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 neoplastic 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 neoplastic disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage comprising affinity ligands for the expression products of the tumor markers, to corresponding methods, and to the use of said tumor markers for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed cancer stage to a more progressed cancer stage. The present invention further relates to a corresponding

immunoassay, to a method of identifying an individual for eligibility for a neoplastic disease therapy as well as to a pharmaceutical composition based on the inhibition of the expression of said tumor markers. BACKGROUND OF THE INVENTION

Neoplasms are abnormal masses of tissue as a result of neoplasia, i.e. an abnormal proliferation of cells. The growth of such clones of cells exceeds, and is

uncoordinated with, that of the normal tissues around it. Neoplasms or neoplastic swellings usually cause lumps or tumors. Generally, a neoplasm can be benign, potentially malignant (constituting a pre-cancer condition), or malignant (constituting a cancer condition or indication a cancer disease). Typically, benign neoplasms include uterine fibroids and melanocytic nevi, which normally do not transform into cancer. Potentially malignant neoplasms may include carcinoma in situ, which may not invade and destroy tissue, but can, given enough time, transform into a cancer. The third type of neoplasms is a malignant neoplasm, i.e. a cancer in which a group of cells display uncontrolled growth, leading to the invasion of tissue and sometimes to metastasis. These three malignant properties of cancers differentiate them from benign tumors and potentially malignant neoplasms, which are self- limited and do not invade or metastasize. Among men, the three most commonly diagnosed cancers are prostate, lung and colorectal cancer in developed countries. Particularly prostate cancer is the most common malignancy in European males. In 2002 in Europe, an estimated 225,000 men were newly diagnosed with prostate cancer and about 83,000 died from this disease.

Several different proteins or peptides have been associated with neoplastic diseases, in particular with cancer development. For instance, phosphodiesterase PDE7 has been shown to be linked to chronic lymphocytic leukemia. Yet, for many cancer types or cancer progression forms, in particular for early developmental stages, e.g. between benign tumors, premalignant states and early malignant states of a neoplastic disease, there is no adequate marker molecule available. One of the few examples of an early cancer marker is maspin, whose up-regulation is believed to precede the transition from premalignant prostate lesions to prostate cancer.

Despite these developments, the early detection of cancer is still largely based on the use of traditional medical approaches, e.g. breast-self examination, Pap tests or digital rectal examination in the case of prostate cancer. A further diagnostic perspective for early prostate cancer is based on the determination of prostate-specific antigen (PSA). However, PSA is not prostate cancer-specific and can be raised due to other circumstances, leading to a large number of false-positives. Furthermore, there will be an unpredictable number of false- negatives who later develop prostate cancer in the presence of a "normal" or traditional PSA test.

Therefore, there is a need for the provision of a new and effective, alternative diagnosis perspective for the detection, monitoring and prognostication of neoplastic diseases, in particular of early cancer forms, e.g. early prostate cancer developments. There is in particular a need for early tumor markers that can increase the diagnostic specificity and differentiate between benign and malignant neoplastic diseases and that can improve the traditional PSA test results.

SUMMARY OF THE INVENTION

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

The above objective is accomplished by the identification of novel tumor markers from samples of patients afflicted with neoplastic diseases. Using tissue or bodily fluid samples, in particular serum samples, of patients representing different progression stages of neoplastic diseases novel tumor markers could be identified which were shown to be differentially expressed, depending on the stage of progression of the neoplastic disease. The identification was achieved via the detection of autoantibodies derived from bodily fluid samples on an array of human proteins. In particular, it could be demonstrated by the present inventors that the novel molecular tumor markers are significantly up-regulated when comparing samples from patients having a less progressed stage of a neoplastic disease to samples from patients having a more progressed stage of the disease. These molecular markers are in certain embodiments of the present invention complemented by additional diagnostic factors such as a patient's age or the outcome of a patient's digital rectal examination. These markers are accordingly considered as markers for the prediction of neoplastic disease, in particular for the prediction of early cancer forms and a decision tool for the stratification of certain neoplasm or cancer surveillance regimes, as well as for the prognosis and monitoring of neoplasm or cancer progression. Diagnostic methods and uses based on the tumor markers of the present invention can thus advantageously be employed for (i) detecting and diagnosing neoplastic disease forms, in particular forms of neoplastic diseases of prostate cancer, (ii) prognosticating neoplastic disease forms, in particular neoplastic diseases of the prostate, (iii) monitoring of neoplastic disease progression towards more progressed disease stages, e.g. early malignant cancer forms, in particular of prostate cancer, and (iv) distinguishing between less and more progressed forms of neoplastic diseases, in particular less and more progressed forms of neoplastic diseases of the prostate, and most advantageously less and more progressed neoplastic disease forms of the prostate wherein the less progressed stage is a benign prostate tumor and the more progressed stage is a prostate cancer of stage < T2 (UICC 2002 classification). Finally, pharmaceutical compositions based on these tumor markers will provide novel therapeutic avenues in the treatment of cancer, in particular prostate cancer.

The present invention, thus relates in a first aspect to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified by increase (i.e. increased or up-regulated) when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least one molecular tumor marker selected from Table 1, Table 2 or Table 3. 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, or all molecular tumor markers of Table 1 and/or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or all of the molecular tumor markers of Table 2 and/or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or all of the molecular tumor markers of Table 3.

In another preferred embodiment of the present invention the p-value of the expression modification of said group of tumor markers is 0.007 or lower.

In a further embodiment of the present invention the group of tumor markers comprises at least 2 molecular tumor markers corresponding to tumor marker #1 to #2 of Table 1, or at least 3 molecular tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 4 molecular tumor markers corresponding to tumor marker #1 to #4 of Table 1, or at least 5 molecular tumor markers corresponding to tumor marker #1 to #5 of Table 1, or at least 6 molecular tumor markers corresponding to tumor marker #1 to #6 of Table 1, or at least 7 molecular tumor markers corresponding to tumor marker #1 to #7 of Table 1, or at least 8 molecular tumor markers corresponding to tumor marker #1 to #8 of Table 1, and/or at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #120 of Table 2 and/or at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #50 of Table 3.

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

(i) at least molecular marker # 1 of Table 1 (FGFR3) and at least one additional marker of Table 1, Table 2 or Table 3; or

(ii) at least molecular marker # 2 of Table 1 (SEPP1) and at least one additional marker of Table 1, Table 2 or Table 3; or

(iii) at least molecular marker # 3 of Table 1 (C9orf97) and at least one additional marker of Table 1, Table 2 or Table 3; or

(iv) at least molecular marker # 4 of Table 1 (PBK) and at least one additional marker of Table 1, Table 2 or Table 3; or

(v) at least molecular marker # 5 of Table 1 (MAPK9) and at least one additional marker of Table 1, Table 2 or Table 3; or

(vi) at least molecular marker # 6 of Table 1 (AKT3) and at least one additional marker of Table 1, Table 2 or Table 3; or (vii) at least molecular marker # 7 of Table 1 (AKT2) and at least one additional marker of Table 1, Table 2 or Table 3; or

(viii) at least molecular marker # 8 of Table 1 (PRAME) and at least one additional marker of Table 1, Table 2 or Table 3; or

(ix) at least molecular marker # 9 of Table 1 (PR CB2) and at least one additional marker of Table 1, Table 2 or Table 3.

In a further preferred embodiment of the present invention the group of tumor markers as defined above additionally comprises protein specific antigen (PSA) as molecular marker. In a particularly preferred embodiment of the present invention said PSA is total PSA, or free PSA, or complexed PSA and/or the ratio of free and total PSA and/or the ratio of complexed and total PSA.

In a further preferred embodiment of the present invention the group of tumor markers as defined above comprises a patient's age and/or the outcome of a patient's digital rectal examination (DRE) as additional diagnostic factor.

In another preferred embodiment of the present invention the specificity for detection of the progression of a neoplastic disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above is at least 30% and the sensitivity for detection of the progression of a neoplastic disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above is at least 90%.

In another preferred embodiment of the present invention the specificity for detection of the progression of a neoplastic disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above is at least 20%> and the sensitivity for detection of the progression of a neoplastic disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above is at least 95%.

In yet another preferred embodiment the group of tumor markers comprises at least one marker combination of selected from the combinations shown in Table 4 (i.e. of combinations 1 to 90, as indicated in column A of Table 4) or shown in Table 5 (i.e. of combinations 1 to 102, as indicated in column A of Table 5).

In a particularly preferred embodiment of the present invention the group of tumor markers comprises at least one of the following combinations of molecular markers and/or of additional diagnostic factors:

(i) AKT2 and FGFR3 and PSA; or (ii) AKT3 and FGFR3 and PSA; or

(iii) AKT2 and DRE and PSA; or

(iv) C9orf97 and SEPP1 and PSA; or

(v) AKT3 and DRE and PSA; or

(vi) C9orf97 and FGFR3 and PBK ; or

(vii) C9orf97 and PRAME and SEPP 1 ; or

(viii) C9orf97 and PBK and Patient Age; or

(ix) FGFR3 and PRAME and SEPP 1 ; or

(x) C9orf97 and PBK and DRE; or

(xi) AKT2 and FGFR3 and MAPK9 and DRE and PSA; or

(xii) AKT2 and MAPK9 and Patient Age and DRE and PSA; or

(xiii) AKT3 and FGFR3 and Patient Age and DRE and PSA; or

(xiv) AKT2 and C9orf97 and SEPP1 and DRE and PSA;or

(xv) C9orf97 and FGFR3 and PBK and SEPP1 and PSA; or (xvi) AKT2 and AKT3 and MAPK9 and DRE and PSA;; or

(xvii) AKT3 and FGFR3 and MAPK9 and DRE and PSA; or

(xviii) AKT2 and AKT3 and FGFR3 and DRE and PSA; or

(xix) FGFR3 and PBK and SEPP 1 and DRE and PSA; or

(xx) C9orf97 and PBK and SEPP1 and DRE and PSA.

In a further aspect the present invention relates to a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand for the expression product(s) or protein(s) of said molecular tumor marker or a group of tumor markers as defined above.

In a preferred embodiment of the present invention said peptide affinity ligand is an antibody.

In a further 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 neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the step of determining the level of a molecular 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 molecular 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 molecular tumor marker or the group of tumor markers as defined above in one or more samples of a less progressed stage of the same neoplastic disease.

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

(a) determining the level of a molecular 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 molecular tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease; and

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

In a further aspect the present invention relates to the use of said tumor marker or a group of tumor markers as defined above as a marker for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease.

In yet another aspect the present invention relates to an immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or for detecting, diagnosing, monitoring or prognosticating the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the steps of

(a) testing in a sample obtained from an individual for the expression of a molecular 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 or stage of a neoplastic disease or the progression of the neoplastic based on the results obtained in step (c),

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

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

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

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

(b); and

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

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

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

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

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

In a preferred embodiment said immunoassay or said methods as defined above comprise the additional step of determining the level of prostate specific antigen (PSA) and/or of determining the patient's age; and/or of determining the outcome of the patient's DRE. In a particularly preferred embodiment said PSA is total PSA, or free PSA, or complexed PSA and/or the ratio of free and total PSA and/or the ratio of complexed and total PSA. In addition, or in the alternative in said immunoassay or said methods the tested individual has shown an initial negative biopsy result.

In an additional aspect the present invention relates to a pharmaceutical composition for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of a molecular tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of:

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

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

(c) a dominant negative form of a protein of a molecular 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 molecular tumor marker as defined above;

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

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

(g) a siRNA specific for a molecular tumor marker as defined above;

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

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

In a preferred embodiment said antibody as described in the context of a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as mentioned herein above, or in the context of a pharmaceutical composition as mentioned herein above, is an antibody against the expression product or protein or a fragment thereof, of a molecular tumor marker or group of tumor markers as mentioned above. In a particularly preferred embodiment, said antibody is an autoantibody against a the expression product or protein or a fragment thereof of a molecular tumor marker or group of tumor markers as defined above.

In a further preferred embodiment of the present invention said autoantibody is an autoantibody against the expression product or protein of a molecular tumor marker or group of tumor markers as defined above, wherein said expression product or protein comprises an amino acid sequence as indicated in section G) of Table 1, 2 or 3, or any fragment thereof.

In another aspect the present invention relates to a vaccine for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 1, Table 2 or Table 3 or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) or G) of Table 1 , Table 2 or Table 3 or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) or G) of Table 1 , Table 2 or Table 3 or any fragment thereof.

An especially preferred embodiment of the present invention relates to the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, or the above mentioned pharmaceutical compositions or vaccine, wherein said neoplastic disease is a neoplastic disease of the prostate.

In a particularly preferred embodiment of the present invention said less progressed stage of the neoplastic disease as mentioned in the context of the above defined tumor marker or group of tumor markers, the above defined compositions, the above defined methods, the above defined uses, the above defined immunoassays, or the above defined pharmaceutical compositions or vaccine is a benign prostate tumor, and said more progressed stage of the neoplastic disease is a prostate cancer of stage < T2 (UICC 2002 classification).

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 depicts a list of clinical samples (and corresponding clinical groups) used for the experiments described in the present application. The group number in the following Figures refer to the group numbers in Fig. 1.

Fig. 2 shows the statistical distribution of sample collection dates (A) and age distribution (B) of clinical samples.

Fig. 3 indicates the statistical distribution of PSA values of the clinical samples with maximally 100 ng/ml.

Fig. 4 indicates the statistical distribution of prostate volumes (A) or total size of cancer tissue in the biopsy core (B) of the clinical samples.

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

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

Fig. 7 gives an overview over the ProtoArray® Immune Response Biomarker Profiling assay controls. On the left side a negative control image is shown, in which the array was incubated with buffer followed by the detection reagent. The inset shows the pattern of control proteins including Alexa Fluor®647-conjugated anti-mouse antibody (boxes in the lower row) a gradient of human IgG (boxes in the upper row) and a mouse anti- biotin antibody (boxes in the middle row). The murine antibody is recognized by the Alexa Fluor®647-conjugated anti-human antibody used for detection.

Fig. 8 (A-N) gives an illustrative overview over the AUC under a ROC of tumor markers according to the present invention for discrimination between clinical insignificant from significant prostate cancer. Please note that table entries #159 & #160 of Table 1, below are represented by only a single ROC curve (plot #159). Plots #160 to #200 of Fig. 8 therefore correspond to table entries #161 to #201 of Table 1.

Fig. 9 (A-Y) gives an illustrative overview over the expression values of tumor markers according to the present invention over the five clinical groups studied (see Fig 1 for details). Please note that table entries #159 & #160 of Table 1, below are represented by only a single data plot (plot #159). Plots #160 to #200 of Fig. 9 therefore correspond to table entries #161 to #201 of Table 1.

Fig. 10 depicts the process of selection of antigens for the testing of the antigens on targeted arrays for marker verification purposes.

DETAILED DESCRIPTION OF EMBODIMENTS

It was shown that tumor markers as depicted in Table 1 , Table 2 or Table 3 are differentially expressed, depending on the stage of progression of a neoplastic disease when testing samples of patients. In particular, it was demonstrated by the present inventors that tumor markers and tumor marker combinations are statistically significantly up-regulated when comparing samples from a less progressed stage to a more progressed stage of a neoplastic disease. Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.

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

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

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

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

If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.

Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

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

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

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

As has been set out above, a first aspect of the present invention pertains to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified by increase (up-regulated) when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least one molecular tumor marker selected from the following Table 1 :

Table 1

The present invention further relates to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified by increase (up-regulated) when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least one molecular tumor marker selected from the following Table 2:

Table 2

The present invention further relates to a tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified by increase (up-regulated) when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least one molecular tumor marker selected from the following Table 3. Table 3 partially overlaps with Table 1. Identical markers are indicated by the same SEQ ID NO.

Table 3

* Note: table entries #159 & #160 are represented by the same measurement data as the corresponding antigen sequence of the protein arrays is matching two different gene symbols and two different swissprot ID's (cyclin D & CDK4). The p-values for these entries are therefore the same, and both entries are represented by only a single ROC curve (see Fig 8, plot #159) and data plot (see Fig 9, plot #159).

The term " molecular marker" or " molecular tumor marker", as used herein, relates to a gene, genetic unit, antigen or sequence (a nucleotide sequence or amino acid or protein sequence) as defined in Table 1 , Table 2 or Table 3 whose expression level is modified, i.e. increased, in a neoplastic cell, or in a neoplastic tissue, in particular a cancerous cell, or in a cancerous tissue or in any type of sample comprising neoplastic cells, in particular cancerous cells or neoplastic, in particular cancerous tissues or portions or fragments thereof, in comparison to a control level or state.

The term also refers to any expression product of said genetic unit or sequence or variants or fragments thereof, as well as homologues or derivatives thereof. The term specifically refers to the genes, genetic units, sequences, proteins, protein sequences, antibodies, antigens, homologues and/or derivatives thereof indicated as marker # 1 to # 9 in Table 1, marker #1 to #120 in Table 2, or marker #1 to #201 in Table 3, having the nucleotide or amino acid sequences of SEQ ID NOs: 1 to 980, respectively, wherein the nucleotide sequences, corresponding amino acid sequences, or derivatives or fragments thereof as used for the underlying immune response experiments are comprised as indicated in Table 1, 2 or 3, or as derivable from the Examples. The term also comprises corresponding genomic sequences which can be obtained from any suitable database or information depositories, e.g. the Genbank database. 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 "molecular marker" or " molecular 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 molecular tumor marker also antibodies which specifically bind to proteins comprising the amino acid sequence as indicated in section E) of Table 1, 2 or 3, more preferably to proteins comprising the amino acid sequence as indicated in section G) of Table 1, 2 or 3, or to epitopes derived from the amino acid sequences indicated in sections E) or G) of Table 1, 2 or 3.

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

Nucleic acid sequences according to the present invention may be, for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section D) of Table 1, 2 or 3, amino acid sequences may be at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section E) or section G) of Table 1, 2 or 3, nucleic acid sequences encoding amino acid sequences may be at least 75%>, 80%>, 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section E) or section G) of Table 1, 2 or 3, or amino acid sequences may be encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%o or 99%> identical to the sequence as set forth in section D) of Table 1, 2 or 3. Further, any variants, mutants and functional domains of the nucleic acid molecules, proteins and polypeptides as mentioned above may be encompassed. The term " molecular tumor marker gene" or " molecular 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 mRNA transcripts encoding a molecular tumor marker as indicated in Table 1, 2 or 3, preferably cDNA molecules.

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

The terms "protein" or "polypeptide" are used herein to designate a produced or naturally occurring polypeptide or a recombinant polypeptide corresponding to the molecular tumor marker as mentioned in Table 1, 2 or 3. The term "protein" according to the present invention is to be seen as being interchangeably with the term "polypeptide". The polypeptides or proteins may be encoded by any of the abovementioned nucleic acid molecules. The polypeptides or proteins may further be glycosylated or may be non- glycosylated or may otherwise by modified. In addition, polypeptides or proteins may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

The term "marker" or "tumor marker", as used herein, relates to a marker comprising a molecular tumor marker as defined herein above, as well as one or more additional diagnostic factors. The term "diagnostic factor" as used herein means factors known to be of significance in the identification, progression, development or diagnosis of a neoplastic disease. Such a factor may be, for example, a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE). Further suitable diagnostic factors would be known to the skilled person and may also be considered.

One or more of these diagnostic factors may be determined, assessed and rated according to suitable rating schemes. It is particularly preferred to convert the diagnostic factor into numerical data according to schemes and procedures known in the art. Further details may be derived from qualified publications or textbooks, e.g. from Kranse et al, The Prostate, 68,1674-80 (2008); Roobol et al, Eur Urol, 57(l),79-85 (2009); Roobol et al, Eur Urol, 182(5), 2112-20 (2009).

The term "neoplastic" as used in the context of the present invention refers to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, as well as any of a number of characteristic structural and/or molecular features. A "neoplastic cell" is accordingly understood as a cell having specific structural properties, lacking differentiation and in many instances, being capable of invasion and metastasis. Thus the term "neoplastic", "neoplastic disease", "neoplastic disease state" etc. refers to all diseases, disorders, clinical manifestations and/or symptoms characterized by uncontrolled, abnormal cellular proliferation, be it benign or malign in nature, that is, these terms refer to all benign hyperplasia, benign growths, benign lesions, benign tumors and the like as well as to all pre-malignant growths, swellings and lesions, malignant tumors, cancers, malignant cancers, metastases, metastasing cancers and the like.

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

The term "progression of a neoplastic disease" as used herein relates to a switch between different stages of development or genesis of a neoplastic disease and principally refers to a situation in which the neoplastic disease becomes worse and/or spreads in the body. Explicitly encompassed in this term is also the transition of healthy tissue and/or cells into a benign tumor, and preferably the transition of a benign tumor into a malignant lesion/growth/hyperplasia or cancer due to uncontrolled, abnormal proliferation of cells and featuring any of a number of characteristic structural and/or molecular features.

Any changes that are associated with a worsening of the disease, i.e. be it the nature of the transformation (healthy to benign tumor/lesion/growth/hyperplasia, benign tumor/lesion/growth/hyperplasia to malignant tumor or cancer), aggressiveness of the cancer, the localization of the tumor and or /cancerous cells, the occurrence of metastases, presentation of additional or more profound clinical symptoms, recurrence of a tumor after treatment, decreased survival rate, are typically translated into a progression of stages of a given neoplastic disease.

Any worsening of the disease can thus be translated into a switch into the next stage of a given neoplastic disease. Any worsening of a neoplastic disease beyond the stage of a benign tumor, a benign lesion, benign growth, or hyperplasia is understood as a "cancer".

Different classifications exists for such switches, e.g. stages 0 and I to IV of the TNM classification, preferably the TNM classification system for prostate cancer as defined herein below, or any other stage or sub-stage of any suitable graduating or scoring system, starting from a healthy condition up to a terminal cancer scenario. Typically such switches are accompanied by a modification of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, preferably an increase or decrease of the expression level in a test sample in comparison to a previous test sample from the same individual, a test sample from an individual having been diagnosed with a certain type of a neoplastic disease as defined herein above and/or a stage or state of a neoplastic disease as defined herein above, or a value derivable from an information depository on expression data etc.

Progression of a neoplastic disease as defined herein above may further be determined, checked, crosschecked or independently be diagnosed etc. according to the "Gleason score". To determine the Gleason score, typically a grade is assigned to the most common tumor pattern, and a second grade to the next most common tumor pattern. The two grades are added together to get a Gleason score. The Gleason grade is also known as the Gleason pattern or Gleason sum. The Gleason grade may range from 1 to 5, with 5 having the worst prognosis. The Gleason score typically ranges from 2 to 10, with 10 having the worst prognosis. Methods to apply the Gleason score system, corresponding assessment techniques etc. would be known to the person skilled in the art. The scoring system may be used, for example, in order to verify, check or fine-tune the diagnosis, detection, indication of stages or monitoring according to the present invention.

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

Preferably, the term includes different early proliferation/developmental stages or levels of tumor development in the organism between (and excluding) a non-cancerous cell state and (including) an advanced malignant 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 defined herein below. The term also includes stages before TNM stage 0, e.g.

developmental stages in which cancer biomarkers known to the person skilled in the art show a modified expression or expression pattern. In a specific embodiment of the present invention a less progressed stage may be a healthy state. In a further specific embodiment of the present invention a less progressed sate may also be a stage of malignant tumor development, e.g. malignant prostate cancer stages. Particularly preferred are less progressed stages of benign tumor development, e.g. benign prostate tumors.

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 a tumor marker known to the person skilled in the art like PCA3 in comparison to a corresponding less progressed stage as defined herein above. Preferably, the term relates to a worsened disease state of a neoplastic disease as defined herein above, e.g. cancer or a malignant tumor in comparison to a less progressed stage as defined herein above. For example, if the less progressed stage is a stage 0 or a healthy state, the more progressed stage may be any higher or more advanced stage, e.g. a stage of any one of stage I, II, III or IV. Preferably, a "more progressed stage" as used herein may be the next worse stage if starting from a less progressed stage as defined herein above. For example, if the less progressed stage is a stage 0 or healthy or a benign tumor, the more progressed stage may be a stage I etc. The "next worse stage" may be reflected by any of the known staging and/or grading systems known to the person skilled in the art. Preferably, the "next worse stage" refers to the staging system provided by the UICC 2002 classification, more preferably the TNM classification for prostate cancer provided herein below. In a specific embodiment of the present invention the staging or grading of a tissue may optionally or additionally be determined, checked, crosschecked or independently be diagnosed by classical staging methods known to the person skilled in the art, e.g. via histological approaches, imaging methods etc.

In a specific embodiment of the present invention a more progressed stage may be determined by a comparison of the expression level of a molecular tumor marker as indicated in Table 1 , 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 in samples from subjects whose disease state is known. Furthermore, the control level can be derived from a database of expression levels or patterns from previously tested subjects or cells. Moreover, the control level may be multiple control levels whose control levels are determined from multiple reference samples. The control level may accordingly be derived from experimental approaches or from a database of expression levels from previously tested subjects, tissues or cells or from any suitable source of information known to the person skilled in the art. In a specific embodiment of the present invention, the control level can be determined from a reference sample derived from a subject who has been diagnosed to suffer from a certain neoplastic disease as defined herein above, preferably cancer, and wherein the stage and development of the neoplastic disease has been determined, as well as from healthy individuals.

In the context of the present invention, a control level determined from a biological sample that is known not to be neoplastic is called "normal control level". In another embodiment of the present invention, the control level can be from a neoplastic biological sample, e.g. a sample from a subject for which the neoplastic disease as defined herein above, preferably cancer, or prostate cancer, was diagnosed independently, it may be designated as "neoplastic control level". Alternatively, reference samples may comprise material derived from cell lines, e.g. immortalized cancer cell lines, or be derived from tissue xenografts. Preferably, material derived from prostate cancer cell lines or material derived from tissue xenografts with human prostate tissue, in particular with benign and tumor- derived human prostate tissue, may be comprised in a reference sample according to the present invention. Examples of cancer cell lines to be used comprise cells lines PC346P, PC346B, LNCaP, VCaP, DuCaP, PC346C, PC3, DU145, PC346CDD, PC346Flul,

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

Correspondingly obtained values and information may also be combined, normalized and statistically processed according to any suitable technique or method known to the person skilled in the art. By comparing a control level to a measured expression level a modification of the expression may be registered, which may accordingly be used for the determination of the more progressed stage of a neoplastic disease as defined herein above. For such comparison processes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more different control levels may be determined or assessed. Accordingly, the less progressed stage of a neoplastic disease as defined herein above may be determined by 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 neoplastic disease, e.g. cancer. In an even more preferred embodiment of the invention said comparison process comprises the determination of control levels obtained from a sample of an individual afflicted with a neoplastic disease, e.g. cancer, wherein the sample is representative of stages of the histological four-, three-, two-layer grading as per the guidelines of the American Joint Commission on Cancer, as defined herein above.

The comparison processes may further be combined with a comparison with the indications in sections H), and I) of Table 1, 2 or 3, wherein section H) indicates the averaged detected amount or expression of the marker in the less progressed stage, and section I) indicates the averaged detected amount or expression of the marker in the more progressed stage. Particularly preferred are control levels determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state is/are known to be prostate tumors, e.g. benign prostate tumors, or prostate cancer, more preferably prostate cancer of stage < T2 (UICC 2002 classification) or of stage >T3 (UICC 2002 classification), or prostate cancer of stage >T3 (UICC 2002 classification) and showing a serum level of PSA of about <10 ng/ml.

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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, 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 an increase or up-regulation of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 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 molecular tumor marker(s) or group of tumor markers is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated, for example, in sections I) (more progressed stage) and H) (less progressed stage) of Table 1, 2 or 3.

In a preferred embodiment of the present invention the expression of the molecular tumor marker(s) or group of tumor markers is increased (up-regulated) when comparing the expression in the more progressed stage to the expression in the less progressed stage, as indicated in section I) vs. section H) of Table 1, 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 between a situation to be analyzed, e.g. a situation derivable from a patient's sample, and a reference point, which could either be a control level derivable from any suitable tumor, e.g. a prostate tumor, or neoplastic disease stage known to the person skilled in the art, e.g. a healthy state, a benign tumor stage or the expression of a less progressed stage as defined herein above. Expression levels are deemed to be "increased" when the gene expression of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, e.g. in a sample to be analyzed, differs by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from a control level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to a control level or the expression level of a less progressed stage as defined herein above.

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, or molecular tumor markers of Table 1. 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, or all molecular tumor markers indicated in Table 1.

In a further preferred embodiment of the present invention the tumor marker or group of tumor markers comprises the first 1, 2, 3, 4, 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 or 120 molecular tumor markers of Table 2.

In a particularly preferred embodiment of the present invention the tumor marker or group of tumor markers comprises at least 1, 2, 3, 4, 6, 7, 8, 9, 11, 1,2, 13, 14, 16, 17, 18, 19, 21, 22, 23, 24, 26, 27, 28, 29, 31, 32, 33, 34, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 51, 52, 53, 55, 56, 57, 58, 59, 61, 62, 63, 64, 66, 67, 68, 69, 71, 72, 73, 74, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or all molecular tumor markers indicated in Table 2.

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, 23, 24, 25, 27, 29, 30, 32, 35, 37, 40, 42 45, 47, 50, 52, 55, 57 or 60 molecular tumor markers of Table 3

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, 46, 47, 48, 49, 50 or all molecular tumor markers indicated in Table 3.

In another preferred 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, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200 or all of the molecular tumor markers of Table 3.

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, 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 or 166 of the molecular tumor markers indicated in Table 3. The group may further comprise any sub-grouping or combinations of these markers.

In a particularly preferred embodiment the group of tumor markers may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, or all molecular tumor markers of Table 1 and/or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or all of the molecular tumor markers of Table 2, and/or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or all of the molecular tumor markers of Table 3.

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 0.00007, 0.0008, 0.0001, 0.00015, 0.0002, 0.00025, 0.0003, 0.00035, 0.0004, 0.00045, 0.0005, 0.00055, 0.0006, 0.00065, 0.0007, 0.0008, 0.0009, 0.001, 0.0011, 0.0012, 0.0013, 0.0014, 0.005, 0.06, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, or lower as indicated in section J) of Table 1, 2 or 3. Preferred are groups of markers which show p- values of the expression of between about 0.00007 and about 0.004, particularly preferred are groups of markers having a p-value of expression of 0.007 or lower, as indicated in section J) of Table 1, 2 or 3. The term "p-value" is a measure of the probability that a variant would assume a value greater than or equal to the observed value strictly by chance and is expressed by the following term: P (z > z ₀ b _se rved). Thus, in the context of the present invention, the p- value may be seen as a measure of statistical significance.

In another embodiment of the present invention the group of tumor markers comprises at least 2 molecular tumor markers corresponding to tumor marker #1 to #2 of Table 1, or at least 3 molecular tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 4 molecular tumor markers corresponding to tumor marker #1 to #4 of Table 1, or at least 5 molecular tumor markers corresponding to tumor marker #1 to #5 of Table 1, or at least 6 molecular tumor markers corresponding to tumor marker #1 to #6 of Table 1, or at least 7 molecular tumor markers corresponding to tumor marker #1 to #7 of Table 1, or at least 8 molecular tumor markers corresponding to tumor marker #1 to #8 of Table 1.

In another embodiment the group of tumor markers comprises at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker#l to #30, #30 to #60, #60 to #90, #90 to #120, #1 to #60, #60 to #120 or #1 to #120 of Table 2 and/or at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #120 of Table 2 and/or at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #50 of Table 3.

In yet another embodiment the group of tumor marker comprises at least 1, 2,

3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #50 of Table 3.

In another embodiment of the present invention the group of tumor markers comprises at least 2 molecular tumor markers corresponding to tumor marker #1 to #2 of Table 1, or at least 3 molecular tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 4 molecular tumor markers corresponding to tumor marker #1 to #4 of Table 1, or at least 5 molecular tumor markers corresponding to tumor marker #1 to #5 of Table 1, or at least 6 molecular tumor markers corresponding to tumor marker #1 to #6 of Table 1, or at least 7 molecular tumor markers corresponding to tumor marker #1 to #7 of Table 1, or at least 8 molecular tumor markers corresponding to tumor marker #1 to #8 of Table 1 and at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #120 of Table 2.

Table 3.

In another embodiment of the present invention the group of tumor markers comprises at least 2 molecular tumor markers corresponding to tumor marker #1 to #2 of Table 1, or at least 3 molecular tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 4 molecular tumor markers corresponding to tumor marker #1 to #4 of Table 1, or at least 5 molecular tumor markers corresponding to tumor marker #1 to #5 of Table 1, or at least 6 molecular tumor markers corresponding to tumor marker #1 to #6 of Table 1, or at least 7 molecular tumor markers corresponding to tumor marker #1 to #7 of Table 1, or at least 8 molecular tumor markers corresponding to tumor marker #1 to #8 of Table 1 and at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #50 of Table 3.

In another embodiment the group of tumor markers comprises at least 2 molecular tumor markers corresponding to tumor marker #1 to #2 of Table 1, or at least 3 molecular tumor markers corresponding to tumor marker #1 to #3 of Table 1, or at least 5 molecular tumor markers corresponding to tumor marker #1 to #5 of Table 1, or at least 9 molecular tumor markers corresponding to tumor marker #1 to #9 of Table 1, or at least 12 molecular tumor markers corresponding to tumor marker #1 to #12 of Table 1, or at least 15 molecular tumor markers corresponding to tumor marker #1 to #15 of Table 1, or at least 17 molecular tumor markers corresponding to tumor marker #1 to #17 of Table 1; and at least 1,

2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #120 of Table 2; and at least 1, 2, 3, 4, 5, 6, 10, 15, 20, 25 or 30 molecular tumor markers selected from tumor marker #1 to #50 of Table 3.

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 3, at least 10 tumor markers corresponding to tumor marker #1 to #10 of Table 3, at least 15 tumor markers corresponding to tumor marker #1 to #15 of Table 3, at least 20 tumor markers corresponding to tumor marker #1 to #20 of Table 3, at least 25 tumor markers corresponding to tumor marker #1 to #25 of Table 3, at least 30 tumor markers corresponding to tumor marker #1 to #30 of Table 3, at least 35 tumor markers corresponding to tumor marker #1 to #35 of Table 3, at least 40 tumor markers corresponding to tumor marker #1 to #40 of Table

3, at least 45 tumor markers corresponding to tumor marker #1 to #45 of Table 3 or at least 50 tumor markers corresponding to tumor marker #1 to #50 of Table 3. 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, tumor markers #1 to #100, tumor markers #1 to #105, tumor markers #1 to #110, tumor markers #1 to #115, tumor markers #1 to #120, tumor markers #1 to #125, tumor markers #1 to #130, tumor markers #1 to #135, tumor markers #1 to #140, tumor markers #1 to #145, tumor markers #1 to #150, tumor markers #1 to #155, tumor markers #1 to #160, tumor markers #1 to #165, tumor markers #1 to #170, tumor markers #1 to #175, tumor markers #1 to #180, tumor markers #1 to #185, tumor markers #1 to #190, tumor markers #1 to #195, tumor markers #1 to #200 of Table 3. The group of tumor marker according to the present invention may also comprise tumor markers #1, #3, #5, #7 and #9 of Table 3, #2, #4, #6, #8, and #10 of Table 3, #3, #5, #7, #9 and #11 of Table 3, #4, #6, #8, #10, and #12 of Table 3, #5, #7, #9, #11 and #13 of Table 3, #6, #8, #10, #12 and #14 of Table 3, #7, #9, #11, #13 and #15 of Table 3, #8, #10, #12, #14and #16 of Table 3, #9, #11, #13, #15 and #17 of Table 3, #10, #12, #14, #16 and #18 of Table 3, #11, #13, #15, #17 and #19 of Table 3, #12, #14, #16, #18 and #20 of Table 3, #13, #15, #17, #19 and #21 of Table 3 etc.

In another preferred embodiment of the present invention the group of tumor markers may comprise:

(i) at least molecular marker # 1 of Table 1 (FGFR3) and at least one additional marker of Table 1, Table 2 or Table 3; or

(ii) at least molecular marker # 2 of Table 1 (SEPP1) and at least one additional marker of Table 1, Table 2 or Table 3; or

(iii) at least molecular marker # 3 of Table 1 (C9orf97) and at least one additional marker of Table 1, Table 2 or Table 3; or

(iv) at least molecular marker # 4 of Table 1 (PBK) and at least one additional marker of Table 1, Table 2 or Table 3; or

(v) at least molecular marker # 5 of Table 1 (MAPK9) and at least one additional marker of Table 1, Table 2 or Table 3; or

(vi) at least molecular marker # 6 of Table 1 (AKT3) and at least one additional marker of Table 1, Table 2 or Table 3; or

(vii) at least molecular marker # 7 of Table 1 (AKT2) and at least one additional marker of Table 1, Table 2 or Table 3; or

(viii) at least molecular marker # 8 of Table 1 (PRAME) and at least one additional marker of Table 1, Table 2 or Table 3; or

(ix) at least molecular marker # 9 of Table 1 (PRKCB2) and at least one additional marker of Table 1, Table 2 or Table 3.

In another embodiment of the present invention the group of tumor markers may comprise:

(i) at least molecular marker # 1 of Table 1 (FGFR3) and optionally at least one additional marker of Table 1, Table 2 or Table 3, and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or

(ii) at least molecular marker # 2 of Table 1 (SEPP1) and optionally at least one additional marker of Table 1, Table 2 or Table 3 and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or

(iii) at least molecular marker # 3 of Table 1 (C9orf97), and optionally at least one additional marker of Table 1, Table 2 or Table 3; and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or (iv) at least molecular marker # 4 of Table 1 (PBK), and optionally at least one additional marker of Table 1, Table 2 or Table 3; and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or

(v) at least molecular marker # 5 of Table 1 (MAPK9), and optionally at least one additional marker of Table 1, Table 2 or Table 3; and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or

(vi) at least molecular marker # 6 of Table 1 (AKT3) and optionally at least one additional marker of Table 1, Table 2 or Table 3; and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or

(vii) at least molecular marker # 7 of Table 1 (AKT2), and optionally at least one additional marker of Table 1, Table 2 or Table 3; and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or

(viii) at least molecular marker # 8 of Table 1 (PRAME), and optionally at least one additional marker of Table 1, Table 2 or Table 3; and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker; or

(ix) at least molecular marker # 9 of Table 1 (PR CB2) and optionally at least one additional marker of Table 1, Table 2 or Table 3;and one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE); and/or PSA as further molecular marker.

In a further embodiment any of the above described tumor marker groupings may comprise one or more additional diagnostic factors, such as, for example a biopsy result, a patient's histological statement, a patient's age, a patient's vitality, the outcome of superficial examinations, e.g. the outcome of a digital rectal examination (DRE). In a preferred embodiment of the present invention any of the above described tumor marker groups may comprise a patient's age. In another preferred embodiment of the present invention any of the above described tumor marker groups may the outcome of a patient's digital rectal examination (DRE) as additional diagnostic factor. In yet another preferred embodiment of the present invention any of the above described tumor marker groups may comprise a patient's age and the outcome of a patient's digital rectal examination (DRE) additional diagnostic factor.

The term "patient's age" refers to the number of years of a patient's life. These numbers may be used as numerical data within a combination of molecular markers, e.g. expression data obtained from molecular tumor markers as defined herein above. According to suitable procedures this numerical data may be weighted and assessed according to the type or stage of disease. In a specific embodiment of the present invention the high number of years of a patient's life may have a positive effect on the overall significance of a diagnostic/prognostic statement associated with a group of tumor markers as defined herein.

The term "digital rectal examination" or "DRE" as used herein refers to an digital inspection of a patient's rectum whereby lumps, enlargements, or areas of hardness that might indicate cancer, can be identified. The outcome of a patient's digital rectal examination (DRE) may be classified according to suitable numerical schemes for DRE. For example a numerical scheme of DRE results may comprise the following values:

0 = Unknown; 1 = Symmetrical benign; 2 = Asymmetrical benign; 3 = Palpable nodule; 4 = Other palpable abnormality; 5 = Equivocal; 6 = Frank carcinoma; and 7 = Other. This scheme is preferably employed in the context of the present invention. However, further alternative schemes known to the person skilled in the art are also envisaged.

In a further embodiment any of the above described tumor marker groupings or groups may additionally comprise PSA as a further molecular marker. Thus, in a specific embodiment of the present invention any of the above indicated groupings of tumor markers, as well as groupings of tumor markers with additional diagnostic factors such as a patient's age and/or DRE, may additionally comprise PSA as further molecular marker.

The term "PSA" as used herein refers to the gene, transcript, or expression product, e.g. protein, of human prostate specific antigen. Corresponding sequences would be known to the person skilled in the art and can be obtained from public data repositories such as Genbank or the EMBL database. The present invention thus envisages the assessment of PSA at different levels, e.g. at the level of mR A or the level of protein. Furthermore, the condition of PSA may be determined, e.g. whether PSA protein is free circulating in the blood/serum, whether it is complexed to other factors or proteins, how much PSA is present totally etc. Furthermore, the presence of PSA gene or transcript variants or mutants may be determined and is correspondingly considered as further molecular marker within the context of the present invention.

In a particularly preferred embodiment PSA may be total PSA protein, free PSA protein, complexed PSA protein and/or the ratio of free and total PSA protein or the ratio of complexed and total PSA protein, benign PSA, nicked PSA, proPSA, truncated forms of proPSA in particular -2proPSA or any ratios and/or combinations of these values. The determination of PSA may be carried out according to any suitable methods known to the skilled person, e.g. via PCR tests, via antibody tests, e.g. by using monoclonal antibodies, via immune sandwich assays, ELISA, or PJA. An example of a suitable test is the elecsys and cobas immunoassay. The determined amount of PSA may be compared to suitable controls e.g. from healthy subjections, earlier taken samples, etc. or to values known form the prior art, or to database entries etc.

In a particularly preferred embodiment of the present invention, the group of tumor markers comprises at least one marker combination shown in the following Table 4 (e.g. of combinations 1 to 90, as indicated in column A of Table 4; the identity of the markers and or their sequences may be derived from Table 1, Table 2 or Table 3, where the corresponding SEQ ID NOs. are indicated; alternatively, public database entries as indicated in column C of Table 4 may be used in order to obtain suitable marker sequences):

Table 4:

PSA & DRE & A T3 PV3185

(P B gamma) & PV3145 90 37 FGFR3 & SEPP1 BC040075.1

PSA & DRE & A T2

PV3184

(PKB beta) &

PV3620 90 36 MAPK9 (JNK2) &

BC040075.1

SEPP1

PSA & DRE & AKT3

PV3185

(PKB gamma) &

PV3145 90 36 FGFR3 & MAPK9

PV3620

(J K2)

PSA & DRE & AKT3 PV3185

(PKB gamma) & PV3145 90 36 FGFR3 & PBK NM 018492.2

PSA & DRE & BC022958.1

C9orff7 & PBK & NM 018492.2 90 36 SEPP1 BC040075.1

PSA & DRE & BC022958.1

C9orf97 & FGFR3 & PV3145 90 36 PBK NM 018492.2

BC022958.1

DRE & C9orff 7 & PV3145

90 36 FGFR3 & PBK & NM 018492.2

SEPP1 BC040075.1

PSA & DRE & AKT2

PV3184

(PKB beta) & AKT3

PV3185 90 35 (PKB gamma) &

PV3620

MAPK9 (J K2)

PSA & DRE & PV3145

FGFR3 & MAPK9 PV3620 90 35 (J K2) & SEPP1 BC040075.1

PSA & Patient Age&

BC022958.1

DRE & C9orff 7 & 90 35

PV3145

FGFR3

PSA & DRE & AKT3

PV3185

(PKB gamma) &

PV3620 90 35 MAPK9 (J K2) &

BC040075.1

SEPP1

PSA & Patient Age&

DRE & AKT2 (PKB PV3184

90 34 beta) & AKT3 (PKB PV3185

gamma)

PSA & Patient Age&

BC022958.1

DRE & C9orff 7 & 90 34

BC040075.1

SEPP1

PSA & Patient Age&

PV3145

DRE & FGFR3 & 90 34

PV3620

MAPK9 (J K2)

BC022958.1

Patient Age& C9orf97 PV3145

90 34 & FGFR3 & PBK & NM 018492.2

SEPP1 BC040075.1

PV3184

DRE & AKT2 (PKB BC022958.1

90 34 beta) & C9orff7 & NM 018492.2

PBK & SEPP1 BC040075.1

PSA & DRE & AKT2

PV3184

(PKB beta) & AKT3

PV3185 90 34 (PKB gamma) &

BC040075.1

SEPP1

PSA & DRE & AKT2 PV3184

(PKB beta) & FGFR3 PV3145 90 34 & SEPP1 BC040075.1 PSA & A T2 (P B PV3184

beta) & C9orff7 & BC022958.1

80 95 24

MAP 9 (JNK2) & PV3620

PB NM 018492.2

PSA & Patient Age &

PV3185

81 DRE & A T3 (PKB 95 24

BC040075.1

gamma) & SEPP1

PSA & DRE & AKT2

PV3184

(PKB beta) &

82 BC022958.1 95 24

C9orff7 & MAPK9

PV3620

(J K2)

PSA & DRE & AKT2 PV3184

83 (PKB beta) & FGFR3 PV3145 95 24

& PRKCB2 P2251

PSA & DRE & AKT2 PV3184

84 (PKB beta) & FGFR3 PV3145 95 24

& SEPP1 BC040075.1

PSA & DRE & AKT3 PV3185

85 (PKB gamma) & PV3145 95 24

FGFR3 & PBK NM 018492.2

PSA & DRE & BC022958.1

86 C9orff7 & MAPK9 PV3620 95 24

(J K2) & SEPP1 BC040075.1

PSA & DRE & PV3145

87 FGFR3 & MAPK9 PV3620 95 24

(J K2) & PRAME BC039731.1

PV3184

PSA & AKT2 (PKB BC022958.1

88 95 24

beta) & C9or»7 & NM 018492.2

PBK & SEPP1 BC040075.1

PSA & C9orf97 &

BC022958.1

FGFR3 PV3145 &

89 PV3620 95 24

MAPK9 (J K2) &

NM_018492.2

PBK

BC022958.1

DRE & C9orff 7 & PV3620

90 95 24

MAPK9 (J K2) & BC039731.1

PRAME & SEPP1 BC040075.1

The combinations provided in Table 4 may comprise further molecular markers or additional diagnostic factors, e.g. one or more of the molecular markers of Table 1, 2 or 3.

In a further preferred embodiment the present invention also envisages at least one marker combination derivable from the following Table 5 (e.g. of combinations 1 to 102, as indicated in column A of Table 5; the identity of the markers and or their sequences may be derived from Table 1, Table 2 or Table 3, where the corresponding SEQ ID NOs. are indicated; alternatively, public database entries as indicated in column C of Table 5 may be used in order to obtain suitable marker sequences): Table 5

BC008624.1

N/A-BC008624.1 &

NM 013230.1

CD24 & PPP2R4 & 90 39

NM 021131.1

CYP1A1 & ADCKl

NM 000499.2

NM 021821.2

MRPS35 & CREM &

BC017117.1

ADCKl & ITPA & 90 39

NM 020421.1

COL9A1

NM 033453.2

NM 021821.2

MRPS35 & CD24 &

NM 013230.1

PPP2R4 & AMN1 & 90 39

NM 021131.1

ADCKl

NM 207337.1

NM 021821.2

MRPS35 & CD24 &

NM 013230.1

PPP2R4 & ADCKl & 90 39

NM 021131.1

COL9A1

NM 020421.1

NM 021821.2

MRPS35 & CD24 &

NM 013230.1

CYP1A1 & ADCKl 90 39

NM 000499.2

& COL9A1

NM 020421.1

NM 021821.2

MRPS35 & PPP2R4

NM 021131.1

& AMN1 & ADCKl 90 39

NM 207337.1

& COL9A1

NM 020421.1

NM 021821.2

MRPS35 & PPP2R4

NM 021131.1

& ADCKl & ITPA & 90 39

NM 020421.1

CASC2

NM 033453.2

NM 021821.2

MRPS35 & PPP2R4

NM 021131.1

& ADCKl & CASC2 90 39

NM 020421.1

& COL9A1

NM 201377.1

NM 021821.2

MRPS35 & AMN1 &

NM 207337.1

GNG4 & ADCKl & 90 39

NM 004485.2

COL9A1

NM 020421.1

NM 021821.2

MRPS35 & AMN1 &

NM 207337.1

ADCKl & CASC2 & 90 39

NM 020421.1

COL9A1

NM 201377.1

BC017117.1

CREM & MLF1IP &

BC031520.1

PPP2R4 & ADCKl & 90 39

NM 021131.1

CASC2

NM 020421.1

BC031520.1

MLF1IP & CD24 &

NM 013230.1

PPP2R4 & CYP1A1 90 39

NM 021131.1

& ADCKl

NM 000499.2

BC031520.1

MLF1IP & CD24 &

NM 013230.1

AMN1 & GNG4 & 90 39

NM 207337.1

ADCKl

NM 004485.2

BC031520.1

MLF1IP & CD24 &

NM 013230.1

ADCKl & ITPA & 90 39

NM 020421.1

COL9A1

NM 033453.2

BC031520.1

MLF1IP & AMN1 &

NM 207337.1

CYP1A1 & GNG4 & 90 39

NM 000499.2

LUC7L2

NM 004485.2 BC031520.1

MLF1IP & CYP1A1

NM 000499.2

& ADCKl & CASC2 90 39

NM 020421.1

& COL9A1

NM 201377.1

BC031520.1

MLF1IP & CYP1A1

NM 000499.2

& ADCKl & CASC2 90 39

NM 020421.1

& LUC7L2

NM 201377.1

NM 013230.1

CD24 & PPP2R4 &

NM 021131.1

CYP1A1 & ADCKl 90 39

NM 000499.2

& COL9A1

NM 020421.1

NM 013230.1

CD24 & AMN1 &

NM 207337.1

CYP1A1 & ADCKl 90 39

NM 000499.2

& COL9A1

NM 020421.1

NM 013230.1

CD24 & AMN1 &

NM 207337.1

ADCKl & ITPA & 90 39

NM 020421.1

COL9A1

NM 033453.2

NM 021131.1

PPP2R4 & AMN1 &

NM 207337.1

CYP1A1 & ADCKl 90 39

NM 000499.2

& CASC2

NM 020421.1

NM 021131.1

PPP2R4 & AMN1 &

NM 207337.1

ADCKl & CASC2 & 90 39

NM 020421.1

COL9A1

NM 201377.1

BC008624.1

N/A - BC008624.1 &

NM 021821.2

MRPS35 & MLF1IP 90 37

BC031520.1

& ADCKl & CASC2

NM 020421.1

BC008624.1

N/A-BC008624.1 &

BC031520.1

MLF1IP & AMN1 & 90 37

NM 207337.1

CYP1A1 & ADCKl

NM 000499.2

BC008624.1

N/A-BC008624.1 &

BC031520.1

MLF1IP & AMN1 & 90 37

NM 207337.1

ADCKl & CASC2

NM 020421.1

BC008624.1

N/A-BC008624.1 &

BC031520.1

MLF1IP & CYP1A1 90 37

NM 000499.2

& ADCKl & CASC2

NM 020421.1

NM 021821.2

MRPS35 & CREM &

BC017117.1

AMN1 & ITPA & 90 37

NM 207337.1

COL9A1

NM 033453.2

NM 021821.2

MRPS35 & MLF1IP

BC031520.1

& ADCKl & ITPA & 90 37

NM 020421.1

COL9A1

NM 033453.2

NM 021821.2

MRPS35 & CD24 &

NM 013230.1

ADCKl & ITPA & 90 37

NM 020421.1

COL9A1

NM 033453.2

NM 021821.2

MRPS35 & PPP2R4

NM 021131.1

& GNG4 & ADCKl 90 37

NM 004485.2

& CASC2

NM 020421.1 NM 021821.2

MRPS35 & AMN1 &

NM 207337.1

ADC 1 & ITPA & 90 37

NM 020421.1

COL9A1

NM 033453.2

NM 021821.2

MRPS35 & CYP1A1

NM 000499.2

& ADC 1 & ITPA & 90 37

NM 020421.1

COL9A1

NM 033453.2

BC017117.1

CREM & MLFIIP &

BC031520.1

AMN1 & ADC 1 & 90 37

NM 207337.1

COL9A1

NM 020421.1

BC017117.1

CREM & MLFIIP &

BC031520.1

CYPlAl & ADC 1 90 37

NM 000499.2

& ITPA

NM 020421.1

BC017117.1

CREM & CD24 &

NM 013230.1

ADC 1 & ITPA & 90 37

NM 020421.1

COL9A1

NM 033453.2

BC031520.1

MLFIIP & CD24 &

NM 013230.1

PPP2R4 & ADC 1 & 90 37

NM 021131.1

COL9A1

NM 020421.1

BC031520.1

MLFIIP & CD24 &

NM 013230.1

AMN1 & ADC 1 & 90 37

NM 207337.1

ITPA NM 020421.1

BC031520.1

MLFIIP & CD24 &

NM 013230.1

CYPlAl & CASC2 & 90 37

NM 000499.2

COL9A1

NM 201377.1

BC031520.1

MLFIIP & PPP2R4 &

NM 021131.1

AMN1 & ADC 1 & 90 37

NM 207337.1

CASC2

NM 020421.1

BC031520.1

MLFIIP & PPP2R4 &

NM 021131.1

AMN1 & CASC2 & 90 37

NM 207337.1

COL9A1

NM 201377.1

BC031520.1

MLFIIP & AM 1 &

NM 207337.1

CYPlAl & ADC 1 90 37

NM 000499.2

& LUC7L2

NM 020421.1

BC031520.1

MLFIIP & AM 1 &

NM 207337.1

ADC 1 & CASC2 & 90 37

NM 020421.1

COL9A1

NM 201377.1

BC031520.1

MLFIIP & AM 1 &

NM 207337.1

ADC 1 & COL9A1 90 37

NM 020421.1

& LUC7L2

BC015409.1

NM 013230.1

CD24 & PPP2R4 &

NM 021131.1

AMN1 & CYPlAl & 90 37

NM 207337.1

ADC 1

NM 000499.2

NM 013230.

CD24 & PPP2R4 &

NM 021131.1 1

AMN1 & CYPlAl & 90 37

NM 207337.1

CASC2

NM 000499.2 NM 013230.1

CD24 & PPP2R4 &

NM 021131.1

AMN1 & ADCKl & 90 37

NM 207337.1

CASC2

NM 020421.1

NM 013230.1

CD24 & PPP2R4 &

NM 021131.1

ADCKl & CASC2 & 90 37

NM 020421.1

COL9A1

NM 201377.1

NM 013230.1

CD24 & ADCKl &

NM 020421.1

ITPA & COL9A1 & 90 37

NM 033453.2

LUC7L2

BC015409.1

NM 207337.1

AMN1 & CYP1A1 &

NM 000499.2

ADCKl & ITPA & 90 37

NM 020421.1

CASC2

NM 033453.2

NM 207337.1

AMN1 & CYP1A1 &

NM 000499.2

ADCKl & ITPA & 90 37

NM 020421.1

COL9A1

NM 033453.2

NM 207337.1

AMN1 & GNG4 &

NM 004485.2

ITPA & CASC2 & 90 37

NM 033453.2

COL9A1

NM 201377.1

BC008624.1

N/A-BC008624.1 &

NM 021821.2

M PS35 & MLF1IP 90 35

BC031520.1

& CASC2 & COL9A1

NM 201377.1

BC008624.1

N/A-BC008624.1 &

NM 021821.2

MRPS35 & ADCKl 90 35

NM 020421.1

& CASC2 & COL9A1

NM 201377.1

BC008624.1

N/A-BC008624.1 &

BC031520.1

MLF1IP& AMN1 & 90 35

NM 207337.1

CASC2 & LUC7L2

NM 201377.1

BC008624.1

N/A-BC008624.1 &

BC031520.1

MLF1IP & ADCKl & 90 35

NM 020421.1

CASC2 & COL9A1

NM 201377.1

NM 021821.2

MRPS35 & CREM &

BC017117.1

MLF1IP & CASC2 & 90 35

BC031520.1

COL9A1

NM 201377.1

NM 021821.2

MRPS35 & MLF1IP

BC031520.1

& AMN1 & ADCKl 90 35

NM 207337.1

& COL9A1

NM 020421.1

NM 021821.2

MRPS35 & CD24 &

NM 013230.1

PPP2R4 & ADCKl & 90 35

NM 021131.1

CASC2

NM 020421.1

NM 021821.2

MRPS35 & CD24 &

NM 013230.1

PPP2R4 & CASC2 & 90 35

NM 021131.1

COL9A1

NM 201377.1

NM 021821.2

MRPS35 & CD24 &

NM 013230.1

AMN1 & GNG4 & 90 35

NM 207337.1

ADCKl

NM 004485.2 NM 021821.2

MRPS35 & PPP2R4

NM 021131.1

74 & AMN1 & ITPA & 90 35

NM 207337.1

COL9A1

NM 033453.2

NM 021821.2

MRPS35 & PPP2R4

NM 021131.1

75 & CYP1A1 & GNG4 90 35

NM 000499.2

& ADC 1

NM 004485.2

NM 021821.2

MRPS35 & PPP2R4

NM 021131.1

76 & GNG4 & ADC 1 90 35

NM 004485.2

& COL9A1

NM 020421.1

NM 021821.2

MRPS35 & AMN1 &

NM 207337.1

77 GNG4 & ADC 1 & 90 35

NM 004485.2

ITPA NM 020421.1

NM 021821.2

MRPS35 & CYP1A1

NM 000499.2

78 & ADC 1 & CASC2 90 35

NM 020421.1

& GL01

NM 201377.1

NM 021821.2

MRPS35 & CYP1A1

NM 000499.2

79 & ADC 1 & CASC2 90 35

NM 020421.1

& COL9A1

NM 201377.1

BC017117.1

CREM & ADC 1 &

NM 020421.1

80 ITPA & CASC2 & 90 35

NM 033453.2

COL9A1

NM 201377.1

BC031520.1

MLFIIP & CD24 &

NM 013230.1

81 ADC 1 & CASC2 & 90 35

NM 020421.1

COL9A1

NM 201377.1

BC031520.1

MLF1IP& PPP2R4 &

NM 021131.1

82 AMN1 & ADC 1 & 90 35

NM 207337.1

COL9A1

NM 020421.1

BC031520.1

MLFIIP & PPP2R4 &

NM 021131.1

83 ADC 1 & CASC2 & 90 35

NM 020421.1

COL9A1

NM 201377.1

BC031520.1

MLFIIP & AM 1 &

NM 207337.1

84 CYP1A1 & GNG4 & 90 35

NM 000499.2

ADC 1

NM 004485.2

BC031520.1

MLFIIP & AM 1 &

NM 207337.1

85 CYP1A1 & ITPA & 90 35

NM 000499.2

CASC2

NM 033453.2

BC031520.1

MLFIIP & AM 1 &

NM 207337.1

86 GNG4 & ADC 1 & 90 35

NM 004485.2

LUC7L2

NM 020421.1

BC031520.1

MLFIIP & AM 1 &

NM 207337.1

87 ADC 1 & ITPA & 90 35

NM 020421.1

CASC2

NM 033453.2

BC031520.1

MLFIIP & AM 1 &

NM 207337.1

88 ADC 1 & CASC2 & 90 35

NM 020421.1

LUC7L2

NM 201377.1 BC031520.1

MLF1IP & CYP1A1

NM 000499.2

89 & ADC 1 & ITPA & 90 35

NM 020421.1

CASC2

NM 033453.2

BC031520.1

MLF1IP & CYP1A1

NM 000499.2

90 & ADC 1 & ITPA & 90 35

NM 020421.1

COL9A1

NM 033453.2

BC031520.1

MLF1IP & ADC 1 &

NM 020421.1

91 ITPA & CASC2 & 90 35

NM 033453.2

GLOl

NM 201377.1

BC031520.1

MLF1IP & ADC 1 &

NM 020421.1

92 ITPA & COL9A1 & 90 35

NM 033453.2

LUC7L2

BC015409.1

NM 013230.1

CD24 & PPP2 4 &

NM 021131.1

93 GNG4 & ADC 1 & 90 35

NM 004485.2

COL9A1

NM 020421.1

NM 013230.1

CD24 & CYP1A1 &

NM 000499.2

94 GNG4 & ADC 1 & 90 35

NM 004485.2

COL9A1

NM 020421.1

NM 013230.1

CD24 & CYP1A1 &

NM 000499.2

95 ADC 1 & ITPA & 90 35

NM 020421.1

COL9A1

NM 033453.2

NM 013230.1

CD24 & GNG4 &

NM 004485.2

96 ADC 1 & ITPA & 90 35

NM 020421.1

COL9A1

NM 033453.2

NM 021131.1

PPP2R4 & AMN1 &

NM 207337.1

97 CYP1A1 & CASC2 & 90 35

NM 000499.2

COL9A1

NM 201377.1

NM 021131.1

PPP2R4 & AMN1 &

NM 207337.1

98 GNG4 & CASC2 & 90 35

NM 004485.2

COL9A1

NM 201377.1

NM 021131.1

PPP2R4 & AMN1 &

NM 207337.1

99 ITPA & CASC2 & 90 35

NM 033453.2

COL9A1

NM 201377.1

NM 021131.1

PPP2R4 & CYP1A1

NM 000499.2

100 & ITPA & CASC2 & 90 35

NM 033453.2

COL9A1

NM 201377.1

NM 207337.1

AMN1 & CYP1A1 &

NM 000499.2

101 ADC 1 & CASC2 & 90 35

NM 020421.1

COL9A1

NM 201377.1

NM 000499.2

CYP1A1 & GNG4 &

NM 004485.2

102 ADC 1 & ITPA & 90 35

NM 020421.1

COL9A1

NM 033453.2

The combinations provided in Table 5 may comprise further molecular markers or additional diagnostic factors, e.g. one or more of the molecular markers of Table 1, 2 or 3. In another preferred embodiment of the present invention the specificity for detection of the progression of a neoplastic disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above may be at least 20, 21, 22, 23, 24, 25, preferably at least 26, 27, 28, 29, more preferably at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 % with a sensitivity for detection of the progression of a neoplastic disease from a less progressed stage to a more progressed stage of a group of tumor markers as defined above of at least 85%, preferably 90%>. These values may be associated with any grouping of markers of Table 1, Table 2 and/or Table 3 as well as the above mentioned additional diagnostic factors. It is preferred that the sensitivity and specificity values as indicated above may be associated with combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more markers, more preferably with combinations of 3 or 5 markers.

In another preferred embodiment of the present invention the specificity for detection of the progression of a neoplastic disease from a less progressed stage to a more progressed stage of the group of tumor markers as defined above may be at least 19, 20, 21, 22, 23, preferably at least 24, 25, , more preferably at least 26, 27, 28, 29, 30%> with a sensitivity for detection of the progression of a neoplastic disease from a less progressed stage to a more progressed stage of a group of tumor markers as defined above of at least 92%o, preferably 95%>. These values may be associated with any grouping of markers of Table 1, Table 2 and/or Table 3 as well as the above mentioned additional diagnostic factors. It is preferred that the sensitivity and specificity values as indicated above may be associated with combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more markers, more preferably with combinations of 3 or 5 markers.

The specificity and sensitivity of a group or combination of markers according to the present invention may be determined according to suitable procedures known to the person skilled in the art. Preferably, the specificity and sensitivity may be determined as explained in Example 8. The methodology and the corresponding results are also envisaged as further embodiments of the present invention.

In particularly preferred embodiments the specificity and sensitivity may be a specificity and/or sensitivity as indicated in sections D) or E) of Table 4 or 5, or of corresponding Tables in the Examples. The group or combination of tumor markers may, thus, have any of the sensitivities or specificities as indicated in sections D) or E) of Table 4 or 5 or of corresponding Tables in the Examples.

In a further preferred embodiment the group of tumor markers may comprise at least one of the following combinations of elements, i.e. molecular markers and/or of additional diagnostic factors:

(i) AKT2 and FGFR3 and PSA; or

(ϋ) AKT3 and FGFR3 and PSA; or

(iii) AKT2 and DRE and PSA; or

(iv) C9orf97 and SEPP1 and PSA; or

(v) AKT3 and DRE and PSA; or

(vi) C9orf97 and FGFR3 and PBK ; or

(vii) C9orf97 and PRAME and SEPP1; or

(viii) C9orf97 and PBK and Patient Age; or

(ix) FGFR3 and PRAME and SEPP1; or

(x) C9orf97 and PBK and DRE; or

(xi) AKT2 and FGFR3 and MAPK9 and DRE and PSA; or

(xii) AKT2 and MAPK9 and Patient Age and DRE and PSA; or

(xiii) AKT3 and FGFR3 and Patient Age and DRE and PSA; or

(xiv) AKT2 and C9orf97 and SEPP1 and DRE and PSA;or

(XV) C9orf97 and FGFR3 and PBK and SEPP1 and PSA; or

(xvi) AKT2 and AKT3 and MAPK9 and DRE and PSA;; or

(xvii) AKT3 and FGFR3 and MAPK9 and DRE and PSA; or

(xviii) AKT2 and AKT3 and FGFR3 and DRE and PSA; or

(xix) FGFR3 and PBK and SEPP1 and DRE and PSA; or

(XX) C9orf97 and PBK and SEPP1 and DRE and PSA.

Further envisaged are combinations of combination (i) to (xx), as well as groupings of these combinations with any other marker of Tables 1 , 2 or 3, and/or with an additional diagnostic factor as mentioned above.

In a further aspect the present invention relates to the use of a tumor marker or group of tumor markers as defined herein above as a marker for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed of a neoplastic disease to a more progressed stage of a neoplastic disease, or prognosticating the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease.

The term "diagnosing a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease" as used herein means that a subject or individual may be considered to be suffering from a more progressed neoplastic disease as defined herein above, e.g. a more progressed prostate cancer, when the expression level of a molecular 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. Furthermore, in certain embodiments of the invention, the determination of additional diagnostic factors, such as a patient's age, the outcome of DRE, or the determination of PSA may also be considered and/or combined with the determined expression levels and the corresponding results. 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 molecular 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%o, 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 of said expression level.

In a further embodiment, an additional similarity in the overall gene expression pattern of a group of tumor markers according to the present invention between a sample obtained from a subject and a control sample or a sample corresponding to a less progressed disease state of a neoplastic disease as defined herein above as described herein above, may indicate that the subject is suffering from a more progressed disease stage of the neoplastic disease as defined herein above. In another embodiment of the present invention, the diagnosis may be combined with the elucidation of additional cancer biomarker expression levels, in particular prostate cancer biomarkers. Suitable biomarkers, in particular prostate cancer biomarkers, would be known to the person skilled in the art. For example, the expression of biomarkers like PC A3 may be tested.

The term "detecting a neoplastic disease associated with a progression from a less progressed stage to a more progressed stage" as used herein means that the presence of a neoplastic disease or disorder as defined herein above in an organism, which is associated with a more progressed stage may be determined or that such a neoplastic disease or disorder as defined herein above may be identified in an organism. The determination or identification of a more progressed neoplastic disease or disorder as defined herein above may be accomplished by a comparison of the expression level of the molecular tumor marker or group of tumor markers of the present invention in a sample from a patient or individual to be analyzed and the expression level of a control level as defined herein above, wherein said control level corresponds to the expression level of said more progressed neoplastic disease or disorder as defined herein above. Furthermore, in certain embodiments of the invention, the determination of additional diagnostic factors, such as a patient's age, the outcome of DRE, or the determination of PSA may also be considered and/or combined with the determined expression levels and the corresponding results. In a preferred embodiment of the present invention a more progressed stage of a neoplastic disease as defined herein above may be detected if the expression level of the molecular tumor marker or group of tumor markers is similar to an expression level of a more progressed stage of the neoplastic disease as defined herein above. The expression level of the more progressed stage of the neoplastic disease as defined herein above may be independently established, e.g. from sample depositories, value databases etc. as mentioned herein above.

The term "graduating a neoplastic disease associated with a progression from a less progressed stage to a more progressed stage" as used herein means that the clinical stage, phase, grade or any other suitable sub-step of features related to the neoplastic disease as defined herein above, such as the progression defined herein above, the transition from a benign to a malignant tumor, the grade of malignancy, the grade of tissue damage to noncancerous tissue, the grade of the extent of tumor growth, the grade of aggressiveness of a tumor, the grade of metastasizing and all other useful and suitable parameters of a cancerous disease or disorder in an organism may be determined in an organism. Furthermore, in certain embodiments of the invention, the determination of additional diagnostic factors, such as a patient's age, the outcome of DRE, or the determination of PSA may also be considered and/or combined with the determined values and the corresponding results.

In a preferred embodiment the graduating of a more progressed stage of the neoplastic disease as defined herein above may be accomplished by a comparison of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 of the present invention in a sample from a patient or individual to be analyzed and a control level as defined herein above, or the expression level of a less progressed stage as defined herein above of the neoplastic disease as defined herein above. In a further preferred embodiment the graduating of a more progressed stage may be

accomplished by a comparison of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 of the present invention in a sample from a patient or individual to be analyzed and any of the cancer cells, cancer tissues, tumor biopsies, or the cancerous control levels mentioned above. In a further preferred embodiment the graduating of a more progressed stage of the neoplastic disease as defined herein above may be accomplished by a comparison of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 of the present invention in a sample from a patient or individual to be analyzed and the above mentioned cancer cells, cancer tissues, tumor biopsies, or the neoplastic control levels being derived from or representative for a less progressed stage of the neoplastic disease as defined herein above. In a further preferred embodiment the graduating of a more progressed stage of the neoplastic disease as defined herein above, preferably cancer, may be accomplished by a comparison of the expression level of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 of the present invention in a sample from a patient or individual to be analyzed and the above mentioned cancer cells, cancer tissues, tumor biopsies, or the neoplastic control levels being derived from or representative for a more progressed stage of the neoplastic disease as defined herein above, preferably cancer.

The term "monitoring a neoplastic disease associated with a progression from a less progressed stage to a more progressed stage" as used herein relates to the

accompaniment of a diagnosed or detected, more progressed neoplastic disease or disorder as defined herein above, e.g. during a treatment procedure or during a certain period of time, typically during 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other period of time. The term "accompaniment" means that states of disease as defined herein above and, in particular, changes of these states of disease may be detected by comparing the expression level of the molecular tumor marker or group of tumor markers of the present invention in a sample to a control level as defined herein above or to the expression level of an established, e.g. independently established cancer cell or cell line, or to corresponding database values in any type of periodical time segment, e.g. every week, every 2 weeks, every month, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 month, every 1.5 year, every 2, 3, 4, 5, 6, 7, 8,9 or 10 years, during any period of time, e.g. during 2 weeks, 3 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years, respectively. The established, e.g.

independently established, cancer cell or cell line giving rise to a control level may be derived from samples corresponding to different stages of neoplastic development, e.g. the stages as mentioned herein above. In a preferred embodiment of the present invention the term relates to the accompaniment of a diagnosed more progressed prostate cancer.

The term "prognosticating a neoplastic disease associated with a progression form a less progressed stage to a more progressed stage" as used herein refers to the prediction of the course or outcome of a diagnosed or detected more progressed stage of the neoplastic disease as defined herein above, e.g. during a certain period of time, during a treatment or after a treatment. The term also refers to a determination of chance of survival or recovery from the disease, as well as to a prediction of the expected survival time of a subject. A prognosis may, specifically, involve establishing the likelihood for survival of a subject during a period of time into the future, such as 6 months, 1 year, 2 years, 3 years, 5 years, 10 years or any other period of time.

The term "progression from a less progressed stage to a more progressed stage" as used herein relates to a switch between different stages of the development or genesis of a neoplastic disease, as defined herein above. Such a progression may be a development in small steps, e.g. from a certain stage to the next, or may alternatively be a development skipping one or more such steps, e.g. from stage I to stage III of the TNM classification. A progression from a less progressed stage to a more progressed stage may be considered as being detected or diagnosed if the expression level of a molecular tumor marker or group of tumor markers according to the present invention is modified, e.g.

increased 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 or other diagnostic values of the tumor marker or group of tumor markers at a first point in time and at a second point in time after the above indicated period of time.

In a particularly preferred embodiment of the present invention the term "progression from a less progressed stage to a more progressed stage" relates to a switch from a benign prostate tumor state to a malignant prostate cancer state, as defined herein.

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

In a further embodiment the present invention relates to the diagnosis and detection of a predisposition for developing a more progressed stage of the neoplastic disease as defined herein above. A "predisposition for developing a more progressed stage of the neoplastic disease as defined herein above, preferably cancer" in the context of the present invention is a state of risk of developing a more progressed stage neoplastic disease as defined herein above. Preferably a predisposition for developing a more progressed stage may be present in cases in which the expression level of the molecular tumor marker or group of tumor markers of the present invention as defined herein above is above a normal control level as defined herein above, e.g. a reference expression level derived from tissues or samples of a subject which are evidently healthy or other reference diagnostic values. The term "above" in this context relates to an expression level of the molecular tumor marker or group of tumor markers which is increased by about 2% to 20% in comparison to such a control level, preferably increased by about 15%.

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

Thus, a predisposition for a more progressed stage of the neoplastic disease as defined herein above, e.g. cancer, may be considered as being diagnosed or detected if one of the above depicted situations is observed.

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

In the context of the present invention, the terms "diagnosing" and "prognosticating" are also intended to encompass predictions and likelihood analyses. Tumor markers or groups of tumor markers may accordingly be used clinically in making decisions concerning treatment modalities, including therapeutic intervention or diagnostic criteria such as a surveillance for the disease. According to the present invention, an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease. Alternatively, the present invention may be used to detect neoplastic cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.

A subject or individual to be diagnosed, monitored or in which a more progressed neoplastic disease as defined herein above, a progression towards such a neoplastic disease as defined herein above, or a predisposition to such a neoplastic disease as defined herein above, is to be detected or prognosticated according to the present invention is an animal, preferably a mammal, more preferably a human being.

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 neoplastic disease as defined herein above, preferably cancer of the progression towards such a neoplastic disease as defined herein above, preferably cancer. For example, a tumor marker or group of tumor markers according to the present invention may be used as a marker for diagnosing, detecting, monitoring or prognosticating pre-malignant growths, swellings and lesions, malignant tumors, malignant cancers, metastases and the like or the progression towards more progressed states of such neoplastic disease and disorders as defined above, for example malignant, hormone- sensitive prostate cancer and/or the progression towards more progressed cancer states, in approaches like 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 neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand for the expression product(s) or protein(s) of said molecular tumor marker or a group of tumor markers as defined above.

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

The term "peptide affinity ligand for the protein of a molecular tumor marker or a group of tumor markers" as used herein refers to a peptide molecule being able to specifically bind to the proteins of the molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3. The peptide molecule may preferably be able to specifically bind to a protein or polypeptide comprising the amino acid sequence(s) as set forth in section E) or section G) of Table 1 , 2 or 3. The peptide affinity ligand may also be able to

specifically bind to a protein or polypeptide comprising an amino acid sequence encoded by a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o or 99% identical to the sequence as set forth in section E) or section G) of Table 1 , 2 or 3 or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section E) or section G) of Table 1 , 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3, generated by the expression of the corresponding genomic sequence according to Table 1 , 2 or 3. More preferably, the term relates to a processed transcript of the molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 as defined herein above, e.g. via the sequence(s) as set forth in section D) 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 D or F) of Table 1 , 2 or 3.

The term "protein of a molecular 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) or section G) of Table 1 , 2 or 3 or any domain, epitope, oligopeptide, or peptide derivable there from.

In a preferred embodiment of the present invention the composition of the present invention may comprise one or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid and/or peptide affinity ligands selected from the group consisting of a set of oligonucleotides specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, a probe specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, an aptamer specific for the expression product or for the protein of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, an antibody specific for the protein the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 and an antibody variant specific for the protein of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3. The composition of the present invention may, for example, comprise a set of oligonucleotides specific for the expression product the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 and/or a probe specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.

The term "oligonucleotide specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3" as used herein refers to a nucleotide sequence which is complementary to the sense- or antisense-strand of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3. Preferably, the oligonucleotide is complementary to the DNA sequence(s) shown in section D) of Table 1, 2 or 3, or to the complementary DNA sequence of the sequence shown in section D) of Table 1, 2 or 3. The oligonucleotide sequence may also be complementary to a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in section D) of Table 1 , 2 or 3 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%>, 85%, 90%>, 91%>, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in section E) or section G) of Table 1, 2 or 3. The oligonucleotide may have any suitable length and sequence known to the person skilled in the art, as derivable from the sequence(s) shown in section D) of Table 1, 2 or 3 or its complement. 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 may be defined with the help of software tools known to the person skilled in the art.

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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3. Corresponding information on genomic loci can be derived from any suitable database or information depositary, e.g. by using the data provided in section F) of Table 1, 2 or 3. An oligonucleotide usable as a forward primer may be localized at the boundary between exonic and intronic sequences. Such boundary positions may be determined with the help of any suitable tool, based on the information which can be derived from any suitable database or information depositary, e.g. by using the data provided in section F) of Table 1, 2 or 3.

The term "probe specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3" as used herein means a nucleotide sequence which is complementary to the sense- or antisense-strand of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3. Preferably, the probe is complementary to the DNA sequence(s) depicted in section D) of Table 1, 2 or 3 or to the complementary DNA sequence of the sequence(s) shown in section D) of Table 1, 2 or 3. The probe sequence may also be complementary to a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in the sequences indicated in section D) of Table 1, 2 or 3 or a DNA sequence encoding an amino acid sequence being at least 75%>, 80%>, 85%>, 90%>, 91%>, 92%>, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as shown in section E) or section G) of Table 1, 2 or 3. The probe may have any suitable length and sequence known to the person skilled in the, as derivable from the sequence(s) shown in section D) or its/their complement. 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 molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 may be defined with the help of software tools known to the person skilled in the art. The composition of the present invention may additionally or alternatively comprise an aptamer specific for the expression product or protein of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3.

The term "aptamer specific for the expression product of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3" as used herein refers to a short nucleic acid molecule, e.g. RNA, DNA, PNA, CNA, HNA, LNA or ANA or any other suitable nucleic acid format known to the person skilled in the art, being capable of specifically binding to the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, preferably the DNA molecule with (a) sequence(s) as shown in section D) of Table 1, 2 or 3. More preferably, the nucleic acid aptamer molecule may specifically bind to a DNA sequence(s) shown in section D) of Table 1, 2 or 3 or a double stranded derivative thereof. The nucleic acid aptamer according to the present invention may also bind to an RNA molecule corresponding to the transcript(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, preferably an RNA molecule corresponding to the DNA sequence(s) as shown in section D) of Table 1, 2 or 3. The nucleic acid aptamer may further be capable of specifically binding to a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as shown in section D) of Table 1, 2 or 3 or a DNA sequence encoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as shown in section D) of Table 1 , 2 or 3 or RNA molecules corresponding to these sequences. A nucleic acid aptamer according to the present invention may further be combined with additional moieties, e.g. with interacting portions like biotin or enzymatic functionalities like ribozyme elements.

The term "aptamer specific for the protein of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3" as used herein refers to (a) short peptide(s) capable of interacting and specifically binding the protein(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3. The peptide aptamer(s) may preferably be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (the) amino acid sequence(s) as shown in section E) or section G) of Table 1, 2 or 3. The peptide aptamer(s) may also be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (an) amino acid sequence(s) encoded by (a) DNA sequence(s) being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as shown in section D) of Table 1, 2 or 3 or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as set forth in shown in section E) or section G) of Table 1, 2 or 3. 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 molecular tumor marker or group of tumor markers as defined herein, e.g. specific for one or more of the protein(s) of the molecular tumor marker according to Table 1, 2 or 3. 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 or 3 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 section G) of Table 1, 2 or 3, or a part or fragment of said sequence. In a further embodiment of the present invention such an antibody may be an autoantibody, e.g. an autoantibody against the expression product or protein of a molecular tumor marker or group of tumor markers as defined herein above, more preferably an autoantibody against the expression product or protein of a molecular tumor marker or group of tumor markers wherein said expression product or protein comprises an amino acid sequence as indicated in section G) of Table 1, 2 or 3, or comprises a fragment of said amino acid sequence, e.g. a peptide of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids length.

In another aspect the invention thus relates to an antibody specific for one or more of the molecular tumor markers according to Table 1, 2 or 3. Preferably, such an antibody specifically binds to a protein or polypeptide having or comprising the amino acid shown in section E) or section G) of Table 1, 2 or 3, or any derivative, fragment etc. thereof as defined herein above. Such antibodies are contemplated for any application, use, method, composition, immunoassay, screening method and pharmaceutical compositions as defined in the present application. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i. e.

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 molecular tumor markers of the present invention, preferably of the sequence as shown in section E) or section G) of Table 1, 2 or 3. Particularly preferred are antibodies binding to epitopes or antigens comprised in or comprising the amino acid sequence as mentioned in section G) of Table 1, 2 or 3, or parts of said sequence, e.g. fragments of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids. 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 molecular tumor marker according to Table 1, 2 or 3" as used herein refers to the immunospecific detection and binding of an antibody to an antigenic epitope as defined herein above. The term "specifically binding" excludes nonspecific binding but does not necessarily exclude cross-reactivity with other antigens, in particular with antigens comprising the same antigenic epitope detected by the present antibody.

In a preferred embodiment antibodies of the invention include autoantibodies, preferably human autoantibodies, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, Fab' fragments, fragments produced by a Fab expression library, F(ab')2, Fv, disulfide linked Fv, minibodies, diabodies, scFv, sc(Fv)2, whole immunoglobulin molecules small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins, camelized antibodies, V _HH containing antibodies, anti-idiotypic (anti-Id) antibodies (including, e. g., anti-Id antibodies to antibodies of the invention) and epitope-binding fragments of any of the above.

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.

The term "autoantibody" as used herein refers to an antibody generated by the immune system that is directed against one or more of the individual's own proteins.

Autoantibodies are normally generated by the immune system against the body's normal constituents or proteins, for example in reaction to the overexpression of said proteins or sudden appeance of said proteins after cellular modifications or transitions, e.g. during the development of a neoplastic disease or cancer. In the context of the present invention autoantibodies are envisaged which typically detect a molecular tumor marker protein as defined in Table 1, 2 or 3. The invention thus envisages antibodies, in particular human autoantibodies against the molecular tumor marker proteins as defined in Table 1, 2 or 3.

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 molecular tumor marker protein as defined in Table 1, 2 or 3, more preferably bound by a human autoantibody against a molecular tumor marker protein as defined in Table 1, 2 or 3.

In a further embodiment the present invention encompasses a screening method to identify antigen or epitope sequences bound by an antibody, in particular a human autoantibody against a molecular tumor marker protein as defined in Table 1, 2 or 3. Further envisaged are the epitope sequences and/or antigen sequences derived from a screening procedure. In particular embodiment the present invention also envisages antigen or epitope sequences which are bound, recognized, specifically recognized or detected by an autoantibody as defined herein. Such antigens or epitopes may preferably be derived from a molecular tumor marker or group of tumor marker sequences as depicted in Table 1, 2 or 3. The antigen or epitope sequences encompassed by the present invention may, for example, comprise, have, or alternatively consist of amino acids 1-10, 11-20, 21-30, 31-40, 41-50, 51- 60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170, 171-180, 181-190, 191-200, 201-210 etc. or any other specific stretch of amino acids of the N- or C-terminus of a protein or expression product of the molecular tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2 or 3, preferably of the sequence as shown in section E) or section G) of Table 1, 2 or 3.

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 molecular tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2 or 3, preferably of the sequence as shown in section E) or section G) of Table 1, 2 or 3. The epitope or antigen according to the present invention may further comprise or consist of additional amino acid stretches or fragments of any length or derived from any position, e.g. N-terminus, C-terminus, middle, specific domain, specified exposed region etc. of the protein or expression product of the molecular tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2 or 3, preferably of the sequence as shown in section E) or section G) of Table 1, 2 or 3. 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 molecular tumor markers or group of tumor markers of the present invention, i.e. as defined in Table 1, 2 or 3, preferably of the sequence as shown in section E) or section G) of Table 1, 2 or 3.

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

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 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 molecular tumor marker according to Table 1, 2 or 3, in particular against a protein having an amino acid sequence as indicated in section E) or section G) of Table 1 , 2 or 3 may be comprised in the composition or may be used diagnostically .

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

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. ³ H, ¹⁴ C, ³² P, ³³ P, ³⁵ S, ¹²⁵ I, ⁿ C, ¹³ N, ¹⁵ 0, ¹⁸ F, ⁶⁴ Cu, ⁶² Cu, ¹²⁴ I, ⁷⁶ Br, ⁸² Rb, ⁶⁸ Ga or ¹⁸ F) 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and which can be detected by an apparatus positioned outside the human or animal body. Preferably, such contrast agents are suitable for use in magnetic resonance imaging (MRI) or magnetic photon imaging (MPI). The term "specifically interacting" refer to the property of a molecular compound to preferentially interact with the molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 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 or 3 or the amino acid sequence(s) shown in section E) or section G) of Table 1, 2 or 3 or derivatives or homologous variants thereof as defined herein above. Preferred contrast agents are aptamers specific for the expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 or for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 as well as antibodies specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. Contrast agents, aside from their property of being capable of specifically recognizing the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 will in addition typically comprise a further molecule which is detectable by the specific detection technology used.

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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, for the preparation of a composition for diagnosing, detecting, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage to a more progressed stage or the progression from a less progressed stage to a more progressed stage or a predisposition for a more progressed stage, as described herein above. The composition is preferably for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed cancer stage to a more progressed cancer stage or the progression from a less progressed cancer stage to a more progressed cancer stage or a predisposition for a more progressed cancer stage in an individual, e.g. a patient or subject to be analyzed or examined.

In a preferred embodiment the present invention relates to the use of a set of oligonucleotides specific for the expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or a probe specific for the expression product(s) the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably specific for a nucleic acid sequence having a sequence as indicated in section D) of Table 1, 2 or 3 or being complementary to such a sequence, for the preparation of a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage, as described herein above.

In another preferred embodiment the present invention relates to the use of an aptamer specific for the expression product(s) or protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably a protein having an amino acid sequence as shown in section E) or section G) of Table 1, 2 or 3, for the preparation of a composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease, as described herein above. The set of

oligonucleotides is preferably for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease in an individual, e.g. a patient or subject to be analyzed or examined.

In a further preferred embodiment the present invention relates to the use of an antibody specific for protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or an antibody variant specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 for the preparation of a composition for diagnosing, detecting, monitoring or prognosticating neoplastic disease associated with a progression from a less progressed stage of said neplastsic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease as described herein above.

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

In another aspect the present invention relates to a diagnostic kit for diagnosing, detecting, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease or a predisposition for a more progressed stage of said neoplastic disease, comprising a set of oligonucleotides specific for the expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, a probe specific for the expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or an aptamer specific for the expression product(s) or protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or an antibody specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and an antibody variant specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. Typically, the diagnostic kit of the present invention contains one or more agents allowing the specific detection of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. The agents or ingredients of a diagnostic kit may, according to the present invention, be comprised in one or more containers or separate entities. The nature of the agents is determined by the method of detection for which the kit is intended. Where detection at the mR A expression level of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, i.e. via the expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be comprised. Where detection at the protein level of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 is intended, the agents to be comprised may be antibodies (including autoantibodies) or compounds containing an antigen-binding fragment of an antibody or antibody variants specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, as described herein above. In addition or alternatively an aptamer specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be comprised.

Alternatively, a diagnostic kit may comprise a contrast agent as defined herein above. In a further embodiment of the present invention, a kit according to the present invention may be a companion diagnostics test kit. The presence of specific proteins may also be detected using other compounds that specifically interact with the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, e.g. specific substrates or ligands. Preferably, a diagnostic kit of the present invention contains detection reagents for expression product(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 or the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may comprise accessory ingredients like a PCR buffers, dNTPs, a polymerase, ions like bivalent cations or monovalent cations, hybridization solutions etc. A diagnostic kit for the detection of protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may also comprise accessory ingredients like secondary affinity ligands, e.g. secondary antibodies, detection dyes and any other suitable compound or liquid necessary for the performance of a protein detection based known to the person skilled in the art. Such ingredients are known to the person skilled in the art and may vary depending on the detection method carried out.

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

In another aspect the present invention relates to a method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprising at least the step of determining the level of a molecular tumor marker or a group of tumor markers as defined above, in a sample.

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

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

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

For example, the measurement of the nucleic acid level of the expression of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may be assessed by separation of nucleic acid molecules (e.g. R A or cDNA) obtained from the sample in agarose or polyacrylamide gels, followed by hybridization with molecular tumor marker specific oligonucleotide probes as defined herein above, e.g. oligonucleotide probes comprising fragments of the sequences indicated in section D) of Table 1, 2 or 3, or complementary sequences thereof. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be detected in a DNA array or microarray approach. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 of the present invention or known 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 molecular tumor marker associated polynucleotide may be used as probe for the DNA array, as long as the polynucleotide allows for a specific distinction between the molecular tumor marker expression and the expression of other genes. Typically, cDNAs, PCR products, and oligonucleotides are useful as probes. For example, a fragment comprising 5'- or 3'-portions of the molecular tumor markers or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, e.g. of the sequences indicated in section D) of Table 1, 2 or 3 may be used as a probe. The DNA array or microarray may comprise probes of one or more of the molecular tumor marker of Table 1, 2 or 3, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 etc. or all of the tumor markers or any combination of said markers. 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 or 3. In addition to the determination of the expression of molecular tumor markers according to Table 1, 2 or 3 also the determination of the expression of other genes, e.g. additional biomarker or cancer marker genes is envisaged by the present invention.

A DNA array- or microarray-based detection method typically comprises the following steps: (1) Isolating mRNA from a sample and optionally converting the 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 may be detected in a quantitative RT-PCR approach, preferably in a real-time PCR approach following the reverse transcription of the mRNA transcript(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.

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 molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 as defined herein above, serve as internal probes which are used in conjunction with a pair of opposing primers that flank the target region of interest, preferably a set of oligonucleotides specific for the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 as defined herein above. 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 molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 as defined above of about 22 to 30 bases that is labeled on both ends with a FRET pair. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, have no guanine (G) at the 5' end adjacent to the reporter dye in order to avoid quenching of the reporter fluorescence after the probe is degraded. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or of any fragments, homologues or derivates derived thereof may be carried out via any suitable detection technique known in the art. Preferably, the protein level of molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and derivatives thereof may be determined immunologically, e.g. by using an antibody specific for the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an antibody as defined herein above. In a particularly preferred embodiment of the present invention the presence and/or amount of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be determined with an antibody specifically binding to or detecting a protein or antigen or epitope thereof of the proteins as indicated in section G) of Table 1, 2 or 3, or any fragment thereof, or any epitope comprised therein, as defined herein above. Further preferred are autoantibodies according to the present invention. Alternatively, antibody variants or fragments as defined herein above may be used.

The present invention also envisages the use of peptide affinity ligands like ap tamers specific for the protein(s) of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 as defined herein above.

Determination of the protein levels of the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 can be accomplished, for example, by the separation of proteins from a sample on a polyacrylamide gel, followed by identification of the protein(s) of molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 using specifically binding antibodies in a Western blot analysis. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be placed on a support and be immobilized. Proteins derived from samples or tissues to be analyzed may subsequently be mixed with the antibodies. A detection reaction may then be carried out, e.g. with a second affinity ligand as defined herein above, preferably with a specific antibody.

Furthermore, within the context of the present invention a molecular tumor marker expression product or protein or the expression product or protein of a group of molecular tumor marker, preferably as derivable from Table 1, 2 or 3 (e.g. as defined in sections E) and/or section G)), which are or can specifically be bound by an autoantibody or a group of autoantibodies as mentioned herein above may be placed on a support and/or be immobilized. The present invention in particular envisages an array of peptide sequences, protein sequences or expression products comprising one or more of the molecular tumor markers or groups of tumor marker as defined herein above. The array may be provided in any suitable form known to the person skilled in the art and may comprise one or more suitable control elements, proteins or antibodies as would be known to the person skilled in the art. The bound elements in the array may further be of any suitable length, comprising peptides/proteins of e.g. 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8 amino acids length or any other suitable length.

In a further, preferred embodiment one or more antibodies, in particular autoantibodies present in a sample or tissue to be analyzed may be mixed with the presented peptides, proteins, epitopes etc., e.g. the proteins, peptides etc. immobilized on an array. In an additional embodiment a detection reaction may then be carried out, e.g. with a second affinity ligand as defined herein above, preferably with a specific antibody. The present invention also encompasses an autoantibody bound or detected in such an approach.

Immunological tests which may be used in the context of the present invention, in particular for the diagnostic purposes of the present invention, include, for example, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassay like RIA (radio-linked immunoassay), ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence

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

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

The determination of the biological activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be carried out by employing molecular or enzymatic assays specific to the corresponding function or functions of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, e.g. of a molecular tumor marker protein having a sequence as indicted in section E) or section G) of Table 1, 2 or 3. Suitable techniques would be known to the person skilled in the art. An inhibition of the activity may be carried out by any suitable means known to the person skilled in the art, preferably via the use of suitable antisense nucleotides, siR A molecules or miR A molecules, more preferably via specifically hybridizing antisense nucleotides, specific siRNA or miRNA molecules, e.g. as described herein below.

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

The level of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may also be detected in methods involving histological or cell-biological procedures. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 in a cell may, for instance, be detected or determined by removing cells to be tested from samples as defined herein above. Also tissue sections or biopsy samples may be used for these methods.

Subsequently, affinity ligands for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be applied, preferably antibodies or aptamers. Typically, such affinity ligands are labeled, preferably with fluorescent labels as defined herein above. Such a procedure allows for the detection of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, 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 molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 may be determined by suitable molecular imaging techniques, e.g. magnetic resonance imaging (MRI) or magnetic photon imaging (MPI), and/or by using suitable contrast agents, e.g. contrast agents as defined herein above.

In a further preferred embodiment of the present invention a method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, or the progression form a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprises the additional step of comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the molecular tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease. The expression level of the molecular tumor marker or the group of tumor markers of a less progressed stage of the same neoplastic disease may be an expression level which may be determined at the same time and/or under similar or comparable conditions as the test sample by using (a) sample(s) previously collected and stored from a subject/subjects whose disease state, is/are known or from the same subject at an earlier point in time or an expression level corresponding to a stage of said neoplastic disease or form of said neoplastic disease, whose disease state or stage is known including different

proliferation/developmental stages of the neoplastic disease as defined herein above, or levels of development of benign hyperplasia, benign growths, benign lesions, benign tumors and the like as well as to all pre-malignant growths, swellings and lesions, malignant tumors, malignant cancers, metastases and the like, in particular such diseases and/or disorders of the prostate in the organism, as described herein above

The term "comparing" as used herein refers to any suitable method of assessing, calculating, evaluating or processing of data. In a preferred embodiment of the present invention the determination of the presence or amount of (an) expression product(s), e.g. (a) transcript(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or of (a) protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, is accomplished by the measurement of nucleic acid or protein levels or by the determination of the biological activity of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. Thus, the expression level(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may be determined by a method involving the detection of an mR A encoded by the genes encoding for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, or the according cDNA sequence, e.g. the sequences indicated in section D) of Table 1, 2 or 3, the detection of the protein(s) encoded by the transcript of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or the detection of the biological activity of the protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, e.g. of the proteins having the sequences indicated in section E) or section G) of Table 1, 2 or 3. For example, the measurement of the nucleic acid level of expression of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may be assessed by separation of nucleic acid molecules as described herein above. In yet another embodiment as a further, additional step a decision on the presence or stage of a more progressed stage of the neoplastic disease as defined herein above, or the progression towards said stage of said neoplastic disease may be based on the results of the comparison step. A more progressed stage of said neoplastic disease may be diagnosed or prognosticated or a progression towards said more progressed stage of said neoplastic disease may be diagnosed or prognosticated in said method according to the corresponding definitions provided herein above in the context of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.

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

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

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

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

The determination of the level of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be carried out according to steps as defined herein above. Preferably, the determination may be carried out as measurement of a nucleic acid level or protein level according to the herein above described options for such measurements. In one embodiment, steps a), b), c) and/or d) of this method of diagnosis may be performed outside the human or animal body. A more progressed stage of a neoplastic disease may be graduated in said method according to the corresponding definitions provided herein above in the context of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.

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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; and

(b) comparing the expression as determined in step (a) to a control level. The testing for expression of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 may be carried out according to steps as defined herein above. Preferably, the testing may be carried out as measurement of protein levels of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, more preferably according to the herein above described options for such measurements. The term "control level" as used in the context of the method of data acquisition refers to the expression of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or other suitable markers in a control or the expression level of said marker(s) in a less progressed stage of a neoplastic disease, as defined herein above. The status, nature, amount and condition of the control level may be adjusted according to the necessities.

A comparison of the expression to a control level may be carried out according to any suitable method of assessing, calculating, evaluating or processing of data and particularly aims at the detection of differences between two data sets. A statistical evaluation of the significance of the difference may further be carried out. Suitable statistical methods are known to the person skilled in the art. Obtained data and information may be stored, accumulated or processed by suitable informatics or computer methods or tools known to the person skilled in the art and/or be presented in an appropriate manner in order to allow the practitioner to use the data for one or more subsequent deduction or conclusion steps.

In another embodiment the present invention relates to a method of detecting, diagnosing, monitoring or prognosticating a neoplastic disease as defined herein above associated with a more progressed stage of said neoplastic disease or the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprising at least the steps of:

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

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

(c) determining the difference in the expression of steps (a) and (b); and

(d) deciding on the presence of a neoplastic disease as defined herein above in particular of a more progressed stage of said neoplastic disease or on the

progression of said neoplastic disease, in particular from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease based on the results obtained in step (c).

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

In another aspect the present invention relates to an immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease as defined herein above associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, or for detecting, diagnosing, graduating, monitoring or prognosticating the progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease comprising at least the steps

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

(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 or stage of said neoplastic disease or the progression of said neoplastic disease based on the results obtained in step (c), wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a molecular tumor marker or a group of tumor markers as defined above.

The testing for expression of the molecular tumor marker or group of tumor markers according to Table 1 , 2 or 3 may be carried out according to steps as defined herein above. Preferably, the testing may be carried out as measurement of protein levels of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3, more preferably according to the herein above described options for such measurements. 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 molecular tumor marker according to Table 1, 2 or 3 as laid out above, e.g. (a) commercially available antibody/antibodies against (a) protein(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. A neoplastic disease as defined herein above in particular a more progressed cancer stage, may be diagnosed or prognosticated or a progression of a neoplastic disease as defined herein above, in particular from a less progressed to a more progressed stage of said neoplastic disease, may be diagnosed or prognosticated in said immunoassay according to the corresponding definitions provided herein above in the context of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 as marker(s) for said neoplastic disease.

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

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

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

(b); and

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

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

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 molecular tumor marker or group of tumor markers of the invention. If the testing is carried out in the same sample, a single detection or a multiplex detection approach may be performed.

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 molecular 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 maker or group of tumor markers and the expression in the same test sample for a reference gene are compared, e.g. after normalization against a further gene as normalization reference. According to the outcome of the comparison the test sample is indicated as providing a similar expression as the reference gene, an increased expression in comparison to the reference gene, or an reduced expression in comparison to the reference gene.

According to the classification of the expression results an individual may be considered to be eligible for a neoplastic disease therapy when the expression level of a molecular tumor marker as defined herein above or in Table 1, 2 or 3, or the expression level of a groups of tumor markers as defined herein above is increased. The expression level is deemed to be "increased" when the molecular tumor marker gene expression, or the expression of the group of molecular tumor marker genes in the test sample is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the corresponding molecular tumor marker gene expression, or the expression of the corresponding group of tumor markers in a control sample, or elevated at least 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the molecular tumor marker expression, or the expression of the group of tumor markers in a control sample; or when the molecular tumor marker or group of tumor marker gene expression is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%), 25%o, 30%), 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, the 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. In a further aspect the present invention relates to an immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:

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

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

(c) determining the difference in expression of a molecular tumor marker or group of tumor markers as defined above of step (a) and the expression of a molecular tumor marker or group of tumor markers as defined above and/or the reference gene in step (b); and

(d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an increased level of molecular tumor marker or group of tumor marker expression.

The testing of the expression of a molecular tumor marker or group of tumor markers may preferably be carried out via the determination of the amount of molecular tumor marker protein or the determination of the molecular tumor marker protein/expression product activity level. Preferred is the determination of the amount of molecular tumor marker proteins or peptides with the help of specific antibodies binding said molecular tumor marker protein or expression product, e.g. an autoantibody as defined herein. 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 molecular tumor marker or group of tumor markers. If the testing is carried out in the same sample, a single detection or a parallel or multiplex detection approach may be performed. Preferably, for a parallel or multiplex detection differently labeled primary or secondary antibodies may be used.

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 molecular tumor marker or group of tumor markers may be combined.

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

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

The term "stratifying an individual or cohort of individuals to neoplastic disease therapy" as used herein means that an individual is identified as pertaining to a group of similar individuals, whose optimal therapy form is a neoplastic disease therapy, preferably a therapy against an early prostate cancer form in accordance with the outcome of the expression test as described herein above, in particular in accordance with encountered difference in the molecular tumor marker or group of tumor marker expression level and a reference gene or the molecular tumor marker or group of tumor marker expression level in different samples. According to the determination of the expression difference an individual may be identified as pertaining to a group of similar individuals whose optimal therapy form is a neoplastic disease therapy when the molecular tumor marker or group of tumor marker expression levels are increased. The expression level is deemed to be "increased" when the molecular tumor marker or group of tumor marker gene expression in the test sample is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to the molecular tumor marker or group of tumor marker expression in a control sample, or at least elevated 0.1 fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more in comparison to the molecular tumor marker or group of tumor marker expression in a control sample; or when the molecular tumor marker or group of tumor marker gene expression is elevated by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 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.

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

Ultrasound ablation), or thermal ablation or any type of combination therapy of at least two of the above mentioned treatment forms either in direct combination, or used in a subsequent form.

Typically, an individual considered to be eligible for neoplasstic disease therapy due to an increased expression of a molecular tumor marker or group of tumor marker of the present invention may be deemed to be suffering from a neoplastic disease or be prone to develop a neoplastic disease in the future, e.g. within the next 1 to 24 months. A correspondingly identified or stratified individual may be treated with a pharmaceutical composition according to the present invention, e.g. as defined herein below. In a further embodiment a correspondingly identified individual may be treated with a pharmaceutical composition according to the present invention in combination with an additional cancer therapy. The term "additional cancer therapy" refers to any types of cancer therapy known to the person skilled in the art. Preferred are cancer therapy forms known for prostate cancer. The term includes, for example, all suitable forms of chemotherapy, radiation therapy, surgery, antibody therapies etc.

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

In a further embodiment of the present invention the classification method for eligibility or the immunoassay for stratification as described herein above may also be used for monitoring the treatment of an individual, e.g. an individual being classified as suffering from a neoplastic disease. The monitoring process may be carried out as expression determination or protein detection over a prolonged period of time, e.g. during or after treatment sessions, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months, or 1, 2, 3 or more years. The determination steps may be carried out in suitable intervals, e.g. every week, 2 weeks, 3 weeks, every month, 2 months, 3 months, 6 months, 12 months etc. In a further embodiment of the present invention any treatment scheme as mentioned herein may be adjusted, e.g. enforced or attenuated, or altered in any suitable manner in

correspondence with the results of the monitoring process.

In a particularly preferred embodiment of the present invention the reference gene is a housekeeping gene or a phosphodiesterase gene. In human organisms, examples of "housekeeping genes" include inter alia β-actin, glycerinaldehyde 3 -phosphate

dehydrogenase (GAPDH), porphobilinogen deanimase (PBGD), and ribosomal protein PI . Apart from these genes any other suitable gene may be used as a house-keeping gene, as long as the gene shows an expression or transcription on a steady, non-modified level, in particular during different stages of neoplastic disease development, more preferably during different stages of prostate cancer development. Expression data of a house-keeping gene may be obtained from one or more samples of the same individual or from more individuals, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 1000, 5.000, 10.000 or more. Expression data may also be obtained from databases or from data collections available to the person skilled in the art.

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

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

In a particularly preferred embodiment of the present invention the above mentioned method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease, or for identifying an individual for eligibility for a neoplastic disease therapy or the above mentioned immunoassay for detecting, diagnosing, monitoring or prognosticating a neoplasticdisease or for stratifying an individual or cohort of individuals with a neoplastic disease comprises the additional step of determing the level of PSA, and/or of determining the patient's age; and/or of determining the outcome of the patient's DRE as defined herein above.

The determination of the level of PSA may include the analysis of the PSA gene, transcript, or expression product, e.g. protein, of human prostate specific antigen. Furthermore, the condition of PSA may be determined, e.g. whether PSA protein is free circulating in the blood/serum, whether it is complexed to other factors or proteins, how much PSA is present totally etc. Furthermore, the presence of PSA gene or transcript variants or mutants may be determined. In a particularly preferred embodiment the amount of total PSA protein, of free PSA protein, of complexed PSA protein and/or the ratio of the amount of free and total PSA protein or the ratio of the amount of complexed and total PSA protein, and/or the amount or presence of benign PSA, nicked PSA, proPSA, of truncated forms of proPSA in particular -2proPSA or any ratios and/or combinations of these values may be determined. The determination of PSA may be carried out according to any suitable methods known to the skilled person, e.g. via PCR tests, via antibody tests, e.g. by using monoclonal antibodies, via immune sandwich assays, ELISA, or RIA. An example of a suitable test is the elecsys and cobas immunoassay. The determined amount of PSA may be compared to suitable controls e.g. from healthy subjections, earlier taken samples, etc. or to values known form the prior art, or to database entries etc.

Preferred is the determination of the level of PSA in blood. Thus, if the level of PSA in blood is encountered to be of a ranger of about 2 to 5 ng/ml or more, preferably of about 2.2 to 4.8 ng/ml or more, 2.4 to 4.4 ng/ml or more, 2.6 to 4.2 ng/ml ore more or 2.8 to 4.0 ng/ml or more, more preferably of about 2.5 to 4 ng/ml or more, an individual may be considered to be suffering from a neoplastic disease, or be likely to develop a neoplasstic disease in the near future, i.e. within the next 1, 2, 3, 4, 5, 6, 12, 14, 48 months. The testing for expression of a molecular tumor marker or group of tumor markers may be carried out according to steps as defined herein above. These values may further be combined with the expression level of any of the molecular tumor markers or groups of tumor marker according to the present invention, or with additional diagnostic factors as defined herein.

In a further preferred embodiment of the present invention the immunoassay for stratifying or the method of identifying an individual for eligibility for a neoplastic disease therapy may be carried out in cases in which the tested individual showed an initial negative biopsy result. Such a negative biopsy result may be derived from any known cancer detection methods, in particular histological analyses of samples, and/or the determination of PSA levels. Additionally, or alternatively, further biomarkers may also have lead to an initial negative biopsy result. In such a case either the already used sample for the biopsy may be employed for the determination of the expression level of a molecular tumor marker or group of tumor markers as defined herein, or a further sample may be derived from such an individual, preferably a serum sample.

In a further preferred embodiment of the present invention the method of identifying an individual for eligibility for a neoplastic disease therapy based on the expression of a molecular tumor marker or group of tumor markers or the immunoassay for stratifying an individual or cohort of individuals as described herein above may be combined with the determination of the level of prostate specific antigen (PSA) in cases in which the tested individual showed an initial negative biopsy result as mentioned herein above. The determination of PSA is preferably carried out in a different sample.

In a further, particularly preferred embodimentof the present invention the measurement of a marker, or of group or combination of markers as defined above, i.e. from Table 1, 2 or 3, in a patient serum sample may be used to identify a patient with prostate cancer although the initial biopsy gave a negative result. The detection of the marker or group of markers as defined above, which may be carried out as mentioned herein above, wherein the detected expression level is above a given threshold as defined herein, e.g. in Table 1, 2 or 3 or derivable from the Examples, indicates a >95%, >90%, >85%, >80%, >75%, >70%, >65%, or >60% probability that a follow-up prostate biopsy will deliver a positive result.

The detection or measurement of the molecular tumor marker, or of the group or combination of markers as defined herein, i.e. derivable from Table 1, 2 or 3, may further be used to monitor the potential re-currence of a prostate cancer after a primary treatment, e.g. surgery, or radiotheary. The tumor marker detection or measurement, including the determination of additonal diagnostic factors such as DRE may be performed every 3, or every 6, or every 12 months after the patient has been treated. If the expression level of the molecular tumor marker, or group of tumor markers as defined herein is going to pass a threshold as defined herein, e.g. in Table 1, 2 or 3 or derivable from the Examples, by 5%, 10%, 15%, 20%), 25%o, 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 neoplastic, e.g. cancerous epithelial cell or an epithelial cell derived from tissue suspected to be neoplastic, e.g. cancerous. The sample used in the context of the present invention should preferably be collected in a clinically acceptable manner, more preferably in a way that nucleic acids (in particular RNA) or proteins are preserved.

Alternatively, the biological sample may contain a cell population derived from a glandular tissue, e.g. the sample may be derived from the prostate of a male individual. Additionally, cells may be purified from obtained body tissues and fluids if necessary, and then used as the biological sample. In a specific embodiment of the present invention the content of a biological sample may also be submitted to an enrichment step. For instance, a sample may be contacted with ligands specific for the cell membrane or organelles of certain cell types, e.g. prostate cells, functionalized for example with magnetic particles. The material concentrated by the magnetic particles may subsequently be used for detection and analysis steps as described herein above or below.

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

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

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

A subject or individual to be diagnosed, monitored or in which a neoplastic disease as defined herein above, a progression of said neoplastic disease or predisposition for said neoplastic disease is to be detected or prognosticated according to the present invention is an animal, preferably a mammal, more preferably a human being.

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

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

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

The term "a compound indirectly inhibiting the activity of a molecular tumor marker " as used herein refers to a compound which is capable of decreasing the activity of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 by an interaction with a direct interactor of the molecular tumor marker according to Table 1, 2 or 3 ("indirect interactor") or via an indirect working pathway not involving an interaction with of the molecular tumor marker according to Table 1, 2 or 3. Such a compound may be any direct interactor of an interactor of the molecular tumor marker according to Table 1, 2 or 3, e.g. of a molecular tumor marker protein having a sequence as indicated in section E) or section G) of Table 1, 2 or 3. The effect conveyed by the direct interactor of an interactor of the molecular tumor marker according to Table 1, 2 or 3 may be either negative if the interactor of the molecular tumor marker or group of tumor markers according to Table 1, 2 or 3 itself has a negative effect on the activity of the molecular tumor marker according to Table 1, 2 or 3, or negative, if the interactor of the molecular tumor marker according to Table 1, 2 or 3 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. Typically negatively working indirect interactors may have an inhibitory effect on activators of the molecular tumor marker according to Table 1, 2 or 3. Examples of such interactors are enzymatic activities degrading activators of the molecular tumor marker according to Table 1, 2 or 3, or proteins capable of binding and quenching activators of the molecular tumor marker according to Table 1, 2 or 3. Alternatively, such interactors may positively modulate activities leading to a degradation of the molecular tumor marker according to Table 1, 2 or 3, e.g. proteinases. Further examples and their

implementation would be known to the person skilled in the art.

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

The "nucleic acid encoding and expressing a dominant negative form of a protein of a molecular 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3. Thus the term refers to a nucleic acid encoding (a) variant(s) of the molecular tumor marker according to Table 1, 2 or 3, which comprises an antimorphic modification, in particular which adversely affects the molecular tumor marker according to Table 1, 2 or 3 of the invention. Typically, such a behavior may occur if the antimorphic variant can interact with the molecular tumor marker according to Table 1, 2 or 3, but blocks some aspect of its function. Preferably, such variants may comprise or lack specific domains of the molecular tumor marker according to Table 1, 2 or 3, e.g. one or more protein-protein interacting or dimerization domains, complex assembly domains, one or more membrane-associated domains etc. This is particularly of importance in a protein that functions as a dimer or multimer. If, for example, one part of that protein complex is mutant in some functional aspect of the multimer but is still able to form the multimer it may have a dominant effect on the other wildtype portions of the complex, and a negative effect if the mutation prevents the complex from carrying out its normal function. 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 or 3 and/or functional assays of the proteins and or polypeptides of the invention comprising the amino acid sequence as indicated in section E) or section G) of Table 1, 2 or 3 or derivatives thereof.

The term "miRNA specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3" refers to a short single- stranded RNA molecule of typically 18-27 nucleotides in length, which regulate gene expression of one or more of the molecular tumor marker according to Table 1, 2 or 3.

miRNAs are encoded by genes from whose DNA they are transcribed but are not translated into a protein. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3" refers to nucleic acids corresponding to the sequences indicated in section D) of Table 1, 2 or 3 or the complementary strand thereof. Preferably, the antisense molecule of the invention comprises a sequence complementary to at least a portion of a molecular tumor marker expression product according to the present invention. While antisense molecules complementary to the coding region sequence of molecular tumor marker expression products may be used, those complementary to the transcribed and untranslated region are preferred. 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 or 3 may be used to design an antisense RNA or DNA oligonucleotide of from about 6 to 50 nucleotides in length. Preferably, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides in length.

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

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

complementary to the 3' untranslated sequences of 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, miRNA- or siRNA molecule, may comprise at least one modified base moiety which is selected from the group including 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethyl-aminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methyl guanine, 3 -methyl cytosine, 5-methylcytosine, N6-adenine, 7- methyl guanine, 5-methylaminomethyluracil, 5-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 R A in which the strands run parallel to each other.

The term "siRNA specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3" refers to a particular type of antisense-molecules, namely small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway to negatively regulate gene expression of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3" as used herein refers to (a) short peptide(s) capable of interacting and specifically binding the protein(s) of the molecular tumor marker according to Table 1, 2 or 3. The peptide aptamer(s) may preferably be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (the) amino acid sequence(s) as indicated in section E) or section G) of Table 1, 2 or 3. The peptide aptamer(s) may also be able to specifically bind to (a) protein(s) or polypeptide(s) comprising (an) amino acid sequence(s) encoded by (a) DNA sequence(s) being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o or 99% identical to the sequence(s) as indicated in section D) of Table 1, 2 or 3, or to a protein or polypeptide comprising an amino acid sequence being at least 75%, 80%>, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as indicated in section E) or section G) of Table 1, 2 or 3. 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) or section G) of Table 1, 2 or 3 and to reduce the biological activity and/or the enzymatic activity of these/this protein(s) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), 90%), 95%) or by at least 98%> or 99% when compared to a control level obtained from an untreated sample.

A "small molecule capable of specifically binding to the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3" as used herein refers to a small organic compound that is preferably biologically active, i.e. a biomolecule, but is preferably not a polymer. Such an organic compound may have any suitable form or chemical property. The compound may be a natural compound, e.g. a secondary metabolite or an artificial compound, which has been designed and generated de novo. In an embodiment of the present invention a small molecule is capable of blocking the interaction between the molecular 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3" 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or an antibody variant specific for said protein, e.g. an antibody or antibody variant as defined herein above.

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

The skilled person would also be aware of the possibility to target and destroy neoplastic and/or cancer cells and tissue by virtue of conjugated antibodies specific for the tumor markers. Thus, in a specific embodiment of the present invention the antibody or fragment thereof as defined herein above may be conjugated to a therapeutic or cytotoxic agent. The term "therapeutic agent" refers to any compound, drug, small molecule or medicament, which is able to confer a therapeutic effect to a cell, a tissue or the entire organism. Examples of such agents are known to the person skilled in the art. The term "cytotoxic agent" refers to any compound, drug, small molecule which is able to confer a toxic effect to a cell or a tissue. Such agents may, for example, comprise compounds which activate endogenous cytotoxic effector systems, as well as radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. The term may also include radioisotopes known in the art, additional antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, R Ase, alpha toxin, ricin, abrin,

Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. The term also refers to cytotoxic produgs. By "cytotoxic prodrug" is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the invention include glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,

daunorubicin, and phenoxyacetamide derivatives of doxorubicin.

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

In a further embodiment of the present invention such an antibody may be an autoantibody as mentioned above, e.g. an autoantibody against the expression product or protein of a molecular tumor marker or group of tumor markers as defined herein above, more preferably an autoantibody against the expression product or protein of a molecular tumor marker or group of tumor markers wherein said expression product or protein comprises an amino acid sequence as indicated in section G) of Table 1, 2 or 3, or comprises a fragment of said amino acid sequence, e.g. a peptide of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids length. In another aspect the present invention relates to a pharmaceutical composition for use in, or for the treatment or prevention of a neoplastic disease, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, wherein said neoplastic disease implies the increased (up-regulated) expression of a molecular tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of: (a) a compound directly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an antagonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (e) a miR A specific for a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (f) an antisense molecule of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (g) a siR A specific for a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (h) an aptamer specific for the expression product of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (i) a small molecule or peptido mimetic capable of specifically binding to the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; and (j) an antibody specific for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or an antibody variant specific for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an agonist of said molecular tumor marker enzymatic activity;(b) a compound indirectly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and (e) a miR A inhibitor specific for a miR A of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.

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

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

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

Alternatively, an indirect stimulation of the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may be conveyed by compounds activating, protecting or sustaining the expression of the endogenous gene(s) of the molecular tumor marker according to Table 1, 2 or 3. Examples of such compounds are specific transcription factors of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, specific stabilizing activities of the mR A(s) of the the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or splice factors specific for the the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. Further examples and their implementation would be known to the person skilled in the art.

The "protein of a molecular tumor marker" comprised in the pharmaceutical composition may be a protein of the molecular tumor marker according to Table 1, 2 or 3 as defined herein above. In particular, it may be a protein being encoded by splice variant of the molecular tumor marker according to Table 1, 2 or 3. More preferably, it may have the amino acid sequence as set forth in section E) of Table 1, 2 or 3. The "protein of a molecular 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 or 3 and amino acid sequences being encoded by nucleotide sequences being at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as indicated in section D) of Table 1, 2 or 3. In a further embodiment of the invention the homologous variants of the molecular tumor marker according to Table 1, 2 or 3 may additionally or alternatively have a similar or identical localization pattern as the molecular tumor marker according to Table 1, 2 or 3 within a cell or within a tissue type. The term "biologically active equivalent of a molecular tumor marker" as used herein refers to a protein of the molecular tumor marker according to Table 1, 2 or 3 which is capable of performing all or a majority of the individual functions of the molecular tumor marker according to Table 1, 2 or 3. In a further embodiment of the invention the biologically active equivalents of the molecular tumor marker according to Table 1, 2 or 3 may additionally or alternatively have a similar or identical localization pattern as the molecular tumor marker according to Table 1, 2 or 3 within a cell or within a tissue type. Biologically active equivalents of the molecular tumor marker according to Table 1, 2 or 3 may also comprise variants of the molecular tumor marker according to Table 1, 2 or 3 as defined herein above.

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

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

A suitable vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. The vectors may preferably comprise one or more of the nucleotide sequences indicated in section D) of Table 1, 2 or 3.

Polynucleotides encoding the molecular tumor markers according to Table 1, 2 or 3 or biologically active equivalents of the molecular tumor markers according to Table 1, 2 or 3 may be joined to a vector or carrier containing a selectable marker for propagation in a host. A corresponding polynucleotide insert may be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, or the PSA promoter. 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 molecular tumor marker proteins according to Table 1 , 2 or 3 or biologically active equivalents of the molecular tumor markers according to Table 1 , 2 or 3 of the invention can be chemically synthesized using techniques known in the art, e.g. by using a peptide synthesizer.

The "nucleic acid encoding and expressing the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3" comprised in the pharmaceutical composition as defined herein above refers to any suitable carrier element, e.g. as described herein above, comprising an expressable gene of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3. Preferably, such a carrier element may comprise the sequence(s) as indicated in section E) of Table 1 , 2 or 3. Such a carrier element may also comprises nucleotide sequences showing a high degree of homology to the tumor markers according to Table 1 , 2 or 3, e.g. nucleic acid sequences being at least 60%, 70%>, 75%, 80%>, 85%, 90%>, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence(s) as indicated in section D) of Table 1 , 2 or 3 or nucleic acid sequences encoding amino acid sequences being at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o or 99% identical to the sequence(s) as indicated in section E) of Table 1 , 2 or 3.

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

The polynucleotide encoding the molecular tumor marker according to Table 1 , 2 or 3 may preferably be joined to a vector containing a selectable marker for propagation in a human cell. 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 may be provided via living therapeutics. The term "living therapeutic" means that the tumor markers according to Table 1, 2 or 3 or biologically active equivalents of the molecular tumor markers according to Table 1, 2 or 3 as defined herein above are expressed in any suitable live carrier. 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, retroviruses 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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3" comprised in the pharmaceutical composition as defined herein above refers to a nucleic acid molecule encoding a nucleic acid sequence complementary to a miRNA or microRNA molecule of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. The term "complementary" as used herein refers to a perfect complementary between the miRNA inhibitor nucleic acid (sense molecule) and the miRNA (antisense molecule) without any mismatch, as well as situations in which the nucleic acid contains any base mismatches and/or additional or missing nucleotides in comparison to the miRNA molecule. 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 tranfection 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 molecular tumor marker according to Table 1, 2 or 3. Examples of demethylation agents to be used in the context of the present invention are 5-aza-2'-deoxycytidine and 5-azacytidine, which reactivate genes inappropriately silenced by structural chromatin changes that involve DNA methylation and which can reverse these changes and, therefore, restore principal cellular pathways. This typically results in gene re-expression and reversion of some aspects of the transformed state. 5-azacytidine and 5-aza-2'-deoxycytidine typically inactivate DNA cytosine C5- methyltransferases through the formation of stable complexes between the 5- aza-2'-deoxycytidine residues in DNA and the enzyme, thereby mimicking a stable transition state intermediate when bound to the methyltransferase enzyme.

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

In a another aspect the present invention relates to a pharmaceutical composition for the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer associated with a progression form a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, wherein said neoplastic disease implies the decreased (down-regulated) expression of a molecular tumor marker or group of tumor markers as defined above, comprising at least one element selected from the group of: (a) a compound directly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an agonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3; (c) a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and (e) a miRNA inhibitor specific for a miRNA of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3. Such pharmaceutical composition preferably comprises elements as defined herein above. In a preferred embodiment of the invention a pharmaceutical composition according to the present invention, e.g. as defined herein above, may further comprise additional compounds being active against neoplastic and/or cancer cells. Furthermore, in another embodiment of the invention the pharmaceutical composition may further comprise hormone-inhibitors, preferably anti-androgens or androgen antagonists like spironolactone, cyproterone acetate, flutamide, nilutamide, bicalutamide, ketoconazole, finasteride or dutasteride.

The skilled person is aware of the fact that also a situation in which a given tumor marker of the invention is up-regulated in a neoplastic disease while in parallel another tumor marker of the invention is down-regulated. It is thus an aim of the present invention to provide also pharmaceutical compositions wherein the pharmaceutical composition contains any combination of such elements as laid out above, e.g. the compounds, proteins, dominant negative proteins, nucleic acids, miRNAs, siRNAs, antisense RNAs, aptamers, antibodies, peptidomimetics and small molecules, and wherein the composition contains said elements being capable of down-regulating at least one tumor marker according to Table 1, 2 or 3 and up-regulating at least one other tumor marker according to Table 1, 2 or 3. It is preferred to avoid conflicting and/or opposite functionalities or overlapping functional spectra of the elements of the pharmaceutical compositions as defined herein above, e.g. if the tumor markers have similar functionalities. Due to the different identity of the tumor marker, in such situations the use of highly specific elements like antibodies, siRNAs etc. is envisaged.

In another preferred embodiment of the invention the pharmaceutical relates to the pharmaceutical compositions as laid out in the present description for the treatment of a neoplastic disease as defined herein above, preferably cancer, more preferably prostate cancer.

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

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

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

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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, a miRNA inhibitor specific for miRNA(s) of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, an antagomir, a demethylation agent specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, a peptidomimetic specific for the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 with one or more cells which express the molecular tumor marker according to Table 1, 2 or 3 as (a) transcript(s), (b) detecting the expression level of said sequence; and (c) indentifying an interacting molecule by observing binding or a modulation or reduction of the expression level of the molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.

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

In a further aspect the present invention relates to a pharmaceutical composition as defined herein above for use in, or for the treatment or prevention of a neoplastic disease, preferably cancer.

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

Further, in yet another aspect, the present invention relates to the use of (a) a compound directly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an agonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and (e) a miRNA inhibitor specific for a miRNA of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 for the preparation of a pharmaceutical composition for the neoplastic disease as defined herein above, preferably cancer, preferably for the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease.

In another aspect the present invention relates to a method of treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, in particular the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, comprising the administration of (a) a compound directly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an antagonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (e) a miR A specific for a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (f) an antisense molecule of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (g) a siR A specific for a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (h) an aptamer specific for the expression product of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (i) a small molecule or peptido mimetic capable of specifically binding to the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; and/or (j) an antibody specific for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or an antibody variant specific for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 to an individual, in particular to an individual suffering from a neoplastic disease as defined herein above, preferably cancer or being prognosticated to develop said neoplastic disease.

In another aspect the present invention relates to a method of treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, in particular the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease, comprising the administration of (a) a compound directly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an agonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and (e) a miRNA inhibitor specific for a miRNA of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 to an individual, in particular to an individual suffering from a neoplastic disease as defined herein above, preferably cancer or being prognosticated to develop said a neoplastic disease.

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

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

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

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

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

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

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

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

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 molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 or biologically active equivalents thereof as defined herein above may be provided as fusion proteins. The fusion partner may be provided at the N- or C- terminus.

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 a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least one element selected from the group consisting of (a) a compound directly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an antagonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly inhibiting the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (c) a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a dominant negative form of a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (e) a miR A specific for a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (f) an antisense molecule of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (g) a siR A specific for a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (h) an aptamer specific for the expression product of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 or for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; (i) a small molecule or peptido mimetic capable of specifically binding to the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3; and j) an antibody specific for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3 and/or an antibody variant specific for the protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3; or at least one element selected from the group consisting of (a) a compound directly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3, preferably an agonist of said molecular tumor marker enzymatic activity; (b) a compound indirectly stimulating or modulating the activity of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3; (c) a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 or a biologically active equivalent thereof; (d) a nucleic acid encoding and expressing a protein of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1 , 2 or 3 and (e) a miR A inhibitor specific for a miRNA of a molecular tumor marker or group of tumor markers as mentioned herein above or according to Table 1, 2 or 3.

A medical kit that can be used in the context of the administration of the pharmaceutical composition as defined herein above. In particular, a kit according to the present invention may be used for the treatment or prevention of a neoplastic disease as defined herein above, preferably cancer, associated with a progression from a less progressed stage of said neoplastic disease to a more progressed stage of said neoplastic disease.

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

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

In another aspect the present invention relates to a vaccine comprising the expression product or protein, or any fragment thereof, of a molecular tumor marker or group of tumor markers as defined herein. 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 1, 2 or 3, more preferably as defined in section G) of Table 1, 2 or 3, or any fragment, variant, derivative or modified form thereof, having a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 300, 350 or 400 amino acids or the entire length or any other suitable length.

In a further embodiment the vaccine may comprise a nucleic acid molecule encoding a molecular tumor marker of the present invention or a group of tumor markers as defined above, e.g. a nucleic acid molecule comprising a nucleotide sequence as indicated in section D) of Table 1, 2 or 3, and/or a vector comprising said nucleic acid molecule, a host cell comprising said vector, an antibody as defined herein, in particular an autoantibody according to the present invention, or a CTL specific for an antigen as defined herein. Thus, a vaccine according to the present invention may, for example, comprise polypeptides or proteins of varying length comprising the amino acids sequence of SEQ ID NOs: 202 to 602, a nucleotide sequence encoding such a polypeptide, e.g. the nucleotide sequences of SEQ ID NOs: 1 to 201, or of amino acid sequences or nucleotide sequences as defined in Table 2, an expression vectors capable of expressing the polypeptide or comprising said nucleic acid or fragments thereof, e.g. DNA plasmid vectors, viral vectors etc., host cells expressing such a polypeptide, preferably host cells expressing the polypeptide at the surface of the cell, or secrete the polypeptide. 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 molecular tumor marker antigens or proteins of the present invention may be present either separately or in combination.

In specific aspect the present invention relates to a vaccine for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 1, 2 or 3, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) or G) of Table 1, 2 or 3, or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) or G) of Table 1, 2 or 3, or any fragment thereof.

The expression product, polypeptide or antigen comprised in the vaccine may comprise one epitope or various epitopes, e.g. a MHC I and/or a MHC II epitope or two or more copies of each or combinations thereof. Corresponding, i.e. encoding nucleic acid molecules may be provided, preferably in the form of DNA or R A 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 sections E) or G) of Table 1, 2 or 3. Subsequently, such a CTL may perform a cytotoxic reaction, e.g. via the release of the cytotoxins perforin and granulysin, or induce an appoptotic cell reaction, e.g. via the interaction with cell-surface molecules, e.g. Fas proteins expressed on a target cell. The making of CTLs typically comprises the presentation of antigens, preferably of MHC I specific antigenic peptides, to T cells, preferably to CD8+ T cells. Preferably, the antigen or antigenic peptide may be presented by a dendritic cell (DC), more preferably by a MHC I molecule present on a dendritic cell.

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

PBMCs to be used may be derived from blood obtained from blood donors.

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

In a specific embodiment the present invention relates to such CTLs, as well as to a method of making CTLs specific for the antigen or molecular tumor marker expression product or protein, or group of molecular tumor marker expression products or proteins of the present invention, e.g. comprising the amino acid sequence as indicated in sections E) or G) of Table 1, 2 or 3, which comprises the step of stimulating autologous T cells in vitro with dendritic cells loaded with a peptide derived from the antigen or a fragment thereof. This procedure may be carried out according to any suitable procedure known in the art.

Typically, the following procedure may be used: non-adherent PBMCs (preferably in an amount of 2 x 10 ⁶ 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 sections E) or G) of Table 1, 2 or 3, may be present in a peptide, polypeptide, protein, polyprotein or particle comprising one, two or more epitopes, or as a recombinant string of epitopes or in the context of the native target antigen, or in the form of a mixture or combination of the mentioned entities.

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

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

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

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

In a particularly preferred embodiment the present invention relates to a vaccine as defined herein, or a CTL as defined herein, for the treatment or prevention of a neoplastic disease, preferably for the treatment or prevention of a cancer disease, more preferably of a cancer disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a cancer disease.

If a vaccine according to the present invention is for the treatment or prevention of a neoplastic or cancer disease, it may preferably comprise, in addition to antigens or epitopes as mentioned above, additional antigens or epitopes derived from or representing known tumor associated antigens (TAAs). Furthermore the string of epitopes or multiple epitopes of a corresponding vaccine may include one or more epitopes derived from or representing a known tumor associated antigen (TAA). Examples of such as TAAs which may be included in vaccines of the present invention are MAGE antigen, a SSX antigen family member, NY-ESO-1, Melan-A/MART-1, g lOO, tyrosinase, tyrosinase-related protein 1 (TRP1), TRP2, CEA, PSA, Her2/neu, p53, MUC1, PRAME, sarcosin (N-methylglycin), CA-125 (Carbophydrate antigen- 125) or survivin. The sequence and identity of these and further suitable TAAs would be known to the person skilled in the art or can be derived from a suitable textbook.

In yet another preferred embodiment the present invention envisages a method of inducing an immune response in an individual comprising administering to said subject a therapeutically effective amount of an antigen, vector, epitope etc. as mentioned above. The term "immune response" refers to a therapeutic immune response beneficial for the subject or individual. Such an immune response may be a passive or active immunization, or it may be a short-term or long-term immunoprotection. Preferably the immune response is an immune response against a neoplastic disease, e.g. a cancer disease. The term "immune response against a neoplastic disease" means that a neoplastsic cell or tissue, e.g. a cancerous cell or tissue, may be attacked by components of the immune system, e.g. by CTLs or antibodies, that such a cell or tissue may be reduced in its size or modified in its structure by the mentioned entities or that such cell or tissue may be eliminated by the mentioned entities.

In a further, particularly preferred embodiment the present invention also envisages a method of identifying an individual for eligibility for neoplastic disease therapy or treatment or a corresponding immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease as defined above, wherein the detection of an increased level of expression of a molecular tumor marker or group of tumor markers as described herein above, leads to, or is used for the preparation of a vaccine or of a method of vaccination comprising an antigen or epitope linked to the detected, increased molecular tumor marker or group of tumor markers of the present invention. The present invention thus also encompasses a personalized vaccine or method for vaccination based on the outcome of a method of identifying an individual for eligibility for a neoplastic disease therapy, or a corresponding immunoassay for stratifying an individual or cohort of individuals with such a disease. The term "linked to" as used herein means that the molecular tumor marker or group of tumor markers encountered to be increased according to parameters as defined herein may be used in the form of a protein sequences, e.g. comprising or comprised in the amino acid sequence as indicated in sections E) or G) of Table 1, 2 or 3, or in the form of a nucleotide sequence, e.g. comprising or comprised in the nucleic acid sequence as indicated in section D) of Table 1, 2 or 3. 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 molecular tumor markers as defined herein, or in combination with known markers or TAA as described above.

In a further preferred embodiment the present invention relates to a method for screening for immunogenic epitopes of any of the molecular tumor marker or group of tumor marker expression products or proteins, e.g. a protein comprising or comprised in an amino acid sequence as mentioned in sections E) or G) of Table 1, 2 or 3. Such a method may preferably comprise:

(i) Immobilization of an antigen, expression product or protein of the present invention, e.g. a protein comprising or comprised in amino acid sequence as mentioned in sections E) or G) of Table 1, 2 or 3; preferably an antigen may be used which is detected or identified according to a method of identifying an individual for eligibility for neoplastic disease treatment or a corresponding immunoassay for stratifying an individual or cohort of individuals with such a disease, or according to any other detection or diagnosis method of the present invention as described herein. The immobilization may be carried out according to any suitable method known to the person skilled in the art and also comprises the employment of pre-manufactured protein arrays etc.

(ii) Incubation with an individual's sample, preferably an individual's serum, e.g. the sample or serum which was initially tested positive with regard to the antigen of (i), in particular the antibody or autoantibody present in said serum. The incubation may be carried out according to any suitable method or procedure known to the person skilled in the art, e.g. according to parameters known for the interaction between immobilized antigens and antibodies.

(iii) Competing the interaction of the antigen, expression product or protein and the serum ingredients, i.e. antibody by addition of short protein fragments or peptides of said antigen to the incubation reaction of (ii) in one or more separate reactions. The term

"short protein fragments or peptides" as used here means peptides or fragments of a length of about 10 to 30 amino acids, preferably of about 15 to 25 amino acids, more preferably of about 15 to 20 amino acids of the initially immobilized antigen, expression product or protein, e.g. a protein comprising or comprised in amino acid sequence as mentioned in sections E) or G) of Table 1, 2 or 3. The peptides or protein fragments may additionally be modified, varied or derivatized according to suitable methods known in the art.

(iv) Measurement of the interaction between the immobilized antigen and serum ingredients, e.g. an antibody, in particular an autoantibody, during the competition reaction(s). Such a measurement may be carried out according to any suitable method or procedure known to the person skilled in the art.

(v) Selection of peptides or protein fragments as defined in (iii) which attenuate or impede an interaction between the immobilized antigen and serum ingredients, e.g. an antibody, in particular an autoantibody. The term "attenuate" as used herein refers to a weakening of the interaction between the immobilized antigen and serum ingredients in comparison to an interaction reaction between the immobilized antigen and serum ingredients in the presence of a control peptide which has no sequence or structure similarity with the antigen and/or which is not able to influence said interaction, by 90%, 80%>, 70%>, 60%>, 50%>, 40%), 30%o, 20%) or 15%. The term "impede" as used herein refers to a complete or almost complete abolition of the interaction between the immobilized antigen and serum ingredients in comparison to an interaction reaction between the immobilized antigen and serum ingredients in the presence of a control peptide which has no sequence or structure similarity with the antigen and/or which is not able to influence said interaction.

Peptides or protein fragments and their corresponding amino acid sequences, as well as derived or modified variants thereof, which are selected or identified according to the above described method, are also encompassed in a particularly preferred embodiment of the present invention.

Such peptides or protein fragments may preferably be used for any of the diagnostic or prognostic methods or assays according to the present invention. They may for example be synthetically produced, immobilized on substrates as described herein above, employed in detection procedures etc. Furthermore, such peptides or protein fragments may preferably be used for vaccination methods as described herein above, or be comprised in a vaccine according to the present invention. More than one such fragment may be provided in the form of a string of proteins as described above. The present invention thus also refers to a personalized vaccine comprising antigenic compounds or protein fragments identified as being detected by an individual's autoantibody. Such vaccine may preferably be used for the treatment of a neoplastic disease.

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

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 molecular tumor marker protein or expression product as defined in

Section E) or G) of Table 1, 2 or 3 may be conjugated to carrier elements such as a bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc.

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

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, SRL1 72, Virosomes and other Virus-like particles, YF-17DBCG, Aquila's QS21 stimulon, Detox Quil, Superfos, Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant, pertussis toxin (PT), E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S 109, and PT-K9/G129, MF59, or saponin adjuvants.

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

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

A vaccine usually may be provided, for example, in ready-to-use syringes having a volume of from 0.01 to 1 ml, preferably 0.1 to 0.75 ml, of the concentrated solution, or suspension, respectively.

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

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

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

compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein the neoplastic disease as defined herein above is a neoplastic disease of the prostate. In a further specific embodiment of the present invention said neoplastic disease of the prostate is a benign prostate tumor or a malignant prostate tumor, e.g. prostate cancer.

The terms "neoplastic disease of the prostate", "benign prostate tumor" and "prostate cancer" refer to different stages and developmental transitions starting form a healthy prostate tissue and/or cells over benign tumor/lesion/growth/hyperplasia, to cancers of the prostate gland. A "neoplastic disease", "prostate tumor" or "prostate cancer" as used herein thus denotes a developmental stage of prostate tissue which can be classified according to the TNM classification by the International Union Against Cancer (UICC). Preferably, the terms relates to a classification of prostate tissue pursuing the following T - Primary Prostate Tumor classification schedule:

TX. Primary tumor cannot be assessed

TO. No evidence of primary tumor

Tl . Clinically inapparent tumor not palpable or visible by imaging

Tla. Tumor incidental histological finding in 5% or less of tissue resected

Tib. Tumor incidental histological finding in more than 5% of tissue resected

Tic. Tumor identified by needle biopsy (e.g., because of elevated PSA)

T2. Tumor confined within prostate (Tumor found in one or both lobes by needle biopsy, but not palpable or visible by imaging, is classified as Tic)

T2a. Tumor involves one half of one lobe or less

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

T2c. Tumor involves both lobes

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

T3a. Extracapsular extension (unilateral or bilateral)

T3b. Tumor invades seminal vesicle(s)

T4. Tumor is fixed or invades adjacent structures other than seminal vesicles: bladder neck, external sphincter, rectum, levator muscles, or pelvic wall Nl . Tumor invades regional lymph node(s)

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

Mlb. Tumor invades bone(s)

Mlc. Tumor invades other site(s) G Histopathological Grading

GX. Grade cannot be assessed

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

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

10),

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

Stage I: Tla; N0; M0; Gl

Stage II: Tla; NO; M0; G2, 3-4, or

Tib. c; NO; M0; any G, or

T1. T2; N0; M0; any G

Stage III T3; NO; M0; any G

Stage IV T4; NO; M0; any G, or

Any T; Nl; M0; any G, or

Any T; Any N; Ml; any G.

Thus, the term "benign prostate tumor" as used herein particularly refers to a prostate tumor which lacks all three of the malignant properties of a cancer, i.e. does not grow in an unlimited, aggressive manner, does not invade surrounding tissues, and does not metastasize. Typically, a benign prostate tumor implies a mild and non-progressive prostate neoplastic or swelling disease lacking the invasive properties of a cancer. Furthermore, benign prostate tumors are typically encapsulated, and thus inhibited in their ability to behave in a malignant manner. A benign tumor or a healthy condition may be determined by any suitable, independent molecular, histological or physiological method known to the person skilled in the art.

In contrast thereto the term "prostate cancer" as used herein relates to a cancer of the prostate gland in the male reproductive system, which occurs when cells of the prostate mutate and begin to multiply out of control. Typically, prostate cancer is linked to an elevated level of prostate-specific antigen (PSA). In one embodiment of the present invention the term "prostate cancer" relates to a cancer showing PSA levels above 4.0. In another embodiment the term relates to cancer showing PSA levels above 2.0. The term "PSA level" refers to the concentration of PSA in the blood in ng/ml. A "prostate cancer" as used herein may further have one of the following grade of Gleason score: Grade 1 (the cancerous prostate closely resembles normal prostate tissue. The glands are small, well- formed, and closely packed), Grade 2 (the tissue still has well- formed glands, but they are larger and have more tissue between them), Grade 3 (the tissue still has recognizable glands, but the cells are darker. At high magnification some of these cells have left the glands and are beginning to invade the surrounding tissue), Grade 4 (the tissue has few recognizable glands. Many cells are invading the surrounding tissue), Grade 5 the tissue does not have recognizable glands. There are often just sheets of cells throughout the surrounding tissue). The grading typically follows the Gleason grading as established by the ASCP .

Typically, prostate cancer is further linked to an elevated level of prostate- specific antigen (PSA). In one embodiment of the present invention the term "prostate cancer" relates to a cancer showing PSA levels above 4.0. In another embodiment the term relates to cancer showing PSA levels above 2.0, preferably 2.5 or 3.0. The term "PSA level" refers to the concentration of PSA in the blood in ng/ml.

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

compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above.

In a further specific embodiment of the present invention the less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above and said more progressed stage of the neoplastic disease can be any one of the clinically classifiable stages, grades, and/or substeps of prostate cancer as defined herein above.

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

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

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.

In a further specific embodiment of the present invention the more progressed stage of the neoplastic disease may imply a prostate cancer stage of Tl, Tla, Tib, Tic, T2, T2a, T2b or T2c„ preferably <T2 (UICC 2002 classification), but not stage T3 or higher (UICC 2002 classification).

In further specific embodiments of the present invention the more progressed stage of the neoplastic disease may imply a prostate cancer of stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification).

In a further specific embodiment of the present invention the less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above and said more progressed stage of the neoplastic disease may imply a prostate cancer of stage of of Tl, Tla, Tib, Tic, T2, T2a, T2b or T2c„ preferably <T2 (UICC 2002 classification), but not stage T3 or higher (UICC 2002 classification).

In a further specific embodiment of the present invention the less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above and said more progressed stage of the neoplastic disease may imply a prostate cancer of stage of T3, T3a, T3b or T4, preferably T3 (UICC 2002 classification).

A further particularly preferred embodiment relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc, wherein said more progressed stage of the neoplastic disease can be any one of the clinically classifiable stages, grades, and/or substeps of prostate cancer as defined herein above, wherein said more progressed stage of the neoplastic disease shows a serum level of PSA of >2 ng/ml, > 2.5 ng/ml, >3 ng/ml,> 3.5 ng/ml, >4 ng/ml, >4.5 ng/ml,> 5 ng/ml, >5.5 ng/ml, >6 ng/ml, >7 ng/ml, >8 ng/ml, >9 ng/ml, >9.5 ng/ml.

A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said more progressed stage of the neoplastic disease can be any one of the clinically classifiable stages, grades, and/or substeps of prostate cancer as defined herein above, wherein said less progressed stage of the neoplastic disease has a tumor volume of between about <0.05 ml and about 3.0 ml, e.g. <0.1 ml, <0.2 ml, <0.3 ml, <0.4 ml, <0.5ml, <0.6 ml, <0.7 ml, <0.8ml, <0.9 ml, <1.0 ml, <1.1 ml, <1.2ml, <1.3 ml, <1.4 ml, <1.5ml, <1.6 ml, <1.7 ml, <1.8ml, <1.9 ml or <2.0 ml. A further particularly preferred embodiment relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of the neoplastic disease is a benign prostate tumor and wherein said more progressed stage of the neoplastic disease can be any one of the clinically classifiable stages, grades, and/or substeps of prostate cancer as defined herein above, wherein said prostate cancer shows a serum level of PSA of >2 ng/ml, > 2.5 ng/ml, >3 ng/ml,> 3.5 ng/ml, >4 ng/ml, >4.5 ng/ml,> 5 ng/ml, >5.5 ng/ml, >6 ng/ml, >7 ng/ml, >8 ng/ml, >9 ng/ml, >9.5 ng/ml.

A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc., wherein said less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above and said more progressed stage of the neoplastic disease can be any one of the clinically classifiable stages, grades, and/or substeps of prostate cancer as defined herein above, wherein said prostate cancer has a tumor volume of between about <0.05 ml and about 3.0 ml, e.g. <0.1 ml, <0.2 ml, <0.3 ml, <0.4 ml, <0.5ml, <0.6 ml, <0.7 ml, <0.8ml, <0.9 ml, <1.0 ml, <1.1 ml, <1.2ml, <1.3 ml, <1.4 ml, <1.5ml, <1.6 ml, <1.7 ml, < 1.8ml, <1.9 ml or <2.0 ml.

A further, particularly preferred embodiment the present invention relates to any of the above mentioned tumor marker or group of tumor markers, the above mentioned compositions, the above mentioned methods, the above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc, wherein said more progressed stage of a neoplastic disease is a prostate cancer is of stage < T2 or < T3 (UICC 2002 classification) as defined herein above.

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 above mentioned use, the above mentioned immunoassay, kits or the above mentioned pharmaceutical compositions etc, wherein said less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above, and said more progressed stage of the neoplastic disease is a prostate cancer of stage < T2 (UICC 2002 classification) as defined herein above. In a further specific embodiment of the present invention the less progressed stage of the neoplastic disease is a benign prostate tumor as defined herein above and said more progressed stage of the neoplastic disease is a prostate cancer of stage > T2(UICC 2002 classification); Gleason Score > 6; Prostate Cancer Volume > 0.5 ml.

In specific embodiments of the present invention the more progressed stage of prostate cancer may imply a stage Tl, Tla, Tib, Tic, T2, T2a, T2b or T2c, preferably Tl, or higher (UICC 2002 classification) as defined herein above.

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 - Host Immune Response Profiling (HIRP) - Preparation of samples

Host Immune Response Profiling (HIRP)

Host Immune Response Profiling is a technology that allows the identification of host immune reactions against body proteins that are changed in its characteristics due the development, or the progression, or the treatment of a disease condition. The change in characteristics may mean a change in expression of a protein, the change in

cellular/extracellular location of a protein, the mutation of a protein, the aberrant excretion of a protein, the fragmentation of a protein, the aberrant fusion of a protein to another protein, the mutation of a protein, etc. The immune system of the host is able to detect such changes via an innate or humoral immune reaction against any sort of non-normal body proteins. In that sense the host immune system picks up any change in e.g. cellular proteins that occur while a disease may develop and is therefore able to potentially pick up this process way before the disease is going to manifest in the form of clinical symptoms. Further, as the progression of a disease is often associated with increased proliferation of cells (particularly in any type of tumor disease) this technology is especially suited to detect early signs of progression of disease thereby providing significant value for the prognosis of the future development of a given disease.

The technology works by the immobilization of body proteins that were previously expressed in a vertebrate cellular expression system, like insect cells, or in mammalian cells. After expression the proteins are purified, spotted and immobilized onto a solid support (e.g., glass slides, 96-well Microtiter plates), and subsequently incubated with human serum of plasma of a disease patient, or of a normal/healthy control. The binding of antibodies in the blood sample to the immobilized proteins is detected be a secondary fluorescence-labeled antibody which is directed against, and specific for the constant part of human IgG. Such antibodies are typically called auto-antibodies as they react against body- owned proteins. This detection step ensures that only auto-antibodies that are present in patient/control blood against body proteins will be detected. The reaction patterns against patient samples can then be compared to corresponding patterns of control samples to select those that are able to discriminate between different sample groups (e.g., healthy vs. disease). The strength of this technique is the blood-based detection of events that are typically closely linked to changes on the cellular level as a disease starts to develop or starts to progress. Auto-antibodies may typically be developed by the host against cellular proteins derived from the diseased tissue, and which are secreted at small levels into the circulation during apoptosis or necrosis of cells, or that are unnaturally secreted into the circulation as full or fragmented proteins. As such proteins do not typically present in the circulation this may - together with the change in other protein characteristics like protein mutations - lead to a host auto-immune reaction against a body-owned protein. The immune system hereby acts as an amplification mechanism for potentially only very small amounts of proteins derived from diseased tissue that otherwise would be very difficult - if not impossible - to detect. In that way, Host Immune Response Profiling provides a significant potential to unravel novel blood-based markers that are useful for the diagnosis and prognosis of human diseases.

Clinical human plasma samples

In order to implement the Host Immune Response Profiling clinical samples were prepared. These 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&4) men with localised prostate cancers, and 5) 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 biopsies (i.e., presumably men with a benign prostate disease). Group 3 men were selected based on PSA levels >3 ng/ml and cancer positive biopsy and pre- and post-treatment stages T2 or less, and Gleason scores 6 or less, and a prostate cancer volume of <0.5 ml. Group 4 men were selected based on PSA levels >3 ng/ml and cancer positive biopsy and pre -treatment stages T2 or less, post- treatment stages >T2, Gleason scores >7, and a prostate cancer volume >0.5 ml. Group 5 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 - Quantifying 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 H ₂ 0.

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) H ₂ 0 (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

ProtoArray Assays

A total of one hundred and twenty (120) serum samples from 5 groups (healthy controls, benign disease, low-grade cancer, locally advanced cancer, and metastatic cancer, see Example 1 and Fig. 1 to 6) were profiled on ProtoArray® Human Protein Microarrays v4.1 (Invitrogen Inc) containing more than 8,000 human proteins. Sera were profiled at a 1 :500 dilution, utilizing one ProtoArray® Human Protein Microarray per sample. Two lots of arrays were used in this assay, and a negative control assay for each of the two lots was run in parallel with the samples as described below. Samples from each of the five groups were divided evenly between the two lots of arrays used in this study (lots HA20174 and HA20179 were used).

Assay Controls

In the negative control assays, a ProtoArray® Human Protein Microarray from each of the two lots used in this service was treated in an identical manner to the

experimental assays, except that it was incubated with buffer containing no serum prior to incubation with the Alexa Fluor®647-anti-human IgG detection reagent. A small percentage (less than 0.5%) of the proteins exhibited significant signals in the negative control assay due to interaction with the detection reagent and were eliminated from the analysis. Figure 2 shows a representative negative control image displaying the regular pattern of control proteins spotted in one subarray. The purpose of these control proteins is to provide reference points for data acquisition and analysis. The Alexa Fluor® conjugated antibody (boxed in white) allows proper alignment of the spotfinding software for data acquisition. The anti- biotin antibody (boxed in green) serves as a control for another ProtoArray® application, but is recognized by the anti-human IgG antibody used for detection in this assay. Each

ProtoArray® subarray contains a gradient of human IgG (boxed in red), which serves as a control for proper performance of the detection reagent. Anti- human IgG antibody is also spotted as a gradient in every subarray. This antibody binds to IgG present in the serum sample and serves as a control for proper assay performance.

Assay Performance

In this study, a total of one hundred and twenty (120) serum samples divided into five groups were profiled on Proto Array® Human Protein Microarrays v4.1. Array images showed good signal-to background levels in all samples tested, although elevated background was seen for samples 3, 27, and 77. As shown above, the control features on the array serve as an indicator of assay performance; however, data quality can also be influenced by the scanning and data acquisition steps. To optimize this parameter, scanner settings were selected such that maximal signals on the array were sub-saturated, thus ensuring that the full dynamic range of the scanner was utilized. Maximizing the dynamic range of the scanner without increasing background to unacceptable levels is also critical. As illustrated in Table 6, the maximum group average background signal observed across the five groups was 437 relative fluorescence units (RFU) in group 2, with the cumulative average background across all samples equal to 419. Maximal signals were observed at >65,000 RFU for all of the samples, indicating a dynamic range of greater than 2 logs.

Together, these data indicate that the experiments and data acquisition were performed under optimal conditions.

Some samples profiled in this study exhibited globally low signal intensity across the array. One of the control protein features present on the array is anti-human IgG, printed in every subarray as a gradient. Signals arising from the anti-human IgG gradient are used to identify proper scanning parameters, such that the signals at the highest concentration of anti-human IgG are just below saturation.

Table 7, below shows the mean signals for the spots corresponding to the highest concentration of anti-human IgG on each array. Arrays profiled with samples 46, 60, 61, 62, 84, 108, and 109 had mean signal intensities that were greater than two standard deviations below the mean for all arrays (highlighted). The low signals may be the result of lower IgG levels or differences in immunoglobulin binding proteins in the corresponding samples. To ensure the use of the highest quality data set, those samples highlighted in Table 3 were excluded from the analysis and candidate autoantigen tables in this report.

Table 6 (average background fluorescence values reported in relative fluorescence units (RFUs) for all serum samples profiled):

SUBSTITUTE SHEET RULE 26 Table 7 (average fluorescence for spots corresponding to the highest concentration in the gradient of anti-human IgG. Signal intensity values reported in relative fluorescence units (RFUs) for all serum samples profiled):

Data analysis

The array data were analyzed as follows: The raw results (fluorescence from duplex, neighboring spots) were converted to semi-ready data by averaging the intensities of the two spots. The resulting file contained 8302 rows for the different spotted proteins and 113 columns for the samples that had passed the quality control (described before) . As a final step, all arrays were normalized to have the same median intensity. We found this an essential step as the median intensities of the arrays varied by more than a factor of two in either direction before normalization.

In order to find the most relevant proteins with respect to the clinical question, the proteins were ranked according to their p-value from a one-sided Wilcoxon test comparing groups 3 and 4 (signal in 4 greater than in 3). For visual inspection, for each protein the normalized data of all patients were plotted, ordered by group (see Fig. 9); the full and dashed line indicate the median and the mean normalized intensity per patient group, respectively. The top-left number indicates the rank of the protein according to the Wilcoxon test, with number one the protein with the lowest p-value. The PSA values measured clinically for each patient were added as a separate row to the data as a benchmark, also PSA could be detected (gene name KLK3) which appears with rank 42.

To investigate which marker proteins are statistically significant, the distribution of the p-values for the individual proteins was compared with the average distribution of 1000 randomized sets. The number found is larger than the largest number found in 50-90% of the random sets. It can thus be concluded that roughly the top 100-500 proteins seem significant at the 90% level. The calculation was done in R with 1000 randomized sets (samples from groups 2, 3 and 4 shuffled), and took around 10 hours to run on a Dell dual-core workstation (1.5 GB RAM, 2.4 GHz clock frequency).

The quality of a clinical test is commonly described in terms of sensitivity and specificity; these depend on the threshold chosen. As the threshold goes down, the true positive rate increases, but so does the false positive rate. This is typically shown by means of a Receiver-Operating-Curve (ROC). The area under the curve (AUC) represents the accuracy of the test, averaged over all thresholds. The ROC's and their AUC's were calculated for all proteins, for discrimination between groups 3 and 4. Fig 8 shows the ROC's for the proteins with rank 1 to 200. With increasing rank, the AUC drops as expected.

Example 5 - Identification of men with localized, and locally advanced prostate cancer

SUBSTITUTE SHEET RULE 26 Screening with PSA (Prostate Specific Antigen) has resulted in a marked increase in the number of newly diagnosed prostate cancers. PSA is a protein, found in the blood. It is produced exclusively by the prostate, but not exclusively by prostate cancer. An elevated concentration of PSA (>3 ng/ml) can be related to the presence of prostate cancer, but may also point toward other benign conditions of the prostate, such as benign

enlargement (BPH) or infection/inflammation.

Above the age of 50 however approximately 2 in 10 men (20%) have an elevated PSA value (www.erspc.org). The elevated value can point toward prostate cancer but may also be related to benign prostatic enlargement, a urinary tract infection or an infection of the prostate. In a PSA range of 3-10 ng/ml, several biopsies have to be performed to find one prostate cancer patient.

80% of men above 50 have a normal (not elevated) PSA value. The PSA test can miss prostate cancer. Men with a completely normal PSA test can have a (usually small) prostate cancer. This applies to at least 1 in 100 men (1%).

Thus, it is clear that novel markers with improved prostate cancer specificity are needed to provide more effective diagnostics to better discriminate between benign and malignant prostate tumors, and/or to identify cancers in men with not elevated PSA values.

The measurement of a marker, or a combination of markers from Table 1 , 2 or 3 in a patient serum sample is used to identify a patient with prostate cancer. The

measurement of the marker, or the combinations of markers above a given threshold hereby indicates a >95%, >90%, >85%, >80%, >75%, >70%, >65%, or >60% chance that a prostate biopsy will deliver a positive result.

The measurement of the marker, or a combination of markers from Table 1 , 2 or 3 is further used to monitor the potential re-currence of the prostate cancer after primary treatment like surgery, or radiotheary. The marker(s) measurement is performed every 3, or every 6, or every 12 months after the patient has been treated. If the marker, or marker combination measurement is going to pass a given threshold by 5%, 10%>, 15%, 20%, 25%, or 30% further investigations or treatment will be started. Example 6 - Identification of men with elevated PSA and first negative biopsy for

stratification to re -biopsy Screening with PSA (Prostate Specific Antigen) has resulted in a marked increase in the number of newly diagnosed prostate cancers. PSA is a protein, found in the blood. It is produced exclusively by the prostate, but not exclusively by prostate cancer. An elevated concentration of PSA (>3 ng/ml) can be related to the presence of prostate cancer, but may also point toward other benign conditions of the prostate, such as benign

enlargement (BPH) or infection/inflammation.

Above the age of 50 however approximately 2 in 10 men (20%) have an elevated PSA value (www.erspc.org). The elevated value can point toward prostate cancer but may also be related to benign prostatic enlargement, a urinary tract infection or an infection of the prostate. In a PSA range of 3-10 ng/ml, several biopsies have to be performed to find one prostate cancer patient. This leads to a high rate of negative biopsies which can be either due to the fact that the patient does not have prostate cancer but rather a benign type of disease, or due to the fact that the biopsy procedure has missed the cancer that is actually present. The biopsy has a reported false-negative rate of up to 25%. As a consequence, there is a significant number of men who have a negative initial, 1 ^st biopsy but who may later on be diagnosed with prostate cancer on a 2 ^nd , or 3 ^rd biopsy procedure indicating that first biopsy procedure may have missed the cancer. Therefore, it would be of major clinical importance to have a biomarker test that stratifies patients towards a 2 ^nd biopsy after initial negative biopsy in case the test indicates a high likelihood of presence of prostate cancer.

The measurement of a marker, or a combination of markers from Table 1 , 2 or 3 in a patient serum sample is used to identify a patient with prostate cancer although the initial biopsy gave a negative result. The measurement of the marker, or the combinations of markers above a given threshold hereby indicates a >95%, >90%, >85%, >80%, >75%, >70%, >65%, or >60%> chance that a follow-up prostate biopsy will deliver a positive result.

The measurement of the marker, or a combination of markers from Table 1 , 2 or 3 is further used to monitor the potential re-currence of the prostate cancer after primary treatment like surgery, or radiotheary. The marker(s) measurement is performed every 3, or every 6, or every 12 months after the patient has been treated. If the marker, or marker combination measurement is going to pass a given threshold by 5%, 10%, 15%, 20%, 25%, or 30% or more, further investigations or treatment will be started. Example 7- Selection of antigens for the testing of such antigens on targeted arrays for marker verification purposes To further verify markers 213 antigens were selected from the initial discovery that was performed on 120 Prostate Cancer (PCa) patient derived blood samples. The process is outlined in Figure 10.

The 120 blood samples were coming from 5 clinically defined patient groups ranging from normal/healthy patients over patients with a benign prostate disease to finally those patients with significant and advanced prostate cancer:

Group 1) Control, very low PSA (#24)

Group 2) PSA>3ng/ml, 2x negative Biopsy (#24)

Group 3) PSA>3ng/ml, pathol. Gleason 6 & pT2 & TVol<0.5 ml (#24)

Group 4) PSA>3ng/ml, pathol. Gleason >=7 or >pT2 or Tvol>0.5 ml (#24)

Group 5) PSA>3ng/ml, advanced disease (#24)

Those samples were used to identify marker candidates as described above. The data was statistically analyzed for discriminating features between samples from patients with benign disease (patient group 2) and samples drawn from patients with indolent or aggressive Prostate Cancer disease (patient groups 3 & 4). For each antigen a p-value and a ROC (Receiver Operator Curve) was calculated as quantitative parameters to discriminate between the two patient groups (indolent vs. aggressive). The antigens were ranked according to p-value and the size of the Area Under the Curve (AUC) of the ROC. Subsequently 213 candidates were selected to build a targeted antigen array for testing of additional patient samples (see Example 8 below).

Example 8 - Marker verification and identification of 3- and 5-parameter signatures to discriminate indolent from aggressive prostate cancer with high sensitivity

In order to define marker/antigen signatures that are able to discriminate between benign versus indolent/non-aggressive & significant/aggressive Prostate Cancer (PCa) we analyzed in total 329 blood samples from PCa patients. The samples were spread over 4 different independent data sets (see Table 8). Table 8

Table 8: Numbers of benign vs. indolent & aggressive PCa spread of four independent data sets. Samples in each data set were selected such that the diagnostic specificity of PSA at a diagnostic sensitivity was corresponding to a historical value demonstrated on larger sample collections of ca 20%.

For each of the data sets 213 antigens were analyzed on their power to discriminate between indolent and aggressive prostate cancer patients. The clinical characteristics of the patients are:

1 - (corresponding to group 2 above) Benign -> PSA >3 & <10 ng/ml PSA; 2x negative biopsy, cancer free >5 years

2 - (corresponding to group 3 above) Indolent PCa -> PSA >3 ng/ml; Gleason <6; Stage <T2; no recurrent disease > 10 years median follow-up

3 - (corresponding to group 4 above) Significant PCa -> PSA >3 ng/ml; Gleason >6; Stage >T2; recurrent disease > 10 years median follow-up

To determine this power the diagnostic specificity was calculated at a diagnostic sensitivity of 90%>. Subsequently, individual antigens were selected based on their overall performance on all four analyzed data sets. Only markers were selected with a minimum specificity of >20% on at least 3 of the 4 data sets to continue with.

The diagnostic sensitivity is defined as the probability of the identification of a disease condition; the sensitivity is expressed as a percentage as it also indicates how many disease cases out 100 is a test procedure able to correctly identify. The value 100-sensitivity is defined as the False-Negative Rate (FNR) as this number indicates the percentage of disease cases that are missed by a diagnostic test or procedure. For instance, a diagnostic sensitivity of a test procedure of 90% indicates that that 90%> of disease cases out of a tested population can be correctly identified by the test procedure as having the disease condition. The diagnostic specificity is defined as the probability of the identification of a non-disease condition (vs. a disease condition) by a diagnostic test/procedure; the specificity is expressed as a percentage as it also indicates how many non-disease cases out 100 is a test procedure able to correctly identify. The value 100-sensitivity is defined as the False-Negative Rate (FPR) as this number indicates the percentage of non-disease cases that are incorrectly classified as having the disease. For instance, a diagnostic specificity of a test procedure of 50% indicates that that 50% of non-disease cases out of a tested population can be correctly identified by the test procedure as not having the disease condition.

The following Tables 9 and 10 provide an overview over the data points of DATA set 1, DATA set 2 (both in Table 9), and DATA set 3 and DATA set 4 (both in Table 10), respectively, measured, the derived p-values and the derived specificity at 90%>

sensitivity of all 213 candidates.

Table 9

A) B) C) D) E)

Marker Swisspr. Marker Function / DATA 1 DATA 2

No or Acces. Descriptt

code Code

Mean Mean

Mean Mean

Value Value

Value p-Value Specif, Value p-Value Specif, detect, detect,

detect, 2 vs. at 90% detect, 2 vs.3& at 90% amount amount

amount 3&4 sensitiv. amount 4 sensitiv.

Group Group3

Group 2 Group 2

3&4 &4

A - PSA 4,25 5,81 0,0053 13 6,14 10,16 0,0336 23

B - Patient Age 64,00 61,77 0,0563 0 65,90 62,06 0,0003 0

C - DRE 1,00 1,48 0,0558 0 1,13 1,74 0,0001 0

1 P00519 ABL1 E255K 4882,23 1830,24 0,2083 21 3473,17 4200,23 0,4517 16

2 P00519 ABL1 G250E 7929,88 3390,84 0,1545 13 4076,37 4589,33 0,6115 3

3 P00519 ABL1 T315I 15694,07 13245,80 0,6101 4 2991,80 3450,77 0,5003 13

Q96GD4 Aurora B 1334,47 1321,15 0,9234 17 518,62 590,47 0,1846 10

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SUBSTITUTE SHEET RULE 26 exosome

84 Q96B26 component 8 5089,99 6947,46 0,0951 17 849,20 906,29 0,6436 16

(EXOSC8)

Regulator of

85 Q96LZ7 microtubule 281,39 448,27 0,0034 34 1561,01 1755,38 0,3480 6 dynamics protein 2

F-box and leucine-

86 Q96IG2 rich repeat protein 580,44 820,60 0,0295 21 1049,87 1255,20 0,2786 10

20 (FBXL20)

F-box/WD repeat-

87 Q9UKB1 containing protein 1716,57 1561,49 0,5144 30 6635,01 7600,73 0,5381 20

11

fibroblast growth

88 Q9NSA1 3750,04 2706,16 0,4453 21 2168,62 2114,58 0,8722 10 factor 21 (FGF21)

89 P22607 FGFR3 393,17 339,69 0,5322 21 464,27 531,91 0,0743 20 hypothetical

90 Q8NE77 protein FLJ11171 414,31 618,46 0,0021 30 987,53 1047,75 0,4532 3

(FLJ11171)

hypothetical

91 Q9H5C5 protein FLJ23584 1420,41 1796,93 0,0150 21 2441,54 2594,30 0,5425 13

(FLJ23584)

UDP-galactose-4-

92 Q14376 epimerase (GALE), 984,11 1008,18 0,8750 17 1264,82 1323,94 0,6855 16 transcript variant 1

golgi associated,

gamma adaptin ear

containing, ARF

93 Q9UJY4 2190,66 2454,42 0,6882 4 2510,70 2222,22 0,6445 0 binding protein 2

(GGA2), transcript

variant 2

GIY-YIG domain

containing 2

94 Q6NTG6 1084,84 1100,56 0,8985 26 2291,88 2570,72 0,4414 23

(GIYD2),

transcript variant 2

glyoxalase I

95 Q04760 1671,31 2523,10 0,1423 26 2693,35 2831,03 0,8333 6

(GLOl)

Guanine

nucleotide-binding

96 P09471 303,17 498,46 0,0086 17 1146,95 1183,43 0,6961 13 protein G(o)

subunit alpha

guanine nucleotide

binding protein (G

97 P50150 protein), gamma 4 1276,33 2250,20 0,1614 30 5285,44 3427,73 0,3770 3

(GNG4), transcript

variant 3

glutamate receptor,

98 P48058 ionotrophic, 162,99 314,55 0,0215 26 2079,56 2140,67 0,6847 0

AMPA 4 (GRIA4)

99 P34947 GRK5 1690,42 2316,77 0,0389 17 1392,36 1439,86 0,8939 6

Q8WTQ

00 GRK7 1732,81 2753,67 0,2237 13 1869,65 1782,77 0,8807 3 7

hyaluronan

01 000219 synthase 3 (HAS3), 491,90 738,48 0,0015 34 1402,52 1857,76 0,0544 13 transcript variant 2

hemoglobin, zeta

02 P02008 1432,27 2133,64 0,3806 17 969,74 1146,10 0,5728 13

(HBZ)

Homeobox-03 Q6NT76 containing protein 714,72 910,04 0,2155 21 3301,98 3851,81 0,7194 13

1

homeobox

04 Q6NT76 containing 1 1433,02 1661,23 0,4194 13 2588,22 4161,54 0,2920 6

(HMBOXl)

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SUBSTITUTE SHEET RULE 26 Table 10

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SUBSTITUTE SHEET RULE 26 Guanine

nucleotide-binding

96 P09471 757,09 715,45 0,4724 6 280,33 299,55 0,4126 11 protein G(o)

subunit alpha

guanine nucleotide

binding protein (G

97 P50150 protein), gamma 4 1005,99 2440,03 0,3482 13 2899,97 1059,31 0,1646 7

(GNG4), transcript

variant 3

glutamate receptor,

98 P48058 ionotrophic, 1463,45 1274,61 0,1654 6 569,39 594,00 0,6088 18

AMPA 4 (GRIA4)

99 P34947 GRK5 726,46 1600,41 0,1626 10 740,85 985,04 0,6820 11

Q8WTQ

100 GRK7 841,79 1048,16 0,1536 23 418,57 455,58 0,7739 18

7

hyaluronan

101 000219 synthase 3 (HAS3), 947,35 836,17 0,2496 16 338,14 478,18 0,0200 11 transcript variant 2

hemoglobin, zeta

102 P02008 519,60 517,40 0,9862 16 281,58 307,47 0,7310 11

(HBZ)

Homeobox-

103 Q6NT76 containing protein 1593,16 1585,89 0,9740 16 1010,90 1331,99 0,5955 11

1

homeobox

104 Q6NT76 containing 1 2393,34 2202,42 0,7943 13 962,21 1131,80 0,7684 3

(HMBOXl)

homer homolog 3

105 Q9NSC5 (Drosophila) 3865,57 5300,54 0,1936 3 1213,14 2109,88 0,0940 3

(HOMER3)

homeobox B5

106 P09067 1909,05 1732,28 0,3932 10 745,83 831,32 0,2719 14

(HOXB5)

Ig mu chain C

107 P01871 1401,13 1222,06 0,1403 3 610,67 606,25 0,9194 11 region

Inhibitor of

nuclear factor

108 Q14164 793,79 747,58 0,5690 10 325,96 345,88 0,5570 18 kappa-B kinase

subunit epsilon

IMP (inosine

monophosphate)

109 P20839 dehydrogenase 1 2794,49 3999,07 0,2041 16 801,57 1324,18 0,0885 18

(IMPDH1),

transcript variant 2

integrator complex

110 Q68E01 5282,83 4101,20 0,5277 10 994,59 1602,49 0,1897 14 subunit 3 (INTS3)

interferon

111 014896 regulatory factor 6 986,75 1288,76 0,2811 13 398,36 554,68 0,1487 7

(IRF6)

iron-sulfur cluster

assembly 2

112 Q86U28 1374,56 1434,50 0,8570 16 372,48 558,29 0,1865 29 homolog (S.

cerevisiae) (ISCA2)

inosine

triphosphatase

(nucleoside

113 Q9BY32 triphosphate 671,02 611,53 0,4564 3 342,18 351,63 0,8629 22 pyrophosphatase)

(ITPA), transcript

variant 1

JmjC domain-

114 Q9H9V9 containing protein 1113,16 1082,82 0,7729 16 383,80 443,51 0,2261 3

4

Kv channel

interacting protein

115 Q9NS61 322,01 366,70 0,4473 16 109,60 160,72 0,0781 14

2 (KCNIP2),

transcript variant 5

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transcript variant 1

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SUBSTITUTE SHEET RULE 26 zinc finger protein

210 095789 258 (ZNF258), 822,75 972,20 0,4432 13 812,76 464,95 0,2379 14 transcript variant 2

zinc finger protein

211 Q8TD17 398 (ZNF398), 1771,88 1729,38 0,9046 16 581,57 603,21 0,8430 14 transcript variant 2

Q5JUW zinc finger protein

212 816,79 759,21 0,5831 16 354,11 374,01 0,6570 14

0 673 (ZNF673)

zinc ribbon domain

containing 1

213 Q9P1U0 (ZNRD1), 955,18 1435,59 0,1507 6 706,64 1047,09 0,3666 14 transcript variant

b

Finally, the data of each selected marker for the four different data sets were combined in a linear fashion for each possible combination. To generate 3- or 5 antigen signatures we selected 9 antigens (see Table 11 below).

Table 11

Table 11 : Selected marker candidates including PSA, Patient Age, & DRE to generate 3- or 5- antigen signatures.

Further, next to these selected antigen markers the PSA values were included for each patient, the patient age, as well as the DRE (Digital Rectal Examination) as additional parameters. The PSA value (typically a value from 1-30 representing the

concentration of this protein in patients per Milliliter blood) was scaled for instance with a factor of 100 to achieve similar numeric values as for the selected antigen marker candidates. In the same way the patient age was scaled e.g. with a factor of 10. The DRE result is

SUBSTITUTE SHEET RULE 26 typically represented by a single digit number from 1-5 each representing different pathophysiological conditions of the prostate. A typical numerical representation of a DRE results also used here would be: l=Symmetrical benign; 2= Asymmetrical benign; 3=Palpable nodule; 4=Other palpable abnormality; 5=Equivocal; 6=Frank carcinoma; 7=Other

In order to include these values into the calculation of marker combinations the DRE numeric values were scaled according to PSA and patient age by a factor of 10 or 100. In consequence, 12 marker candidates were included to build either 3- marker/antigen signatures, or 5-marker/antigen signatures for the purpose to discriminate between benign vs. indolent & aggressive prostate tumors.

All linear combinations of the antigens were generated (220

combinations in total for 3-marker/antigen signatures and 792 combinations on total for 5- marker/antigen signatures). For each 3- or 5~marker/antigen combination the diagnostic specificity at a diagnostic sensitivity of 90% and 95% was calculated. The antigen combinations were subsequently ranked according to their highest specificity at 90/95%) sensitivity (see Tables 12-15 below). For comparative reasons PSA, Patient Age, as well as the results of the DR examination (DRE) were included to the analysis as individual markers as well as a combination of these three parameters.

Table 12: 3 Marker/ Antigen Combinations with a minimal diagnostic specificity of 30% at a diagnostic sensitivity level of >90%> (for comparative purposes the performance data of PSA, Patient Age, and DRE as single parameters were included (see rows 1-3)):

SUBSTITUTE SHEET RULE 26 Table 12

SUBSTITUTE SHEET RULE 26 Table 13: 3 Marker/ Antigen Combinations with a minimal diagnostic specificity of 20% at a diagnostic sensitivity level of >95%> (for comparative purposes the performance data of PSA, Patient Age, and DRE as single parameters were included (see rows 1-3)):

Table 13

SUBSTITUTE SHEET RULE 26 Table 14: 5 Marker/ Antigen Combinations with a minimal diagnostic specificity of 30% at a diagnostic sensitivity level of >90%> (for comparative purposes the performance data of PSA, Patient Age, and DRE as single parameters were included (see rows 1-3)):

Table 14

SUBSTITUTE SHEET RULE 26

SUBSTITUTE SHEET RULE 26 Table 15: 3 Marker/ Antigen Combinations with a minimal diagnostic specificity of 24% at a diagnostic sensitivity level of >95%> (for comparative purposes the performance data of PSA, Patient Age, and DRE as single parameters were included (see rows 1-3)):

Table 15

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SUBSTITUTE SHEET RULE 26 As can be seen from Tables 12-15 numerous combinations of antigens with or without combination with PSA, and/or Patient Age, and/or DRE have been defined, which perform significantly better compared to PSA, Patient Age, or DRE alone or in combination to discriminate between benign versus benign vs. indolent & aggressive prostate tumors based on the measurement of such antigens in a patient blood sample as described here.

It is of particular importance and relevance for the clinical application that the identified marker combinations are able to identify Prostate Cancer at a very high diagnostic sensitivity of >90%, and at the same time discriminate between benign and malignant prostate tumors with specificity of >30% (3 marker/antigen signatures), or a specificity of >40% (5 marker/antigen signatures). Further, if the diagnostic sensitivity level is increased to >95% with very low cancer cases missed the specificity is still at a minimal level of >20% (3 marker/antigen signatures), or a specificity of >30% (5 marker/antigen signatures). As comparison, the max specificity which can be achieved by using PSA, Patient Age or DRE is 20% (at 90% sensitivity), or 9% (at 95% sensitivity).

The identified marker/antigen combinations are able to achieve a max specificity of 41% (at 90% sensitivity), and 33% (at 95% sensitivity) respectively. Such it is possible by using the disclosed marker combinations to increase the diagnostic specificity at 90%) diagnostic sensitivity by a factor of 2 over the currently used gold standard PSA (20%> vs. 41% for a 5-marker/antigen signature). In case the diagnostic sensitivity is increased to 95% the diagnostic specificity can even be increased by a factor of 3 over the currently used gold standard PSA (9% vs. 30% for a 5-marker/antigen signature).

Example 9 - Marker verification and identification of 3- and 5-parameter signatures to discriminate indolent from aggressive prostate cancer with high sensitivity for patients with PSA range 4-10 ng/ml

In order to define marker/antigen signatures that are able to discriminate between indolent/non-aggressive vs. significant/aggressive Prostate Cancer (PCa) 329 blood samples from PCa patients were analyzed by the methods described above. To select antigen combinations for the suitable for patient with a PSA range 4-10 ng/ml 80 out of the 329 samples were selected for further data processing (see Table 16).

SUBSTITUTE SHEET RULE 26 Table 16: Numbers of indolent vs. aggressive PCa spread of four independent data sets (DATA l to DATA 4). NOTE that for the further processing of the data only those patient samples with PSA 4-10 ng/ml were selected:

Table 16

SUBSTITUTE SHEET RULE 26 The patient samples were derived from patient cohorts with the following characteristics: DATA_1

i) Control, very low PSA (GROUP _1 )

ii) PSA>3ng/ml, 2x negative Biopsy (GROUP _2)

Hi) PSA>3ng/ml, pathol. Gleason 6 & pT2 & TVol<0.5 ml (GROUP_3)

iv) PSA>3ng/ml, pathol. Gleason > = 7 or >pT2 or Tvol>0.5 ml (GROUP _4)

v ) PSA>3ng/ml, advanced disease ( GRO UP_5 ) DATA_2, DATA_3, & DATA_4

i) Benign -> PSA >3 & <10 ng/ml PSA; 2x negative biopsy, cancer free >5 years) (GROUP _2)

ii) Indolent PCa -> PSA >3 ng/ml; Gleason <6; Stage <T2; no recurrent disease > 10 years median follow-up (GROUP _3)

Hi) Significant PCa -> PSA >3 ng/ml; Gleason >6; Stage >T2; recurrent disease > 10 years median follow-up (GROUP _4)

For each of the data sets we analyzed 213 antigens on their power to discriminate between benign versus indolent & aggressive prostate cancer patients for the selected patient samples.

As the data were derived from independently measured data we performed as a first step a 'global normalization' for the different data sets, i.e. all intensities from each data set were divided by the median value of all intensities for that data set. Subsequently all data sets were merged into a single data set which was used to define marker/antigen

combinations.

For all antigens the AUC (Area Under the Curve) of a ROC (Receipher Operator Curve) to separate indolent from aggressive Prostate Cancer was determined, and the antigens were ranked according to the size of the AUC (largest to smallest). For the generation of 5 -antigen signatures we selected the top 15 antigens according to their AUC rank (see Table 17).

SUBSTITUTE SHEET RULE 26 Table 17: Selected antigen marker candidates to generate 5 -antigen signatures:

Table 17

SUBSTITUTE SHEET RULE 26 The diagnostic sensitivity is defined as the probability of the identification of a disease condition; the sensitivity is expressed as a percentage as it also indicates how many disease cases out 100 is a test procedure able to correctly identify. The value 100-sensitivity is defined as the False-Negative Rate (FNR) as this number indicates the percentage of disease cases that are missed by a diagnostic test or procedure. For instance, a diagnostic sensitivity of a test procedure of 90% indicates that that 90%> of disease cases out of a tested population can be correctly identified by the test procedure as having the disease condition. The diagnostic specificity is defined as the probability of the identification of a non-disease condition (vs. a disease condition) by a diagnostic test/procedure; the specificity is expressed as a percentage as it also indicates how many non-disease cases out 100 is a test procedure able to correctly identify. The value 100-sensitivity is defined as the False-Negative Rate (FPR) as this number indicates the percentage of non-disease cases that are incorrectly classified as having the disease. For instance, a diagnostic specificity of a test procedure of 50%) indicates that that 50%> of non-disease cases out of a tested population can be correctly identified by the test procedure as not having the disease condition.

The Tables 9 and 10 above provide an overview over the data points of DATA set 1, DATA set 2 (both in Table 9), and DATA set 3 and DATA set 4 (both in Table 10), respectively, measured, the derived p-values and the derived specificity at 90%> sensitivity of all 213 candidates.

Finally, the data of each selected antigen were combined in a linear fashion for each possible combination of 5 antigens. Next for each sample the sum of the intensities for all unique combinations of 5 antigens from the top-ranked 15 antigens and the ROC curves for each of these combinations was calculated. From these curves, the diagnostic specificity at 90%) diagnostic sensitivity was determined. These values were used to rank the combinations on their ability to separate the groups. Combinations with a diagnostic specificity above 40% are shown in Table 18a, and 18b.

Table 18a: 5- Marker/ Antigen Combinations with a minimal diagnostic specificity of >35% at a diagnostic sensitivity level of >90%>:

SUBSTITUTE SHEET RULE 26 o o

Seasi ttvi ty >9€ %

Speeificity 35% Specificity

COMBINATIONS

MLF1IP-BC031520. ί <& AMN1-NM_207337J & CYP1A1-NM_0 Q499.2 & AD€ 1-NM_02042L1 & COL9A1 48 MRPS35-NM_021821.2 & PPP2R4-NM_021 31.1 & CYPIA1- M_000499.2 & ADC l-NM_0202i .1 & COL A1

MKPS35-NM_02 82i .2 & MLF I IP-BC031520, 1 & AD€Kl-NM_02042I.i & CASC2- M_201377.1 & COL9A1 43 MRPS35-NM_Q21821.2 & CD24-lSiM_013230.1 & GNG4-NM_004485.2 & ADCK1-NM_020421.1 & COL9A1 43 MLF1IP-BC03I52Q.1 & CD24-NM_0I3230.1 & AMN1-N _207337.1 & CYP1A1-NMJ3O0499.2 & CASC2 43 MLF1∑P-BC031520.1 & AMN1-NM _. 207337.1 & CYPl A1-NM_000499.2 & ADCKl-mi_020421.1 & CASC2 43 N/A-BC0O8624.1 & MLF1 IP-BC031520.1 & CYPlAi-NM_000499.2 & GNG4-NM_004485.2 & ADCK1 44 MRPS35-NM_0 1821.2 & PPP2R4-NM_021131.1 & ADCKl-NM_020421,i & ITP A-NM _0343.2 & COL9A1 4J MLF1IP-BC031520.1 & CD24-NM_013230.1 & AMN1-NM_207337.1 & CYP1 A1-NM_000499.2 & ADC 1 il MLFHP-BC0 520.1 & CD24- M 013230.1 & CYPlAi-NM_000499.2 & ADC 1-NM_020421.1 & COL9A1 44 MLF1IP-BC031520.1 & ADCK1-NM_020421.1 & ITPA-NMJ333453.2 & CASC2-NM_201377.1 & COL9A1 41 PPP2R4-NM_021131.1 & AMN1-NM_2G7337.1 & CYP1A!-NM_000499,2 & ADCKl-NM_02042i.I & COL9AI 4J_ N/A-BC0OS624.1 & MLFHP-BC031520,1 & ADC 1-NM_020421.1 & CASC2-NM_201377.1 & GLOi 39 N/A-BC0OS624.1 &€D24-NM_ i323iU & PPP2R4-NMJ321131,1 & CYPiAl-NM_000499.2 & ADCK1 39 MRPS35-NM_Q2 21.2 & CREM-BC01 117.1 & ADCK1-NM_020421.1 & ITP A-NM_033453.2 & COL9A1 39 MRPS3S-NM 021821.2 & CD24-NM_013230.1 & PPP2R4-NM_02113 .1 & AM 1-NM_207337.1 &ADCK1 39 MRPS35-NM_021821.2 & CD24-$iM_01323Q.l & PPP2R4- M_0211 1.1 & ADC 1-NM_02042 .1 & COL9A1 39 MRPS35-NM_021821.2 & CD24-NM_013230.1 & CYPl A1- M 0G0499.2 & ADCK1-NM_020421.1 & COL9A1 39 MRPS35-NM_02 821.2 & PPP2R4-NM_021131.1 & AM i-NM_207337.1 & ADC l-NM_02042i J & COL9A1 39 MRPS35- M_021821.2 & PPP2R4-NM_021131.1 & ADCK1-NM_020421.1 & ΓΓΡΑ-ΝΜ_033453.2 & CASC 2 39 MRPS35-NM_021821.2 & PPP2R4-NM_02 ^' i 131.1 & ADC 1-NM_02042 L1 & CASC2-NM_201377.1 & COL9A1 39 MRPS35- MJ321821.2 & AMN1-NM_2073371 & GNG4-KM_004485.2 & ADCK1-N¾^i_02 421.1 & COL9A1 39 MRPS35- M_02182L2 & AM 1-NM.207337.1 & ADCK1-NM_02O421.1 & CASC2-NM_2 1377.1 & COL9A1 39 CREM-BC017117.1 & MLF IP-BC031520, 1 & PPP2R4-NM_021131.1 & ADC .!- M_02042 .1 & CASC2 39 MLF1 IP-BC031520.1 & CD24-NMJ) 13230.1 & PPP2R4-NM_02i 131.1 &. CYP1A1-NM_000499.2 & ADCK1 39 MLF1 IP-BC031520.1 & CD24-NM 013230.1 & A N1-NM_207337.1 & G G4-NM_O04485.2 & ADCKl 39 MLFHP-BC0 520.1 & CD24-NM_013230.1 & ADCKi-NM_02042i .1 & ΪΤΡΑ- Ινί_033453.2 &COL9A1

MLF1IP-BC03520.1 &AMN1- M 207337.1 & CYPl A -NM 000499.2 & G G4-NM 004485.2 & LUC7L2 39

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MLF1IP-BC0 1520.1 & CYP1 A1-NM 000499.2 & ADCK 1-NM 020421.1 & CASC2-NM_201377.1 & COL9A1

MLF1IP-BC0 1520.1 & CYP1AI-NM_000499.2 & ADCK 1-NM_020421.1 & CASC2-NM_201377.1 & LUC7L2

CD24-NMJ) 13230.1 & PPP2R4-NM 021131.1 & CYP1A1 -NM 000499.2 & ADCK 1-NM 020421.1 & COL9A1 39 CD24-NM_013230.1 & AMN1-NM_207337.1 & CYPi A1-NM_000499.2 & ADCK1-NM_020421.1 & COL9A1 39 CD24-NM 013230.1 & AMN1-NM 207 37.1 & ADCK 1-NM 020421.1 & ITPA-NM_033453.2 & COL9A1 39 PPP2R4-NM_021 131.1 & AMN1-NM_207337,1 & CYP1A1-NM_000499.2 & ADCK1-NM_020421.1 & CASC2 39 PPP2R4-NM 021131.1 & AMN1-NM 207337.1 & ADCK1-NM 020421.1 & CASC2-NM_201377.1 & COL9A1 39 N/A-BC008624.1 & MRPS35-NM_021821.2 & MLF1IP-BC031520.1 & ADCK1 -NM 020421.1 & CASC2

N/A-BC008624.1 & MLF1IP-BC031520.1 & AMN1-NM_207337.1 & CYPI A1-NM_000499.2 & ADCK1

N/A-BC008624.1 & MLF1IP-BC031520.1 & AMN1-NM_207337.1 & ADC 1-NM_020421.1 & CASC2

N/A-BC008624.1 & MLF1IP-BC031520.1 & CYP1A1-NM 000499.2 & ADCKl -NM 020421.1 & CAS 2 37 MRPS35-NM 02I821.2 & CREM-BCO 17117.1 & AMN1-NM 207337.1 & ΓΧΡΑ-ΝΜ 033453.2 & COL9A1 37 MRPS35-NM_021821.2 & MLF1IP-BC031520.1 & ADCK1 -NM 020421.1 & ΓΤΡΑ-ΝΜ 033453.2 & COL9A1 MRPS 5-NM 0 1821.2 & CD24-NM 013230.1 & ADCK1-NM_020421.1 & ITPA-NM 0334 3.2 & COL9A1 MRPS35-NM_021821.2 & PPP2R4-NM_021 131.1 & GNG4-NM_004485.2 & ADCK1-NM_020421.1 & CASC2 37 MRPS35-NM 02I821.2 & AMN1-NM 207337.1 & ADCK1-NM_020421.1 & ITPA-NM 0 453.2 & COL9A1 MRPS35-NM_021821.2 & CYP1A1-NM_000499.2 & ADCK1-NM_020421.1 & ITPA-NM 033453.2 & COL9A1

CREM-BC0171 17.1 & MLF1 IP-BC031520.1 & AMN1-NM_207337.1 & ADCK 1-NM_020421.1 & COL A1

CREM-BC0171 17.1 & MLF1IP-BC031520.1 & CYP1A1-NM_000499.2 & ADCK1-NM_020421.1 & ITPA 37 CREM-BCO 171 17.1 & CD24-NM 013230.1 & ADCK 1-NM 020421.1 & ITPA-NM 033453.2 & COL9A1 37 MLF1IP-BC031520.1 & CD24-NM_ 13230.1 & PPP2R4-NM_021 131.1 & ADCK1-NM_020421.1 & COL9A1 MLF1IP-BC031520.1 & CD24-NM 013230.1 & AMN1-NM 207337.1 & ADCK1-NM 020421.1 & ITPA

MLF1IP-BC0 1520.1 & CD24-NM 013230.1 & CYP1A1-NM 000499.2 & CASC2-NM 201377. 1 & COL9A1 37 MLF1IP-BC031520. 1 & PPP2R4-NM 0 1 131.1 & AMN1-NM 207337.1 & ADCK 1 -NM 020421.1 & CASC2 37 MLF1IP-BC031520.1 & PPP2R4-NM_021131.1 & AMN1-NM_207337.1 & CASC2-NM_201377.1 & COL9A1 MLF1IP-BC031520.1 & AMN1-NM_207337.1 & CYP1A1-NM_000499.2 & ADCK 1-NM 020421.1 & LUC7L2 MLF 1 IP-BCO 1520.1 & AMNI-NM_207337.1 & ADCK 1-NM_020421 ,1 & CASC2-NM_201377.1 & COL9A1 MLF1IP-BC031520.1 & AMN1-NM_207337.1 & ADCK1-NM 020421 ,1 & COL9A1-BC015409.1 & LUC7L2 37 CD24-NM 01 230.1 & PPP2R4-NM 021 131.1 & AMNl-NM 207337.1 & CYPI Al-NM 000499.2 & ADCK 1 37

o o

Sensitivity>90%

Specif icity>35% Specificity

COMBINATIONS ^' _

CD24-NM_013230.1 & PPP2R4-NM_02i 131.1 & AMN1-NM_207337.1 & CYP1A1-NM_000499.2 & CASC2 7

CD24-NMJ) 13230.1 & PPP2R4-NM 021131.1 & AMN1-NM 207337.1 & ADCK1-NM 0 0421.1 & CASC2 37

CD24-NM_ 13230.1 & PPP2R4-NM_02 1 1.1 & ADCKl-NM_02042i .1 & CASC2-NM_201377,1 & COL9A1 7

CD24-NM 013230.1 & ADCKI-NM_02042L1 & iTPA-NM_033453.2 & COL9A1-BC015409.1 & LUC7L2 37

AMN1-NM_207337.1 & CYP1 A1-NM 0004 .2 & ADCK1-NM_020421.1 & ΓΤΡΑ-ΝΜ 33453.2 & CASC2 37

AMN1-NM_207337.1 & CY 1 A1-NM 000499.2 & ADC 1-NM 020421.1 & ITPA-NM 033453.2 & COL9A1 7

AMN1-NM_207337.1 & GNG4-NM_004485.2 & ITPA-NM 033453.2 & CASC2-NM_201377.1 & COL9A1 37

NA-BC008624.1 & MRPS35-NM_021821.2 & MLF1IP-BC031520.1 & CASC2-NM_201377,1 & COL9A1 35

N/A-BC008624.1 & MRPS35-NM 021821.2 & ADCK1-NM 02 421.1 & CASC2-NM 201377.1 & COL9A1 35

N/A-BC008624.1 & MLF1IP-BC031520.1 & AMN1-NM 207337.1 & CASC2-NM 201377.1 & LUC7L2 35

N/A-BC008624.1 & MLF1IP-BC031520.1 & ADCK1-NM_020421.1 & CASC 2-NM_201377.1 & COL9A1 35

MRPS35-NM_021821.2 & CREM-BC017117.1 & MLF1IP-BC031520.1 & CASC2-NM 20 377.1 & COL9A1 35

MRPS35-NM 021821.2 & MLF 1 IP-BC031 20.1 & AMN1-NM_207337.1 & ADCK1-NM 020421.1 & COL9A1 35

MRPS35-NM_021821.2&CD24-NMJ)13230.1 & PPP2R4-NM 021131.1 & ADCK1-NM 020421.1 & CASC2 35

MRPS35-NM_021821.2 & CD24-NMJ313230.1 & PPP2R4-NM_021131,1 & CASC2-NM_201 77.1 & COL A1 35

MRPS35-NM_021821.2 & CD24-NM 013230.1 & AMN1-NM 207337.1 & GNG4-NM 004485.2 & ADCK1 35

MRPS35-NM 021821.2 & PPP2R4-NM 021131.1 & AMN1-NM_207337.1 & ITPA-NM 033453.2 & COL9A1 35

MRPS35-NM_021821.2 & PPP2R4-NM 021131.1 & CYP1 A1-NM 000499.2 & GNG4-NM 004485.2 & ADCK1 35

MRPS35-NM_021821.2 & PPP2R4-NM 021131.1 & GNG4-NM 004485.2 & ADCK1-NM_02042L1 & COL9A1 35

MRP S 35 -NM 021821.2 & AMN1-NM 207337.1 & GNG4-NM 004485.2 & ADCK1-NM 020421.1 & ITPA 35

MRPS35-NM 021821.2 & CYP1 A1-NM 000499.2 & ADCK1-NM 020421.1 & C ASC2-NM 201377.1 & GLOl 35

MRPS35-NM 021821.2 & CYP1A1-NM 000499.2 & ADCK1-NM 020421.1 & CASC2-NM 201377.1 & COL9A1 35

CREM-BC017117.1 & ADCK1-NM_020421.1 & ITPA-NM_033453.2 & CASC2-NM 201377.1 & COL9A1 35

MLF 1IP-BC031520.1 & CD24-NM 013230.1 & ADCK1-NM_020421.1 & CASC2-NM 20 377.] & COL9A1 35

MLF 1IP-BC031520.1 & PPP2R4-NM_021131.1 & AMN1-NM 207337.1 & ADCK1-NM 020421.1 & COL9A1 35

MLF 1IP-BC031520.1 & PPP2R4-NM 021131.1 & ADCK1-NM 020421.1 & CASC2-NM 201377.1 & COL A1 35

MLF 1IP-BC031520.1 & AMN1-NM 207337.1 & CYP1 A1-NM 000499.2 & GNG4-NM 004485.2 & ADCK1 35

MLF 1IP-BC031520.1 & AMNl-NM 207337.1 & CYP1A1-NM 000499.2 & ITPA-NM 033453.2 & CASC2 35

O o

MLF 1IP-BC031520.1 <& AMN1-NM_207337.I & GNG4-NM_004485.2 & ADCK 1-NM_020421.1 & LUC7L2 MLF i IP-BC031520.1 & AMN1-NM 207337.1 & ADCK1-NMJ32042I .1 & ITPA-NM 033453.2 & CASC2

MLF 1 IP-BC0 1520.1 & AMN1-NM_207337.1 & ADCK1-NM_020421.1 & CASC2-NM_201377.1 & LUC7L2 MLF 1 IP-BC03 1520.1 & CYP1A1-NM_000499.2 & ADCK1-NMJ)20421.1 & TTPA-NM 033453..2 & CASC2 MLF I IP-BC031520.1 & CYP1A1 -NM_000499.2 & ADCK1-NM_020421.1 & ITPA-NM_033453.2 & C0L9A1 MLF Ϊ IP-BC03 1520.1 & ADCK!-NM_020421.1 & ITPA-NM_033453.2 & CASC2-NM_2 1377.1 & GLOl MLF 1 IP-BC0 1520.1 & ADCK1-NM_020421.1 & ITPA-NM_033453.2 & COL9A1-BC015409, 1 & LUC7L2 CD24-NM 013230. & PPP2R4-NM 021 131.1 & GNG4-NM 004485.2 & ADCKl -NM 020421.1 & C0L9A 1 CD24-NMJS 13230.1 & CYP 1A1-NM_000499.2 & GNG4-NM_004485.2 & ADCK 1-NM_020421.1 & C0L9A1 CD24-NM 013230.1 & CYP 1 A1-NM 000499.2 & ADCKl-NM 020421.1 & ITPA-NM 033453.2 & C0L9A1 CD24-NM_013230.I & GNG4-NM_004485.2 & ADCK 1-NM_020421.1 & ITPA-NM_0334 3.2 & C0L9A1 PPP2R4-NM 02I I31.1 & AMN1-NM_207337.1 & CYP 1 A 1-NM_000499.2 & CASC2-NM 201377J & C0L9A 1 PPP2R4-NM 02I I31.1 & AMN1-NM_207337.1 & GNG4-NM 004485.2 & CASC2-NM_201377.1 & C0L9A1 PPP2 4-NM_021131.1 & AMNl -NM_207337.1 & ITPA-NM_033453.2 & CASC2-NM_ 201377.1 & C0L9A1 PPP2R4-NM_0211 1.1 & CYP1A1-NM_000499.2 &:ITPA-NM_033453.2 & CASC2-NM_20I377,1 & COL9A1 AMN1-NM_207337.1 & CYP1A -NM_000499.2 & ADCK 1-NM_020421.1 & CASC2-NM_ 201377.1 & C0L9A1 CYP! A1-NM 000499.2 & GNG4- M 004485.2 & ADCKl-NM 020421.1 & ITPA-NM 033453.2 & C0L9A 1

As can be seen from Tables 18a and 18b numerous combinations have been defined that perform significantly better compared to PSA, Patient Age, or DRE alone (see Tables above) and which can be used to discriminate between benign versus indolent & aggressive Prostate tumors based on the measurement of such antigens in a patient blood sample as described herein.

It is of particular importance and relevance for the clinical application that the identified marker/antigen combinations are able to identify Prostate Cancer at a very high diagnostic sensitivity of >90%, and at the same time discriminate between benign versus indolent and & Prostate tumors with specificity of >35%. As comparison, the max specificity which can be achieved by using PSA, Patient Age or DRE is 20% (at 90%> sensitivity) (see Tables Example 2).

The identified marker/antigen combinations are able to achieve a max specificity of 48% (at 90% sensitivity). Such it is possible by using the disclosed marker combinations to increase the diagnostic specificity at 90% diagnostic sensitivity by a factor of 2.5 over the currently used gold standard PSA (20% vs. 48% for a 5-marker/antigen signature).

SUBSTITUTE SHEET RULE 26 In some embodiments, the present invention relates to the following items:

Item 1 : A tumor marker or group of tumor markers associated with the progression of a neoplastic disease from a less progressed stage to a more progressed stage, wherein the expression of the tumor marker or group of tumor markers is modified by increase (up- regulated) when comparing the expression of the tumor marker or group of tumor markers in the less progressed stage to the expression in the more progressed stage, wherein said tumor marker or group of tumor markers comprises at least one tumor marker selected from

Table 3.

Item 2: The group of tumor markers of item 1, 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, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or all of the tumor markers of Table 3.

Item 3 : The group of tumor markers of any one of item 1 or 2, wherein the p-value of the expression modification is 0.007 or lower.

Item 4: The group of tumor markers of any one of items 1 to 3, wherein the group comprises at least 5 tumor markers corresponding to tumor marker #1 to #5 of Table 3, at least 10 tumor markers corresponding to tumor marker #1 to #10 of Table 3, at least 15 tumor markers corresponding to tumor marker #1 to #15 of Table 3, at least 20 tumor markers corresponding to tumor marker #1 to #20 of Table 3, at least 25 tumor markers corresponding to tumor marker #1 to #25 of Table 3, at least 30 tumor markers corresponding to tumor marker #1 to #30 of Table 3, at least 35 tumor markers corresponding to tumor marker #1 to #35 of Table 3, at least 40 tumor markers corresponding to tumor marker #1 to #40 of Table 3, at least 45 tumor markers corresponding to tumor marker #1 to #45 of Table 3 or at least 50 tumor markers corresponding to tumor marker #1 to #50 of Table 3. Item 5: A composition for diagnosing, detecting, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid affinity ligand and/or a peptide affinity ligand

SUBSTITUTE SHEET RULE 26 for the expression product(s) or protein(s) of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4.

Item 6: The composition of item 5, wherein said nucleic acid affinity ligand or peptide affinity ligand is modified to function as an imaging contrast agent.

Item 7: A method for detecting, diagnosing, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the step of determining the level of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4 in a sample.

Item 8: The method of item 7, wherein the determining step is accomplished by the measurement of nucleic acid or protein level(s) or by the determination of the biological activity of said tumor marker or group of tumor markers.

Item 9: The method of item 8, wherein said method comprises the additional step of comparing the measured nucleic acid or protein level(s) or the measured biological activity to a control level, wherein said control level is the expression level of the tumor marker or the group of tumor markers in one or more samples of a less progressed stage of the same neoplastic disease.

Item 10: The method of item 9, wherein said method is a method of graduating neoplastic disease, comprising the steps of

(a) determining the level of a tumor marker or a group of tumor markers as defined in any one of items 1 to 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,

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

SUBSTITUTE SHEET RULE 26 (c) deciding on the stage or developmental status of a neoplastic disease based on the results obtained in step (b).

Item 11 : Use of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4 as a marker for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease.

Item 12: An immunoassay for detecting, diagnosing, graduating, monitoring or prognosticating a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, or for detecting, diagnosing, monitoring or prognosticating the progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease comprising at least the steps

(a) testing in a sample obtained from an individual for the expression of a tumor marker or a group of tumor markers according to any one of items 1 to 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

(d) deciding on the presence or stage of a neoplastic disease or the progression of the neoplastic disease based on the results obtained in step (c),

wherein said testing steps are based on the use of an antibody specifically binding (a) protein(s) of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4.

Item 13: A method of identifying an individual for eligibility for a neoplastic disease therapy comprising:

(a) testing in a sample obtained from an individual for the expression of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4;

(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4;

SUBSTITUTE SHEET RULE 26 (c) classifying the levels of expression of step (a) relative to levels of step

(b); and

(d) identifying the individual as eligible to receive a neoplastic disease therapy where the individual's sample is classified as having an increased level of expression of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4.

Item 14: An immunoassay for stratifying an individual or cohort of individuals with a neoplastic disease comprising:

(a) testing in a sample obtained from an individual for the expression of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4;

(b) testing in said sample for the expression of a reference gene and/or in a control sample for the expression of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4;

(c) determining the difference in expression of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4 of steps (a) and the expression of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4 and/or the reference gene in step (b); and

(d) stratifying an individual or cohort of individuals to a neoplastic disease therapy based on the results obtained in step (c), where the individual's sample has an increased level of expression of a tumor marker or a group of tumor markers as defined in any one of items 1 to 4.

Item 15: The method of any one of items 7 to 10 or 13, or the immunoassay of item 12 or 14, wherein said method or immunoassay comprises the additional step of determining the level of prostate specific antigen (PSA) and/or wherein the tested individual showed an initial negative biopsy result.

Item 16: A pharmaceutical composition for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, wherein said neoplastic disease implies the increased (upregulated) expression of a tumor marker or group of tumor markers as defined in any one of items 1 to 4, comprising at least one element selected from the group of:

SUBSTITUTE SHEET RULE 26 (a) a compound directly inhibiting the activity of a tumor marker as defined in any one of items 1 to 4, preferably an antagonist of said tumor marker enzymatic activity;

(b) a compound indirectly inhibiting the activity of a tumor marker as defined in any one of items 1 to 4;

(c) a dominant negative form of a protein of a tumor marker as defined in any one of items 1 to 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 as defined in any one of items 1 to 4;

(e) a miR A specific for a tumor marker as defined in any one of items 1 to 4;

(f) an antisense molecule of a tumor marker as defined in any one of items

1 to 4;

(g) a siRNA specific for a tumor marker as defined in any one of items 1 to 4;

(h) an aptamer specific for the expression product of a tumor marker as defined in any one of items 1 to 4 or for the protein of a tumor marker as defined in any one of items 1 to 4;

(i) a small molecule or peptidomimetic capable of specifically binding to the protein of a tumor marker as defined in any one of items 1 to 4; and

j) an antibody specific for the protein of a tumor marker as defined in any one of items 1 to 4 and/or an antibody variant specific for the protein of a tumor marker as defined in any one of items 1 to 4. Item 17: The composition for diagnosing, detecting, graduating, monitoring or prognosticating of item 5 or the pharmaceutical composition of item 16, wherein said antibody is an autoantibody against the expression product or protein or a fragment thereof, of a tumor marker or group of tumor markers as defined in any one of items 1 to 4. Item 18: The composition or pharmaceutical composition of item 17, wherein said autoantibody is an autoantibody against the expression product or protein of a tumor marker of group of tumor markers as defined in any one of items 1 to 4, wherein said expression product or protein comprises an amino acid sequence as indicated in section G) of Table 3, or any fragment thereof.

SUBSTITUTE SHEET RULE 26 Item 19: A vaccine for the treatment or prevention of a neoplastic disease associated with a progression from a less progressed stage of a neoplastic disease to a more progressed stage of a neoplastic disease, comprising a nucleic acid molecule comprising a nucleic acid sequences as indicated in section D) of Table 3, or any fragment thereof, or an expression product, protein or antigen comprising an amino acid sequence as indicated in section E) or G) of Table 3, or any fragment thereof, or a CTL specific for an antigen derived from an expression product or protein comprising an amino acid sequence as indicated in section E) or G) of Table 3, or any fragment thereof.

Item 20: The tumor marker or group of tumor markers of any one of items 1 to 4, the composition of item 5 or 6, 17 or 18, the method of any one of items 7 to 10, 13 or 15, the use of item 11, the immunoassay of item 12, 14 or 15, the pharmaceutical composition of item 16, 17 or 18, or the vaccine of item 19, wherein said neoplastic disease is a neoplastic disease of the prostate.

Item 21 : The tumor marker or group of tumor markers, composition, method, use, immunoassay, pharmaceutical composition or vaccine of item 20, wherein said less progressed stage of the neoplastic disease is a benign prostate tumor, and said more progressed stage of the neoplastic disease is a prostate cancer of stage < T2 (UICC 2002 classification).

SUBSTITUTE SHEET RULE 26