defined herein above, preferably cancer, comparable to the one that the subject or individual has been treated for or diagnosed for, based on the expression modification of the tumor marker or a group of tumor markers as defined above. A risk of recurrence may, for example, be present when a tumor marker or group of turmor markers as defined herein above shows a modified expression level, e.g. an increased or decreased expression level in comparison to a less progressed stage, in particular a non-malignant stage, benign tumor stage or a healthy stage, although histological markers, cell shapes etc. or other tumor biomarker, e.g. PSA, show no modification, or a different modification, e.g. an opposite modification or a less pronounced increase or decrease of expression level(s).

The testing for expression of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 may be carried out according to steps as defined herein above. Preferably, the testing may be carried out as measurement of protein levels of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4, more preferably according to the herein above described options for such measurements. As controls or control samples controls as defined herein above may be used.

In a particularly preferred embodiment the testing steps may be based on the use of an antibody specifically binding to a tumor marker according to Table 1, 2, 3 or 4 as laid out above, e.g. (a) commercially available antibody/antibodies against (a) protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to

Table 1, 2, 3 or 4. 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 tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 as cancer marker(s).

(a) testing in a sample obtained from an individual for the expression of an up-regulated tumor marker or group of tumor markers as defined above, or as indicated in section I) of Table 1, 2 or 3;

(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of said up-regulated tumor marker or group of tumor markers as defined above; (c) classifying the levels of expression of step (a) relative to levels of step

(b); and

(a) testing in a sample obtained from an individual for the expression of a down-regulated tumor marker or group of tumor markers as defined above, or as indicated in section J) of Table 1 , 2 or 3 ;

The level of a tumor marker or group of tumor markers may be determined on the nucleic acid, protein or activity level as described herein above. Preferred is the determination of the amount of tumor marker protein and/or transcript(s). In addition the level of a reference gene in a sample may be determined.

The term "reference gene" as used herein refers to any suitable gene, e.g. to any steadily expressed and continuously detectable gene, gene product, expression product, protein, peptide or protein variant in the organism of choice. The term also includes gene products such as expressed proteins, peptides, polypeptides, as well as modified variants thereof. The invention hence also includes reference proteins derived from a reference gene.

Also encompassed are all kinds of transcripts derivable from the reference gene as well as modifications thereof or secondary parameters linked thereto. Alternatively or additionally, other reference parameters may also be used for reference purposes, e.g. metabolic concentrations, cell sizes etc.

Testing for the expression of a reference gene may be carried out in the same sample used for the determination of the tumor marker or group of tumor markers of the invention. If the testing is carried out in the same sample, a single detection or a multiplex detection approach may be performed.

For the performance of the multiplex detection the concentration of primers and/or probe oligonucleotides may be modified. Furthermore, the concentration and presence of further ingredients like buffers, ions etc. may be modified, e.g. increased or decreased in comparison to manufacturers' indications.

Alternatively, the testing for the expression of a reference gene may be carried out in a different sample, preferably a control sample. Preferably, such a "control sample" may be a control sample from the same individual as the test sample, or a control sample derived from a different source or individual. The control sample may further be either a sample derived from the same tissue, preferably prostate tissue, or be derived from a different tissue type. Examples of preferred alternative tissue types are stromal prostate tissue, bladder epithelial tissue and urethra epithelial tissue. Furthermore, the testing of the test sample for the expression of a reference gene and the testing of control sample for the expression of a tumor marker or group of tumor markers may be combined.

In a further embodiment the control sample may also be tested for the expression of the reference gene. In case more than one sample was tested for the expression of a reference gene, the obtained expression results may be compared and/or averaged or normalized according to any suitable statistical method known to the person skilled in the art.

The term "classifying the levels of expression of step (a) relative to levels of step (b)" as used herein means that the expression in a test sample for a tumor maker or group of tumor markers according to the invention and the expression in a control sample for a tumor maker or group of tumor markers as defined herein are compared, e.g. after

normalization against a suitable normalization references. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the control sample, an increased expression in comparison to the control sample, or an reduced expression in comparison to the control sample. The term further means that the expression in a test sample for a tumor marker and the expression in the same test sample for a reference gene are compared, e.g. after normalization against a further gene as normalization reference. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the reference gene, an increased expression in comparison to the reference gene, or an reduced expression in comparison to the reference gene.

According to the classification of the expression results an individual may be considered to be eligible for a cancer disease therapy when the expression level of an up- regulated tumor marker as defined herein above, or as indicated in section I) of Table 1, 2 or 3, or the expression level of a group of tumor markers as defined herein above is increased. The expression level is deemed to be "increased" when the tumor marker gene expression, or the expression of the group of tumor marker genes in the test sample is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding tumor marker gene expression, or to the expression of the corresponding group of tumor markers in a control sample, or elevated at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the tumor marker expression, or the expression of the group of tumor markers in a control sample; or when the tumor marker or group of tumor marker gene expression is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the expression of a reference gene in a control sample, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more elevated in comparison to the expression of a reference gene. In a specific embodiment, the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes. Alternatively, instead of the gene expression level the amount of protein present, or the amount of detecting antibodies present may be measured. For these alternative determination procedures the above indicated parameters also indicate an increase of expression.

According to the classification of the expression results an individual may also be considered to be eligible for a cancer disease therapy when the expression level of a down- regulated tumor marker as defined herein above, or as indicated in section J) of Table 1, 2, or 3, or the expression level of a group of tumor markers as defined herein above is decreased. The expression level is deemed to be "decreased" when the tumor marker gene expression, or the expression of the group of tumor marker genes in the test sample is lowered by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding tumor marker gene expression, or to the expression of the corresponding group of tumor markers in a control sample, or lowered at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the tumor marker expression, or to the expression of the group of tumor markers in a control sample; or when the tumor marker or group of tumor marker gene expression is lowered by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the expression of a reference gene in a control sample, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more lowered in comparison to the expression of a reference gene. In a specific embodiment, the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes. Alternatively, instead of the gene expression level the amount of protein present, or the amount of detecting antibodies present may be measured. For these alternative determination procedures the above indicated parameters also indicate a decrease of expression.

In another aspect the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a cancer disease comprising:

(a) testing in a sample obtained from an individual for the expression of an up-regulated tumor marker or group of tumor markers as defined above or as indicated in section I) of Table 1, 2 or 3;

(a) testing in a sample obtained from an individual for the expression of a down-regulated tumor marker or group of tumor markers as defined above or as indicated in section J) of Table 1, 2 or 3;

(c) determining the difference in expression of said down-regulated tumor marker or group of tumor markers as defined herein above of steps (a) and the expression of said down-regulated tumor marker or group of tumor markers as defined above and/or the reference gene in step (b); and (d) stratifying an individual or cohort of individuals to a cancer disease therapy based on the results obtained in step (c), where the individual's sample has an decreased level of expression of an down-regulated tumor marker or group of tumor markers as defined above.

The testing of the expression of a tumor marker or group of tumor markers may preferably be carried out via the determination of the amount of tumor marker protein or the determination of the tumor marker protein/expression product activity level. Preferred is the determination of the amount of tumor marker proteins or peptides with the help of specific antibodies binding said tumor marker protein or expression product. Alternatively, the immunoassay may be carried out with any other suitable agent or be combined with the determination of other entities. For example, the assay may be combined with the detection of the presence or amount of nucleic acids, or enzymatic testing methods as described herein. In addition the level of a reference gene as described herein above in a sample may be determined. Testing for the expression of a reference gene may be carried out in the same sample used for the determination of the tumor marker or group of tumor markers. If the testing is carried out in the same sample, a single detection or a parallel or multiplex detection approach may be performed. Preferably, for a parallel or multiplex detection differently labeled primary or secondary antibodies may be used.

Alternatively, the testing for the expression of a reference gene may be carried out in a different sample, preferably a control sample. Preferably, such a control sample may be a control sample from the same individual as the test sample, or a control sample derived from a different source or individual. The control sample may further be either a sample derived from the same tissue, preferably prostate tissue, or be derived from a different tissue type. Examples of preferred alternative tissue types are stromal prostate tissue, bladder epithelial tissue and urethra epithelial tissue. Furthermore, the testing of the test sample for the expression of a reference gene and the testing of control sample for the expression of a tumor marker or group of tumor markers may be combined.

The term "determining the difference in expression of a tumor marker or a group of tumor markers of step (a) and the expression of a tumor marker or group of tumor markers and/or the reference gene in step (b)" as used herein means that the expression in a test sample for the tumor marker or group of tumor markers and the expression in a control sample for the tumor marker or group of tumor markers are compared, e.g. after

normalization against a suitable normalization references. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the control sample, an increased expression in comparison to the control sample, or an reduced expression in comparison to the control sample. The term further means that alternatively or additionally the expression in a test sample for a tumor marker or group of tumor markers and the expression in the same test sample for a reference gene are compared, e.g. after normalization against a further gene as normalization reference. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the reference gene, or a difference in the expression. The difference may be either an increased expression in comparison to the reference gene, or an reduced expression in comparison to the reference gene.

The term "stratifying an individual or cohort of individuals to 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 tumor marker or group of tumor marker expression level and a reference gene or the tumor marker or group of tumor marker expression level in different samples.

According to the determination of the expression difference an individual may be identified as pertaining to a group of similar individuals whose optimal therapy form is a cancer disease therapy when the expression levels of the up-regulated tumor marker or group of tumor markers as defined herein above, or as indicated in section I) of Table 2 or 3, are increased. The expression level is deemed to be "increased" when the gene expression of the up-regulated tumor marker or group of tumor marker as defined herein above, or as indicated in section I) of Table 1, 2 or 3, is elevated in the test sample by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to said tumor marker or group of tumor marker expression in a control sample, or at least elevated 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to said tumor marker or group of tumor marker expression in a control sample; or when the tumor marker or group of tumor marker gene expression is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the expression of a reference gene in a control sample, or is elevated at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more elevated in comparison to the expression of a reference gene. In a specific embodiment, the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes.

According to the determination of the expression difference an individual may also be identified as pertaining to a group of similar individuals whose optimal therapy form is a cancer disease therapy when the expression levels of the down-regulated tumor marker or group of tumor marker as defined herein above, or as indicated in section J) of Table 1 , 2 or 3, are decreased. The expression level is deemed to be "decreased" when the gene expression of the down-regulated tumor marker or group of tumor markers as defined herein above, or as indicated in section J) of Table 1, 2 or 3, is lowered in the test sample by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to said tumor marker or group of tumor marker expression in a control sample, or at least lowered 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to said tumor marker or group of tumor marker expression in a control sample; or when the tumor marker or group of tumor marker gene expression is lowered by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the expression of a reference gene in a control sample, or is lowered at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more lowered in comparison to the expression of a reference gene. In a specific embodiment, the expression of a reference gene may also be normalized or adjusted to the expression of additional genes or markers, e.g. housekeeping genes.

An individual being considered to be eligible for a cancer disease therapy or being stratified to a cancer disease therapy as described herein above may receive any suitable therapeutic treatment for this disease form known to the person skilled the art. In particular, the term " cancer disease therapy" as used herein refers to any suitable cancer disease therapy, preferably prostate cancer related therapy known to the person skilled in the art, and preferably includes includes surgical castration by removal of the testes as the main organ of male sex hormone production, chemical castration by e.g., suppression of generation of androgens or by inhibition of the androgen receptor activity, cytotoxic, chemotherapy, targeted therapy (e.g. targeting cellular proteins with chemical molecules to suppress or stimulate their activity), radiation therapy (External Beam Radiation Therapy,

Brachytherapy), Cryotherapy, focal therapies like HIFU ablation (High Frequency Ultrasound ablation), or thermal ablation or any type of combination therapy of at least two of the above mentioned treatment forms either in direct combination, or used in a subsequent form.

Typically, an individual considered to be eligible for a cancer disease therapy due to an increased expression of a tumor marker or group of tumor marker of the present invention may be deemed to be suffering from a 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. In a further embodiment a correspondingly identified individual may be treated with a pharmaceutical composition according to the present invention in combination with an additional cancer therapy. The term "additional cancer therapy" refers to any types of cancer therapy known to the person skilled in the art. Preferred are cancer therapy forms known for prostate cancer. The term includes, for example, all suitable forms of chemotherapy, radiation therapy, surgery, antibody therapies etc.

Alternatively, a correspondingly identified or stratified individual may also be treated solely with one or more cancer therapies such as a chemotherapy, radiation therapy, surgery, antibody therapies etc. Preferred are cancer therapies typically used for prostate cancer.

In a further embodiment of the present invention the classification method for eligibility or the immunoassay for stratification as described herein above may also be used for monitoring the treatment of an individual, e.g. an individual being classified as suffering from a 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.

In a further embodiment of the present invention any treatment scheme as mentioned herein may be adjusted, e.g. enforced or attenuated, or altered in any suitable manner in correspondence with the results of the monitoring process.

In a particularly preferred embodiment of the present invention the reference gene is a housekeeping gene. In human organisms, examples of "housekeeping genes" include inter alia β-actin, glycerinaldehyde 3-phosphate dehydrogenase (GAPDH), porphobilinogen deanimase (PBGD), and ribosomal protein PI . Apart from these genes any other suitable gene may be used as a house-keeping gene, as long as the gene shows an expression or transcription on a steady, non-modified level, in particular during different stages of 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.

Accordingly normalization and/or comparison with GAPDH or PBGD may preferably be used for the methods of identifying or the immunoassays for discriminating or stratifying individuals. Corresponding determination steps may either be carried out in separate reactions, or, particularly preferred in multiplex reactions. For the performance of the multiplex detection the concentration of primers and/or probe oligonucleotides may be modified. Furthermore, the concentration and presence of further ingredients like buffers, ions etc. may be modified, e.g. increased or decreased in comparison to manufacturers' indications.

In a further embodiment of the present invention the method of identifying an individual for eligibility for a cancer disease therapy based on the expression of a tumor marker or group of tumor markers or the immunoassay for stratifying an individual or cohort of individuals as described herein above may further be combined with one or more similar identification methods, based on the expression of one or more different bio markers.

Preferred is the determination of the level of prostate specific antigen (PSA), more preferably in blood. Thus, if the level of PSA in blood is encountered to be of a range of about 2 to 5 or more ng/ml, preferably of about 2.2 to 4.8 ng/ml or more, 2.4 to 4.4 ng/ml or more, 2.6 to 4.2 ng/ml ore more or 2.8 to 4.0 ng/ml or more, more preferably of about 2.5 to 4 ng/ml or more, an individual may be considered to be suffering from a cancer disease, or be likely to develop a cancer disease in the near future, i.e. within the next 1, 2, 3, 4, 5, 6, 12, 14, 48 months.The testing for expression of a tumor marker or group of tumor markers may be carried out according to steps as defined herein above.

In a further embodiment of the present invention the measurement of a marker, or of a group or combination of markers as defined above, i.e. from Table 1, 2, 3 or 4, in a patient blood or 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. Preferably, the measurement of a tumor marker, or of a group or combination of tumor markers, e.g. those as defined in Table 1, 2, 3 or 4, may be performed in a serum or blood sample of a patient before a biopsy is performed. The measurement of a tumor marker, or of a group or combination of tumor markers, e.g. those as defined in Table 1, 2, 3 or 4, 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 which is taken, for example, before a biopsy procedure is performed. 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%, >55%, or >50% probability that the identified cancer disease, in particular prostate cancer disease is a significant disease and is going to progress in the future. The measurement of the tumor marker, or the group or combination of tumor markers as defined above below a given threshold as defined herein, e.g. in Table Table 1, 2 or 3 or derivable from the Examples, indicates a >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% probability that in case a cancer disease, in particular a prostate cancer is present it is an insignificant disease and is not going to progress in the future. The test or measurement of a marker, or of a group or combination of markers as defined above, i.e. from Table 1, 2, 3 or 4, in a patient serum sample may preferably be carried out before a biopsy was taken, e.g. as first step in a cancer diagnosis or screening.

The detection or measurement of the tumor marker, or of the group or combination of markers as defined herein, i.e. derivable from Table 1, 2, 3 or 4, may further be used to monitor the potential re-currence of a cancer, in particular prostate cancer, after a primary treatment, e.g. surgery, or radiotheary. The tumor marker detection or measurement may be performed every 3, or every 6, or every 12 months after the patient has been treated. If the expression level of the tumor marker, or group of tumor markers as defined herein is going to pass a threshold as defined herein, e.g. in Table 1, 2, 3 or 4 or derivable from the Examples, by 5%, 10%, 15%, 20%>, 25%, or 30% or more, further investigations or treatment may be started.

In a preferred embodiment of the present invention the diagnosing, detecting, monitoring or prognosticating as mentioned above is to be carried out on a sample obtained from an individual.

The term "sample obtained from an individual" as used herein relates to any biological material obtained via suitable methods known to the person skilled in the art from an individual, as laid out above. The sample used in the context of the present invention should preferably be collected in a clinically acceptable manner, more preferably in a way that nucleic acids (in particular R A) or proteins are preserved.

The biological samples may include body tissues and/or fluids, such as blood, or blood components like serum or plasma, sweat, sputum or saliva, semen and urine, as well as feces or stool samples. Furthermore, the biological sample may contain a cell extract derived from or a cell population including an epithelial cell, preferably a 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.

Alternatively, the biological sample may contain a cell population derived from a glandular tissue, e.g. the sample may be derived from the prostate of a male individual. Additionally, cells may be purified from obtained body tissues and fluids if necessary, and then used as the biological sample. Samples, in particular after initial processing, may be pooled. However, also non-pooled samples may be used.

In a specific embodiment of the present invention the content of a biological sample may also be submitted to an enrichment step. For instance, a sample may be contacted with ligands specific for the cell membrane or organelles of certain cell types, e.g. prostate cells, functionalized for example with magnetic particles. The material concentrated by the magnetic particles may subsequently be used for detection and analysis steps as described herein above or below.

In a specific embodiment of the invention, biopsy or resections samples may be obtained and/or used. Such samples may comprise cells or cell lysates. Furthermore, cells, e.g. tumor cells, may be enriched via filtration processes of fluid or liquid samples, e.g.

blood, urine, sweat etc. Such filtration processes may also be combined with enrichment steps based on ligand specific interactions as described herein above.

In a particularly preferred embodiment of the present invention a sample may be a tissue sample, a urine sample, a biopsy sample, a urine sediment sample, a blood sample, a serum sample, a plasma sample, a saliva sample, a semen sample, or a sample comprising circulating tumor cells.

A subject or individual to be diagnosed, monitored or in which a 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.

In a specific embodiment of the present invention the obtaining step of a sample may be included as a first or additional step in any of the herein mentioned methods, uses or approaches.

In another aspect the present invention relates to a pharmaceutical composition comprising at least one element selected from the group of: (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table

1, 2, 3 or 4; (e) a miR A specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ,

2, 3 or 4; (g) a siR A specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (h) an aptamer specific for the expression product of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (i) a small molecule or peptidomimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; and (j) an antibody specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or an antibody variant specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4.

The term "a compound directly inhibiting the activity of a tumor marker" as used herein refers to a compound which is capable of decreasing the activity of a tumor marker according to Table 1, 2, 3 or 4. Such a compound may be any direct interactor of the tumor marker according to Table 1, 2, 3 or 4, which has negative influence on the catalytic activity of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4. Such a compound may preferably be an antagonist of the catalytic activity of the tumor marker according to Table 1, 2, 3 or 4, e.g. of a tumor marker protein having a sequence as indicated in section E) of Table 1, 2, 3 or 4.

The term "a compound indirectly inhibiting the activity of a tumor marker " as used herein refers to a compound which is capable of decreasing the activity of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 by an interaction with a direct interactor of the tumor marker according to Table 1, 2, 3 or 4 ("indirect interactor") or via an indirect working pathway not involving an interaction with of the tumor marker according to Table 1, 2, 3 or 4. Such a compound may be any direct interactor of an interactor of the tumor marker according to Table 1, 2, 3 or 4, e.g. of a tumor marker protein having a sequence as indicated in section E) of Table 1, 2, 3 or 4. The effect conveyed by the direct interactor of an interactor of the tumor marker according to Table 1, 2, 3 or 4 may be either negative if the interactor of the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 itself has a negative effect on the activity of the tumor marker according to Table 1, 2, 3 or 4, or negative, if the interactor of the tumor marker according to Table 1, 2, 3 or 4 has a positive effect on the activity of said tumor marker.

Alternatively, such negatively working indirect integrators may provoke a modification of the binding behavior of directly binding proteins, leading to a decreased activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Typically negatively working indirect interactors may have an inhibitory effect on activators of the tumor marker according to Table 1, 2, 3 or 4. Examples of such interactors are enzymatic activities degrading activators of the tumor marker according to Table 1, 2, 3 or 4, or proteins capable of binding and quenching activators of the tumor marker according to Table 1, 2, 3 or 4. Alternatively, such interactors may positively modulate activities leading to a degradation of the tumor marker according to Table 1, 2, 3 or 4, e.g. proteinases. Further examples and their implementation would be known to the person skilled in the art.

Alternatively, an indirect inhibition of the activity of the tumor marker according to Table 1, 2, 3 or 4 may be conveyed by compounds deactivating, interfering or disrupting the expression of the endogenous gene(s) of said tumor markers. Examples of such compounds are specific interactors of transcription factors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 that inhibit and/or preclude binding of transcription factors and the basal transcription machinery to the promoters of the tumor marker according to Table 1, 2, 3 or 4, specific destabilizing activities of the mR A(s) of the tumor marker according to Table 1, 2, 3 or 4 or factors inhibiting the splicing factors specific for the tumor marker according to Table 1, 2, 3 or 4. Further examples and their implementation would be known to the person skilled in the art.

The "nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker" as used herein refers to any nucleic acid capable of expressing a mutant form of a naturally occurring protein or polypeptide of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Thus the term refers to a nucleic acid encoding (a) variant(s) of the tumor marker according to Table 1, 2, 3 or 4, which comprises an antimorphic modification, in particular which adversely affects the tumor marker according to Table 1, 2, 3 or 4 of the invention. Typically, such a behavior may occur if the antimorphic variant can interact with the tumor marker according to Table 1, 2, 3 or 4 but blocks some aspect of its function. Preferably, such variants may comprise or lack specific domains of the tumor marker according to Table 1, 2, 3 or 4, e.g. one or more protein-protein interacting or dimerization domains, complex assembly domains, one or more membrane-associated domains etc. This is particularly of importance in a protein that functions as a dimer or multimer. If, for example, one part of that protein complex is mutant in some functional aspect of the multimer but is still able to form the multimer it may have a dominant effect on the other wildtype portions of the complex, and a negative effect if the mutation prevents the complex from carrying out its normal function. Thus, especially in the case of tumor markers of the present invention which homomultimerize, such a dominant- negative form can specifically block the action of the wild-type tumor-marker from which it was derived. Tests to identify dominant negative variants include appropriate genetic screenings, for instance readout-systems based on the expression of nucleic acids comprising the nucleotide sequence as indicated in section D) of Table 1, 2, 3 or 4 and/or functional assays of the proteins and or polypeptides of the invention comprising the amino acid sequence as indicated in section E) of Table 1, 2, 3 or 4 or derivatives thereof.

The term "miR A specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4" refers to a short single-stranded R A molecule of typically 18-27 nucleotides in length, which regulate gene expression of one or more of the tumor marker according to Table 1, 2, 3 or 4. miR As are encoded by genes from whose DNA they are transcribed but are not translated into a protein. In a natural context miRNAs are first transcribed as primary transcripts or pri-miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). After integration into an active RISC complex, miRNAs may base pair with their complementary mRNA molecules and inhibit translation or may induce mRNA degradation by the catalytically active members of the RISC complex, e.g. argonaute proteins. Mature miRNA molecules are typically at least partially

complementary to mRNA molecules corresponding to the expression product of the present invention, and fully or partially down-regulate gene expression. Preferably, miRNAs according to the present invention may be 100% complementary to their target sequences. Alternatively, they may have 1, 2 or 3 mismatches, e.g. at the terminal residues or in the central portion of the molecule. miRNA molecules according to the present invention may have a length of between about 18 to 27 nucleotides, e.g. 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides. Preferred are 21 to 23 mers. miRNAs having 100% complementarity may preferably be used for the degradation of nucleic acids according to the present invention, whereas miRNAs showing less than 100% complementarity may preferably be used for the blocking of translational processes.

The term " antisense molecule of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4" refers to nucleic acids corresponding to the sequences indicated in section D) of Table 1, 2, 3 or 4 or the

complementary strand thereof. Preferably, the antisense molecule of the invention comprises a sequence complementary to at least a portion of a tumor marker expression product according to the present invention. While antisense molecules complementary to the coding region sequence of tumor marker expression products may be used, those complementary to the transcribed and untranslated region are preferred. Generally, antisense technology can be used to control, i.e. reduce or abolish gene expression through antisense DNA or RNA, or through triple-helix formation. In one embodiment, an antisense molecule may be generated internally by the organism, for example intracellularly by transcription from an exogenous sequence. A vector or a portion thereof may be transcribed, producing an antisense nucleic acid of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense molecule.

Corresponding vectors can be constructed by recombinant DNA technology methods known to the person skilled in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells, e.g. vectors as defined herein above.

In another embodiment, the antisense molecule may be separately administered. As an example, the 5' coding portion of a nucleic acid according to the present invention, e.g. of the sequence indicated in section D) of Table 1, 2, 3 or 4 may be used to design an antisense R A or DNA oligonucleotide of from about 6 to 50 nucleotides in length. Preferably, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides in length.

The antisense nucleic acids of the invention typically comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion of an RNA transcript" as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex or triplex formation in the case of double stranded antisense nucleic acids. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex or triplex. A person skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Preferably antisense molecules complementary to the 5' end of the transcript, e.g., the 5' untranslated sequence up to and including the AUG initiation codon may be used in for the inhibition of translation. In a further preferred embodiment, sequences

complementary to the 3' untranslated sequences of mRNAs may also be used.

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. g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane or the blood- brain barrier hybridization triggered cleavage agents or intercalating agents. The molecule may accordingly be conjugated to another molecule, e. g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense molecule or antisense oligonucleotide, miR A- or siR A molecule, may comprise at least one modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethyl-aminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methyl guanine, 3 -methyl cytosine, 5-methylcytosine, N6-adenine, 7- methyl guanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, and 2,6-diaminopurine. The molecule may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2- fluoroarabinose, xylulose, and hexose. In another embodiment, the molecule comprises alternatively or additionally at least one modified phosphate backbone, e.g. a

phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In another embodiment, the antisense molecule, e.g. the antisense oligonucleotide may be an alpha-anomeric oligonucleotide, i.e. an oligonucleotide which forms specific double-stranded hybrids with complementary RNA in which the strands run parallel to each other.

The term "siRNA specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4" refers to a particular type of antisense-molecules, namely small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway to negatively regulate gene expression of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. These siRNA molecules can vary in length and may be between about 18-28 nucleotides in length, e.g. have a length of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides. Preferably, the molecule has a length of 21, 22 or 23 nucleotides. The siRNA molecule according to the present invention may contain varying degrees of complementarity to their target mRNA, preferably in the antisense strand. siRNAs may have unpaired overhanging bases on the 5' or 3' end of the sense strand and/or the antisense strand. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region. Preferably the siRNA may be double-stranded wherein the double-stranded siRNA molecule comprises a first and a second strand, each strand of the siRNA molecule is about 18 to about 23 nucleotides in length, the first strand of the siRNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA via RNA interference, and the second strand of said siRNA molecule comprises nucleotide sequence that is complementary to the first strand.

Methods for designing suitable siRNAs directed to a given target nucleic acid are known to person skilled in the art. Furthermore, antagonistic siRNA molecules may be obtained according to methods of identifying antagonists as described herein.

The term "aptamer specific for the expression product or specific for the protein of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4" as used herein refers to (a) short peptide(s) capable of interacting and specifically binding the protein(s) of the tumor marker according to Table 1 , 2, 3 or 4. The peptide aptamer(s) may preferably be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (the) amino acid sequence(s) as indicated in section E) of Table 1, 2, 3 or 4. The peptide aptamer(s) may also be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (an) amino acid sequence(s) encoded by (a) DNA sequence(s) being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as indicated in section D) of Table 1, 2, 3 or 4, or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%>, 85%, 90%>, 91%>, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as indicated in section E) of Table 1, 2, 3 or 4. Typically, (a) peptide aptamer(s) is/are a variable peptide loop, comprising for example, 10 to 20 amino acids. In the context of the present invention the peptide aptamer(s) may preferably be attached at one or both ends to a scaffold structure. The scaffold structure may be any molecule, preferably a protein, which has good solubility properties. Suitable scaffold molecules would be known to the person skilled in the art. A preferred scaffold molecule to be used in the context of the present invention is the bacterial protein thioredoxin-A. The aptamer peptide loop may preferably be inserted within a reducing active site of the scaffold molecule. Alternatively, staphylococcal protein A and domains thereof and derivatives of these domains, such as protein Z or lipocalins may be used as scaffold structures in the context of the present invention. Peptide aptamers may be generated according to any suitable method known to the person skilled in the art, e.g. via yeast two-hybrid approaches.

In a preferred embodiment the above mentioned peptide aptamer is capable to bind to a protein or polypeptide of the invention corresponding to the sequences indicated in section E) of Table 1, 2, 3 or 4 and to reduce the biological activity and/or the enzymatic activity of these/this protein(s) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or by at least 98% or 99% when compared to a control level obtained from an untreated sample.

A "small molecule capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4" as used herein refers to a small organic compound that is preferably biologically active,

1. e. a biomolecule, but is preferably not a polymer. Such an organic compound may have any suitable form or chemical property. The compound may be a natural compound, e.g. a secondary metabolite or an artificial compound, which has been designed and generated de novo. In an embodiment of the present invention a small molecule is capable of blocking the interaction between the tumor marker protein(s) and its interactor(s). Methods and techniques for the identification and preparation of small molecules as well as assays for the testing of small molecules are known to the person skilled in the art.

The term "peptidomimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 ,

2, 3 or 4" in the context of the present invention refers to a small protein-like chain designed to mimic a peptide and capable of binding (a) protein(s) of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Such a peptidomimetic may arise from a modification of an existing peptide, e.g. a peptide or peptide aptamer as defined herein above, in order to alter the molecule's properties. A peptidomimetic may arise from a modification which changes the molecule's stability or binding capability. These modifications typically involve changes to the peptide that will not occur naturally. For example, a peptidomimetic according to the present invention may have altered peptide backbones or may comprise non-natural amino acids. Methods and techniques for the preparation of peptidomimetics as well as assays for the testing of peptidomimetics are known to the person skilled in the art.

A pharmaceutical composition according to the present invention may also comprise an antibody specific for the protein(s) of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or an antibody variant specific for said protein, e.g. an antibody or antibody variant as defined herein above.

In a preferred embodiment such an antibody or antibody fragment may be capable of inhibiting the biological activity and/or enzymatic activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4.

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. Thus, in a specific embodiment of the present invention the antibody or fragment thereof as defined herein above may be conjugated to a therapeutic or cytotoxic agent. The term

"therapeutic agent" refers to any compound, drug, small molecule or medicament, which is able to confer a therapeutic effect to a cell, a tissue or the entire organism. Examples of such agents are known to the person skilled in the art. The term "cytotoxic agent" refers to any compound, drug, small molecule which is able to confer a toxic effect to a cell or a tissue. Such agents may, for example, comprise compounds which activate endogenous cytotoxic effector systems, as well as radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. The term may also include radioisotopes known in the art, additional antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, R Ase, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. The term also refers to cytotoxic produgs. By "cytotoxic prodrug" is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the invention include glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubicin, and phenoxyacetamide derivatives of doxorubicin.

In a preferred embodiment of the present invention the pharmaceutical composition may comprise, or may additionally comprise, an antibody or a group of antibodies specific for the expression product or protein of a tumor marker or group of tumor markers as defined herein, e.g. specific for one or more of the protein(s) of the tumor marker according to Table 1, 2, 3 or 4. Particularly preferred is an antibody which specifically binds to an expression product, protein or peptide comprising the amino acid sequence as indicated in section E) of Table 1, 2, 3 or 4, or a fragment of said amino acid sequence, e.g. a peptide of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids length.

In a another aspect the present invention relates to a pharmaceutical composition for use in, or for the treatment or prevention of a 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 above, comprising at least one element selected from the group of: (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (e) a miR A specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (g) a siRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (h) an aptamer specific for the expression product of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (i) a small molecule or peptido mimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; and (j) an antibody specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or an antibody variant specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Such pharmaceutical composition preferably comprises elements as defined herein above.

In another aspect of the present invention relates to a pharmaceutical comprising at least one element selected from the group of: (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and (e) a miR A inhibitor specific for a miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4.

The term "a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4" as used herein refers to a compound which is capable of increasing the activity of one or more of the tumor marker according to Table 1, 2, 3 or 4. Such a compound may be any direct interactor of the tumor marker according to Table 1, 2, 3 or 4, which has positive influence on the catalytic activity of the tumor marker according to Table 1, 2, 3 or 4. Such a compound may preferably be an agonist of the catalytic activity of the tumor marker according to Table 1, 2, 3 or 4.

The term "a compound indirectly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4" as used herein refers to a compound which is capable of increasing the activity of the tumor marker according to Table 1, 2, 3 or 4 by an interaction with a direct interactor of the tumor marker according to Table 1, 2, 3 or 4 ("indirect interactor") or via an indirect working pathway not involving an interaction with of the tumor marker according to Table 1, 2, 3 or 4. Such a compound may be any direct interactor of an interactor of the tumor marker according to Table 1, 2, 3 or 4, e.g. of the protein(s) of a tumor marker or group of tumor markers according to section E) of Table 1, 2, 3 or 4. The effect conveyed by the direct interactor of an interactor of the tumor marker according to Table 1, 2, 3 or 4 may be either positive if the interactor of the tumor marker according to Table 1, 2, 3 or 4 itself has a positive effect on the activity of the tumor marker according to Table 1, 2, 3 or 4, or negative, if the interactor of the tumor marker according to Table 1, 2, 3 or 4 has a negative effect on the activity of the tumor marker according to Table 1, 2, 3 or 4.

Alternatively, such positively working indirect integrators may provoke a modification of the binding behavior of directly binding proteins, leading to an increased activity of the tumor marker according to Table 1, 2, 3 or 4. Typically negatively working indirect interactors may have an inhibitory effect on inhibitors of the tumor marker according to Table 1, 2, 3 or 4. Examples of such interactors are enzymatic activities degrading inhibitors of the tumor marker according to Table 1, 2, 3 or 4, or proteins capable of binding and quenching inhibitors of the tumor marker according to Table 1, 2, 3 or 4. Alternatively, such interactors may inhibit activities leading to a degradation of the tumor marker according to Table 1, 2, 3 or 4, e.g. proteinase inhibitors. Further examples and their implementation would be known to the person skilled in the art.

Alternatively, an indirect stimulation of the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be conveyed by compounds activating, protecting or sustaining the expression of the

endogenous gene(s) of the tumor marker according to Table 1, 2, 3 or 4. Examples of such compounds are specific transcription factors of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, specific stabilizing activities of the mR A(s) of the the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or splice factors specific for the the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Further examples and their implementation would be known to the person skilled in the art.

The "protein of a tumor marker" comprised in the pharmaceutical composition may be a protein or peptide of the tumor marker according to Table 1, 2, 3 or 4 as defined herein above. In particular, it may be a protein or peptide being encoded by splice variant of the tumor marker according to Table 1, 2, 3 or 4. More preferably it may have the amino acid sequence as set forth in section E) of Table 1, 2, 3 or 4. The "protein of a tumor marker " as used in this context also comprises amino acid sequences being at least 60%, 70%>, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences as set forth in section E) of Table 1, 2, 3 or 4 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, 3 or 4. In a further embodiment of the invention the homologous variants of the tumor marker according to Table 1, 2, 3 or 4 may additionally or alternatively have a similar or identical localization pattern as the tumor marker according to Table 1, 2, 3 or 4 within a cell or within a tissue type.

The term "biologically active equivalent of a tumor marker" as used herein refers to a protein of the tumor marker according to Table 1, 2, 3 or 4 which is capable of performing all or a majority of the individual functions of the tumor marker according to Table 1, 2, 3 or 4. In a further embodiment of the invention the biologically active equivalents of the tumor marker according to Table 1, 2, 3 or 4 may additionally or alternatively have a similar or identical localization pattern as the tumor marker according to Table 1, 2, 3 or 4 within a cell or within a tissue type. Biologically active equivalents of the tumor marker according to Table 1, 2, 3 or 4 may also comprise variants of the tumor marker according to Table 1, 2, 3 or 4 as defined herein above.

The proteins of the tumor marker according to Table 1, 2, 3 or 4 or

biologically active equivalents of the tumor marker according to Table 1, 2, 3 or 4 according to the present invention may be produced recombinantly by any suitable method known to the person skilled in the art. The present invention, thus, also encompasses methods for the production of the tumor marker proteins according to Table 1, 2, 3 or 4 or biologically active equivalents of the tumor markers according to Table 1, 2, 3 or 4.

Accordingly, the present invention contemplates vectors containing the polynucleotides encoding the tumor markers according to Table 1, 2, 3 or 4 or biologically active equivalents of the tumor markers according to Table 1, 2, 3 or 4 as defined herein above, host cells, and the production of the tumor markers according to Table 1, 2, 3 or 4 or biologically active equivalents of the tumor markers according to Table 1, 2, 3 or 4 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, 3 or 4.

Polynucleotides encoding the tumor markers according to Table 1, 2, 3 or 4 or biologically active equivalents of the tumor markers according to Table 1, 2, 3 or 4 may be joined to a vector or carrier containing a selectable marker for propagation in a host. A corresponding polynucleotide insert may be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, or the PSA promoter. Other suitable promoters are known to the person skilled in the art. The expression constructs may further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated. The polypeptides or proteins may be glycosylated or may be non-glycosylated or may otherwise by modified. In addition, polypeptides or proteins may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Furthermore, the polypeptide, protein or peptide may be modified by acetylation, pegylation, hesylation, formylation, phosphorylation, amidation, derivatization by known

protecting/blocking groups, specific chemical cleavage, proteolytic cleavage, a linkage to a cellular ligand or other protein or hapylation, i.e. a fusion with a glycine-rich homo-amino- acid polymer (HAP), etc. Such modifications may be carried out by suitable techniques known to the person skilled in the art. Additionally, the polypeptide, peptide or variant may contain one or more non-classical amino acids.

In addition, the tumor marker proteins according to Table 1, 2, 3 or 4 or biologically active equivalents of the tumor markers according to Table 1, 2, 3 or 4 of the invention can be chemically synthesized using techniques known in the art, e.g. by using a peptide synthesizer.

The "nucleic acid encoding and expressing the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4" comprised in the pharmaceutical composition as defined herein above refers to any suitable carrier element, e.g. as described herein above, comprising an expressable gene of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Preferably, such a carrier element may comprise the sequence(s) as indicated in section E) of Table 1, 2, 3 or 4. Such a carrier element may also comprises nucleotide sequences showing a high degree of homology to the tumor markers according to Table 1, 2, 3 or 4, e.g. nucleic acid sequences being at least 60%, 70%, 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, 3 or 4 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) of Table 1, 2, 3 or 4. Alternatively, the carrier may comprise the genomic sequence of the tumor marker according to Table 1, 2, 3 or 4.

Furthermore, biologically active equivalents of the tumor markers according to Table 1, 2, 3 or 4 as defined herein above may be comprised in a carrier of the present invention.

The polynucleotide encoding the tumor marker according to Table 1, 2, 3 or 4 may preferably be joined to a vector containing a selectable marker for propagation in a human cell. In a preferred embodiment the polynucleotide insert may be operatively linked to a PSA promoter.

In one embodiment of the present invention nucleic acids encoding and expressing the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 may be provided via living therapeutics. The term "living therapeutic" means that the tumor markers according to Table 1, 2, 3 or 4 or biologically active equivalents of the tumor markers according to Table 1, 2, 3 or 4 as defined herein above are expressed in any suitable live carrier. Accordingly, the present invention relates to corresponding polynucleotides which are suitable for expression in a living cell. The present invention also relates to vectors containing such polynucleotides, appropriate host cells, and the production of polypeptides by recombinant techniques in said host cells.

The term "live carrier" relates to any appropriate living host cell or virus known to the person skilled in the art. Representative examples of appropriate hosts include, but are not limited to, bacterial cells such as Escherichia coli or Lactobacillus, fungal cells, such as yeast cells, protozoa, insect cells, or animal cells. Preferably, the term relates to attenuated bacteria, attenuated fungal cells or attenuated protozoa. Representative examples of appropriate viruses include viruses of the group of adenoviruses, retrovirues or

lentiviruses, preferably attenuated viruses of the group of adenoviruses, retroviruses or lentiviruses. In a preferred embodiment, probiotic bacterial cells, in particular probiotic Escherichia coli or Lactobacillus cells may be used. More preferably, cells of Escherichia coli Nissle 1973 and even more preferably cells of Lactobacillus casei or Lactobacillus zeae 393 may be used.

The "miRNA inhibitor specific for miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4" comprised in the pharmaceutical composition as defined herein above refers to a nucleic acid molecule encoding a nucleic acid sequence complementary to a miRNA or microRNA molecule of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4. The term "complementary" as used herein refers to a perfect complementary between the miRNA inhibitor nucleic acid (sense molecule) and the miRNA (antisense molecule) without any mismatch, as well as situations in which the nucleic acid contains any base mismatches and/or additional or missing nucleotides in comparison to the miRNA molecule. In other embodiments, the two molecules comprise one or more base mismatches or differ in their total numbers of nucleotides (due to additions or deletions). In further embodiments, the "complementary" miRNA inhibitor nucleic acid molecule comprises at least ten contiguous nucleotides showing perfect complementarity with a sequence comprised in the miRNA molecule.

Typically miRNA inhibitor nucleic acid molecules are naturally occurring DNA- or RNA-molecules or synthetic nucleic acid molecules comprising in their sequence one or more modified nucleotides which may be of the same type or of one or more different types.

It is, for example, envisaged by the present invention that such a miRNA inhibitor nucleic acid molecule comprises at least one ribonucleotide backbone unit and at least one deoxyribonucleotide backbone unit. Furthermore, the miRNA inhibitor nucleic acid molecule may contain one or more modifications of the RNA backbone into 2'-0-methyl group or 2'-0-methoxyethyl group (also referred to as "2'-0-methylation"), which prevented nuclease degradation in the culture media and, importantly, also prevented endonucleolytic cleavage by the RNA-induced silencing complex nuclease, leading to irreversible inhibition of the miRNA. Another possible modification, which is functionally equivalent to 2'-0- methylation, involves locked nucleic acids (LNAs) representing nucleic acid analogs containing one or more LNA nucleotide monomers, as defined herein above.

Another class of silencers of miRNA expression to be used in the context of the present invention comprises chemically engineered oligonucleotides named "antagomirs", which represent single-stranded RNA molecules conjugated to cholesterol. The molecules may comprise between 19 and 25 nucleotides. Preferably, the molecule comprises 20, 21, 22, 23 or 24 nucleotides. More preferably, the molecule comprises 23 nucleotides.

In another embodiment of the present invention miRNA inhibitors as defined herein above may be provided in the form of expression vectors to be introduced into tissue or cells. Alternatively, such vectors may also be introduced in living therapeutics as defined herein above.

Typically, RNAs may be produced from transgenes provided in the form of trans fection or transient expression vectors or carriers. For instance, competitive miRNA inhibitors may be provided as transcripts expressed from strong promoters, containing more than one, preferably multiple, tandem binding sites to a microRNA of interest.

In a specific embodiment of the present invention a demethylation agent may be comprised in the pharmaceutical composition according to the present invention. The term "demethylation agent" as used herein refers to an agent capable of demethylating chromatine structures, preferably promoter regions, more preferably the promoter(s) of the tumor marker according to Table 1, 2, 3 or 4. Examples of demethylation agents to be used in the context of the present invention are 5-aza-2'-deoxycytidine and 5-azacytidine, which reactivate genes inappropriately silenced by structural chromatin changes that involve DNA methylation and which can reverse these changes and, therefore, restore principal cellular pathways. This typically results in gene re-expression and reversion of some aspects of the transformed state. 5-azacytidine and 5-aza-2'-deoxycytidine typically inactivate DNA cytosine C5- methyltransferases through the formation of stable complexes between the 5-aza-2'- deoxycytidine residues in DNA and the enzyme, thereby mimicking a stable transition state intermediate when bound to the methyltransferase enzyme.

A further agent, which may be comprised in a pharmaceutical composition according to the present invention, either per se or in combination with 5-aza-2'- deoxycytidine and/or 5-azacytidine, is trichostatin A (TSA).

In a another aspect the present invention relates to a pharmaceutical composition for use in, or for the treatment or prevention of a 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 tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and (e) a miRNA inhibitor specific for a miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Such

pharmaceutical composition preferably comprises elements as defined herein above.

In an preferred embodiment of the invention a pharmaceutical composition according to the present invention, e.g. as defined herein above, may further comprise additional compounds being active against cancer cells. Furthermore, in another embodiment of the invention the pharmaceutical composition may further comprise hormone-inhibitors, preferably anti-androgens or androgen antagonists like spironolactone, cyproterone acetate, flutamide, nilutamide, bicalutamide, ketoconazole, finasteride or dutasteride. The skilled person is aware of the fact that also a situation in which a given tumor marker of the invention is up-regulated in a cancer diasease while in parallel another tumor marker of the invention is down-regulated. It is thus an aim of the present invention to provide also pharmaceutical compositions wherein the pharmaceutical composition contains any combination of such elements as laid out above, e.g. the compounds, proteins, dominant negative proteins, nucleic acids, miR As, siR As, 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, 3 or 4 and up-regulating at least one other tumor marker according to Table 1, 2, 3 or 4. It is preferred to avoid conflicting and/or opposite functionalities or overlapping functional spectra of the elements of the pharmaceutical compositions as defined herein above, e.g. if the tumor markers have similar functionalities. Due to the different identity of the tumor marker, in such situations the use of highly specific elements like antibodies, siRNAs etc. is envisaged.

In another preferred embodiment of the invention the pharmaceutical relates to the pharmaceutical compositions as laid out in the present description for the treatment of cancer, particularly prostate cancer.

In a particularly preferred embodiment of the present invention the above described pharmaceutical compositions is for the manufacture of a medicament for the treatment of cancer, particularly prostate cancer.

In a most preferred embodiment of the present invention the pharmaceutical compositions and medicaments of the present invention are capable of reducing the tumor volume of a given prostate carcinoma by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), 90%), or at least 95% when compared to an untreated control.

In a further embodiment the present invention also envisages screening procedures and methods for the identification of an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, a compound directly stimulating or modulating the activity of the tumor marker according to Table 1, 2, 3 or 4, an agonist of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 enzymatic activity, a miRNA inhibitor specific for miRNA(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, an antagomir, a demethylation agent specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, or a peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. Such screening procedures may comprise the steps of (a) producing cells which express the tumor marker or group of tumor markers according to Table 1, 2, 3 or 4 as a polypeptide either as secreted protein or on the cell membrane or as intracellular component, (b) contacting the polypeptide produced in step (a) with a test sample potentially containing an interacting molecule, e.g. an aptamer specific for the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, a compound directly

stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, an agonist of enzymatic activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, a peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, and (c)

an interacting molecule by observing binding and/or inhibition or modulation of the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4.

Alternatively, such screening procedures may comprise the steps of (a) contacting a test sample potentially containing a directly or indirectly interacting molecule, e.g. an aptamer specific for the transcript(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, a miR A inhibitor specific for miRNA(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, an antagomir, a demethylation agent specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, a peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 with one or more cells which express the tumor marker according to Table 1, 2, 3 or 4 as (a) transcript(s), (b) detecting the expression level of said sequence; and (c) indentifying an interacting molecule by observing binding or a modulation or reduction of the expression level of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4.

The present invention also encompasses an aptamer specific for the expression product(s) or protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, a compound directly stimulating or modulating the activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, an agonist of the enzymatic activity of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, a miR A inhibitor specific for miR A(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, an antagomir, a demethylation agent specific for of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and a peptidomimetic specific for the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, obtainable or obtained by a screening procedure or method as described herein above.

In a further aspect the present invention relates to a pharmaceutical composition as defined herein above for the treatment or prevention of cancer.

Further, in yet another aspect, the present invention relates to the use of (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4; (e) a miRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4; (g) a siRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (h) an aptamer specific for the expression product of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (i) a small molecule or peptidomimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; and/or j) an antibody specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or an antibody variant specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 for the preparation of a pharmaceutical composition for the treatment or prevention of cancer, preferably 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.

Further, in yet another aspect, the present invention relates to the use of (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and (e) a miR A inhibitor specific for a miR A of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 for the preparation of a

pharmaceutical composition for the treatment or prevention of cancer, preferably 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.

In another aspect 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 tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (e) a miRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (g) a siRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (h) an aptamer specific for the expression product of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4; (i) a small molecule or peptidomimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; and/or (j) an antibody specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or an antibody variant specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 to an individual, in particular to an individual suffering from cancer or being prognosticated to develop cancer.

In another aspect 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 tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an agonist of said tumor marker enzymatic activity; (b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and (e) a miRNA inhibitor specific for a miRNA of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2, 3 or 4 to an individual, in particular to an individual suffering from cancer or being prognosticated to develop cancer.

A pharmaceutical composition according to the present invention may be administered to a patient, subject or individual with the help of various delivery systems known to the person skilled in the art, e.g., via encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction may be topical, enteral or parenteral and may include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, inhalational, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e. g., oral mucosa, rectal and intestinal mucosa, etc.) or by inhalation and may be administered together with other biologically active agents. Administration can be systemic or local. A preferred method of local administration is by direct injection.

In another embodiment the pharmaceutical composition may be delivered directly to internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the site of interest. The pharmaceutical composition may also be administered to disease sites at the time of surgical intervention. In yet another embodiment, the composition can be delivered in a controlled release system.

Preferably the pharmaceutical composition is in a form, which is suitable for oral, local or systemic administration. In a preferred embodiment the pharmaceutical composition is administered locally, orally or systemically.

In a further embodiment the pharmaceutical composition comprises a therapeutically effective amount of the ingredients of the pharmaceutical composition of the present invention as defined herein above and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such a carrier is pharmaceutically acceptable, i.e. is non-toxic to a recipient at the dosage and concentration employed.

Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.

The pharmaceutical composition of the invention can be formulated as neutral or salt forms.

Preferably, the pharmaceutical composition may be administered directly or in combination with any suitable adjuvant known to the person skilled in the art. The composition of the present invention can be administered to an animal, preferably to a mammal. "Mammal" as used herein is intended to have the same meaning as commonly understood by one of ordinary skill in the art. Particularly, "mammal" encompasses human beings.

The term "administered" means administration of a therapeutically effective dose of the aforementioned composition. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered, preferably this effect is induction and enhancement of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

The concentration of the active ingredients or compounds of a pharmaceutical composition according to the present invention may be further adjusted to the intended dosage regimen, the intended usage duration, the exact amount and ratio of all ingredients of the composition and further factors and parameter known to the person skilled in the art.

The active agents or compounds according to the present invention may be administered alone or in combination with other treatments. In a preferred embodiment the pharmaceutical composition of the present invention may be administered in combination with an anti-hormone treatment, e.g. an anti-androgen treatment.

The pharmaceutical composition of the present invention can also comprise any suitable preservative known to the person skilled in the art.

Furthermore, the preparations according to the invention may also comprise compounds, which have an antioxidative, free-radical scavenger, antierythematous, antiinflammatory or antiallergic action, in order to supplement or enhance their action.

In another preferred embodiment of the present invention active components of the pharmaceutical composition as defined herein above may be fused to a suitable carrier protein, e.g. to Ig Fc receptor proteins or polymeric Ig receptors. Preferably the protein(s) of the tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or biologically active equivalents thereof as defined herein above may be provided as fusion proteins. The fusion partner may be provided at the N- or C-terminus.

If the pharmaceutical composition according to the present invention is to be administered in the form of a live cell or living therapeutic as defined herein above, transformed and prepared cells may be administered to a patient in any suitable form known to the person skilled in the art. Preferably living therapeutics may be administered in the form of a composition comprising a microorganism, e.g. a Lactobacillus as described above, in an amount between 10^ to 1012 cells, preferably 10^ to 10^ cells.

In a further preferred embodiment of the present invention the ratio between two or more ingredients in the pharmaceutical composition or medicament may be suitably adjusted according to the skilled person's knowledge. Suitable assays may optionally be employed to help identify optimal ratios and/or dosage ranges for ingredients of pharmaceutical compositions of the present invention. The precise dose and the ratio between the ingredients of the pharmaceutical composition as defined herein above to be employed in the formulation will, inter alia, depend on the route of administration, and the exact type of disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses or ingredient ratios may be extrapolated from dose-response curves derived from in vitro or (animal) model test systems.

A typical dose can be, for example, in the range of 0.001 to 1000 μg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.

In another aspect the present invention relates to a medical kit for the treatment or prevention of cancer, comprising at least one element selected from the group consisting of (a) a compound directly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an antagonist of said tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (c) a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (e) a miR A specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (f) an antisense molecule of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (g) a siRNA specific for a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (h) an aptamer specific for the expression product of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (i) a small molecule or peptido mimetic capable of specifically binding to the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; and (j) an antibody specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and/or an antibody variant specific for the protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; or at least one element selected from the group consisting of (a) a compound directly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4, preferably an agonist of said tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4; (c) a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4 and (e) a miR A inhibitor specific for a miR A of a tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2, 3 or 4.

A medical kit that can be used in the context of the administration of the pharmaceutical composition as defined herein above. In particular, a kit according to the present invention may be used for the treatment or prevention of cancer.

The ingredients of a medical kit may, according to the present invention, be comprised in one or more containers or separate entities. They may preferably be formulated as pharmaceutical compositions or medicaments, more preferably they may be formulated as has been described herein above in the context of the pharmaceutical compositions of the present invention, e.g. they may comprise suitable pharmaceutical carriers etc. Particularly preferred are formulations for topical administration as mentioned herein above in the context of pharmaceutical compositions of the invention. The medical kit according to the present invention may optionally also comprise a documentation which indicates the use or employment of the medical kit and its components. Preferably, instructions comprised in the medical kit of the present invention may comprise recommended treatment options, dosage regimens etc. The medical kit may also comprise an instruction leaflet and/or may provide additional information on the use, dosage etc.

The medical kit of the present invention may be administered to a patient according to any suitable dosage regimen known to the person skilled in the art. The medical kit or kit components may preferably be given once a week, more preferably 2 times, 3 times, 4 times, 5 times or 6 times a week and most preferably daily and or 2 times a day or more often, unless otherwise indicated. During progression of the treatment the dosages may be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., several times a day. In a preferred case a response to the treatment may be monitored using herein described methods and further methods known to those skilled in the art and dosages may accordingly be optimized, e.g., in time, amount and/or composition. Progress can be monitored by periodic assessment. It is also envisaged that the medical kit is employed in co-therapy approaches, i.e. in co-administration with other medicaments or drugs, for example antibiotics, antiviral medicaments or IgG or IgA immunoglobulins, anticancer medicaments and, preferably, anti-hormone medicaments, more preferably anti- androgens as mentioned herein above.

In another aspect the present invention relates to a vaccine comprising the expression product or protein, or any fragment thereof, of an up-regulated tumor marker or group of tumor markers as defined herein, or as indicated in section I) of Table 2 or 3, or in section F) of Table 6.

Preferably, the vaccine may comprise a protein or antigen, having, comprising or consisting of an amino acid sequence as defined in section E) of Table 6, or any fragment, variant, derivative or modified form thereof, having a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 300, 350 or 400 amino acids or the entire length or any other suitable length.

Table 6:

D) E) F) G)

B) SEQ ID SEQ ID Up- Down-

A) Uniprot C) NO. NO. amino regulation regulation Index Accession Marker Function

nucleotide acid in Pool c2 in Pool c2 Code

sequence sequence vs. cl vs. cl

Keratin type I cyto skeletal

1 K1C10 267 268 +

10 -

Hypoxia up-regulated

2 HYOU1 159 160 +

protein 1 -

3 HBA Hemoglobin subunit alpha 287 288 + -

Vitamin K-dependent

4 PROC

protein C 475 476 + -

Plasma protease CI

5 IC1 569 570 +

inhibitor -

Vitamin D-binding protein

6 VTDB 49 50 +

precursor -

7 IGHA1 Ig alpha- 1 chain C region 535 536 + -

Procollagen C-

8 PCOC1 19 20 +

endopeptidase enhancer 1 - BST1 ADP-ribosyl cyclase 2 259 260 + -

G Protein Coupled Receptor

GRK7 849 850 + - Kinase 7

C07 Complement component C7 497 498 + -

ADIPO Adiponectin 215 216 + -

Neural cell adhesion

CHL1 239 240 + - molecule LI -like protein

TSP1 Thrombospondin- 1 471 472 + -

SAA4 Serum amyloid A-4 protein 203 204 + -

Monocyte differentiation

CD14 3 4 + - antigen CD 14

HEMO Hemopexin 623 624 + -

MMP2 72 kDa type IV collagenase 449 450 + -

CYTC Cystatin-C 65 66 + -

CXCL7 Platelet basic protein 607 608 + -

EGF-containing fibulin-like

FBLN3 extracellular matrix protein 149 150 + - 1

HBB Hemoglobin subunit beta 279 280 + -

APOA1 Apolipoprotein A-I 211 212 + -

IGHM Ig mu chain C region 219 220 + -

CADH1 Epithelial cadherin 269 270 + -

Ig heavy chain V-II region

HV201 173 174 +

OU -

FRIL Ferritin light chain 595 596 + -

EF2K Elongation factor 2 kinase 237 238 + -

DNA replication licensing

MCM2 43 44 +

factor MCM2 -

DHAK Dihydroxyacetone kinase 31 32 + -

PLD5 Inactive phospholipase D5 119 120 + -

Amiloride-sensitive amine

ABP1 291 292 +

oxidase [copper-containing] - Uncharacterized protein

CC058 369 370 +

C3orf58 -

Lysosome -associated

LAMP2 333 334 +

membrane glycoprotein 2 -

Ig kappa chain V-I region

KV121 553 554 +

Ni -

EGLN Endoglin 605 606 + -

Ig kappa chain V-I region

KV1 19 577 578 +

Wes -

CI 140 Protein C9orfl40 125 126 + -

DMP4 Dentin matrix protein 4 561 562 + -

ENPL Endoplasmin 593 594 + -

Proteasome subunit beta

PSB2 541 542 +

type-2 -

Divalent cation tolerant

CUTA 47 48 +

protein CUTA -

PROM1 Prominin-1 601 602 + -

ATL2 ADAMTS-like protein 2 619 620 + -

Keratin type I cuticular

K1H2 139 140 +

Ha2 -

HPT Haptoglobin 507 508 + -

STRN Striatin 441 442 + -

GRDN Girdin 551 552 + -

Purine nucleoside

PNPH 253 254 +

phosphorylase -

PLEK Pleckstrin 579 580 + -

Ankyrin repeat and

ANKF1 fibronectin type-Ill domain- 241 242 + - containing protein 1

Latent -transforming growth

LTBP2 factor beta-binding protein 479 480 + - 2

CH3L1 Chitinase-3-like protein 1 157 158 + -

Macrophage colony-

CSF1R 335 336 +

stimulating factor 1 receptor -

Beta- 1.4-

B4GT1 433 434 +

galactosyltransferase 1 -

Lipoxygenase homology

LOXH1 603 604 +

domain-containing protein 1 - Mediator of R A

57 MD13L polymerase II transcription 611 612 + - subunit 13 -like

58 A1AG2 Alpha- 1 -acid glycoprotein 2 521 522 +

Glycerol-3-phosphate

59 PCAT1 acyltransferase-like protein 255 256 + - 1

ATPase family AAA

60 ATD2B domain-containing protein 169 170 + - 2B

Armadillo repeat-containing

61 ARMX3 469 470 +

X-linked protein 3 -

62 LEGL Galectin-related protein 627 628 + -

HLA class I

63 1A68 histocompatibility antigen 565 566 + - A-68 alpha chain

Lys o somal -trafficking

64 LYST

regulator 523 524 + -

Eukaryotic translation

65 IF4G2 223 224 +

initiation factor 4 gamma 2 -

2.3 -bispho sphoglycerate

66 PMGE 377 378 +

mutase -

67 MFN1 Mitofusin-1 609 610 + -

68 DPEP1 Dipeptidase 1 205 206 + -

69 IGHG4 Ig gamma-4 chain C region 581 582 + -

Serine or threonine -protein

70 STK4 617 618 +

kinase 4 -

Asialoglycoprotein receptor

71 ASGR2 325 326 +

2 -

The vaccine may preferably comprise a nucleic acid molecule encoding a tumor marker of the present invention or a group of tumor markers as defined above, e.g. a nucleic acid molecule comprising a nucleotide sequence as indicated in section D) of Table 6, and/or a vector comprising said nucleic acid molecule, a host cell comprising said vector, an antibody as defined herein, or a CTL specific for an antigen as defined herein. Thus, a vaccine according to the present invention may, for example, comprise polypeptides or proteins of varying length comprising the or comprised in the amino acids as indicated in section E) of Table 6, or a nucleotide sequence encoding such a polypeptide, an expression vectors capable of expressing the polypeptide or comprising said nucleic acid or fragments thereof, e.g. DNA plasmid vectors, viral vectors etc., host cells expressing such a

polypeptide, preferably host cells expressing the polypeptide at the surface of the cell, or secrete the polypeptide. These components or ingredients may be present either separately or in combination or in any sub-grouping or sub-combination of the mentioned items. For example, one, two, three or more different tumor marker antigens or proteins of the present invention may be present either separately or in combination.

In a specific aspect the present invention relates to a vaccine for the treatment or prevention of a cancer disease associated with a progression from a less progressed stage of a cancer disease to a more progressed stage of a cancer disease, wherein said cancer disease implies the increased (up-regulated) expression of a tumor marker or group of tumor markers as defined above, or as derivable from section I) of Table 2 or 3 or from section F) of Table 6, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 6, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) of Table 6, or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) of Table 6, or any fragment thereof.

The expression product, polypeptide or antigen comprised in the vaccine may comprise one epitope or various epitopes, e.g. a MHC I and/or a MHC II epitope or two or more copies of each or combinations thereof. Corresponding, i.e. encoding nucleic acid molecules may be provided, preferably in the form of DNA or RNA molecules, e.g. DNA vectors or expression vectors. Such vectors may preferably be DNA plasmids or viral vectors. Vectors, in particular viral vectors may be capable of replication or replication- impaired or non-replicating. The term "non-replicating" or "replication-impaired" as used herein means not capable of replication to any significant extent in the majority of normal mammalian cells or normal human cells. Viruses which are non-replicating or replication- impaired may have become so naturally (i.e. they may be isolated as such from nature) or artificially e.g. by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication. Suitable viral vectors for use in a vaccine according to the present invention include non-replicating adenoviruses such as El deletion mutants, vectors based on herpes virus and Venezuelan equine encephalitis virus (VEE). Suitable bacterial vectors include recombinant BCG and recombinant Salmonella and Salmonella transformed with plasmid DNA. Alternative suitable non- viral vectors include lipid-tailed peptides known as lipopeptides, peptides fused to carrier proteins such as KLH either as fusion proteins or by chemical linkage.

In a preferred embodiment a vaccinia virus vector such as MVA or NYVAC may be used. Most preferred is the vaccinia strain modified virus ankara (MVA) or a strain derived therefrom. MVA is a replication impaired vaccinia strain with a good safety record. In most cell types and normal human tissues, MVA does not replicate; limited replication of MVA is observed in a few transformed cell types such as BHK21 cells. Alternatives to vaccinia vectors include pox virus vectors, e.g. avipox vectors such as fowl pox or canarypox vectors. Particularly suitable as an avipox vector is a strain of canarypox known as ALVAC, and strains derived therefrom.

The term "CTL" as used herein means "cytotoxic T lymphocyte" and refers in particular to a CD4+ T lymphocyte, a CD8+ T lymphocyte or a natural killer cell. Preferably, the term refers to a CD8+ T lymphocyte or a natural killer cell. A CTL may be genertad according to any suitable approach for the generation of a CTL, which specifically detects an antigen according to the present invention, preferably a protein having or comprised in a sequence as indicted in section E) of Table 6. Subsequently, such a CTL may perform a cytotoxic reaction, e.g. via the release of the cytotoxins perforin and granulysin, or induce an appoptotic cell reaction, e.g. via the interaction with cell-surface molecules, e.g. Fas proteins expressed on a target cell. The making of CTLs typically comprises the presentation of antigens, preferably of MHC I specific antigenic peptides, to T cells, preferably to CD8+ T cells. Preferably, the antigen or antigenic peptide may be presented by a dendritic cell (DC), more preferably by a MHC I molecule present on a dendritic cell.

Positively reacting T lymphocytes may subsequently be selected, enriched and/or expanded according to suitable methods known to the person skilled in the art. T cells to be used for the production of CTLs according to the present invention may be derived from lymphoid tissue, preferably they may be obtained from a peripheral blood mononuclear cell (PBMC) cell fraction. Typically, PBMCs may be extracted from whole blood using ficoll. Alternatively, PBMC may be extracted from whole blood using a hypotonic lysis. Any other suitable method known to the person skilled in the art may also be used.

PBMCs to be used may be derived from blood obtained from blood donors. Alternatively, autologous PBMCs may be used. For the extraction of autologous cells any suitable method known to the person skilled in the art may be used.

In a specific embodiment the present invention relates to such CTLs, as well as to a method of making CTLs specific for the antigen or tumor marker expression product or protein, or group of tumor marker expression products or proteins of the present invention, e.g. comprising the amino acid sequence as indicated in section E) of Table 6, which comprises the step of stimulating autologous T cells in vitro with dendritic cells loaded with a peptide derived from the antigen or a fragment thereof. This procedure may be carried out according to any suitable procedure known in the art.

Typically, the following procedure may be used: non-adherent PBMCs (preferably in an amount of 2 x 10⁶ mL) may be co-cultered in suitable medium, e.g. in human serum, preferably in Aim-V medium supplemented with 10% pooled human serum, with mature dendritic cells preincubated with peptides derived from the antigen of the present invention. The co-cultivation may be carried out according to suitable parameters, e.g. for 7- 10 days. The preincubation with the peptides may also be carried out according to any suitable parameters known to the person skilled in the art. Preferably, the peptide may be used in a concentration of 50μg/ml. Subsequently, T cells may be collected and preferably restimulated with dendritic cells loaded with peptides derived from the antigen. The restimulation may preferably be carried out once in a week. Furthermore, the medium may be supplemented with additional factors, e.g. with IL-2, IL-7 and/or IL-15.

After a repetition of the stimulation cycle, preferably by 3 to 5 times T-cell lines may be established by limiting-dilutions. Subsequently, T-cell line clones may be expanded in T-cell medium comprising IL-2, IL-7 and/or IL-15, e.g. during 2 weeks. The CTLs may additionally be tested for their biological activity according to known methods, e.g. a cytotoxicity test.

Accordingly obtained CTLs may be stored or further expanded or be used for the preparation of medicaments of pharmaceutical compositions. Any suitable deviation from this protocol based on the knowledge of the skilled person is also envisaged by the present invention.

In a further preferred embodiment an epitope to be comprised in a vaccine may be of varying length. It may be a B-cell, T-cell, MHC I specific, or MHC II specific epitope. It may preferably have a length of about 8 to 10 amino acids in the case of a MHC I specific epitope e.g. 8, 9 or 10 amino acids, it may preferably have a length of about 13 to 17 amino acids in the case of a MHC II epitope, e.g. 13, 14, 15, 16 or 17 amino acids.

The epitope or antigen may be capable of eliciting B-cell or T-cell immune responses. The epitope or antigen may alternatively be capable of eliciting CTL or cytotoxic reactions. For a T-helper cell response, the presentation by MHC II molecules may be required. For T-cell response, the presentation by MHC I molecules may be required.

Antigens, peptides or epitopes as defined herein, may also stimulate NK-cells (natural killer cells) that are effective tumor killing cells and may be used for such an approach. Peptides or epitopes according to the present invention, e.g. as defined herein, may also stimulate dendritic cells for enhancing antigenic stimulation of lymphocytes and may be used for such an approach. The activation of cells, e.g. T-cells, NK cells or dendritic cells, may be tested with suitable tests known to the person skilled in the art. For the detection of T-cell activation an ELISPOT assay as known to the person skilled in the art may be used.

The epitopes of the present invention, e.g. fragments of proteins or peptides comprising or comprised in an amino acid sequence as indicated in section E) of Table 6, may be present in a peptide, polypeptide, protein, polyprotein or particle comprising one, two or more epitopes, or as a recombinant string of epitopes or in the context of the native target antigen, or in the form of a mixture or combination of the mentioned entities.

The term "polyprotein" refers to two or more proteins which may be the same, or preferably different, linked together. Particularly preferred in this embodiment is a recombinant proteinaceous particle such as a Ty virus-like particle (VLP).

The term "epitope string" as used herein refers to a juxtaposition or combination of one or more epitopes, e.g. of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more epitopes. Such a string may comprise solely MHC I specific epitopes or MHC II specific epitopes or a combination of both. The epitopes in a string of epitopes or of multiple epitopes may be linked together without intervening sequences so that unnecessary nucleic acid and/or amino acid material is avoided. Alternatively, the epitopes in a string of epitopes or of multiple epitopes may be linked together with intervening sequences. Such intervening sequences may have a length of 1 to 10 amino acids, preferably 2 to 5 amino acids. They may comprise amino acids without influence on the overall structure of the string, e.g. glycine.

In a further embodiment of the present invention also antigens or fragments of antigens may be presented in a string like manner, e.g. linked together. These strings may or may not have intervening sequences as mentioned above.

In addition the string of epitopes or antigens or multiple epitopes may include one or more epitopes recognised by T helper cells, to augment the immune response generated by the epitope or antigen string. Particularly suitable T helper cell epitopes are ones, which are active in individuals of different HLA types, for example T helper epitopes from tetanus (against which most individuals will already be primed). Particularly preferred is a combination of three T helper epitopes and an epitope according to the present invention. Additionally or alternatively, the epitope string may also include one or more B cell epitopes for stimulating B cell responses and antibody production. Suitable T helper and B cell epitopes are known to the person skilled in the art.

In a particularly preferred embodiment the present invention relates to a vaccine as defined herein, or a CTL as defined herein, for the treatment or prevention of a cancer disease, preferably 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.

If 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). Examples of such as TAAs which may be included in vaccines of the present invention are MAGE antigen, a SSX antigen family member, NY- ESO-1, Melan- A/MART- 1 , gplOO, tyrosinase, tyrosinase-related protein 1 (TRP1), TRP2, CEA, PSA, Her2/neu, p53, MUC1, PRAME, sarcosin (N-methylglycin), CA-125

(Carbophydrate antigen-125) or survivin. The sequence and identity of these and further suitable TAAs would be known to the person skilled in the art or can be derived from a suitable textbook.

In yet another preferred embodiment the present invention envisages a method of inducing an immune response in an individual comprising administering to said subject a therapeutically effective amount of an antigen, vector, epitope etc. as mentioned above. The term "immune response" refers to a therapeutic immune response beneficial for the subject or individual. Such an immune response may be a passive or active immunization, or it may be a short-term or long-term immunoprotection. Preferably the immune response is an immune response against a 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.

In a further, particularly preferred embodiment 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 tumor marker or group of tumor markers as described herein above, leads to, or is used for the preparation of a vaccine or of a method of vaccination comprising an antigen or epitope linked to the detected, increased tumor marker or group of tumor markers of the present invention. The present invention thus also encompasses a personalized vaccine or method for vaccination based on the outcome of a method of identifying an individual for eligibility for a 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 tumor marker or group of tumor markers encountered to be increased according to parameters as defined herein may be used in the form of a protein sequences, e.g. comprising or comprised in the amino acid sequence as indicated in sections E) of Table 6, 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 6. Such antigens or nucleic acid molecules may be provided in different suitable forms, e.g. as full length sequence, or in the form of fragments, in combination with one or more tumor marker s as defined herein, or in combination with known markers or TAA as described above.

A vaccine according to the present invention or vaccine compounds or ingredients as defined herein above may be given, e.g. in the course of a method for immunization or method of treatment of the present invention, once or more than one time, e.g. 2, 3, 4, 5, 6 or more times, preferably 2, 3 or 4 times according to any suitable vaccination scheme known in the art.

In case a vaccine or vaccine ingredient are given more than once a prime and boost administration may be pursued, e.g. a "prime" administration is followed by one or more "boosts" to achieve the desired effects. The same composition or vaccine ingredient can be administered as the prime and as the one or more boosts. Alternatively, different compositions or vaccine ingredients can be used for priming and boosting. Furthermore, priming and boosting compositions or vaccines may comprise different combinations of vaccine ingredients, e.g. first a combination of a DNA plasmid and peptide or polypeptide, followed a DNA plasmid or vice versa etc.

Furthermore, prime and boost compositions may be different in terms of vectors to be used. In a preferred example, the priming composition may be a viral vector and the boosting composition may also be a viral vector, however derived from a different virus. Alternatively, a prime composition may comprise a DNA or plasmid vector and the boost composition may comprise a viral vector, or vice versa. Further preferred are prime boost schemes in which at least one of the vectors is replication-impaired or non-replicating.

A preferred carrier for a vaccine is a molecule that does not itself induce the production of antibodies harmful to the individual receiving the vaccine. Suitable carriers are typically large, slowly metabolized macro molecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Furthermore, the antigen of the present invention, e.g. an antigen comprising or comprised in a tumor marker protein or expression product as defined in Section E) of Table 6 may be conjugated to carrier elements such as a bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc.

The vaccine used according to the invention may also be provided in frozen, freeze-dried or lyophilized form, which may be thawed, or reconstituted, respectively, when needed.

In a further embodiment of the present invention the vaccine may comprise or be mixed with at least one suitable adjuvant. Adjuvants which are preferred for vaccines comprise 1018 IS S, aluminium salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, Amplivax, AS 15, BCG, CP-870893, CpG7909, CyaA, dSLIM, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, SAF, RAS, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRLl 72, Virosomes and other Virus-like particles, YF-17DBCG, Aquila's QS21 stimulon, Detox Quil, Superfos, Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant, pertussis toxin (PT), E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S 109, and PT-K9/G129, MF59, or saponin adjuvants.

Adjuvants may be combined in any suitable form and amount with the pharmaceutical composition, kit or vaccine of the present invention. The use of an adjuvant may be adjusted to the concrete purpose of the treatment. Such a use may vary depending on the target cell or tissue, the administration way, treatment scheme etc.

A vaccine or immunological formulation may contain the immunogenic active substance at any suitable concentration, preferably at low concentrations, such as in an immunogenic amount ranging from 0.01 μg to 10 mg. Depending on the nature of the antigen, epitope, vector, vehicle, antibody etc. or on the presence of additional agents such a carriers or adjuvants, a suitable immunogenic dose may be chosen, e.g. in the range of from 0.01 μg to 750 μg, preferably 100 μg to 500 μg. In a further embodiment the vaccine according to the present invention may be provided in the form of a depot vaccine which is to be delivered to the organism over an extended period of time may. In such a case, the amount of ingredients may be higher such as from at least 1 mg to up to more than 10 mg. A vaccine usually may be provided, for example, in ready-to-use syringes having a volume of from 0.01 to 1 ml, preferably 0.1 to 0.75 ml, of the concentrated solution, or suspension, respectively.

Vaccines of the present invention may be administered to a subject or individual by any suitable method, preferably via injection using either a conventional syringe or a gene gun, such as the Accell® gene delivery system. Delivery of DNA into cells of the epidermis is particularly preferred as this mode of administration provides access to skin-associated lymphoid cells and provides for a transient presence of DNA in the recipient. Both, nucleic acids and/or peptides and/or antibodies can be injected either subcutaneously, epidermally, intradermally, intramucosally such as nasally, rectally and vaginally, intraperitoneally, intravenously, orally or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, needle-less injection,

transcutaneous and transdermal applications. If solids are employed as auxiliary agents for the vaccine formulation, e.g. an adsorbate or a suspended mixture of vaccine ingredient with the auxiliary agent is administered. In special embodiments, the vaccine is administered as a solution, or liquid vaccine, respectively, in an aqueous solvent.

As mentioned above, tumor markers as described herein may be over- expressed or down-regulated upon progression from a less progressed cancer stage to a more progressed cancer stage. Accordingly, pharmaceutically active agents that down-regulate or increase expression of such tumor markers can be used to specifically treat such cancers including prostate cancer. Pharmaceutical compositions comprising such active agents have been described in detail above. However, the knowledge of over-expression or down- regulation of tumor markers can also be used to screen molecular libraries of potentially pharmaceutically active agents for identifying pharmaceutically active agents that may be used to specifically treat or prevent the cancers described herein, such as prostate cancer.

The present invention in one embodiment thus relates to the use of a tumor marker or a group of tumor markers as described herein for identifying pharmaceutically active agents useful in the treatment or prevention of a cancer disease. In a preferred embodiment the present invention relates to the use of a tumor marker or a group of tumor markers as described herein for identifying pharmaceutically active agents useful in the treatment or prevention of prostate cancer.

The present invention thus e.g. also relates to a method for identifying pharmaceutically active agents which can be used to treat or prevent progression from a less progressed cancer stage to a more progressed cancer stage comprising at least the steps of: a) providing a cell;

b) determining expression of a tumor marker or a group of tumor markers as described herein in said cell;

c) subjecting said cell to at least one potentially pharmaceutically active agent;

d) determining expression of a tumor marker or a group of tumor markers as described herein in said cell which has been subjected to at least one potentially pharmaceutically active agent;

e) comparing the expression level of step b) and d);

f) classifying the at least one potentially pharmaceutically active agent as an agent that either increases or decreases expression of a tumor marker or a group of tumor markers as described herein.

In step a) one may use a cell obtained from a healthy human being of from a patient suffering from a disease such as a cancer. One may also use a cultured cell line. In a preferred embodiment, these cells and cell lines are outside the human body

In step c) one may subject such cells and cell lines to libraries of potentially pharmaceutically active agents. Such libraries may be small molecule libraries, aptamer libraries, antibody libraries, siR A libraries, peptide libraries and the like.

Determining expression levels in steps b) and d) may be undertaken as is common in the art.

The potentially pharmaceutically active agents can then be classified as an agent that either increases or decreases expression of a tumor marker or a group of tumor markers as described herein. Such agents may be used directly to treat or prevent progression from a less progressed cancer stage to a more progressed cancer stage or they may be used as hits or lead compounds for further drug development. The accordingly encountered active agents are also envisaged as part of the present invention.

An especially preferred embodiment the present invention relates to any of the 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 cancer is prostate cancer.

The term "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. Preferably, the term relates to the classification of prostate cancer pursuing the following T - Primary Prostate Tumor classification schedule:

TX. Primary tumor cannot be assessed

TO. No evidence of primary tumor

Tl . Clinically inapparent tumor not palpable or visible by imaging Tla. Tumor incidental histological finding in 5% or less of tissue resected Tib. Tumor incidental histological finding in more than 5% of tissue resected Tic. Tumor identified by needle biopsy (e.g., because of elevated PSA) T2. Tumor confined within prostate (Tumor found in one or both lobes by needle biopsy, but not palpable or visible by imaging, is classified as Tic) T2a. Tumor involves one half of one lobe or less

T2b. Tumor involves more than half of one lobe, but not both lobes

T2c. Tumor involves both lobes

T3. Tumor extends through the prostatic capsule (Invasion into the prostatic apex, or into (but not beyond) the prostate capsule, is not classified as T3, but as T2)

T3a. Extracapsular extension (unilateral or bilateral)

T3b. Tumor invades seminal vesicle(s)

T4. Tumor is fixed or invades adjacent structures other than seminal vesicles: bladder neck, external sphincter, rectum, levator muscles, or pelvic wall

Nl . Tumor invades regional lymph node(s)

Mia. Tumor invades non-regional lymph node(s)

Mlb. Tumor invades bone(s)

Mlc. Tumor invades other site(s)

G Histopathological Grading

GX. Grade cannot be assessed

Gl . Well differentiated (slight anaplasia) (Gleason 2-4)

G2. Moderately differentiated (moderate anaplasia) (Gleason 5-6) G3-4. Poorly differentiated/undifferentiated (marked anaplasia) (Gleason 7-

10),

wherein T categories are physical examination, imaging, endoscopy, biopsy, and biochemical tests , N categories are physical examination and imaging tests and M categories are physical examination, imaging, skeletal studies, and biochemical tests, and wherein stages I to IV of prostate cancer correspond to the following scheme:

Stage I: Tla; N0; M0; Gl

Stage II: Tla; NO; MO; G2, 3-4, or

Tib. c; NO; MO; any G, or

T1. T2; N0; MO; any G

Stage III T3; NO; MO; any G

Stage IV T4; NO; MO; any G, or

Any T; Nl; MO; any G, or

Any T; Any N; Ml; any G.

A "prostate cancer" as used herein may further have one of the following grade of Gleason score: Grade 1 (the cancerous prostate closely resembles normal prostate tissue. The glands are small, well- formed, and closely packed), Grade 2 (the tissue still has well- formed glands, but they are larger and have more tissue between them), Grade 3 (the tissue still has recognizable glands, but the cells are darker. At high magnification some of these cells have left the glands and are beginning to invade the surrounding tissue), Grade 4 (the tissue has few recognizable glands. Many cells are invading the surrounding tissue), Grade 5 the tissue does not have recognizable glands. There are often just sheets of cells throughout the surrounding tissue). The grading typically follows the Gleason grading as established by the ASCP.

Typically, prostate cancer is further linked to an elevated level of prostate- specific antigen (PSA). In one embodiment of the present invention the term "prostate cancer" relates to a cancer showing PSA levels above 4.0. In another embodiment the term relates to cancer showing PSA levels above 2.0. The term "PSA level" refers to the concentration of PSA in the blood in ng/ml.

In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the 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 a local prostate cancer with insignificant disease parameters. The term "insignificant disease parameters", as used herein, refers to a clinical tumor stage < T2 and a Gleason Score < 6 and a Prostate Cancer Volume < 0.5 ml.

The term "Prostate Cancer Volume" as used herein refers to a calculated or measured volume of a prostate tumor or prostate swelling, which may be based on the assessment of the extension of the tumor/s welling in three dimensions and a subsequent determination of the volume. For example, such an assessment may employ suitable morphometric reconstruction techniques, as known to the person skilled in the art. Preferably, such a measurement follows any suitable method of cancer volumetric calculations known to the person skilled in the art.

In a further preferred embodiment the present invention relates to any of the abovementioned tumor marker or group of tumor markers, the above mentioned

compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of prostate cancer is a local prostate cancer with significant disease parameters. The term "significant disease parameters", as used herein, refers to a clinical tumor stage > T2 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.

In a further preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned

compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is a local prostate cancer with insignificant disease parameters, as defined herein above and wherein said more progressed stage of prostate cancer is a local prostate cancer with significant disease parameters, as defined herein above.

compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of prostate cancer is a local prostate cancer with insignificant disease parameters 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.

In specific embodiments of the present invention the less progressed stage of prostate cancer may imply a Gleason score of 1, 2, 3, 4, 5, or 6, preferably 6. In further specific embodiments of the present invention the less progressed stage of prostate cancer may imply a stage of Tl, Tla, Tib, Tlc,T2, T2, T2a, T2b or T2c, preferably Tic or T2 (UICC 2002 classification).

In further specific embodiments of the present invention the less progressed stage of prostate cancer may imply a Gleason score of 1, 2, 3, 4, 5, or 6, preferably 6 and a stage of Tl, Tla, Tib, Tlc,T2, T2, T2a, T2b or T2c, preferably Tic or T2 (UICC 2002 classification). In further specific embodiments of the present invention the more progressed stage of prostate cancer may imply a Gleason score of 7, 8, 9 or 10, preferably 7. In further specific embodiments of the present invention the more progressed stage of prostate cancer may imply a stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification).

In specific embodiments of the present invention the more progressed stage of prostate cancer may imply a Gleason score of 7, 8, 9 or 10, preferably 7 and a stage of T2, T2a, T2b, T2c, preferably T2 (UICC 2002 classification).

In specific embodiments of the present invention 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, 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 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.

The following examples and figures are provided for illustrative purposes. It is thus understood that the example and figures are not to be construed as limiting. The skilled person in the art will clearly be able to envisage further modifications of the principles laid out herein.

EXAMPLES Example 1 -Preparation of samples

Clinical human plasma samples

The clinical samples were human plasma drawn and prepared under standardized conditions. Prepared plasma samples were stored in aliquots at -80°C until use.

The characteristics of the clinical samples were chosen such that they would support as much as possible the identification of diagnostic and prognostic biomarkers.

Four groups of unselected men between ages 50-70 had been selected: 1) controls (healthy), 2) men with a benign prostate disease, 3) men with localised prostate cancers, and 4) men with advanced prostate cancers. Group 1 men were selected based on very low PSA values (<0.5 ng/ml), presumably healthy (i.e., no cancer). Group 2 men were selected based on PSA levels >3 ng/ml, but negative biopsy ((i.e., presumably men with a benign prostate disease). Group 3 men were selected based on PSA levels >3 ng/ml and cancer positive biopsy and pre-treatment stages T2 or less. This group was further divided into group 3A -> men with very small cancers (cancer volume <0.5 ml), clinical stage <=T2 (i.e., presumably indolent prostate cancer), vs. group 3B -> men with bigger cancers (cancer volume >0.5 ml), clinical stage >T2 (i.e., presumably significant prostate cancer). Group 4 men were selected on signs of clinical advanced cancer disease (clinical stage >T3).

The Figures 1 to 6 summarize the characteristics of clinical and diagnostic parameters of the plasma samples.

Example 2 - Pooling and quantifying samples Pooling strategy of plasma samples

Since the proteomics standard approach has multiplexing capabilities up to 8 samples and consists of many fractionation steps, the pooling strategy was based on this. Eight pools were randomly constructed from the available samples:

Pool 1 (pool name: al) 15 individual samples, patient group 1 (healthy, controls)

Pool 2 (pool name: a2) 15 individual samples, patient group 1 (healthy, controls)

Pool 3 (pool name: bl) 15 individual samples, patient group 2 (benign prostate disease) Pool 4 (pool name: b2) 15 individual samples, patient group 2 (benign prostate disease) Pool 5 (pool name: cl) 15 individual samples, patient group 3 A (local prostate disease, insignificant disease) Pool 6 (pool name: c2): 15 individual samples, patient group 3B (local prostate disease, significant disease)

Pool 7 (pool name: dl): 15 individual samples, patient group 4 (advanced disease, PSA <10 ng/ml)

Pool 8 (pool name: d2): 15 individual samples, patient group 4 (advanced disease, PSA >10 ng/ml).

A corresponding list of clinical samples is provided in Fig. 1.

Pooling and quantifying samples

15 samples of pool 1 were selected following the list of the clinical samples

(Cambridge samples). Samples were retrieved from the -80°C freezer, thawed quickly and put on ice. 2 μΐ of sample were diluted with 98 μΐ of Milli-Q (dilution factor 50). The sample dilution was mixed and centrifuged. 300 μΐ of Bradford Reagent was pipetted into a new vial. 10 μΐ of the sample dilution were added to the 300 μΐ of Bradford reagent. After20 minutes the samples were measured. The protein concentration was approximately 1.5 mg/ml, so samples needed to be diluted 70 times. The samples were pooled by adding 68 μΐ of each of the 15 samples to one tube. The samples were mixed and spun (end volume was 1020 μΐ) and this procedure was repeated for pools 2-8. Finally, samples were stored at -80°C. Bradford Analysis

A 9000 μg/ml BSA Stock Solution was obtained by dissolving 10.87 mg of BSA (Bovine Serum Albumin) in 1.20777 ml H₂0.

The following standards were prepared from this BSA stock solution:

Cone. BSA Dilution Amount of the BSA Amount of Total Volume ^g/ml) factor Stock Solution (μΐ) H₂0 (μΐ) (μΐ)

100 90 10 890 900

250 36 25 875 900

500 18 50 850 900

750 12 75 825 900

1000 9 100 800 900

1500 6 150 750 900

10 μΐ of Standard were mixed with 300 μΐ of Bradford Reagent.

The standards were measured after 20 minutes of incubation. Concentration BSA Absorbance at 595 nm rc = 0.9972

0 0.000

100 0.057

250 0.154

500 0.306

750 0.501

1000 0.640

1500 0.914

All samples from all pools were measured individually.

Example 3 - Proteomics Workflows

A) One-dimensional Immunodepletion, four-dimensional LC-MALDI MS workflow

General protocol for immunodepletion applied to clinical samples

Four 0.22 μιη spin filters (Agilent Technologies) were washed with 100 μΐ water followed by centrifugation for 10 minutes at 10,000 rpm and 10°C. The samples from pools and human serum 4522 (Sigma) were thawed and kept on ice. The Human Serum was diluted 4 times with buffer A (Agilent Technologies) by mixing 400 μΐ HS with 1200 μΐ Buffer A. Pool 1 and Pool 2 were diluted four times with buffer A (Agilent) by mixing 120 μΐ Pool sample with 360 μΐ Buffer A. The HS and Pool 1 and 2 samples were filtered over a 0.22 μιη Spin filter at 10000 rpm and 4°C for 60 minutes. The samples were depleted using LC-3 Agilent HU-14 column. All unbound fractions (UF) and bound fractions (BF) were collected and stored at -80°C.

Desalting of samples with 5 filters

200 ml of a 100 mM TEAB solution (5 ml 1M TEAB were diluted with 45 ml

Milli-Q) were prepared. All UF samples were thawed (from -80°C to 4°C). 16 sample tubes of 15 ml were prepared and 4,5 ml 100 mM TEAB were added to each tube. The 16 sample tubes were then placed on ice. 4.5 ml sample were added to each tube. 2 tubes per pool were used. The tubes were then placed back on ice. The samples were filtrated over the 5K Spin Concentrators 4ml tubes (Agilent Technologies). Again 2 tubes per pool were used.

First step: 4 ml sample per tube. Centrifugation for 15 minutes with 4000 rpm at 4°C. 3 ml sample were added to the filter tube. Centrifugation for 12 minutes with 4000 rpm at 4°C. 2 ml sample were added to the filter tube. Centrifugation for 15 minutes with 4000 rpm at 4°C. 3 ml of 100 mM TEAB were added to the filter tube. Centrifugation for 20 minutes with 4000 rpm at 4°C. 3 ml 100 mM of TEAB were added to the filter tube.

Centrifugation for 20 minutes with 4000 rpm at 4°C. 3 ml of 100 mM TEAB were added to the filter tube. Centrifugation for 25 minutes with 4000 rpm at 4°C. Centrifugation for another 10 minutes with 4000 rpm at 4°C. The residue from the filter was pipetted to a new tube and the filter washed with TEAB. All UF samples and the filtrate samples were transferred to a -80°C freezer.

Reducing sample volumes with 5 filters to 150 μΐ.

All UF Pool samples were thawed (from -80°C to 4°C), mixed and centrifuged. 8 Millipore Biomax-5K-NMWL Membrane 0.5 ml filters were used in the following procedure. 500 μΐ sample were pipetted in the filter. Centrifugation for 10 minutes at 10000 rpm and 4°C. 300 μΐ sample were added in the filter. Centrifugation for 10 minutes at 10000 rpm and 4°C. 350 μΐ sample were added in the filter (last part of the sample).

Centrifugation for 15 minutes at 10000 rpm and 4°C. Centrifugation for 3 minutes at 10000 rpm and 4°C. Centrifugation samples 1, 2 and 4 for 2 minutes at 10000 rpm and 4°C. The residue (± 50 μΐ) was pipetted from the filter to a new tube and the filter washed with 2 x 50μ1 TEAB. All UF samples and all filtrate samples were stored at -80°C. The protein concentrations were determined using the Bradford assay as described earlier. For each sample 625 μg protein was needed. The volumes were adjusted such that this amount of protein was taken into analysis.

Denaturation / Alkylation / Digestion.

625 μg of each pool sample was taken into analysis and all samples were adjusted to 8 M urea by adding solid urea (± 0.48 mg urea per 1 μΐ sample). The sample volume needed was pipetted to the amount of ureum. The samples were mixed and spun. Note: due to the ureum the volume increases. The pH of each sample needed to be checked (needs to be around 8). A 500 mM TCEP solution (reducing reagent) was prepared. 2.5 μΐ of the 500 mM TCEP solution were added to each sample tube. The samples were mixed and centrifuged. The solutions were mixed for 1 hour at room temperature. A 200 mM IAA solution was prepared. 37 mg IAA were weighed for 1 ml. 20 μΐ of 200 mM IAA were added to each sample tube. The samples were mixed and spun and then left for 30 minutes at room temperature in the dark. 10 tubes of 5 μg Lys-C (Roche) were prepared and the Lys-C of every tube was dissolved in 50 μΐ H₂0. In total: 50 μg Lys-C in 500 μΐ H₂0. 62.5 μΐ of this Lys-C solution were added to each sample tube. Ratio Lys-C : Protein = 1 : 100. The samples were mixed and spun and then kept for 4 hours at 37°C in the dark.

For the trypsin digestion ureum concentration was reduced from 8 M to 2 M by diluting samples 4 times. 15 tubes of 20 μg trypsin were prepared. The trypsin of every tube was dissolved in 20 μΐ 100 mM TEAB. In total: 300 μg trypsin in 300 μΐ TEAB. 25 μΐ of this trypsin solution were added to each sample tube. Ratio Trypsin: Protein = 1 : 25. The samples were mixed and spun (overnight at 37°C).

Protocol for iTRAQ-8-plex labeling

Samples were dried using a speedvac. The samples were dissolved in 300 μΐ

1M TEAB (total sample volume app. 500 μΐ). Samples were sonicated for 5 minutes in an ultrasonic bath to dissolve all proteins. The iTRAQ-8-plex Kit (25 Units) was thawed. This kit contains 8 labels and every label consists in 25 units. 1 Unit is intended for 50 μg proteins. There is one label for each sample. Half of the label was used since samples contain 625 μg total protein.

The samples contain (in total) 500 μΐ 1 M TEAB and urea in water. The percentage organic phase in the sample must be greater than 70%. Therefore the sample must be diluted with IPA. An end volume of 1666 μΐ is necessary to reach 70% organic phase, starting from 500 μΐ. 1666 μΐ - 500 μΐ = 1166 μΐ IPA that had be added to the sample. Each label is dissolved in app. 558 μΐ IPA. The half of that was used (279 μΐ), so 887 μΐ pure IPA was added to the label. 100 μΐ IPA was used to wash all tubes, so there is only 787 μΐ IPA added to the label.

Sample pool 1 was pipetted into one 2 ml tube. Both tubes were rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 787 μΐ IPA were pipetted in a separate tube. 279 μΐ of label 113 were mixed with the 787 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.

Sample pool 2 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 787 μΐ IPA were pipetted in a separate tube. 279 μΐ of label 114 were mixed with the 787 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.

Sample pool 3 was pipetted into one 2 ml tube. Both tubes were rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 787 μΐ IPA were pipetted in a separate tube. 279 μΐ of label 115 were mixed with the 787 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.

Sample pool 4 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 792 μΐ IPA were pipetted in a separate tube. 274 μΐ of label 116 were mixed with the 792 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.

Sample pool 5 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 810 μΐ IPA were pipetted in a separate tube. 256 μΐ of label 117 were mixed with the 810 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.

Sample pool 6 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 740 μΐ IPA were pipetted in a separate tube. 326 μΐ of label 118 were mixed with the 740 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.

Sample pool 7 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 790 μΐ IPA were pipetted in a separate tube. 276 μΐ of label 119 were mixed with the 790 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun.

Sample pool 8 was pipetted into one 2 ml tube. The tube was rinsed with 100 μΐ IPA and this was added to the 2 ml tube. The volume was app. 600 μΐ. 823 μΐ IPA were pipetted in a separate tube. 243 μΐ of label 121 were mixed with the 823 μΐ IPA. The label/IPA mix was combined with the sample in the 2 ml tube. This sample was mixed very well and spun. The samples were incubated for 2 hours at room temperature. After two hours samples were placed in a -80°C freezer.

B) Isoelectric focusing of peptides

SCX-Clean up Analysis

To remove unbound iTRAQ-labels a SCX-clean up analysis was done before performing the IEF of peptides. SCX-Method: SCX PEPT CLEAN.M

Column: PolyLC in.

PolySulfoethyl A

200 x 2.1 mm, 5 μιη, 200 A

Buffers: Buffer A: 25 % ACN in H₂0, pH 2,5 (Formic acid, app. 2 ml per liter).

Buffer B: Buffer A + 1 M KC1 (74.5 gram in 1 liter).

Gradient program time (min). % B flow (ml/min) Max. Pressure (bar)

0 2.5 0.15

10 2.5 0.15

11 75 0.15

30 75 0.15

31 2.5 0.15

51 2.5 0.15

75 % Buffer B means 0.75 M KC1.

Samples were dissolved in 50 μΐ Buffer A. Volume was app. 140 μΐ. 100 μΐ Buffer A were added to reach a volume of 240 μΐ. 240 μΐ were injected. 1 -minute fractions were collected. SCX-fractions 20 through 32 (from 20 minutes to 33 minutes) were collected in one tube for IEF. Samples were stored in a -80°C freezer.

RPLC-Clean up

To remove the salts (introduced in the SCX clean-up step) a normal RPLC clean-up analysis needed to be done.

RPLC-Method: RP CLEAN 03.M

Column: Agilent P.N. 993967-902

Zorbax Eclipse XDB-C18 150 x 4.6 mm, 5 μιη

Buffer A: H₂0 + 0.1 % TFA.

Buffer B: ACN + 0.1 % TFA.

time (min). % B flow (ml/min) Pressure

0.00 5 1.00 200

7.00 5 1.00 200

7.10 75 1.00 200

15.00 75 1.00 200

15.10 5 1.00 200

22.00 5 1.00 200

SCX samples were thawed and speedvacced. Subsequently, samples were dissolved in buffer A., mixed and spun. The samples were sonicated for 5 minutes in an ultrasonic bath to dissolve all peptides. The samples were mixed and spun. 300 μΐ were injected. The fractions were collected in a well plate. An example of a chromatogram is shown in Figure 8. All samples were placed in a -80°C freezer.

Samples were dried separately in the speedvac. The samples were placed back into the -80°C freezer. Preparation of the Peptide Offgel stock solution 1.25X (for PEPTIDE IEF). 3 ml glycerol solution (50%) were added to an empty tube. 300 μΐ of the OFFGEL Ampholyte Buffer were added (pH 3-10), (refrigerator tube 1ml). Milli-Q was added to a total volume of 25 ml. This Peptide O.S.S. was separated into 7 tubes of 2,88 ml and 9 tubes of 0.48 ml and then stored at -20°C for max. 4 months.

120 μΐ H₂0 were added to one 480 μΐ Peptide Offgel Sample Solution (POSS) tube. This is the Peptide IPG Strip Rehydration Solution. 720 μΐ H₂0 were added to one 2880 μΐ POSS. tube. This is the Peptide Offgel Sample Solution.

The 3100 OFFGEL fractionator was prepared (24 strip, pH 3 - 10). The general protocol of the 3100 OFFGEL fractionator was followed. The Peptide Offgel stock solution was used (for PEPTIDE IEF).

The eight iTRAQ labeled samples were thawed. Each sample was dissolved in 300 μΐ POSS. Samples were sonicated for 5 minutes in an Ultrasonic bath to dissolve all peptides. All samples were mixed together in one sample tube (volume increases). All tubes were rinsed with 400 μΐ POSS (3600 μΐ - (8 * 300 μΐ) - increases volume). The sample was mixed well. 150 μΐ of the POSS were pipetted in each IEF fraction. 10 μΐ was used for directly testing the labeling. This 10 μΐ sample was diluted 4 times (30 μΐ Buffer A (H₂0 + 0.1 % TFA) is added) and analyzed by LC-MALDI. IEF of peptides was started using method OG24PE00 (Offgel 24 strip for peptides; 120 kVh, 8000 Volts, 50 μΑ, 200 mW, 100

All fractions were collected and placed in the -80°C freezer. To increase recovery the IEF was loaded for a second time with a new Peptide Offgel Stock Solution

(150 μΐ per fraction). IEF was started again and run it for app. 2 hours.

Start conditions: 8.50h, 50 μΑ, 100 V.

End conditions: 11.05h, 50 μΑ, 300 V.

Fractions collected together with the first fractions.

The fractions were dried in the speedvac and placed in the -80°C freezer.

High pH RP analysis.

As a second dimension separation reversed phase at high pH had to be performed:

RPLC-Method: RP PEPT HIGH PH JB.M

Column: Agilent P.N. 763973-902

Zorbax 300 Extend - CI 8

150 x 4.6 mm, 3.5 μιη

Buffers: Stock solution = 20 mM NH₄FA, pH 10 (lOx solution)

12.5 gram NH₄OH (28%-solution) + 900 ml H₂0 + 1.62 ml FA (99%- solution).

the pH was adjusted to 10,5 and the volume adjusted to 1 liter.

Buffer A: Stock solution was diluted lOx with H₂0.

Buffer B: Stock solution was diluted lOx with ACN.

Gradient: time (min). % B flow (ml/min) Pressure

0.00 5 0.70 200

3.00 5 0.70 200

30.00 60 0.70 200

32.00 60 0.70 200

33.00 90 0.70 200

38.00 90 0.70 200

38.10 5 0.70 200

45.00 5 0.70 200

Fraction Collector: time (min). Trigger Mode Timeslices

0.00 Off 1.00 Time-based 1.00

45.00 Off

Fraction Delay Volume

Above location = 7 mm

Column by Column

Collection Mode = Discrete fractions

Tray = "eppendorf 2.0 ml Tube Rack"

Plate 1, A1-A9, C1-C9, E1-E9 and Plate 3, A1-A9 were used

High pH Column and buffers were installed on LC 3.

Buffer A was purged for 5 minutes (5 ml/min).

Buffer B was purged for 5 minutes (5 ml/min).

The system was purged with column and start conditions (0.7 ml/min and 5% buffer B). Pressure = 106 bar.

The IEF fractions were dried in the speedvac for 16 hours

IEF 1 = fraction 1 and 2 together. 100 μΐ of buffer A were added to fraction 1.

The solution was mixed and spun. The sample was sonicated for 5 minutes in an ultrasonic bath to dissolve all peptides. The solution was mixed and spun. Fraction 1 was pipetted to fraction 2. The tube of fraction 1 was rinsed with 50 μΐ of buffer A. This wash fraction 1 was pipetted to fraction 2. Fraction 2 was mixed and spun. The sample was sonicated for 5 minutes in an ultrasonic bath to dissolve all peptides. The solution was mixed and spun. Fraction 2 was pipetted in the well plate of the autosampler l-A-5 (= 180 μΐ). The tube of fraction 2 was rinsed with 70 μΐ of buffer A. This wash fraction 2 was also pipetted in the Well Plate of the Autosampler l-A-5 (= 250 μΐ). The complete sample was injected. High pH fractions 1-35 were stored in the -80°C freezer. The other samples were treated in the same way as IEF 1 :

IEF 2 = fraction 3 and 4 together. l-A-6, 250 μΐ

The High pH fractions 1-35 were stored in the -80°C freezer.

IEF 3 = fraction 5 and 6 together. l-A-7, 250 μΐ

The High pH fractions 1-35 were stored in the -80°C freezer.

IEF 4 = fraction 7 and 8 together. l-A-8, 250 μΐ

The High pH fractions 1-35 were stored in the -80°C freezer.

IEF 5 = fraction 9 and 10 together. l-A-9, 250 μΐ

The High pH fractions 1-35 were stored in the -80°C freezer.

IEF 6 = fraction 11 and 12 together. l-A-10, 250 μΐ The High pH fractions 1-35 were stored in the -80°C freezer.

IEF 7 = fraction 13 and 14 together. 1-A-l 1, 250 μΐ

The High pH fractions 1-35 were stored in the -80°C freezer.

IEF 8 = fraction 15 and 16 together. 1-A-l, 250 μΐ

The High pH fractions 1-35 were stored in the -80°C freezer.

IEF 9 = fraction 17 and 18 together. l-A-2, 250 μΐ

The High pH fractions 1-35 were stored in the -80°C freezer.

IEF 10 = fraction 19 and 20 together. l-A-3, 250 μΐ

The High pH fractions 1-35 were stored in the -80°C freezer.

IEF 11 = fraction 21 and 22 together. l-A-4, 325 μΐ

The High pH fractions 1-35 were stored in the -80°C freezer.

IEF 12 = fraction 23 and 24 together. l-A-5, 350 μΐ

The High pH fractions 1-35 were stored in the -80°C freezer.

After analysis the column was washed by injection of 175 μΐ of buffer A.

Pooling strategy high pH RP fractions

Since reversed phase at high and low pH are not very orthogonal a pooling strategy had to be developed to assure that peptides will still elute in the complete separation window in the final separation technique of this multi-dimensional workflow; reversed phase at low pH:

The fractions 1 through 34 of IEF 1 were thawed. Volume was reduced by using the speedvac.

The following fractions were combined:

IEF 1 A: fractions 2 - 8 - 14 - 20 - 26 - 32

IEF 1 B: fractions 3 - 9 - 15 - 21 - 27 - 33

IEF 1 C: fractions 4 - 10 - 16 - 22 - 28 - 34

IEF 1 D: fractions 5 - 11 - 17 - 23 - 29

IEF 1 E: fractions 6 - 12 -18 - - 24 - - 30

IEF 1 F: fractions 7 - 13 - 19 - 25 - 31

Every tube was rinsed with 200 μΐ ACN/H₂0 (50/50)

The samples were dried with the speedvac.

The dry samples were placed in the -80°C freezer.

The fractions 1 through 34 of IEF 2 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:

IEF 2 A: fractions 2 - 8- 14- 20- 26- 32

IEF 2 B: fractions 3 - 9- 15- 21 - 27- 33

IEF 2 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 2 D: fractions 5 - 11 - 17 -23 -29

IEF 2 E: fractions 6 - 12 -18- -24- -30

IEF 2 F: fractions 7 - 13 - 19 -25 -31

Every tube was rinsed with 200 μΐ ACN/H₂0 (50/50)

The samples were dried with the speedvac.

The dry samples were placed in the -80°C freezer.

The fractions 1 through 34 of IEF 3 were thawed. Volume was reduced by using the speedvac.

The fractions were combined as follows:

IEF 3 A: fractions 2 - 8- 14- 20- 26- 32

IEF 3 B: fractions 3 - 9- 15 - 21 - 27- 33

IEF 3 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 3 D: fractions 5 - 11 - 17 -23 -29

IEF 3 E: fractions 6 - 12 -18- -24- -30

IEF 3 F: fractions 7 - 13 - 19 -25 -31

Every tube was rinsed with 200 μΐ ACN/H₂0 (50/50)

The samples were dried with the speedvac.

The dry samples were placed in the -80°C freezer.

The fractions 1 through 34 of IEF 4 were thawed. Volume was reduced by using the speedvac.

The fractions were combined as follows:

IEF 4 A: fractions 2 - 8- 14- 20- 26- 32

IEF 4 B: fractions 3 - 9- 15 - 21 - 27- 33

IEF 4 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 4 D: fractions 5 - 11 - 17 -23 -29

IEF 4 E: fractions 6 - 12 -18- -24- -30

IEF 4 F: fractions 7 - 13 - 19 -25 -31

Every tube was rinsed with 200 μΐ ACN/H₂0 (50/50)

The samples were dried with the speedvac.

The dry samples were placed in the -80°C freezer. The fractions 1 through 34 of IEF 5 were thawed. Volume was reduced by using the speedvac.

The fractions were combined as follows:

IEF 5 A: fractions 2 - 8- 14- 20- 26- 32

IEF 5 B: fractions 3 - 9- 15 - 21 - 27- 33

IEF 5 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 5 D: fractions 5 - 11 - 17 -23 -29

IEF 5 E: fractions 6 - 12 -18 -24 -30

IEF 5 F: fractions 7 - 13 - 19 -25 -31

Every tube was rinsed with 200 μΐ ACN/H₂0 (50/50)

The samples were dried with the speedvac.

The dry samples were placed in the -80°C freezer.

The fractions 1 through 34 of IEF 6 were thawed. Volume was reduced by using the speedvac.

The fractions were combined as follows:

IEF 6 A: fractions 2 - 8- 14- 20- 26- 32

IEF 6 B: fractions 3 - 9- 15 - 21 - 27- 33

IEF 6 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 6 D: fractions 5 - 11 - 17 -23 -29

IEF 6 E: fractions 6 - 12 -18- -24 -30

IEF 6 F: fractions 7 - 13 - 19 -25 -31

Every tube was rinsed with 200 μΐ ACN/H₂0 (50/50)

The samples were dried with the speedvac.

The dry samples were placed in the -80°C freezer.

The fractions 1 through 34 of IEF 7 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:

IEF 7 A: fractions 2 -8- 14- 20- 26- 32

IEF 7 B: fractions 3 -9- 15 - 21 - 27- 33

IEF 7 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 7 D: fractions 5 - 11 - 17 -23 -29

IEF 7 E: fractions 6 - 12 -18- -24 -30

IEF 7 F: fractions 7 - 13 - 19 -25 -31

Every tube was rinsed with 200 μΐ ACN/H₂0 (50/50)

The samples were dried with the speedvac. The dry samples were placed in the -80°C freezer.

The fractions 1 through 34 of IEF 8 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:

IEF 8 A: fractions 2 -8- 14- 20- 26- 32

IEF 8 B: fractions 3 -9- 15 - 21 - 27- 33

IEF 8 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 8 D: fractions 5 - 11 - 17 -23 -29

IEF 8 E: fractions 6 - 12 -18 -24 -30

IEF 8 F: fractions 7 - 13 - 19 -25 -31

Every tube was rinsed with 200 μΐ ACN/H₂0 (50/50)

The fractions 1 through 34 of IEF 9 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:

IEF 9 A: fractions 2 -8- 14- 20- 26- 32

IEF 9 B: fractions 3 -9- 15 - 21 - 27- 33

IEF 9 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 9 D: fractions 5 - 11 - 17 -23 -29

IEF 9 E: fractions 6 - 12 -18 -24 -30

IEF 9 F: fractions 7 - 13 - 19 -25 -31

Every tube was rinsed with 200 μΐ ACN/H₂0 (50/50). The samples were dried with the speedvac. The dry samples were placed in the -80°C freezer.

The fractions 1 through 34 of IEF 10 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:

IEF 10 A: fractions 2 -8- 14- 20- 26- 32

IEF 10 B: fractions 3 -9- 15 - 21 - 27- 33

IEF 10 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 10 D: fractions 5 - 11 - 17 -23 -29

IEF 10 E: fractions 6 - 12 -18 -24 -30

IEF 10 F: fractions 7 - 13 - 19 -25 -31

The fractions 1 through 34 of IEF 11 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:

IEF 11 A: fractions 2 - 8 - 14-20-26- 32 IEF 11 B: fractions 3 - 9- 15 - 21 - 27- 33

IEF 11 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 11 D: fractions 5 - 11 - 17 -23 -29

IEF 11 E: fractions 6 - 12 -18- -24- -30

IEF 11 F: fractions 7 - 13 - 19 -25 -31

The fractions 1 through 34 of IEF 12 were thawed. Volume was reduced by using the speedvac. The fractions were combined as follows:

IEF 12 A: fractions 2 -8- 14- 20- 26- 32

IEF 12 B: fractions 3 -9- 15 - 21 - 27- 33

IEF 12 C: fractions 4 - 10 - 16 -22 -28 -34

IEF 12 D: fractions 5 - 11 - 17 -23 -29

IEF 12 E: fractions 6 - 12 -18 -24 -30

IEF 12 F: fractions 7 - 13 - 19 -25 -31

Strong cation exchange chromatography

As a third dimension of peptide separation, strong cation exchange (SCX) chromatography was used:

RPLC-Method: SCX_pept_1911.M

Column: PolyLC inc.

PolySulfoethyl A

200 x 2.1 mm, 5 μιη, 200 A

Buffers: Buffer A: 25 % ACN in H₂0, pH 2,5 (Formic acid, app.2 ml per liter).

Buffer B: Buffer A + 0.5 M KC1 (37.25 gram in 1 liter).

Gradient: time (min). % B flow (ml/min) Pressure

0.00 5 0.15 200

5.00 5 0.15 200

40.00 90 0.15 200

45.00 90 0.15 200

45.10 5 0.15 200

55.00 5 0.15 200 Fraction Collector: time (min). Trigger Mode Fractions (min.)

0.00 Off

1.00 Time-based 1.00

62.00 Off

Fraction Delay Volume = 77 μΐ

Above location = 7 mm

Column by Column

Collection Mode = Discrete fractions

Tray = "Agilent 96"

Samples were thawed and dissolved in 300 μΐ buffer A, mixed and spun.

Samples were sonicated for 5 minutes in an ultrasonic bath to dissolve all peptides, were mix and spun. 300 μΐ of sample were injected onto the SCX column. The fractions were combined in such a way that based on the UV signal 8 new fractions were generated that contain equal amounts of peptide. The same was done for the other 72 samples.

LC-spotter

The final separation step was nano-reversed phase at low pH in combination with spotting the effluent on MALDI plates for off-line mass analyses using MALDI-ToF mass spectrometry. Samples were thawed, dissolved in 70 μΐ Buffer A (H₂0 + 0.1% TFA), mixed and spun. Samples were sonicated for 5 minutes in an ultrasonic bath to fully dissolve all peptides, then mixed and spun. 40 μΐ of these samples were injected and the remainders stored in the -80°C freezer.

MALDI Methods:

MALDI-MS was performed using a 4800 instrument from Applied

Biosystems. MS and MS/MS of the most intense peaks was done in positive ion mode.

Peak extraction and database searching

Centroid peaks were extracted from the raw data using TS2Mascot 1.0.0. Resulting *.mgf files were searched using Mascot 2.2 or Mascot Deamon (for automated searching). Alternatively, the raw data can directly be searched using Protein Pilot 2.0.

B) Two-dimensional Immunodepletion, two-dimensional LC-MALDI MS workflow The first depletion step removes fourteen highly abundant human plasma proteins: albumin, IgG, IgA, IgM, fibrinogen, transferrin, alpha- 1 -antitrypsin, alpha-2- macroglobulin, haptoglobin, apo A-I, apo A-II, alpha- 1 acid glycoprotein, complement C3, and apo B. The subsequent depletion step utilizes a SuperMix column (for details on the immunodepletion procedure applied here, see Genway Inc. San Diego, CA) to deplete the next 80 most abundant proteins. Proteins were extracted from the depleted plasma by reversed-phase capture. The extracted proteins were subjected to reduction, alkylation (of cysteine residues) and digestion with trypsin.

The resulting peptide pools were labeled with the primary amine specific iTPvAQ reagents; combined into eight-plex iTRAQ mixes which contains six primary samples labeled with the iTRAQ reagents that yield marker fragment ions at m/z 114, 115, 116, 118, 119, and 121. Each iTRAQ mix was analyzed by two-dimensional LC-MS/MS (SCX chromatography followed by RP-HPLC; for details of methods see above under (Proteomics Workflow (A)).

Data processing

All raw spectra were stored as binary objects in an Oracle database, and were viewable using the GPS explorer software from ABI, as delivered with the instrument. For identification and quantification of the peptides' spectra, and for assembly of the peptides into proteins, two different software packages were used. Both packages use peaklists from the peak-picking and de-isotoping software that comes with the instrument.

The first package used was Protein Pilot 2.0 from ABI. This software readily delivers protein and peptide reports in a tabular form for further analysis. For details on this program, see ref Ml . It was found that on large datasets (100.000+ spectra), this program shows a remarkable sensitivity. The second package was Mascot, ref M2, in conjunction with an Integra database system and custom Java scripts, all from Matrix Science, London, UK. In this case, the data pipeline is as follows. First, the peaklists are extracted from the instrument database for each jobrun (typically an LC-run with 381 spots), using TS2Mascot (Matrix Science). The resulting *.mgf files (typically each containing a few 100's of MS2 spectra) are imported into the Integra database. The names (*) of the *.mgf files were chosen to indicate which plate and fraction the LC-run is from. In the mgf file, the header above each separate peaklist indicates the spot number. Hence, for each MS-2 spectrum, the exact position on any of the MALDI plates can be retrieved, together with all peak intensities, the resulting peptide assignments by Mascot, the Mascot score and expectation value. These values were extracted from the Integra database using a custom java script. By matching of the spotname and the exact (to 0.001 Da) precursor mass, the strength and signal to noise of the precursor peak in the MS-1 spectrum could be retrieved from the instrument database and included in the resulting 'Mascot MS-2 report'. This report is more complete than the PP report as it has one line for each MS-2 spectrum, and because the peak strengths in MS-1 and MS-2 are available. This was used to correct the ITRAQ 8-plex labels for contributions from immonium -ions with masses of 112 and 120 Da. For matches below the homology score, Mascot reports only the best matching peptide sequence, but not the protein it has taken the sequence from. Therefore, the protein accessions and names were retrieved using the DB- toolkit software. After defining a cut-off value equal to 1 for the maximum acceptable expectation value, the average ITRAQ ratio's per unique peptide were calculated. Average ITRAQ ratio's for the proteins were calculated by averaging over all peptides from the same protein. All protein identifications were made using human sequences from SwissProt 56.0.

Two datasets were processed. These were on the same 8 pooled samples, using the same type of mass spectrometer but different sample depletion and fractionation, as described. Another difference was in the labeling of the pools, as indicated below.

Table 7:

In the protein ITRAQ profiles, the pool names were used. In the supporting peptide reports, the ITRAQ labels were used to identify the samples and table 7 can be used to refer to the correct sample by taking the provenance of the data into account. Table 8, below, lists some of the parameters of the two sets, using Mascot as described.

Data quality

Before starting the data-analysis, the data were inspected in several ways. First of all, inventors expected that for most spectra, the ITRAQ peaks are nearly equal, as most spectra come from peptides of highly abundant proteins. These will most likely not be influenced by the presence of disease, but rather reflect normal physiology. A typical raw spectrum in the ITRAQ region is shown in Figs. 7 and 8. The ITRAQ peaks were nearly always detectable, as shown in Fig. 9, and ratio's were close to 1. It could be confirmed that this was the typical behavior by plotting the ITRAQ peak strengths for the 8 ITRAQ labels against the average over all 8 peaks for each spectrum. To get an idea of the biological and technical variation, the log2 of the ratio of ITRAQ peak strength were plotted over average peak strength for two pairs of samples from clinically equivalent groups, al and a2, and bl and b2. A normal distribution was found, also for samples cl, c2, dl and d2. This again confirmed that markers for disease are rare, as expected.

Also data reproducibility was checked, by going back to some spots and taking the spectrum again. It is clear that reproducibility is good, and the differences measured between equivalent samples such as al and a2 must reflect biological variation or technical variation due to sample preparation such as digestion or labeling.

Two software packages were used for protein identification, as it is known that this can increase the coverage of the proteome under study. This was also found in the present study, see Table 9 below. It was checked that the peptide quantitation by contrast was nearly identical for the two software packages, small differences can be attributed to differences in isotope corrections and peak selections. Mascot Protein Pilot combined

Set A 525 1654

Set B 534 536

Combined set 876 1963 2350

Table 9: Number of unique protein accessions (SwissProt 56.0); in total, 2350 distinct proteins were identified. Protein identification with tandem mass spectrometry is essentially a two-step process. In the first step, the software determines the peptide sequence that best explains a spectrum / peaklist. In the next step, peptides are assembled into proteins. ITRAQ protein ratio's are obtained by averaging the peptide ratio's. Thus, the data that go into the analysis are tables with on each line a protein accession, protein name and the relative (PP) or absolute (Mascot) ITRAQ peak strengths. Depending on the software, there are additional columns; for an assessment of the strength of the protein identification, in the Mascot protein report, the number of supporting spectra and for the PP protein report the 'unused' parameter are used. For each protein identification, the supporting peptide assignments and spectral data are available in a peptide report. Protein and peptide reports for the combined sets A&B are attached.

Example 4 - Data analysis and scoring Data analysis

The data analysis started with either absolute intensities or intensity ratios for each and each sample. First, some preprocessing on these data is performed:

1. In case of absolute intensities, each sample's measurements were first scaled such that they yield the same total intensity.

2. Each protein's intensities/ratios were divided by the average over the eight samples, such that (new) ratios with a mean value of one for each protein are obtained.

3. A log₂ transformation of the ratios resulting from the previous step is then used.

On these log-ratio data, three techniques to identify interesting proteins were applied:

correlation-based, t-tests, and a scoring method. Correlation-based analysis

In the correlation-based analysis, inventors first computed the Pearson correlation for each protein's log-ratio profile with a reference profile. This was only performed for proteins that had no missing ITRAQ values, and the following three reference profiles were used:

To find proteins with a gradually increasing/decreasing profile over the samples, inventors used a reference profile 0, 0, 1, 1, 1.9, 2.1, 2.9, 3.1 for samples al, a2, bl, b2, cl, c2, dl, d2, respectively, i.e. a step from each group to the next one, and a small differentiation between cl and c2, and between dl and d2.

1. To find diagnostic proteins, a reference profile 0, 0, 0, 0, 1, 1, 1, 1, was used i.e. there is a step between sample b2 (benign) and cl (local).

2. To find prognostic proteins, a reference profile 0, 0, 0, 0, 0, 1, 1, 1, was used i.e. there is a step between sample cl (local insignificant) and c2 (local significant).

The closer a protein's Pearson correlation is to -1 or to +1, the better.

However, Pearson correlation alone is not enough; it is also important that the log-ratio values over a protein's eight measurements shows enough variation (otherwise, differences might not be reproducibly measurable). Therefore, also the standard deviation of each protein's profile was computed. Next, for each protein these two figures were combine into one criterion by taking the square of its Pearson correlation, and multiplying that with the protein's standard deviation. The higher this criterion is, the better.

In order to assess the significance of the findings, random label-permutation experiments were performed, i.e. the reference profile was shuffled, and the above analysis was performed again (actually, all possible shuffling of the reference profiles can be checked, as for the three reference profiles above there are only 10080, 70 and 56 unique permutations, respectively). Then, inventors were able to compare the observed number of proteins with a criterion value above a certain threshold on the original data, with the expected number of such proteins obtained on randomized data, to get an idea what the fraction of such proteins is that one gets by chance (i.e. we estimate a false discovery rate).

T-tests

In the second analysis, inventors aimed at finding interesting steps in the proteins' profiles by means of t-tests. Inventors focused on identifying profiles that have different values for the first four samples (al - b2) as compared to the next three (cl - dl), and for the first five (al - cl) compared to the next two (c2 - dl). The reason to omit sample d2 is that it can already be distinguished by its exceptional high PSA scores.

For the t-test, only proteins that had all ITRAQ values for the first seven samples were considered. The p-values for the respective t-tests were computed, and corrected for multiple testing by a method controlling the false discovery rate.

Scoring method

The third method applied is a heuristic scoring method, which allows for missing values. Inventors applied it at first to the intensity data, and interpret a missing value as a very low value. Again, inventors checked for the first four samples (al - b2) as compared to the next three (cl - dl), and for the first five (al - cl) compared to the next two (c2 - dl). For each protein, the heuristic compares each measurement in the first group to each measurement in the second group (i.e. 12 comparisons for al - b2 vs. cl - dl and 10 comparisons for al - cl vs. c2 - dl). A score is given to each of these comparisons:

+4 if the second intensity is over 4 times the first one

+3 if the second intensity is over 2 times the first one

-4 if the first intensity is over 4 times the second one

-3 if the first intensity is over 2 times the second one

If one of the two values is missing, then a score of +/-4 is given if the other intensity is large enough (more than 500), and +/-3 if it is smaller. If both values are missing, or in other cases, a score of 0 is given to this pair of intensities.

Next, all 12 or 10 scores, respectively, are added, and the sums are divided by the maximum attainable score to get a result in the range [-1, +1]. Proteins with a score close to -1 or +1 are now considered as interesting.

The scoring heuristic can also be applied on ratio data, although in that case the intensity information cannot be taken into account in the comparison to a missing value.

Results from the above experiments and test are depicted in Tables 10 and 11. Tables 10 and 11 show proteins that might be useful as tumor markers, in particular prostate cancer biomarkers to improve the differential diagnosis of benign vs. malignant prostate disease as well as providing an individual prognosis of prostate cancer progression, wherein Table 11 includes the data of Table 10 and comprises additional data as well as additional markers. The markers of Table 11 correspond to the markers mentioned in Table 3.

Table 10:

Table 11:

Example 5 - Further assessment of biomarker candidates by quantitative targeted proteomics approaches for verification purposes

Biomarker candidates (see Tables 2 and 3) were further investigated for biomarker verification. Verification in this context means that a selected list of biomarker candidates (i.e. the markers mentioned in Table 1) was further analysed for differential expression on a set of individual patient blood samples. Whereas during the biomarker discovery phase the biomarker candidates were selected based on the measurement of several patient pools (see Examples 1 to 4, above) during the verification process the patient samples that were used to build the pools for discovery were measured individually so finally derived a single quantitative measurement for each patient.

This means that the data base for statistical differential expression analysis has significantly increased compared to the disovery phase described in Examples 1 to 4. During this intitial discovery phase 8 patient pools were analysed such that for most disease stages like benign, indolent, or aggressive disease a single quantitivate measurement was available for statistical data analysis. Compared to this situation after verification there are ~30 individual quantitive measurements - representing 30 different patients - per measured disease stage available for statistical differential expression analysis. This generates more robust data on the differential expression of a biomarker candidate between two clinical groups compared to the intial discovery phase.

Clinical patient plasma samples used for the verification study

In total 150 individual patient plasma divided over 5 clinical patient groups samples were analyzed for quantitative expression values of the candidate markers depicted in Table 1 :

1) Healthy controls: 30 individual samples, patient group 1 (in discovery (Examples 1 to 4) split into two pools a 15 samples called pool al and a2) 2) Benign prostate disease: 30 individual samples, patient group 2 (in discovery (Examples 1 to 4) split into two pools a 15 samples called pool bl and b2)

3) Local prostate disease, insignificant disease: 30 individual samples, patient group 3 (in discovery (Examples 1 to 4) one pool a 15 samples called pool cl) 4) Local prostate disease, significant disease: 30 individual samples, patient group 4 (in discovery (Examples 1 to 4) one pool a 15 samples called pool c2) 5) Advanced disease: 30 individual samples, patient group 5 (in discovery (Examples 1 to 4) split into two pools a 15 samples called pool dl and d2)

In general, the verification process by targeted proteomics was performed using the mass spec method "Multiple Reaction Monitoring" (MRM) as described in more detail below. The principle of the method uses the fact that the protein sequence of any of the biomarker candidates that has been identified during the discovery phase is known. This knowledge can be used to measure representing peptides of each particular protein in a very sensitive and specific as well as quantitative manner. For each biomarker candidate representative tryptic peptides were identified in silico from the protein sequence according to certain criteria (e.g., their flying behavior in mass spec, their length in amino acids, their amino acid composition etc, see below for more details). This ends in a collection of peptides with at least one peptide representative for a biomarker candidate protein. For each peptide an independent MRM assay was developed.

The MRM principle is based on the combination of detection of specific features of a peptide in two coupled quadrupole mass spectrometers separated by a collision cell in which peptides can be fragmented along their amino bond backbone. Any tryptic peptide from a given protein is in the first place characterized by its specific molecular weight which is obvioulsy a priori known from the protein sequence. The first quadrupole piece of the mass spec instrument is able to identify peptides with a very high mass spec accuracy. So the mass spec instrument can select the peptide of interest for further analysis based on information on the peptide mass provided by a user.

In a complex mixture like human serum containing many proteins and consequently many tryptic peptides with similar masses the primary quadrupole will select not only the peptide of interest but along with it some additional other peptides with very similar mass. In order to further select the peptide of interest and exclude the other peptides that are still in the mixture after the selection step done in the primary quadrupole the peptide mix is transferred into the collision cell where all peptides still contained in the mixture are fragmented by the induced collision with air molecules. The energy of the air molecules is chosen such that the peptide is typically fragmented along the waekest bond which normally is the bond between the different amino acids. The peptide fragments are subsequently transferred to the secondary quadrupole. Due to fact that the amino acid sequence of the peptide of interest is known, the size of the fragments induced in the collision cell can be predicted in silico. The mass spec instrument should accordingly only detect fragment masses of a given pre-defined value to make sure that only fragments of the peptide of interest are detected in the scondary quadrupole mass spec. In many cases this so-called MRM transition for a peptide of interest (the combination of the known primary mass of the full length tryptic peptide together with a known mass of a collision fragment of this peptide) is suffcient to specifically detect a given peptide of interest. However, there might also be cases for which several peptides in combination with different collision fragments have to be tested before a good and specific MRM assay is found for a particular starting peptide.

To make the complete process of MRM assay development more effective it is typically started with the use of synthetic peptides that correspond in the amino acid sequence exactly to the in silico identified tryptic representative peptides of a biomarker candidate. These peptides are available in a purified form after synthesis in large amounts and it is therefore possible to test a whole range of parameters to identify the most optimal transition for this particular peptide (for more details see below). So in essence, for every representative biomarker peptide a peptide was synthesized which was used to develop effectively working MRM transitions. Subsequently, the artifical peptides were spiked into the complex mix (in particular human serum) to support the detection of the endogenous peptide of interest. For this the synthetic peptide was labelled with an isotopic variant of a the carbon atom (¹³C instead of the naturally occuring ¹²C). In that sense the chemical and physical characteristics of the synthetic peptides are exactly the same as compared to the characteristics of the endogenous peptides of interest with the only exception that the artifical peptide has a slightyl higher - but exactly defined - molecular weight compared to the endogenous peptides. The identification of the spiked artificial peptides is possible in the mass spec as it is spiked into the complex mix in an appropriate concentration for detection. The detection of the artificial peptides also helps in the detection of the endogenous peptides which can be present in the mix in much less concentration but due to the exact and known mass difference the endogenous peptide can be more easily detected compared to a situation without the presence of a spiked synthetic peptide.

Furthermore, apart from the detection of the endogenous peptides the synthetic peptides also support the quantitation of the endogenous peptide as the amount of spiked artifical peptide is known and is always the same in each sample measured. In that sense at least a relative quantitation of a representative endogenous biomarker peptide across different samples (e.g., different patient samples) can be achieved. In that way a quantitative measure of a biomarker protein - represented by a peptide - was establised across a number of different patient samples derived from different clinical patient groups to allow an assessment whether a protein of interest is differentially expressed between different clinical patient groups and is useful as a biomarker for discrimination between the patient groups.

Another important characteristic of MRM is the high grade of assay

multiplexing that can be achieved with this technology, meaning that not only a single biomarker candidate can be measured and quantified in a given patient sample during a typical assay (e.g., 90 min) but actually a multitude of biomarkers can be measured and quantified within an assay time of 90 min. In practice a multitude of several tens of biomarker candidates can be measured in a quantitative manner in an assay time of 90 min. Consequently, a collection of 150 patient blood samples could be measured for the presence and quantity of the biomarker candidates outlined in Table 1 within a period of ca 4 weeks with the result that each biomarker candidate has an expression value across 150 patient samples.

Quantitative targeted proteomics

Candidate biomarkers weres assessed and analysed in a multiplexed manner based on the use of quantitative targeted proteomics. Quantitative targeted proteomics include a combination of affinity purification strategies followed by mass spectrometry (MS). These techniques are the basis of any targeted or focused, hypothesis driven proteomics experimentation. The targetting is carried out by selecting and enriching for a compound or group of similar compounds by its very nature. Recently growing in popularity is the development of a few novel non-data-dependent MS instrumentation methods, e.g. multiple reaction monitoring (MRM). These may require a priori information to target a particular class of compounds in the mass spectrometer, the main idea being that the instrument is focused on gathering measurements on a select compound or group of compounds. These instrument methodologies in combination with affinity-based methods can even further target and focus one's proteomic experiments.

In particular, multiple reaction monitoring-mass spectrometry (MRM-MS) for targeted quantitative proteomics is an extremely valueable methodoly in this context. MRM- MS is a deviation of selected reaction monitoring (SRM). A typical MRM workflow is depicted in Fig. 11. While its application is novel in the proteomics community, SRM has been utilized for several decades in the toxicology and pharmacokinetics disciplines. SRM transitions are highly specific scans for detecting specific analytes in complex mixtures utilizing, most predominantly, triple quadrupole-based mass spectrometers. The transitions are designed such that the first mass analyzing quadrupole (Ql) is set to transmit a narrow mass window around the desired parent ion and the third quadrupole (Q3, the second mass analyzing quadrupole) is set to transmit a narrow mass window around the desired fragment ion. Fragmentation via collisional induced disassociation (CID) occurs in the second quadrupole (Q2). Therefore, SRM requires two ions to generate a positive result, making it a very specific detection methodology with very low background thereby enhancing sensitivity of detection. Successful SRM transitions depend not only on the ionization efficiency of the parent ion (Ql transmission) but also the fragmentation efficiency of this parent ion and subsequently the intensity of fragment ion (Q3 transmission). Inputting several different SRM transitions for the same or different analytes, multiple transitions can be monitored within one MS run. This is known as MRM (see also Fig. 12).

To increase productivity of the mass spectrometer method, one strives to increase the number of MRMs one can measure in any given experiment. The number of transitions per experiment or time scale is dependent on various factors, including most importantly the mass spectrometer's cycle-time i.e. the time for the instrument to cycle through separation and detection of each transition. The time to analyze each transition is termed 'dwell-time' simply defined as the amount of time where one mass analyzer is detecting and measuring only one ion, and has important consequences for not only the number of transitions one can measure in any given experiment but also the sensitivity of detection. Dwell-time and cycle-time are directly proportional and intimately effect sensitivity. An increase in dwell-time results in a more sensitive measurement. However this more sensitive measurement is at the cost of increasing the cycle-time and therefore decreasing the number of transitions one can measure simultaneously, i.e. the productivity of the MS run. Traditionally, the dwell-time should be optimized such that each transition is being scanned and analyzed at least seven-eight times as it is eluted from the column such that a good LC peak shape can be constructed which is important for quantification.

For protein identification and quantification however, MRM-MS has not been as routinely used due to the additional challenges of method development. Theoretically, it is possible to spike into a sample a standard mixture of proteins in known concentration to perform quantification as is performed in small molecule analyses. However in practice, this does not allow for robust protein quantification because the standard proteins and the analyte proteins may not produce similar responses in the mass spectrometer due to ion suppression and differences in fragmentation. Additionally, recovery rates of the different proteins may be different if there are sample preparation steps prior to MS. This is why isotopically- labeled peptides resulting from protein digestion are used.

Aptly suited to the multiplex capabilities of this MRM strategy, multiple signature peptides representing a particular protein can then be examined in one experiment allowing for the development of a highly selective, sensitive and high throughput quantitative methodology.

Quantification methods of peptide MRM-MS are based on the classic isotope- dilution MS. It allows to measure and quantify metabolites, both endogenous and

pharmacological. A variation of the small molecule isotope-dilution MS techniques, the labeled internal standard peptides are introduced to a protein sample prior to or during the proteolytic step. Both the labeled internal standard and the unlabeled native peptides are analyzed by LC-MRM-MS similar to small molecule isotope-dilution strategies, i.e. the peak areas of the labeled and unlabeled species are related for quantification. The internal standard and the native peptides generated by proteolysis are chemically identical;

chromatographically co-eluting, ionization efficiency, and relative distribution of fragment ions, but are different in mass which allows the mass spectrometer to differentiate the two species from one another and from all other peptides in the matrix. Since the concentration of the internal standard is known, the ratio between the internal standard and the native peptide can be related quantitatively thereby deducing the absolute amount of native peptide.

Furthermore, the stochiometric relationship of the peptide or peptides to the protein allows subsequent quantification of the protein, which is the only step that is unlike the classical isotope-dilution experiments on small molecules.

Use of peptides synthesized with stable isotope-labeled amino acids now is a common strategy for creating peptide internal standards for absolute quantification.

Furthermore, mTRAQ reagent labels may be used wherein the primary amines of peptides (both N-terminus and lysine) are labeled. Light and heavy mTRAQ reagents create a paired label set with a molecular weight difference of 4 Da, which increases the possible difference in each MRM.

Furthermore, a key requirement of the MRM methodology is the ability to distinguish between correct identifications and false positives. Peptides are much larger and more complex than small molecules, share considerable homology and are measured in very complex matrices. Since each fragmentation product of a peptide only provides information about one position in that peptide, it is highly likely to have interference, i.e. other peptides resulting in the same transition as the peptide analyte in question. A schematic representation of peptide fragmentation is shown in Fig. 13 highlighting some possible fragment ions after CID and outlines a comparison of SRM to MRM. By requiring multiple fragmentation product ions in the Q3 which together result in the same chromatographic elution time, the method can be highly specific providing confidence of the peptide identification in the absence of a full-scan tandem mass spectrum.

Different tryptic peptides from the same protein can produce ion currents differing by factors of 10³ in LC-MS/MS experiments, excluding peptides that are not detected at all. This variation is due to multiple factors, including propensity to ionize in the electrospray source, coincidence in elution time with other easily ionizing peptides, efficiency of release during tryptic digestion and the presence of unrecognized post translational modifications (PTM) arising due to biology or artificially during sample preparation. A consistent response is required across the dynamic range of the analyte measured to ensure proper quantification of both native and isotope-labeled peptide pairs. A potential source of interference is in-source fragmentation of abundant peptides where the fragment ions rather than the precursors are the source of interference. This is caused by coincidence of the primary or secondary fragment of the precursor that has the same or nearly the same mass as the analyte transition of interest. This can be a significant issue for quantification and is dependent on the sensitivity level one is attempting to achieve. It is critical to select transition ions that maximize specificity and potentially minimize interferences from co-eluting species that fall within the mass windows and tolerances of the detector. Decreased mass resolution (1000-3000 FWHH) in the triple quadrupole instruments requires careful consideration of peptide separation so as to ensure accurate measurement of the peptide of choice because of the decreased separation of analytes at the same nominal masses. Therefore, in practice, initial LC-MS/MS experiments are performed on the biological sample to obtain preliminary information on the peptide characteristics, both ionization and fragmentation, and also LC retention times. Once the precursor ion and fragmentation characteristics are noted, the precursor ion of interest and multiple fragment ions are chosen for MRM transitions and the sample is transferred to a triple quadrupole instrument. These transitions, based on the experimentally determined fragmentation pattern and CID parameters are further optimized on the triple quadrupole instrument. The sample is then re-analyzed with identical LC conditions to maintain the retention time with the goal of creating a set of MRM transitions with the best qualitative and quantitative properties per peptide of interest.

For this type of analysis hybrid instruments (4000 QTRAP, 5500 QTRAP, Applied Biosystems) are used that are both a triple quadrupole and a linear trap and offer a unique opportunity to design MRM-MS methods. Based on the MIDAS (MRM-initiated detection and sequencing) workflow, when significant signal in a specific MRM transition is detected (both the parent mass in Ql and fragment mass in Q3 are isolated and detected), the instrument switches the third quadrapole to ion trap mode and collects a full scan tandem mass spectrum. This full scan ion trap MS/MS obtained from the targeted detection is submitted to a database search to confirm that the detected peptide is the peptide of interest and be utilized to further optimize the target for Q3. In this way the MIDAS workflow can develop MRM-MS methods in one step, eliminating the need to do preliminary method development confirming selectivity of peptide identification. This workflow comprises, inter alia, the P3 (Proteotypic Peptide Predictor) module, the TIQAM (Targeted Identification for Quantitative Analysis by MRM) module, or other in silico prediction methods based on amino-acid sequence such as MDIP (Minimum Acceptable Detectability for Identified Peptides), APEX (Absolute Protein Expression). The obtained data are gathered into a PeptideAtlas database or a user-derived database where those proteotypic peptides uniquely identifying proteins of interest are assembled to establish MRM transitions based on the validated MS2 spectrum. TIQAM also offers a protein/peptide-centered-view to predict proteotypic peptides, perform in silico digestions, and view the MRM trace further optimizing the method development.

General protocol for immunodepletion applied to clinical samples

Four 0.22 μιη spin filters (Agilent Technologies) were washed with 100 μΐ water. Subsequently they were centrifuged for 10 minutes at 10,000 rpm and 10°C. The samples were thawed from pools and human serum 4522 (Sigma) and put in ice. the Human Serum was diluted 4 times with buffer A (Agilent Technologies) by mixing 400 μΐ HS with 1200 μΐ Buffer A.

Pool 1 and Pool 2 were diluted four times with buffer A (Agilent) by mixing

120 μΐ Pool sample with 360 μΐ Buffer A

The HS and Pool 1 and 2 filter samples were filtered over a 0.22 μιη Spin filter at 10000 rpm and 4°C for 60 minutes. The samples were depleted using LC-3 Agilent HU-14 columns, all unbound fractions (UF) and bound fractions (BF) were collected and stored at -80°C.

Desalting of samples with 5 filters

A 200 ml 100 mM TEAB was prepared by diluting 5 ml 1M TEAB with 45 ml

Milli-Q. all UF samples were thawed from -80°C to 4°C. 16 sample tubes of 15 ml were added to 4,5 ml 100 mM TEAB in each tube. The 16 sample tubes were placed on ice. 4,5 ml sample was added to each tube. 2 tubes were used per pool. The tubes were placed back on ice. the samples were filtrated over 5K Spin concentrator 4ml tubes (Agilent Technologies). 2 tubes per pool were used.

First step: 4 ml sample per tube were centrifuged for 15 minutes with 4000 rpm at 4°C. 3 ml sample were added to the filter tube. The mixture was centrifuged for 12 minutes with 4000 rpm at 4°C. 2 ml sample were added to the filter tube. Subsequently the mixture was centrifuged for 15 minutes with 4000 rpm at 4°C. 3 ml 100 mM TEAB were added to the filter tube. Subsequently the mixture was centrifuged for 20 minutes with 4000 rpm at 4°C. 3 ml 100 mM TEAB were added to the filter tube. The mixture was centrifuged for 20 minutes with 4000 rpm at 4°C. 3 ml 100 mM TEAB were added to the filter tube. The mxiture was centrifuged for 25 minutes with 4000 rpm at 4°Cabnd subsequently centrifuged for another 10 minutes with 4000 rpm at 4°C. The residue was pipetted from the filter to a new tube and the filter was washed with TEAB. All UF samples and the filtrate samples were stored in a -80°C freezer.

Subsequently, all UF Pool samples were thawed from -80°C to 4°C. They were mixed and spinned based on the use of 8 Millipore Biomax-5K-NMWL Membrane 0.5 ml filters. For this purpose 500 μΐ sample were pipetted in the filter and the filter was subsequently centrifuged for 10 minutes at 10000 rpm and 4°C. Then 300 μΐ sample was added in the filter. The filter was centrifuged for 10 minutes at 10000 rpm and 4°C.

Subsequently 350 μΐ sample was added in the filter (last part of the sample). The filter was centrifuged for 15 minutes at 10000 rpm and 4°C and for 3 minutes at 10000 rpm and 4°C. The samples 1, 2 and 4 were centrifuged for 2 minutes at 10000 rpm and 4°C. The residue (± 50 μΐ) was pipetted from the filter to a new tube and the filter was washed with 2 x 50μ1 TEAB. all UF samples and all filtrate samples were stored in the -80°C freezer. Protein concentrations were determined using the Bradford assay as described earlier. For each sample 625 μg protein was needed. The volumes were accordingly adjusted such that this amount of protein could be taken into analysis. Denaturation / Alkylation / Digestion.

625 μg of each pool sample was taken into analysis and all samples were adjusted to 8 M urea by adding solid urea (± 0.48 mg urea per 1 μΐ sample). The sample volume needed was pipetted to the amount of ureum. The samples were mixed and spun. Due to the ureum the volume increased. The pH of each sample was checked to be around 8. A solution of 500 mM TCEP was prepared. 2.5 μΐ of the 500 mM TCEP solution was added to each sample tube. The samples were mixed and spun. The solutions were mixed for 1 hour at room temperature. Subsequently, a solution of 200 mM IAA was prepared, wherein 37 mg IAA was weighed for 1 ml. 20 μΐ of 200 mM IAA was added to each sample tube. The samples were mixed and spun and left for 30 minutes at room temperature in the dark.

10 tubes of 5 μg Lys-C (Roche) were used. For this purpose the Lys-C of every tube was dissolved in 50 μΐ H₂0. In total: 50 μg Lys-C in 500 μΐ H₂0. 62.5 μΐ of this Lys-C solution was added to each sample tube. Ratio Lys-C : Protein = 1 : 100. The samples were mixed and spun and put for 4 hours at 37°C in the dark.

For the trypsin digestion the ureum concentration was reduced from 8 M to 2 M by diluting samples 4 times. 15 tubes of 20 μg trypsin were used, the trypsin of every tube was dissolved in 20 μΐ 100 mM TEAB. In total: 300 μg trypsin in 300 μΐ TEAB. 25 μΐ of this trypsin solution were added to each sample tube. Ratio Trypsin : Protein = 1 : 25. The samples were mixed and spun overnight at 37°C.

Assay validation - Overview

A workflow for rapid, high-throughput, assay development that is a multi-step process based on synthetic peptide libraries, was developed. The workflow is shown in Fig. 14 and described further below.

Shortly, full scan MS/MS spectra were acquired for each target peptide from pools of synthetic peptides. The best (highest xcorr in Sequest) spectrum served as template to derive the optimal transitions and to define the retention times in relation to a set of standard peptides (TR-kit).

These pre-assays were then refined in MRM mode, where the exact apex retention time and the observed relative intensities are measured. This refined assay was called validated assay. In the assay verification step the validated assays were tested in the target matrix - in this case human plasma peptides provided by Philips. Assays that successfully detected the endogenous peptide were called verified assay. The project was accordingly split into two parts:

1. Assay design and validation (synthetic assays)

a. Selecting target peptides for provided protein targets

b. Selection and validation of transitions

c. Calibration of chromatio graphic retention times

2. Assay verification in target matrix

a. Ordering and QC of labeled reference peptides for quantification b. Pre-screen of detectability of endogenous peptides in plasma with

reference peptides spiked into sample in fixed amount. Scoring, manual inspection and pre-decision of likely detected peptides.

c. Re-balancing concentrations of reference peptides according to the intensities measured in b to verify that detected signal is not a contamination of the synthetic peptides. Scoring, manual inspection and final decision of detectability of targets. Assay design and validation - Selection of peptides

Previously multiple candidate biomarkers were discovered of which several were selected for further verification (see Table 1). The protein identifiers were mapped back to the Uniprot/SPROT protein database version June/2010 resulting in unique proteins. These proteins could be mapped back to the repository of synthetic peptides, i.e. the peptides could be matched with protein sequences.

Assay design and validation - Selection and validation of transitions

Peptides were picked from this peptide library and pooled into 7 pools. These pools were measured on a LTQ Orbitrap (see further below) in inclusion list mode. Spectra were searched with Sequest and the prototypical spectrum was selected as preassay for each peptide in each precursor charge state.

Peptides for which a pre-assay could be acquired were measured in scheduled

MRM mode. In each injection retention time peptides (RT-Kit; see further below) were recorded together with the target peptides. The retention times of the target peptides were stored together with the RT-Kit peptides and could be re-calibrated to a different

chromatographic set-up by a simple linear regression.

Assays were scored with mQuest and manually inspected.

Assay verification - Ordering and QC of labeled reference peptides for quantification

For all validated assays stable isotope labeled versions were ordered. After exclusion of peptides that could not be synthesized according to the manufacturers specifications 586 peptides were received.

These peptides were produced by spot-synthesis. For technical reasons these peptides carry a c-terminal tag, which needs to be cleaved off by tryptic digest prior to use (see Material and Methods). Peptides were pooled in 6 plate-pools for spike-in as reference for verification and quantification.

Pools of synthetic reference peptides were measured in MRM mode for quality control and contamination with the non-labeled isoform was determined to be below 0.1% on average. This contamination was a consequence of incomplete labeling of the heavy amino- acid (Lysine or Arginine) that is used for peptide synthesis. Consequently, for the assay verification a ratio light (endogenous)/heavy (spike-in) of less than 0.3%> was considered as limit for verification of the endogenous peptide.

Pre-screen of detectability of endogenous peptides in plasma with reference peptides spiked into sample in fixed amount

A plasma samples pool with a concentration of approx. 0.05 ug/ul was used to verify the presence of the endogenous peptides in the samples. These samples were quality controlled on an Agilent QTOF, injecting a nominal amount of lug. Sample amount for MRM analysis was adjusted based on the total ion-current for this test-sample. In all further experiments a nominal amount of 1 ug was injected. For assay verification, plate pools were spiked into plasma as described in Material and Methods. Transitions for one plate pool were split into 3 parts. Between 400 and and 600 transitions were measured for each injection (4 transitions each for light and heavy).

MRM data was scored and manually verified as described below. Shortly, data was analyzed with mQuest; each signal was inspected manually and judged based on the sub- scores and overall appearance of the signal. Each signal was classified as either detected, likely detected, not detected, or unsure. The classifier unsure was given in cases where the ratio light/heavy was smaller than 0.3% based on the pre-analysis of the heavy reference peptides alone.

Re-balancing concentrations of reference peptides according to the intensities measured

For all assays that were not labeled not detected the intensities of the reference peptides were extracted. A schema was set up to approximately balance the peptide intensities. Peptides were pooled accordingly, digested and spiked into the plasma samples. MRM analysis was performed and analyzed with mQuest and by manual inspection. Synthetic peptides

A large repository of synthetic peptides matching to human proteins was used. Target protein sequences were matched against this repository. All peptides in this repository that matched to the target proteins were used to design validated assays.

The peptide repository was assembled according to the following criteria: Peptides were chosen with a length of 7-24 amino acids. Only fully tryptic peptides, with no missed cleavages were considered. Preferably, peptides that were unique for the targeted protein were selected. In cases where paralogs of proteins showed high homology also peptides matching to more than one iso-form were allowed.

For proteins previously observed in shotgun proteomics experiments three (or in the case of protein kinases up to 10) proteotypic peptides most frequently observed by mass spectrometry were selected based on the information present in a repository of shotgun MS/MS experiments and data from the public proteomics data repository PeptideAtlas.

Selected peptides were synthesized on a microscale using the Spot-synthesis technology, lyophilized in a 96-well plate (~50 nmol of peptide material per well; JPT Peptide Technologies) and used in an unpurified form.

Peptides were picked from 96 well plates, and resuspended in 20% acetonitrile, 1% formic acid. Aliquots (1/500,000 of the starting material) of each peptide were mixed (96-peptide mixes), evaporated on a vacuum centrifuge to dryness, resolubilized in 0.1% formic acid and analyzed.

MS/MS

MS/MS Full scan data of synthetic peptides was aquired on a Eksigent Nano LC system (Eksigent Technologies, Dublin, CA, USA) connected to a hybrid linear ion trap LTQ Orbitrap (Thermo Scientific, Waltham, MA, USA), equipped with a nanoelectrospray ion source (Thermo Scientific). Normalized collision energy was set to 35%. Inclusion lists were generated using precursor m/z of charge states 2+ and 3+ for pools of synthetic peptides. The generated inclusion lists were imported into the global mass list parent ion table of the MS operating software (Xcalibur 2.0 SRI, Thermo Electron). The dynamic exclusion mass window that is also setting the m/z tolerance for the inclusion list masses was narrowed to ±10 ppm for all directed analyses with enabled monoisotopic precursor selection. Ion signals for which no charge could be assigned were also allowed to trigger MS2 scans. The dynamic exclusion time was set to 10 s to acquire multiple MS2 scans for each feature.

Database search

MS/MS spectra were searched against a database consisting of concatenated peptide sequences with the reversed peptide sequence (expect tryptic c-terminus) as decoy entries. Searches were performed allowing for semi-tryptic peptides (owing to the presence of truncated peptides in the synthesis process) with a precursor mass tolerance of 50 ppm. Evaluation was done using the trans-proteomic pipeline after a Sequest search. Peptide Prophet cut-off was set to 1% false discovery rate. Each prototyopic spectrum was in addition checked manually. Pre-assay design

Pre-assays were designed from identified peptides by selecting the highest scoring spectrum (xcorr, SEQUEST) as the prototype spectrum for each peptide/charge state.

This spectrum served as lead spectrum to extract the highest (most intense) peaks as transitions to be validated later in MRM mode. Up to 8 spectrum peaks matching to fragment ions of the b- and y-ion series (match tolerance 0.6 Da, charge states +l,+2) were selected for

MRM analysis. Spectrum peaks without a match to theoretical fragment ions with these constraints (e.g. ions with waterloss) were not considered.

MRM analysis

LC-MS/MS analysis in MRM mode was performed using an EASY-nLC nanoflow HPLC system (Proxeon) coupled to a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray source. Samples were loaded onto a nanoLC column prepared in house by packing PicoFit emitters (360 um OD, 75 um ID, 10 um tip, New Objective) with approx. 11 cm of Magic CI 8 AQ resin (3 um particle size, 200 A pore size, Michrom). Gradient elution was performed using solvents A = 3% acetonitrile/ 0.1% formic acid in water and B = 3% water/0.1% formic acid in acetonitrile according to the following gradient program:

0-30 min 5-35 %B

30-32 min 35-100 %B

32-40 min 100%B

followed by reequilibration to 5%B. The flow rate was set to 300 nL min^"1.

The LC eluent was electrosprayed using an ionization voltage of 1.9 kV. MRM analysis was performed using Ql and Q3 resolutions of 0.7 Da with a cycle time of 2.5s. Collision energies were calculated for the TSQ-Vantage by a linear regression according to the Vendors specifications as. CE reported for the QTRAP 5500 were calculated according to CE=a+b*m/z as indicated below:

MRM data analysis - Conversion of raw MRM data

Raw data from the Thermo TSQ-Vantage was converted to mzXML by the program readw which is part of the Trans-Proteomic Pipeline distribution. Ion traces for all transitions were reconstructed and mapped to an annotated transition list using the Ql and Q3 m/z values that are reported in the mzXML file format. Annotation contained the peptide sequence, charge state of precursor and transition, ion-type (b- or y-ion and position), expected relative intensity, the expected elution time, isotope form (light or heavy) as extracted from the pre-assay (assay validation) or the validated assay (assay verification)

All transition ion traces for one transition group record were binned to provide equal spacing and allow for later scoring with cross-correlation measures. Traces were smoothened by a sliding average with a window size of 3.

Transitions were grouped into transition group records based on their annotation, and peak detection was run to detect up to 5 peak groups on each transition group record of which at most one is selected in the final analysis. Light and heavy labeled versions of a peptide were treated separately but assigned to the same transition group record. Peak groups were constructed by matching co eluting peaks from all transitions for each isotopic form (assay verification) within one transition group.

A peak group is not required to contain peaks from all transitions. In cases where a transition was not represented in the peak group the trace within the boundaries of the peak group was assigned and the maximum intensity within that section was considered as apex. The rationale for that is to avoid missing data by failure of peak extraction Absence of a detectable signal is not treated as missing data but as the noise line being the maximum possible intensity present. In cases where a light and heavy isotopic form was measured peak groups from corresponding iso-forms were paired into peak-group-pairs.

Scoring of MRM peak groups

Peak group pairs were associated with different scores according to the described criteria of co elution, peak shape similarity, intensity and correlation of relative intensities between peak group and assay (expected intensities) or light peak group vs. heavy peak group (assay verification only):

1) Intensity score: total ion current of all peaks constituting a light peak group

2) Intensity correlation: product moment correlation between apex intensities of peaks in light peak group and expected intensities derived from the assay.

3) Co-elution score: a measure of co-elution based on the average shift in the cross- correlation function between each pair of peak traces. Each peak in a light peak group is cross-correlated to each other peak and the apex of the resulting cross-correlation function is taken as elution time difference. This a more global measure of elution characteristics compared to a simple comparison of apex positions, especially in cases where noisy or jagged data is compared. The pair wise comparisons result in a matrix of elution time differences. The mean plus one standard deviation is the reported score.

4) Shape score: a measure of global shape similarity based on the same cross-correlation matrix like for the co-elution score. The property of a normalized cross-correlation that an apex intensity of 1 is an indication for perfect peak shape similarity and smaller values indicate non-corresponding peak shapes was used. The average of the apex intensity in the cross-correlation functions constitutes the shape score.

5) Reference correlation: Product moment correlation between apex intensities of

corresponding light and heavy peaks of a peak group pair. 6) Retention time deviation: difference of expected and measured retention time in seconds.

7) Reference shape score: A measure of global shape similarity. A cross-correlation matrix is generated as described above, but only for corresponding light and heavy transitions. Average cross-correlation function apex intensities is reported as score.

8) Reference co-elution score: Average shift of cross-correlation functions for

corresponding light and heavy transitions.

Solubilization and digestion of stable isotope labeled peptides

Stable isotopical synthetic reference peptides, which are spiked into biological or clinical samples, were synthesized as internal standards for accurate peak detection and for quantification. These heavy-labeled synthetic peptides have the same chemical and physical properties compared to the endogenous peptides - except for the mass - and are thus only distinguishable in a mass spectrometer.

C-terminal arginines and lysines were labeled with stable isotopes (Arg: ¹³C₆, ¹⁵N₄; Lys: ¹³C₆, ¹⁵N₂), resulting in a mass shift of 10 daltons for c-terminal arginine and 8 daltons for c-terminal lysine peptides compared to the light endogenous peptides.

Peptides were provided that were produced by spot-synthesis (original manufacturer JPT (Berlin, Germany)). Spot synthesis is a technology that yields peptides at small scale but at lower costs compared to the classical bead-based synthesis approaches. This makes these peptides suitable for targeted proteomics experiments with many targets at the same time where no absolute quantification is needed. Peptides synthesized with the Spot-synthesis technology carry a c-terminal amino acid tag, which needs to be cleaved off prior usage.

Peptides were provided solubilized in 20% Acetonitrile (ACN), 1% formic acid (FA), 5 nmol/peptide, as stated by JPT Peptide Technologies GmbH. The peptides contained an additional c-terminal amino acid tag and needed to be processed with Trypsin prior usage. The solubilized peptides were stored long term at -20°C or below, preferably split in aliquots. The solubilized peptides were kept cool in a fridge for short term storage.

Additional required material, reagents and solutions:

• Vortexer (Scientific Industries Inc.; Vortex Genie 2)

• Sonication bath (Bandelin; Sonorex Super) Vacuum concentrator

Acetonitrile (Sigma-Aldrich; LC-MS grade; 34967)

Formic acid (Sigma-Aldrich; puriss p. a.; 56302)

Water (Sigma-Aldrich; for gradient elution; 34877)

Ammonium hydrogen carbonate (Sigma-Aldrich; Mltra grade; 09830)

Trypsin (Promega; Sequencing, modified, frozen; V5113)

Trifluoroacetic acid (Sigma-Aldrich; Reagent grade 98%; T6508, in glass ampoμles) MicroSpin C18 spin column, Vydac C18 Silica (The Nest Group, Inc.; SEM SS18V) Methanol (Merck; gradient grade; 1.06007.2500)

Solubilization Buffer 20% (v/v) Acetonitrile, 1% (v/v) formic acid in water. Glass syringes and glass ware were used for concentrated acids in order to avoid

contaminating the sample with polymers. HPLC or higher grade solvents were used. 1M Ammonium hydrogen carbonate buffer. 79.1 mg per ml solution were weighed in. The powder was dissolved with water to the final volume. The buffer was freshly prepared for every usage.

20% (v/v) Trifluoroacetic acid. Add 4 ml water to a glass bottle. Dispense 1 ml concentrated trifluoroacetic acid using a glass syringe to the water. Glass syringes and glass ware were used for concentrated acids in order to avoid contaminating the sample with polymers. HPLC or higher grade solvents were used.

CI 8 washing solution 80%> (v/v) acetonitrile, 0.1 % (v/v) trifluoroacetic acid in water. Glass ware was used for concentrated acids in order to avoid contaminating the sample with polymers. HPLC or higher grade solvents were used.

CI 8 equilibration and washing solution 0.1 % (v/v) trifluoroacetic acid in water. Glass ware was used for concentrated acids in order to avoid contaminating the sample with polymers. HPLC or higher grade solvents were used.

CI 8 elution solution 40%> (v/v) acetonitrile, 0.1 % (v/v) trifluoroacetic acid in water. Glass ware was used for concentrated acids in order to avoid contaminating the sample with polymers. HPLC or higher grade solvents were used.

Buffer A (0.1% FA)

Experimental protocol The nominal concentration of solubilized peptides in 96 well plates (material spotted on membrane as indicated by manufacturer) was 277 pmol/μΐ, 5 nmol total amount, dissolved in 18 μΐ Solubilization Buffer.

The lid of the 96 well plate was carefully removed, and the required amount of each well was pooled to pools of maximum 96 peptides.

It was started with pools of 1 μΐ each of 96 peptides (one plate, the„standard plate-pool") for screening of peptide intensities prior to use as reference peptides.

1M freshly prepared ammonium hydrogen carbonate was added until pH reaches 8-9. Trypsin with a Trypsin: peptide amount ratio between 1 : 100 to 1 :50 (e.g for a standard plate-pool it is recommended to use 0.5 μg Trypsin) was added.

The sample was incubated at 37 °C with gentle shaking (600 rpm) for 2 hours.

The sample was acidified with 20% (v/v) TFA in water to pH 2, and centrifuged at 16Ό00 x g for 1 minute .Glass ware was used for concentrated acids in order to avoid contaminating the sample with polymers. HPLC or higher grade solvents were used. For the clean-up of peptide pools MicroSpin C18 columns were used. A low centrifugation force was used in order to keep optimal contact time between the mobile phase and the stationary C18 phase.

o 200 μΐ Methanol were added, and spun at 100 x g for 1 minute.

o 200 μΐ C18 washing solution was added, and spun at 100 x g for 1 minute. o The previous step was repeated

o 200 μΐ CI 8 washing/equilibration solution were added, and spun at 200 x g for 1 minute.

o The previous step was repeated two times.

o The sample was loaded, spun at 200 x g for 1 minute, the flowthrough was collected.

o The flow-through was re-loaded, spun at 200xg for 1 minute, the flow-through was saved,

o The previous step was repeated.

o The previous step was repeated two times. o 200 μΐ CI 8 elution buffer were added, spun at 200 x g for 1 minute. The flowthrough was saved to a new tube (-> 40%-eluate).

o The previous step was repeated.

o Subsequently it was spun at 1 Ό00 x g for 1 minute, the flowthrough as then combined with 40%-eluate.

• 40%>-eluate was dried to almost complete dryness using vacuum concentrator. In cases where the peptide pool got dried completely, the peptides were resuspended with 50 μΐ 2% (v/v) acetonitrile in water and the the sample was sonicated for 5 minutes.

• It was re-dissolved in solubilization buffer, e.g. for a standard plate-pool re-dissolve in 20 μΐ ("standard plate-pool stock solution")

• The purification was tested by injection of 1 μΐ of standard plate-pool stock solution 5x diluted into Buffer A (the stock solution contained 20% ACN and needed to be diluted before loading on a chromatographic system)

o 1 μΐ standard plate-pool stock was added to 4 μΐ buffer A.

o 1 μΐ of diluted plate pool were injected

RT-kit: Retention time normalization - Provided material

• Isotopically labeled reference peptides for retention time normalization in LC-MS experiment (Product No. A7-2009-1-5E-12).

• Quantity: Approx. 25 pmol/peptide

Equipment and material needed

• Bench-top centrifuge, capable of centrifuging 1.5ml-Sample-tubes

• Bench-top vortexer

• Sonication bath, with floating device for 1.5ml-Sample-tubes

• HPLC-grade or higher quality Acetonitrile and HPLC-grade or higher quality water

• Sequencing grade, high-quality formic acid (FA)

• Solution preparation

• Solubilization Solution 20% (v/v) Acetonitrile / 0.1 % (v/v) Formic Acid / H₂0

• Glass syringes were used while handling concentrated (above 10%> v/v) acid. Solubilization of reference peptides

1. 25 μΐ of prepared solubilization solution were added to the provided sample-tube (1 pmol/μΐ stock solution)

2. The sample-tube shaken on a bench-top vortexer.

3. The solution was spun down using the centrifuge.

4. The sample-tube was sonicated for 5 minutes.

5. The sample-tube was centrifuged at maximum speed for 1 minute.

Storage of solubilized reference peptides

• The solubilized reference peptides were stored in a fridge (4-6 °C) for short-term storage (several weeks).

• Solubilized reference peptides were split in appropriate aliquots, and frozen at max 20 °C for long-term storage.

Adjustment of peptide spiking amount

1. Solubilized reference peptides were spiked in a ratio of 1 : 10 (reference peptides : sample) into a biological sample, representing the target sample as close as possible (e.g. individual sample or a pool of samples). This ratio was adjusted according to the sensitivity of your set-up. Peptides were clearly visible but did not show signs of overloading, i.e. peak tailing.

2. Data for retention time recalibration were acquired in non-time constraint (non- scheduled) MRM mode

Example 6 - Differential Discrimination Analysis

The MRM data generated as described above, which is basically a list of signal intensities as measured by mass spec for the various peptides and their MRM transitions for all patient samples, was processed as follows:

First, the signal intensities of a MRM signal (representing the amount of peptide fragment detected in the secondary quadrupole of the MS machine) in the patient samples were divided by the intensity of the same MRM transition signal of the identical but Carbon- 13 isotopically labeled reference peptide mixed into the same sample before the analysis was performed. From these MRM intensity ratios, the logarithm (base 2) was taken, resulting in 'logratios'. good correlations (r= 0.9 typically) for logratios from different MRM transitions (i.e., representing different collision induced peptide fragments) of the same peptide across the different patient samples could be verified. Therefore, the logratios (typically 3-4 per peptide) were subsequently averaged into a single logratio per peptide for each patient sample.

Before doing a differential discrimination analysis to identify those peptides/proteins that are able to discriminate different clinical patient groups a normalization of the averaged logratio values for each peptide was performed by subtracting the median logratio value across the samples for each peptide. The data were then plotted as scatter plots (see Fig. 15). These scatter plots show the expression values (i.e., the median normalized logratios) of the individual patient samples of the patient groups that were compared to each other. Further, the plots show the sequence of the representative peptide, the p-value for group discrimination as well as the mean and median of the two different groups (dotted and solid line).

The peptides and their corresponding proteins were ranked on their ability to separate the patient groups by using the p-value from a two-sided Wilcoxon test. For peptides to be considered for ranking, at least three finite values in each of the two patient groups were demanded.

Finally, ROC curves for all the peptides/proteins were calculated and plotted in multi-plots, ordered by rank as before (see Fig. 16). The ROC curves show how diagnostic sensitivity and specificity are related. Diagnostic sensitivities and specificities were calculated in the conventional way by varying the discrimination threshold from the lowest signal in the two studies groups (where sensitivity is 100% and specificity is 0%), to the highest signal in the two study groups (where sensitivity is 0% and specificity is 100%) and recording the true positive versus false negative rate. The area under the curve (AUC) was additionally calculated as an indicator of the quality of the group separation and is indicated in the plots (see Fig. 16). A typical AUC for the standard prostate biomarker PSA is around 0.65 for discrimination between benign and malignant prostate disease. As indicated in Fig. 16 most of the biomarkers of the present invention show a significantly higher AUC compared to PSA, which makes it virtually impossible that the results of the present invention were generated by chance and underlines the medical and diagnostic importance of the identified biomarkers. In some embodiments, the present invention relates to the following items:

Item 1 : A tumor marker or group of tumor markers associated with the progression of a 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 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 Table 2.

Item 2: The tumor marker or group of tumor markers of item 1 , wherein the expression of the marker(s) is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section I) of Table 2.

Item 3 : The tumor marker or group of tumor markers of item 1 , wherein the expression of the marker(s) is decreased (down-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section J) of Table 2.

Item 4: The group of tumor markers of any one of items 1 to 3, wherein said group comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or all of the tumor markers of Table 2. Item 5: The group of tumor markers of any one of items 1 to 4, wherein the p-value of the expression modification is 0.0022 or lower.

Item 6: The group of tumor markers of any one of items 1 to 5, wherein the group comprises at least 5 tumor markers corresponding to tumor marker #1 to #5 of Table 2, at least 10 tumor markers corresponding to tumor marker #1 to #10 of Table 2, at least 15 tumor markers corresponding to tumor marker #1 to #15 of Table 2, at least 20 tumor markers corresponding to tumor marker #1 to #20 of Table 2, at least 25 tumor markers corresponding to tumor marker #1 to #25 of Table 2, at least 30 tumor markers corresponding to tumor marker #1 to #30 of Table 2, at least 35 tumor markers corresponding to tumor marker #1 to #35 of Table 2, at least 40 tumor markers corresponding to tumor marker #1 to #40 of Table 2, at least 45 tumor markers corresponding to tumor marker #1 to #45 of Table 2 or at least 50 tumor markers corresponding to tumor marker #1 to #50 of Table 2.

Item 7: 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 6.

Item 8: The composition of item 7, wherein said nucleic acid affinity ligand or peptide affinity ligand is modified to function as an imaging contrast agent.

Item 9: 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 6 in a sample.

Item 10: The method of item 9, 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 11 : The method of item 10, wherein said method is a method of graduating cancer, comprising the steps of

(a) determining the level of a tumor marker or a group of tumor markers as defined in any one of items 1 to 6 in a sample by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers,

Item 12: Use of a tumor marker or a group of tumor markers as defined in any one of items 1 to 6 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 13: 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

(a) testing in a sample obtained from an individual for the expression of a tumor marker or a group of tumor markers according to any one of items 1 to 6;

wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or a group of tumor markers as defined in any one of items 1 to 6.

Item 14: 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 (upregulated) expression of a tumor marker or group of tumor markers as defined in item 2, comprising at least one element selected from the group of: (a) a compound directly inhibiting the activity of a tumor marker as defined in item 2, preferably an antagonist of said tumor marker enzymatic activity;

(b) a compound indirectly inhibiting the activity of a tumor marker as defined in item 2;

(c) a dominant negative form of a protein of a tumor marker as defined in item 2 or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker as defined in item 2;

(e) a miR A specific for a tumor marker as defined in item 2;

(f) an antisense molecule of a tumor marker as defined in item 2;

(g) a siR A specific for a tumor marker as defined in item 2;

(h) an aptamer specific for the expression product of a tumor marker as defined in item 2 or for the protein of a tumor marker as defined in item 2;

(i) a small molecule or peptido mimetic capable of specifically binding to the protein of a tumor marker as defined in item 2; and

(j) an antibody specific for the protein of a tumor marker as defined in item 2 and/or an antibody variant specific for the protein of a tumor marker as defined in item 2.

Item 15: 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 decreased (down-regulated) expression of a tumor marker or group of tumor markers as defined in item 3, comprising at least one element selected from the group of:

(a) a compound directly stimulating or modulating the activity of a tumor marker as defined in item 3, preferably an agonist of said tumor marker enzymatic activity;

(b) a compound indirectly stimulating or modulating the activity of a tumor marker as defined in item 3;

(c) a protein of a tumor marker as defined in item 3 or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing a protein of a tumor marker as defined in item 3; and

(e) a miRNA inhibitor specific for a miRNA of a tumor marker as defined in item 3. Item 16: Use of a tumor marker or a group of tumor markers of any one of items 1 to 6 for identifying pharmaceutically active agents useful in the treatment or prevention of cancer, preferably in the treatment or prevention of prostate cancer.

Item 17: The tumor marker or group of tumor markers of any one of items 1 to 6, the composition of item 7 or 8, the method of any one of items 9 to 11, the use of item 12 or 16, the immunoassay of item 13, or the pharmaceutical composition of item 14 or 15, wherein said cancer is prostate cancer .

Item 18. The tumor marker or group of tumor markers, composition, method, use, immunoassay or pharmaceutical composition of item 17, 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.

Claims

CLAIMS:

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 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 Table 1 or Table 2.

2. The tumor marker or group of tumor markers of claim 1 , wherein the expression of the marker(s) is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section I) of Table 1 or Table 2.

3. The tumor marker or group of tumor markers of claim 1, wherein the expression of the marker(s) is decreased (down-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section J) of Table 1 or Table 2.

4. The group of tumor markers of any one of claims 1 to 3, wherein said group 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 or all tumor markers of Table 1, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or all of the tumor markers of Table 2.

5. The group of tumor markers of any one of claims 1 to 4, wherein the p-value of the expression modification is 0.0006 or lower.

6. The group of tumor markers of any one of claims 1 to 4, wherein the group comprises at least 3 tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 6 tumor markers corresponding to tumor marker #1 to #6 of Table 1, or at least 9 tumor markers corresponding to tumor marker #1 to #9 of Table 1, or at least 12 tumor markers corresponding to tumor marker #1 to #12 of Table 1, or at least 15 tumor markers

corresponding to tumor marker #1 to #15 of Table 1, or at least 18 tumor markers corresponding to tumor marker #1 to #18 of Table 1, or at least 21 tumor markers

7. The group of tumor markers of any one of claims 1 to 4, wherein the group comprises :

(xvi) at least marker # 16 of Table 1 (CD 14) and at least one additional marker of Table 1 or Table 2; or

(xvii) at least marker # 17 of Table 1 (HEMO) and at least one additional marker of Table 1 or Table 2; or

(^xrx) ^at least marker # 19 of Table 1 (MMP2) and at least one additional marker of Table 1 or Table 2; or

8. 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 claims 1 to 7.

9. The composition of claim 8, wherein said nucleic acid affinity ligand or peptide affinity ligand is modified to function as an imaging contrast agent.

10. 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 claims 1 to 7 in a sample.

11. The method of claim 10, 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.

12. The method of claim 11, wherein said method is a method of graduating cancer, comprising the steps of

(a) determining the level of a tumor marker or a group of tumor markers as defined in any one of claims 1 to 7 in a sample by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers,

13. Use of a tumor marker or a group of tumor markers as defined in any one of claims 1 to 7 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.

14. 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 (a) testing in a sample obtained from an individual for the expression of a tumor marker or a group of tumor markers according to any one of claims 1 to 7;

wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or a group of tumor markers as defined in any one of claims 1 to 7.

15. 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 an up-regulated tumor marker or group of tumor markers as defined in claim 2;

(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of a tumor marker or a group of tumor markers as defined in claim 2;

(c) classifying the levels of expression of step (a) relative to levels of step

(b); and

(d) identifying the individual as eligible to receive a cancer disease therapy where the individual's sample is classified as having an increased level of expression of a tumor marker or a group of tumor markers as defined in claim 2.

16. 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 down-regulated tumor marker or group of tumor markers as defined in claim 3;

(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of a tumor marker or a group of tumor markers as defined in claim 3;

(c) classifying the levels of expression of step (a) relative to levels of step

(b); and (d) identifying the individual as eligible to receive a cancer disease therapy where the individual's sample is classified as having a decreased level of expression of a tumor marker or a group of tumor markers as defined in claim 3.

17. An immunoassay for stratifying an individual or cohort of individuals with a cancer disease comprising:

(c) determining the difference in expression of a tumor marker or a group of tumor markers as defined in claim 2 of steps (a) and the expression of a tumor marker or a group of tumor markers as defined in claim 2 and/or the reference gene in step (b); and

(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 claim 2.

18. An immunoassay for stratifying an individual or cohort of individuals with a cancer disease comprising:

(c) determining the difference in expression of a tumor marker or a group of tumor markers as defined in claim 3 of steps (a) and the expression of a tumor marker or a group of tumor markers as defined in claim 3 and/or the reference gene in step (b); and

(d) stratifying an individual or cohort of individuals to a cancer disease therapy based on the results obtained in step (c), where the individual's sample has a decreased level of expression of a tumor marker or a group of tumor markers as defined in claim 3.

19. The method of any one of claims 10 to 12, or 15 to 17, or the immunoassay of any one of claims 14, 17 or 18, wherein said method or immunoassay comprises the additional step of determining the level of prostate specific antigen (PSA).

20. 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 claim 2, comprising at least one element selected from the group of:

(a) a compound directly inhibiting the activity of a tumor marker as defined in claim 2, preferably an antagonist of said tumor marker enzymatic activity;

(b) a compound indirectly inhibiting the activity of a tumor marker as defined in claim 2;

(c) a dominant negative form of a protein of a tumor marker as defined in claim 2 or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing a dominant negative form of a protein of a tumor marker as defined in claim 2;

(e) a miR A specific for a tumor marker as defined in claim 2;

(f) an antisense molecule of a tumor marker as defined in claim 2;

(g) a siR A specific for a tumor marker as defined in claim 2;

(h) an aptamer specific for the expression product of a tumor marker as defined in claim 2 or for the protein of a tumor marker as defined in claim 2;

(i) a small molecule or peptido mimetic capable of specifically binding to the protein of a tumor marker as defined in claim 2; and

(j) an antibody specific for the protein of a tumor marker as defined in claim 2 and/or an antibody variant specific for the protein of a tumor marker as defined in claim 2.

21. 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 decreased (down-regulated) expression of a tumor marker or group of tumor markers as defined in claim 3, comprising at least one element selected from the group of: (a) a compound directly stimulating or modulating the activity of a tumor marker as defined in claim 3, preferably an agonist of said tumor marker enzymatic activity;

(b) a compound indirectly stimulating or modulating the activity of a tumor marker as defined in claim 3;

(c) a protein of a tumor marker as defined in claim 3 or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing a protein of a tumor marker as defined in claim 3; and

(e) a miR A inhibitor specific for a miR A of a tumor marker as defined in claim 3.

22. 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, wherein said cancer disease implies the increased (up-regulated) expression of a tumor marker or group of tumor markers as defined in claim 2, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 6, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) of Table 6, or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) of Table 6, or any fragment thereof.

23. Use of a tumor marker or a group of tumor markers of any one of claims 1 to 7 for identifying pharmaceutically active agents useful in the treatment or prevention of a cancer disease, preferably in the treatment or prevention of prostate cancer.

24. The tumor marker or group of tumor markers of any one of claims 1 to 7, the composition of claim 8 or 9, the method of any one of claims 10 to 12, 15, 16 or 19, the use of claim 13 or 23, the immunoassay of any one of claims 14, 17, 18 or 19, the pharmaceutical composition of claim 20 or 21, or the vaccine of claim 22, wherein said cancer disease is prostate cancer.

25. The tumor marker or group of tumor markers, composition, method, use, immunoassay, pharmaceutical composition or vaccine of claim 24, 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.