WO2010003772A1 - Method for predicting adverse response to erythropoietin in breast cancer treatment - Google Patents

Abstract

The present invention relates to methods for prediction of the adverse response to erythropoietin in cancer treatment, comprising determining, the expression level of at least one gene of interest, said gene being correlated with the expression level status of one of MMP7, MMP1, IGHM, MLPH, ESR1, PGR, RB1, BRCA1, Her-2/neu, EpoR, KRT5, and SPP1.

Description

Description

Method for predicting adverse response to erythropoietin in breast cancer treatment

Field of the invention

The present invention relates to methods for prediction of the therapeutic success of cancer therapy.

Background of the invention

In some neoplastic diseases, particularly gynaecological cancers like breast cancer, the response to neoadjuvant chemotherapy is comparatively low, with only about 20% of patients achieving pathological complete remission (pCR) with no tumor cells left in the breast or lymph nodes; the latter being the strongest prognostic factor for prolonged survival due to treatment benefit to date.

However, a substantial number of patients suffer severe side effects (ADRs) from highly toxic drug combinations (e.g. alopecia due to inclusion of taxols) without additional benefit. In addition, there is a burden on national health systems due to the high cost of some therapies in this regime, especially if the chemotoxic treatments are combined with new targeted treatment options (e.g. Herceptin®, Lapatinib® and Avastin®) . Moreover the new treatment options are related with some severe, probably life threatening side effects (e.g. cardiac toxicities upon combinatorial treatment with Herceptin®, gastrointestinal perforation upon combinatorial treatment with Avastin) .

A better characterization of the respective tumors would thus allow a better selection of the most promising therapy in a given breast cancer patient, in order to avoid unnecessary side effects due to neoadjuvant chemotherapy in those patients which do no not draw any benefit from such therapy anyway.

Response to neoadjuvant chemotherapy is comparatively low with only about 20% patients of breast cancer patients achieving pathological complete remission (pCR) with no tumor cells left in the breast or lymph nodes, which is the strongest prognostic factor for prolonged survival due to treatment benefit to date. Dose dense therapy regimen have been shown to gain survival benefits for patients (Untch et al . , SABCS2007) . However, a substantial number of patients suffer severe side effects (ADRs) from the dose dense usage of highly toxic drug combinations. Most importantly these patients suffer severe anemia resulting in side effects such as fatigue, headaches, tinnitus, cardiac effects. Anemia is monitored by assessment of Hb levels. Blood transfusion or erythropoietin derivatives are applied to reduce these side effects. Blood transfusion bear potential risks of severe infections and only temporarily adjust the Hb level. However, treatment with eryrthropoietin derivatives bear the risk of harm in case these growth factors drive the proliferation, survival and/or oxygenation of tumors thereby diminishing the chemotherapeutic treatment effect.

An open-label, randomized, multicenter Phase 3 study of

Aranesp® (darbepoetin alfa) in 733 neoadjuvant breast cancer patients receiving dose-dense, dose-intense preoperative chemotherapy compared to a standard preoperative chemotherapy regimen ("PREPARE"; Preoperative Epirubicin Paclitaxel Aranesp Studie") was designed and performed to address these issues. In this trial the patients are treated with or without Darbepoietin alfa (Aranesp®) , as a class member of the erythropoietin derivatives. On 30th Novemver 2007 the interim results were published by the investigator and the sponsoring company Amgen showing numerically more deaths in the Aranesp™ treated patients (37/377 vs 50/356) . Histopathological standard procedures (such as IHC) so far failed to identify the population being at risk of the life threatening ADR.

We have performed RNA analysis of selected candidate genes from paraffin embedded tissue biopsies to identify the patients being harmed by an EPO treatment.

It is thus an objective to provide a method for predicting an adverse response to erythropoietin in cancer treatment and to enable clinicians to increase quality of life of patients by treatment with EPO derivatives without potential harm.

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "prediction" as used herein relates to the likelihood that a patient will respond either favourably or unfavourably to a given therapy. Especially, the term "prediction", as used herein, relates to an individual assessment of the malignancy of a tumor, or to the expected survival rate (DFS, disease free survival; OAS, overall survival) of a patient, if the tumor is treated with a given therapy. In contrast thereto, the term "prognosis" relates to an individual assessment of the malignancy of a tumor, or to the expected survival rate (DFS, disease free survival; OAS, overall survival) of a patient, if the tumor remains untreated.

The term "response marker" relates to a marker which can be used to predict the clinical response of a patient towards a given treatment. Response includes direct observation of tumor shrinkage upon neoadjuvant or palliative treatment as evident by e.g. CT-Scans and/or serum biomarkers as well as effects on Disease Free Survival (DFS) , Overall Survival (OAS) , Metastasis Specific Survival (MSS) , Disease Specific Survival and related assessments.

The term "clinical response" of a patient, as used herein, relates to the effectiveness of a certain therapy in a patient, meaning an improvement in any measure of patient status, including those measures ordinarily used in the art, such as overall survival, progression free survival, recurrence-free survival, and distant recurrence-free survival. Recurrence-free survival (RFS) refers to the time (in years) from surgery to the first local, regional, or distant recurrence. Distant recurrence-free survival (DFRS) refers to the time (in years) from surgery and/or initial diagnosis to the first anatomically distant recurrence. The calculation of these measures in practice may vary from study to study depending on the definition of events to be either censored or not considered. The term "response marker" relates to a marker which can be used to predict the clinical response of a patient towards a given treatment.

The term "adverse response" relates to an unfavourable response not in line with the therapeutic goals of a given therapy. It may include any effects from mild to severe, such as, but not limited to, increased discomfort or pain, side effects, such as fever, disproportionate weight loss or weight gain, reduced or impaired metabolic function, cardiovascular function, renal function, neurological function, immunological function, disease recurrence or prolongation and death.

The term "neoplastic disease" refers to a cancerous tissue this includes adenomas and carcinomas, e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma, and pre- malignant conditions, neomorphic changes independent of their histological origin, e.g. papillary serous, mucinous, endometriod, clear cell, ductal, lobular, medullary, mixed origin . The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. The term "cancer" as used herein includes carcinomas, (e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma) and pre-malignant conditions, adenomas, blood cell neoplasms and neomorphic changes independent of their histological origin. The term "cancer" is not limited to any stage, grade, histomorphological feature, invasiveness, aggressiveness or malignancy of an affected tissue or cell aggregation. In particular stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer, primary carcinomas, and all other types of cancers, malignancies and transformations specially associated with gynecologic cancer are included. The terms "neoplastic disease" or "cancer" are not limited to any tissue or cell type they also include primary, secondary or metastatic lesions of cancer patients, and also comprise lymph nodes affected by cancer cells or minimal residual disease cells either locally deposited or freely floating throughout the patients body.

The term "tumor" as used herein, refers to all abnormal masses of tissue preferably exhibiting neoplastic cell growth and proliferation or impaired cell death meachnaisms, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

The term "neoplastic cells" refer to abnormal cells that grow by increased cellular proliferation, altered cell division symmetry or decreased cell death mechanisms more rapidly than normal. As such, neoplastic cells of the invention may be cells of a benign neoplasm or may be cells of a malignant neoplasm.

Furthermore, the term "characterizing the state of a neoplastic disease" is related to, but not limited to, measurements and assessment of one or more of the following conditions: Type of tumor, histomorphological appearance, dependence on external signal (e.g. hormones, growth factors) , invasiveness, motility, state by TNM Classification of Malignant Tumours (TNM) , a cancer staging system developed and maintained by the International Union Against Cancer, or similar, agressivity, malignancy, metastatic potential, and responsiveness to a given therapy.

The term "therapy modality", "therapy mode", "regimen" or "chemo regimen" as well as "therapy regimen" refers to a timely sequential or simultaneous administration of antitumor, and/or anti vascular, and/or immune stimulating, and/or blood cell proliferative agents, and/or radiation therapy, and/or hyperthermia, and/or hypothermia for cancer therapy. The administration of these can be performed in an adjuvant and/or neoadjuvant mode. The composition of such "protocol" may vary in the dose of the single agent, timeframe of application and frequency of administration within a defined therapy window. Currently various combinations of various drugs and/or physical methods, and various schedules are under investigation.

The term "cytotoxic treatment" refers to various treatment modalities affecting cell proliferation and/or survival. The treatment may include administration of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumour agents, including monoclonal antibodies, inhibitors of repair mechanisms and kinase inhibitors. In particular, the cytotoxic treatment may relate to a taxane treatment. Taxanes are plant alkaloids which block cell division by preventing microtubule function. The prototype taxane is the natural product paclitaxel, originally known as Taxol and first derived from the bark of the Pacific Yew tree. Docetaxel is a semi-synthetic analogue of paclitaxel. Taxanes enhance stability of microtubules, preventing the separation of chromosomes during anaphase. Erythropoietin (EPO) is a glycoprotein hormone that is a cytokine for erythrocyte (red blood cell) precursors in the bone marrow. Also called hematopoietin or hemopoietin, it is produced by the liver and kidney, and is the hormone that regulates red blood cell production. It also has other known biological functions, e.g. it is also involved in the wound healing process. See also Jelkmann, W (2007) . "Erythropoietin after a century of research: younger than ever". Eur J Haematol. 78 (3) : 183-205. The term "functional derivative thereof" means any synthetic, artificial, animal peptidic or protein derivative which produces the same desired therapeutic results as EPO. Commercially available forms of Epo include epoietin (Procrit (also known as Eprex and Epogen) , NeoRecormon, darbepoietin (Aranesp) , and PDpoietin.

The term "determining the expression level of a gene on a non protein basis" relates to methods which are not focussed on the secondary gene translation products, i.e proteins, but on other levels of the gene expression, based on RNA and DNA analysis. In one embodiment of this invention the analysis uses mRNA including its precursor forms. An exemplary determinable property is the amount of the estrogen receptor or progesterone receptor RNA, i.e. of the ESRl, ESR2 and/or PGR gene.

The term "expression level" refers, e.g., to a determined level of gene expression. The term "pattern of expression levels" refers to a determined level of gene expression compared either to a reference gene, e.g. housekeeper, or inversely regulated genes, or to a computed average expression value, e.g. in DNA-chip analyses. A pattern is not limited to the comparison of two genes but is more related to multiple comparisons of genes to reference genes or samples. A certain "pattern of expression levels" may also result and be determined by comparison and measurement of several genes disclosed hereafter and display the relative abundance of these transcripts to each other. Similarly, a "gene being correlated with an expression level status" of another gene refers to a gene the expression level of which is found to be correlated with the expression level of another gene in a cohort of samples. This may be both a positive correlation, or, in the alternative, a negative correlation. A gene being correlated with an expression level of another gene may be used in addition or instead of the gene it is correlated to for said objective.

The term "RNA expression level" refers to a determined level of the converted DNA gene sequence information into transcribed RNA, the initial unspliced RNA transcript or the mature mRNA. RNA expression can be monitored by measuring the levels of either the entire RNA of the gene or subsequences.

The term "pattern of RNA expression" refers to a determined level of RNA expression compared either to a reference RNA or to a computed average expression value. A pattern is not limited to the comparison of two RNAs but is more related to multiple comparisons of RNAs to reference RNAs or samples. A certain "pattern of expression levels" may also result and be determined by comparison and measurement of several RNAs and display the relative abundance of these transcripts to each other .

A "reference pattern of expression levels", within the meaning of the invention shall be understood as being any pattern of expression levels that can be used for the comparison to another pattern of expression levels. In a preferred embodiment of the invention, a reference pattern of expression levels is, e.g., an expression level of at least one reference gene, e.g. a housekeeping gene or a mixture of housekeeping genes. In a preferred embodiment of the invention, a reference pattern of expression levels is, e.g., an average pattern of expression levels observed in a group of healthy or diseased individuals, serving as a reference group. However, yet it is another part of the invention, that said genes can be used without comparison to a reference pattern or after normalization to a reference gene or multiple reference genes.

The terms "biological sample" or "clinical sample", as used herein, refer to a sample obtained from a patient. The sample may be of any biological tissue or fluid. Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), tissue, core or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, urine, peritoneal fluid, and pleural fluid, liquor cerebrospinalis, tear fluid, or cells there from. Biological samples may also include sections of tissues such as frozen or fixed sections taken for histological purposes or microdissected cells or extracellular parts thereof. A biological sample to be analyzed is tissue material from a neoplastic lesion taken by aspiration or punctuation, excision or by any other surgical method leading to biopsy or resected cellular material. Such a biological sample may comprise cells obtained from a patient. The cells may be found in a cell "smear" collected, for example, by a nipple aspiration, ductal lavarge, fine needle biopsy or from provoked or spontaneous nipple discharge. In another embodiment, the sample is a body fluid. Such fluids include, for example, blood fluids, serum, plasma, lymph, ascitic fluids, gynecologic fluids, or urine but not limited to these fluids .

By "array" is meant an arrangement of addressable locations or "addresses" on a device. The locations can be arranged in two dimensional arrays, three dimensional arrays, or other matrix formats. The number of locations can range from several to at least hundreds of thousands. Most importantly, each location represents an independent reaction site. Arrays include but are not limited to nucleic acid arrays, protein arrays and antibody arrays. A "nucleic acid array" refers to an array containing nucleic acid probes, such as oligonucleotides, polynucleotides or larger portions of genes. The nucleic acid on the array is preferably single stranded. Arrays wherein the probes are oligonucleotides are referred to as "oligonucleotide arrays" or "oligonucleotide chips." A "microarray, " herein also refers to a "biochip" or "biological chip", an array of regions having a density of discrete regions of at least about 100/cm², and preferably at least about 1000/cm². The regions in a microarray have typical dimensions, e.g., diameters, in the range of between about 10-250 μm, and are separated from other regions in the array by about the same distance.

The term "oligonucleotide" refers to a relatively short polynucleotide, including, without limitation, single- stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNAiDNA hybrids and double-stranded DNAs. Oligonucleotides are preferably single-stranded DNA probe oligonucleotides. Moreover, in context of applicable detection methodologies, the term "oligonucleotide" also refers to nucleotide analogues such as PNAs and morpholinos.

The terms "modulated" or "modulation" or "regulated" or

"regulation" and "differentially regulated" as used herein refer to both upregulation, i.e., activation or stimulation, e.g., by agonizing or potentiating, and down regulation, i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting.

"Primer pairs" and "probes", within the meaning of the invention, shall have the ordinary meaning of this term which is well known to the person skilled in the art of molecular biology. In a preferred embodiment of the invention "primer pairs" and "probes", shall be understood as being polynucleotide molecules having a sequence identical, complementary, homologous, or homologous to the complement of regions of a target polynucleotide which is to be detected or quantified. In yet another embodiment, nucleotide analogues are also comprised for usage as primers and/or probes. Probe technologies used for kinetic or real time PCR applications could be e.g. TaqMan® systems obtainable at Roche Molecular Diagnostics, extension probes such as Scorpion® Primers, Dual Hybridisation Probes, Amplifluor® obtainable at Chemicon International, Inc, or Minor Groove Binders.

"Individually labeled probes", within the meaning of the invention, shall be understood as being molecular probes comprising a polynucleotide, oligonucleotide or nucleotide analogue and a label, helpful in the detection or quantification of the probe. Preferred labels are fluorescent molecules, luminescent molecules, radioactive molecules, enzymatic molecules and/or quenching molecules.

"Arrayed probes", within the meaning of the invention, shall be understood as being a collection of immobilized probes, preferably in an orderly arrangement. In a preferred embodiment of the invention, the individual "arrayed probes" can be identified by their respective position on the solid support, e.g., on a "chip".

The phrase "response", "therapeutic success", or "response to therapy" refers in the neoadjuvant, adjuvant and palliative chemotherapeutic setting to the observation of a defined tumor free or recurrence free or progression free or overall survival time (e.g. 2 years, 4 years, 5 years, 10 years) . This time period of disease free -, recurrence free - or progression free survival may vary among the different tumor entities but is sufficiently longer than the average time period in which most of the recurrences appear. In a neoadjuvant and palliative therapy modality, response may additionally be monitored by measurement of tumor shrinkage and regression due to apoptosis and necrosis of the tumor mass or reduced blood supply due to altered angiogenic events .

The term "recurrence" or " recurrent disease" includes distant metastasis that can appear even many years after the initial diagnosis and therapy of a tumor, or local events such as infiltration of tumor cells into regional lymph nodes, or occurrence of tumor cells at the same site and organ of origin within an appropriate time.

"Prediction of recurrence" or "prediction of therapeutic success" does refer to the methods described in this invention, wherein a tumor specimen is analyzed for e.g. its gene expression, genomic status and/or histopathological parameters (such as TNM and Grade) and/or imaging data and furthermore classified based on correlation of the expression pattern to known ones from reference samples. This classification may either result in the statement that such given tumor will develop recurrence and therefore is considered as a "non responding" tumor to the given therapy, or may result in a classification as a tumor with a prolonged disease free post therapy time.

"Biological activity" or "bioactivity" or "activity" or "biological function", which are used interchangeably, herein mean an effector or antigenic function that is directly or indirectly exerted by a polypeptide (whether in its native or denatured conformation) , or by any fragment thereof in vivo or in vitro. Biological activities include but are not limited to binding to polypeptides, binding to other proteins or molecules, enzymatic activity, signal transduction, activity as a DNA binding protein, as a transcription regulator, ability to bind damaged DNA, etc. A bioactivity can be modulated by directly affecting the subject polypeptide. Alternatively, a bioactivity can be altered by modulating the level of the polypeptide, such as by modulating expression of the corresponding gene.

The term "marker" or "biomarker" refers to a biological molecule, e.g., a nucleic acid, peptide, protein, hormone, etc., whose presence or concentration can be detected and correlated with a known condition, such as a disease state or a combination of these, e.g. by a mathematical algorithm.

The term "marker gene" as used herein, refers to a differentially expressed gene whose expression pattern may be utilized as part of a predictive, prognostic or diagnostic process in malignant neoplasia or cancer evaluation, or which, alternatively, may be used in methods for identifying compounds useful for the treatment or prevention of malignant neoplasia and gynecological cancer in particular. A marker gene may also have the characteristics of a target gene.

"Target gene", as used herein, refers to a differentially expressed gene involved in cancer, e.g. gynecologic cancer, preferably breast cancer, in a manner in which modulation of the level of the target gene expression or of the target gene product activity may act to ameliorate symptoms of malignant neoplasia. A target gene may also have the characteristics of a marker gene.

The term "receptor", as used herein, relates to a protein on the cell membrane or within the cytoplasm or cell nucleus that binds to a specific molecule (a ligand) , such as a neurotransmitter, hormone, or other substance, especially a hormone as estrogen, and initiates the cellular response. Ligand-induced changes in the behavior of receptor proteins result in physiological changes that constitute the biological actions of the ligands.

The term "signalling pathway" is related to any intra- or intercellular process by which cells converts one kind of signal or stimulus into another, most often involving ordered sequences of biochemical reactions out- and inside the cell, that are carried out by enzymes and linked through hormones and growth factors (intercellular) , as well as second messengers (intracellular) , the latter resulting in what is thought of as a "second messenger pathway". In many signalling pathways, the number of proteins and other molecules participating in these events increases as the process emanates from the initial stimulus, resulting in a "signal cascade" and often results in a relatively small stimulus eliciting a large response. The term "small molecule", as used herein, is meant to refer to a compound which has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention to identify compounds that modulate a bioactivity.

When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.

As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids.

The term "hybridization based method", as used herein, refers to methods imparting a process of combining complementary, single-stranded nucleic acids or nucleotide analogues into a single double stranded molecule. Nucleotides or nucleotide analogues will bind to their complement under normal conditions, so two perfectly complementary strands will bind to each other readily. In bioanalytics, very often labeled, single stranded probes are in order to find complementary target sequences. If such sequences exist in the sample, the probes will hybridize to said sequences which can then be detected due to the label. Other hybridization based methods comprise microarray and/or biochip methods. Therein, probes are immobilized on a solid phase, which is then exposed to a sample. If complementary nucleic acids exist in the sample, these will hybridize to the probes and can thus be detected. These approaches are also known as "array based methods". Yet another hybridization based method is PCR, which is described below. When it comes to the determination of expression levels, hybridization based methods may for example be used to determine the amount of mRNA for a given gene.

The term "a PCR based method" as used herein refers to methods comprising a polymerase chain reaction (PCR) . This is an approach for exponentially amplifying nucleic acids, like DNA or RNA, via enzymatic replication, without using a living organism. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations. When it comes to the determination of expression levels, a PCR based method may for example be used to detect the presence of a given mRNA by (1) reverse transcription of the complete mRNA pool (the so called transcriptome) into cDNA with help of a reverse transcriptase enzyme, and (2) detecting the presence of a given cDNA with help of respective primers. This approach is commonly known as reverse transcriptase PCR (rtPCR) . The term "PCR based method" comprises both end-point PCR applications as well as kinetic/real time PCR techniques applying special fluorophors or intercalating dyes which emit fluorescent signals as a function of amplified target and allow monitoring and quantification of the target. Quantification methods could be either absolute by external standard curves or relative to a comparative internal standard.

The term "method based on the electrochemical detection of molecules" relates to methods which make use of an electrode system to which molecules, particularly biomolecules like proteins, nucleic acids, antigens, antibodies and the like, bind under creation of a detectable signal. Such methods are for example disclosed in WO0242759, WO0241992 and WO02097413 filed by the applicant of the present invention, the content of which is incorporated by reference herein. These detectors comprise a substrate with a planar surface which is formed, for example, by the crystallographic surface of a silicon chip, and electrical detectors which may adopt, for example, the shape of interdigital electrodes or a two dimensional electrode array. These electrodes carry probe molecules, e.g. nucleic acid probes, capable of binding specifically to target molecules, e.g. target nucleic acid molecules. The probe molecules are for example immobilized by a Thiol-Gold- binding. For this purpose, the probe is modified at its 5'- or 3 ' -end with a thiol group which binds to the electrode comprising a gold surface. These target nucleic acid molecules may carry, for example, an enzyme label, like horseradish peroxidase (HRP) or alkaline phosphatase. After the target molecules have bound to the probes, a substrate is then added (e.g., α-naphthyl phosphate or 3, 3' 5,5'- tetramethylbenzidine which is converted by said enzyme, particularly in a redox-reaction . The product of said reaction, or a current generated in said reaction due to an exchange of electrons, can then be detected with help of the electrical detector in a site specific manner.

The term "nucleic acid molecule" is intended to indicate any single- or double stranded nucleic acid and/or analogous molecules comprising DNA, cDNA and/or genomic DNA, RNA, preferably mRNA, peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or Morpholino.

The term "stringent conditions" relates to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium) . Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60° C. for longer probes. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide and the like.

The term "fragment of the nucleic acid molecule" is intended to indicate a nucleic acid comprising a subset of a nucleic acid molecule according to one of the claimed sequences. The same is applicable to the term "fraction of the nucleic acid molecule" .

The term "variant of the nucleic acid molecule" refers herein to a nucleic acid molecule which is substantially similar in structure and biological activity to a nucleic acid molecule according to one of the claimed sequences.

The term "homologue of the nucleic acid molecule" refers to a nucleic acid molecule the sequence of which has one or more nucleotides added, deleted, substituted or otherwise chemically modified in comparison to a nucleic acid molecule according to one of the claimed sequences, provided always that the homologue retains substantially the same binding properties as the latter.

The term "derivative" as used herein, refers to a nucleic acid molecule that has similar binding characteristics to a target nucleic acid sequence as a nucleic acid molecule according to one of the claimed sequences

The term "hybridizing counterparts" as used herein, refers to a nucleic acid molecule that is capable of hybridizing to a nucleic acid molecules under stringent conditions.

The term "anamnesis" relates to patient data gained by a physician or other healthcare professional by asking specific questions, either of the patient or of other people who know the person and can give suitable information (in this case, it is sometimes called heteroanamnesis) , with the aim of obtaining information useful in formulating a diagnosis and providing medical care to the patient. This kind of information is called the symptoms, in contrast with clinical signs, which are ascertained by direct examination.

The term "etiopathology" relates to the course of a disease, that is its duration, its clinical symptoms, and its outcome.

Summary of the invention:

The invention is predicated on the surprising finding that patients with ESRl negative, MLPH negative tumors, which in addition also expressed basal like markers such as MMP7 and/or Keratin 5 and/or immune genes have a reduced benefit and/or increased risk of severe side effects under a cytotoxic therapy, e.g. taxane therapy and experience an increased risk of death under a cytotoxic therapy combined with administration of erythropoetin or a functional derivative thereof. Particularly the addition of a erythropoietin derivative caused increased death rates in cancer patients treated with standard regimen.

The invention relates to the method and its embodiments provided in the following numbered paragraphs:

1. A method for predicting an adverse response of a patient suffering from or at risk of developing a neoplastic disease and undergoing at least one given mode of cytotoxic treatment to the administration of erythropoietin or a functional derivative thereof, said method comprising the steps of:

a) obtaining a biological sample from said patient; b) determining, the expression level of at least one gene of interest, said gene being correlated with the expression level status of one of MMP7, MMPl, IGHM, MLPH, ESRl, PGR, RBl, BRCAl, Her-2/neu, EpoR, KRT5, and SPPl in the said sample, c) comparing the expression level determined in (b) with an expression level of at least one reference gene; and d) predicting an adverse response to the administration of erythropoietin or a functional derivative thereof in said patient from the outcome of the comparison in step (C) .

In a preferred embodiment, the genes of interest comprise MMP7. This marker has been shown to give a good prediction of the adverse response to EPO. Further, marker combinations comprising this marker are preferred embodiments of the invention.

In a preferred embodiment, the genes of interest comprise MMP7 and IGHM. This marker combination has been shown to give a good prediction of the adverse response to EPO. Further, marker combinations comprising this combination are preferred embodiments of the invention.

In a preferred embodiment, the genes of interest comprise IGHM. This marker has been shown to give a good prediction of the adverse response to EPO. Further, marker combinations comprising this marker are preferred embodiments of the invention .

In a preferred embodiment, the genes of interest comprise MMPl. This marker has been shown to give a good prediction of the adverse response to EPO. Further, marker combinations comprising this marker are preferred embodiments of the invention. 2. Method of numbered paragraph 1, wherein in step (c) a value of a difference between an expression level value of the at least one gene of interest and the expression level value of the at least one reference gene is determined and a threshold value is defined for said difference, wherein a value of the difference above said threshold value is indicative of a first therapeutic success and a value of the difference below said threshold value is indicative of a second therapeutic success.

3. Method of numbered paragraph 1 or 2, wherein the gene of interest is a gene selected from the group consisting of MMP7, MMPl, MLPH, IGHM, EPO-R alpha, ESRl, PGR, RBl, BRCAl, EpoR, KRT5, Her-2/neu, and SSPl.

4. Method of any one of the preceding numbered paragraphs wherein the reference gene is a housekeeping gene, in particular RPL37a.

5. Method of any one of the preceding numbered paragraphs, wherein the cancer is breast cancer.

6. Method of any one of the preceding numbered paragraphs, wherein the cytotoxic therapy comprises the administration of anthracycline, cyclophosphamide, methotrexate, fluoruracil, Docetaxel (Taxotere®) or Paclitaxel (Taxol®) .

7. Method of any one of the preceding numbered paragraphs, wherein the adverse response to Epo correlated with a negative Estrogen receptor status in the patient undergoing cytotoxic therapy.

8. The method according to any one of the aforementioned numbered paragraphs, wherein the expression level is determined by a) a hybridization based method; b) a PCR based method; c) determining the protein level, d) a method based on the electrochemical detection of particular molecules, and/or by e) an array based method.

9. The method according to any one of the aforementioned numbered paragraphs, characterized in that the expression level of at least one of the said genes is determined with rtPCR (reverse transcriptase polymerase chain reaction) of the gene-related mRNA.

10. The method according to any one of the aforementioned numbered paragraphs, characterized in that the expression level of at least one of the said genes or fragments thereof are detected on protein level.

11. The method according to any one of the aforementioned numbered paragraphs, characterized in that the expression level of at least one of the said ligands of is determined in formalin and/or paraffin fixed tissue samples .

12. The method according to any one of the aforementioned numbered paragraphs, wherein, after lysis, the samples are treated with silica-coated magnetic particles and a chaotropic salt, in order to purify the nucleic acids contained in said sample for further determination.

13. The method according to any one of the aforementioned numbered paragraphs, wherein the gene of interest is a gene correlated with the expression level of MMP7.

14. The method according to numbered paragraph 13, wherein said gene of interest is a gene selected from the group of genes listed in tables 2, 8, 9, and 10. 15. The method according to numbered paragraph 14, wherein said gene of interest is selected from genes from the group of genes listed in tables 8, 9, and 10 having a correlation coefficient with MMP7 of at least 0.25, preferably at least 0.3, preferably at least 0.5.

16. A kit useful for carrying out a method according to any one of the aforementioned numbered paragraphs, comprising at least a primer pair and/or a probe each having a sequence sufficiently complementary to at least one gene as set forth in numbered paragraph 1, above, or tables 1 to 10.

Basically, a deviating expression level of either of the aformentioned genes can have different reasons, these being

• gene amplification of an oncogene (e.g. as frequently seen in Her-2/neu positive tumors) • underexpression or overexpression of the respective gene due to an altered methylation pattern and/or mutations

• altered properties of a transcription factor, a promotor or another factor which leads to an upregulation of the expression level of the said agent .

In a preferred embodiment of the present invention, it is provided that the adverse response to Epo correlated with a negative hormone receptor status in the patient undergoing cytotoxic therapy. Preferrably said hormone receptor is Estrogen receptor 1.

In another preferred embodiment of the present invention, it is provided that the adverse response to Epo correlated with low expression of MLPH and/or PGR and high expression of MMP7 and/or Keratin 5 and/or immune genes in the patient undergoing cytotoxic therapy. In yet another preferred embodiment of the present invention it is provided that the expression level of at least one of the said gene/s is determined with RT-PCR (reverse transcriptase polymerase chain reaction) of the ligand and/or receptor related mRNA.

In yet another preferred embodiment of the present invention it is provided that the expression level of at least one of the said gene/s is determined with mass spectrometry of the ligand and/or receptor related mRNA.

In yet another preferred embodiment of the present invention it is provided that the expression level of at least one of the said gene/s is determined with in situ hybridization methods (FISH or CISH) of the ligand and/or receptor related mRNA.

In yet another preferred embodiment of the present invention it is provided that the expression level of at least one of the said gene/s is determined on protein level. Said methods comprise but are not limited to immunhistochemistry, ELISA formats or mass spectrometry.

In another preferred embodiment of the present invention, it is provided that the expression level of at least one of the said gene/s is determined in formalin and/or paraffin fixed tissue samples.

In yet another preferred embodiment of the present invention, it is provided that the expression level of at least one of the said gene/s is determined in serum, plasma or whole blood samples. In one embodiment, the expression levels relate to secreted proteins (e.g. MMP7 and or MMPl) and/or shedded proteins, derived from extracellular protease activitiy, such as from Matrix Metallo-Proteinase factors. In yet another embodiment, the expression levels relate to protein fragments of said factors (e.g. EpoR extracellular parts) .

In the following, the invention is described in more detail in conjunction with examples and Figures, which show:

Figs. 1 to 12 showing Kaplan-Meyer Plots comparing survival of patients tested for different markers according to the method of the invention in different cohorts, and

Figs. 13 and 14 showing ROC curves for showing the sensitivity and specificity of predictive testing on sample cohorts according to the method of the invention.

Routinely, in tumor diagnosis tissue samples are taken as biopsies form a patient and undergo diagnostic procedures. For this purpose, the samples are fixed in formaline and/or paraffine and are then examined with immunohistochemistry methods. The formaline treatment leads to the inactivation of enzymes, as for example the ubiquitous RNA-digesting enzymes (RNAses) . For this reason, the mRNA status of the tissue (the so called transcriptome) , remains unaffected.

However, the formaline treatment leads to partial depolymerization of the individual mRNA molecules. For this reason, the current doctrine is that formaline fixed tissue samples can not be used for the analysis of the transcriptome of said tissue.

For this reason, it is provided in a preferred embodiment of the present invention that after lysis, the samples are treated with silica-coated magnetic particles and a chaotropic salt, in order to purify the nucleic acids contained in said sample for further determination.

Collaborators of the inventors of the present invention have developed an approach which however allows successful purification of mRNA out of tissue samples fixed in such manner, and which is disclosed, among others, in WO03058649, WO2006136314A1 and DEl 0201084Al, the content of which is incorporated herein by reference.

Said method comprises the use of magnetic particles coated with silica (SiO₂) • The silica layer is closed and tight and is characterized by having an extremely small thickness on the scale of a few nanometers. These particles are produced by an improved method that leads to a product having a closed silica layer and thus entail a highly improved purity. The said method prevents an uncontrolled formation of aggregates and clusters of silicates on the magnetite surface whereby positively influencing the additional cited properties and biological applications. The said magnetic particles exhibit an optimized magnetization and suspension behaviour as well as a very advantageous run-off behaviour from plastic surfaces. These highly pure magnetic particles coated with silicon dioxide are used for isolating nucleic acids, including DNA and RNA, from cell and tissue samples, the separating out from a sample matrix ensuing by means of magnetic fields. These particles are particularly well-suited for the automatic purification of nucleic acids, mostly from biological body samples for the purpose of detecting them with different amplification methods.

The selective binding of these nucleic acids to the surface of said particles is due to the affinity of negatively charged nucleic acids to silica containing media in the presence of chaotropic salts like guanidinisothiocyanate . Said binding properties are known as the so called "boom principle". They are described in the European patent EP819696, the content of which is incorporated herein by reference .

The said approach is particularly useful for the purification of mRNA out of formaline and/or paraffine fixed tissue samples. In contrast to most other approaches, which leave very small fragments behind that are not suitable for later determination by PCR and/or hybridization technologies, the said approach creates mRNA fragments which are large enough to allow specific primer hybridization and/or specific probe hybridization. A minimal size of at least 100 bp, more preferably 200 base pairs is needed for specific and robust detection of target gene expression. Moreover it is also necessary to not have too many inter-sample variations with regard to the size of the RNA fragments to guarantee comparability of gene expression results. Other issues of perturbance of expression data by sample preparation problems relate to the contamination level with DNA, which is lower compared to other bead based technologies. This of particular importance, as the inventors have observed, that DNAse treatment is not efficient in approximately 10% of FFPE samples generated by standard procedures and stored at room temperature for some years before cutting and RNA extraction.

The said approach thus allows a highly specific determination of candidate gene expression levels with one of the above introduced methods, particularly with hybridization based methods, PCR based methods and/or array based methods, even in formaline and/or paraffine fixed tissue samples, and is thus extremely beneficial in the context of the present invention, as it allows the use of tissue samples fixid with formaline and/or paraffine, which are available in tissue banks and connected to clinical databases of sufficient follow-up to allow retrospective analysis.

Candidate genes, enabling subclassification of defined biological motifs (ESRl, PGR, MLPH, MMP7, KRT5, MMPl, Her- 2/neu, IGHM) together with the receptor of Erythropoietin (Gene Symbol: EpoR; OMIM: 133171; RefSeq: NM_000121) have been analyzed RNA level by kRT-PCR technologies in core needle biopsy specimen of breast tumors, which had been formalin fixed (,,FFPE tissue") or were available as fresh tissues. Formalin fixed tissues were available from breast cancer patients (cT2, N0/N1, MO) receiving neoadjuvant chemotherapy of 4 cycles of epirubicin and cyclophosphamide (90/600 mg/m2) followed by 4 cycles paclitaxel (175 mg/m2) . In total, FFPE tissue and respective clinical information was available for 450 paraffin embedded core needle biopsies. Univariate and multivariate analysis of the selected candidate genes (in particular MMP7) did predict reduced benefit from taxane containing chemotherapy in ESRl negative and/or MLPH and/or Her-2/neu negative tumors, which in addition also expressed basal like markers such as MMP7 and Keratin 5 and/or immune genes. Univariate and combinatorial analysis of ESRl, PGR, MLPH,

Her-2/neu, MMP7, Keratin 5 and IGHM status on RNA level were possible and meaningful in FFPE tissues in from core needle biopsies despite highly variable tumor contents. It could be shown that hormone receptor negative tumors develop increased rates of fatal diseases upon treatment with erythropoietin therapies (57% vs. 14%), while hormone receptor positive tumors did not exhibit elevated levels of deaths (11% vs 10%) . By using RNA expression analysis of hormone receptor genes and /or basal like genes, patients can be identified which can be offered benefit from erythropoietin treatment while not being harmed with increased death rates.

It is known, that the expression levels of certain genes correlate. Therefore, in order to determine the expression status of ESRl, MLPH, MMP7, KRT5, MMPl, Her-2/neu, and IGHM according to the invention, the expression of the genes listed in 1 to 10 may be determined to obtain the expression status of the respective gene.

The following genes were identified to discriminate ESRl positive tumors (IHC status 4) from ESRl negative tumors (IHC status 0) by having high expression levels, high variance and fold change levels as identified in fresh tumor tissue.

Table 1: genes that can be used to discriminate ESRl positive tumors from ESRl negative tumors, i.e. genes being correlated with the expression level status of ESRl.

The applicants have analysed these genes and were able to assign the said genes to given biological motifs which are correlated with, and are thus predictive for, subgroups of Estrogen receptor (ESR) negative tumors.

By way of illustration and not by limitation these motifs may be selected from the group comprising at least

• extracellular matrix degradation, • growth factor signallling,

• immune cell infiltration.

• basal markers .

Extracellular Matrix degradation is frequently caused by Matrix Metalloproteinases . For this reason, most preferred genes are part of the Matrix Metalloproteinase gene family, and the Keratin gene family, which both tend to exhibit bimodal distribution of expression values. Such genes are, for example

• Matrix Metallo Proteinases (MMP), particularly MMPl, MMP3, MMP7, MMP9, MMPIl and MMP12, most preferred MMP7

Table 2 : Preferred genes related genes related to extracellular matrix degradation, i.e. genes being correlated with the expression level status of one MMP7, and/or MMPl.

Genes related to growth factor signaling may for example encode for growth factor receptors, growth factor ligands, inhibitors and the like.

Such genes comprise, for example, genes encoding a receptor from the ErbB-family, or a gene correlated with the Progesteron receptor (PGR) status in the said sample.

Table 3: Preferred genes related related to growth factor signaling, i.e. genes being correlated with the expression level status of Her-2/neu.

Genes related to immune cell infiltration may be selected from the following table (listing is not exclusive) :

Table 4: Preferred genes related to immune cell infiltration, i.e. genes being correlated with the expression level status of IGHM.

Genes related to basal markers (the term "basal markers" is derived from the appearance of the respective cells, which is similar to basal cells) may be selected from the the following table (listing is not exclusive) :

Table 5: Preferred genes related to basal markers, i.e. genes being correlated with the expression level status of one of

ESRl, PGR, MLPH, MMP7, KRT5 , MMPl, Her-2/neu, and IGHM.

Out of these, most preferred genes are the following, as listed in table 6:

Table 6: Preferred genes in the context of the present invention .

The expression level of the gene of interest is compared to the expression level of at least one reference gene. It is preferred to use housekeeping genes as internal controls against which the expression level of a gene of interest is compared. Housekeeping genes are control genes, which are selected because of their stable and constant expression in a wide variety of tissues or cells.

Gene RefSe or ID

Table 7 Preferred reference genes in the context of the present invention

It is also possible to determine the expression of combinations of reference genes, either by making multiple determinations and calculating an average value of different reference genes or by making a single determination using probes for different reference genes which yield a combined signal .

The cytotoxic therapy, in particular the chemotherapy may comprise the administration of at least one agent selected from the group consisting of Cyclophosphamid (Endoxan®, Cyclostin®) . Adriamycin (Doxorubicin) (Adriblastin®) , BCNU (Carmustin) (Carmubris®) , Busulfan (Myleran®) , Bleomycin (Bleomycin®) , Carboplatin (Carboplat®) , Chlorambucil (Leukeran®) , Cis-Platin (Cisplatin®) , Platinex (Platiblastin®) , Dacarbazin (DTIC®; Detimedac®) , Docetaxel (Taxotere®) , Epirubicin (Farmorubicin®) , Etoposid (Vepesid®) , 5-Fluorouracil (Fluroblastin®, Fluorouracil®) , Gemcitabin

(Gemzar®) , Ifosfamid (Holoxan®) , Interferon alpha (Roferon®) ,

Irinotecan (CPT 11, Campto®) , Melphalan

(Alkeran®) , Methotrexat (Methotrexat®, Farmitrexat®) ,

Mitomycin C (Mitomycin®) , Mitoxantron (Novantron®) , Oxaliplatin (Eloxatine®) , Paclitaxel (Taxol®) , Prednimustin

(Sterecyt®) , Procarbazin (Natulan®) , Pemetrexed (Alimta®) ,

Ralitrexed (Tomudex®) , Topotecan (Hycantin®) , Trofosfamid

(Ixoten®), Vinblastin (Velbe®) , Vincristin (Vincristin®) ,

Vindesin (Eldisine®) and/or Vinorelbin (Navelbine®) .

In particular the cytotoxic therapy may comprise the administration of Docetaxel (Taxotere®) or Paclitaxel (Taxol®) .

Of the preferred genes used as markers in the context of the present invention listed in Table 6, particularly the basal- like gene MMP7, which is a marker for stem cell like features and indicating WNT signalling activity indicated a subpopulation of the so called "triple negative" breast tumors, which show reduced expression of ESRl, PGR and Her- 2/neu. The negative Spearman correlation of MMP7 with ESRl and MLPH was remarkably high in the cohort tested (r=0.517 p<0.0001; r=-0.435 p<0.0001) and higher than for other basal like markers such as KRT5. The predictive value of MMP7 was particular evident in breast cancer patients treated with the standard chemotherapy regimen (Arm A of the PREPARE trial: treatment with EC-T, i.e. epirubicin/cyclophosphamide-taxol) . Apparently, the dose intensified regimen in the experimental arm of the PREPARE trial (arm B; E-T-CMF, i.e. epirubicin- taxol- cyclophsphamide/methotrexat/fluorouracil) has compensated for the negative effects of Darbepoetin- addition to these higher risk patients. This also indicates the particular responsiveness of basal like tumors (defined by the genes disclosed in this invention) towards specific chemotherapy combinations and dosages. MMP7 was particularly effective describing this group of patients put to a particularly high risk by addition of Darbopoetin even when employing different MMP7 RNA cut-off levels. Differences to housekeeping genes such as RPL37A or GAPDH (Delta CT values of RT-kPCR) could vary between upper and lower quartile to reliably detect the ADR patient population with 99% sensitivity and approximately 50% specificity. Moreover, as ESRl and MLPH are strongly negatively regulated towards MMP7, it was also possible to predict the Darbopoetin ADR in the standard chemotherapy arm (i.e. Arm A of the PREPARE trial) by using two gene ratios referring or not referring to housekeeping gene expressions.

The method disclosed by this invention enables evaluation of the patient prior to the initial treatment to evaluate the type of regimen (standard, e.g. Arm A of the PREPARE trial, or more-harsh dose intensified regimen, e.g. Arm A of the PREPARE trial) and the best way to cope with anaemia that occur with high frequency due to chemotherapy regimen. This is important as the blood banks often cannot offer matched whole blood samples to patients at the time the anemia starts to develop.

However, MMP7, which was originally identified as non- chemoresponse marker in colorectal cancer, is a general marker for invasiveness and WNT-signalling activities. As WNT signalling activities are important also in other cancers and Adverse effects of Erythropoietin derivatives have been observed among others also in head and neck, lymphoid and lung cancer, MMP7 can also be used as a marker for ADRs in these malignancies. Moreover MMP7 positive tumors might also be defined by co-overexpression of other MMP or TIMP family members, components of the WNT signalling pathway (including secreted factors like SFRPl or intracellular factors such as beta Catenin and APC) , genomic and or epigenetic alterations of the p53, BRCAl, BRCA2 and RBl genes such as point mutations, deletions, methylation events. Genome wide analysis in 1055 breast cancer samples was performed by Affymetrix analysis to identify genes co-regulated with MMP7 (Pearson correlation factor of r>0.2) . This identifies additional genes also predictive to indicating tumors responsive to Erythropoietin and derivatives. Alterations of DNA copy number, DNA methylation, different mRNA expressions, different protein expressions of these genes, either a single marker, in combination with each other or in combination with MMP7 can identify patients who are at risk of having an ADR due to treatment with Erythropoietin and derivatives. These gene lists include EGFR and interconnected downstream signalling factors (such as RASSF4), other Keratins (KRT16, KRT17, KRT5, KRT6) , NFkB and Notch Pathway members (TONDU), embryonic segmentation genes such as WNT and engrailed signalling pathway members and interconnected adhesion molecules (e.g. Cadherin3, desmoglein3) , immune genes and chemokines (e.g. IGHD and CXCLl and CXCL2), kallikreins (KLK5, KLK7), extracellular matrix genes (Laminins) , cell cycle genes (GASl) . Instead of measuring the gene expression level of MMP7 itself, it is also possible to measure the expression level of at least one gene having an expression level that is correlated with the expression level of MMP7. The tables 3, 9, 10, and 11 show genes whose expression is correlated with MMP7.

The genes listed in tables 3, 9, 10, and 11 are correlated with the expression level status of MMP7 within the meaning of the present invention. These genes can be used in the method of the invention. Preferrably genes with a correlation coefficient of >0.2, more preferably >0.25, >0.3, >0.35, >0.4, >0.45, >0.5 are used as markers for determing the expression level. The genes were identified by pearson correlation of array based fresh tissue data. For this purpose the Affymetrix (Santa Clara, CA) HG-U133A array and GeneChip System™ was used to quantify the relative transcript abundance in the breast cancer tissues. Starting from 5 μg total RNA, labeled cRNA was prepared using the Roche Microarray cDNA Synthesis, Microarray RNA Target Synthesis (T7) and Microarray Target Purification Kit, according to the manufacturer's instructions. In the analysis settings, the global scaling procedure was chosen which multiplied the output signal intensities of each array to a mean target intensity of 500. Samples with suboptimal average signal intensities (i.e., scaling factors >25) or GAPDH 3 '/5' ratios >5 were relabeled and rehybridized on new arrays. Correlations between MMP7 and the expression data of all other genes was done by using the Genedata Refiner and Genedata Expressionist Analyst software package version Pro 4.0.4.

All transcripts were evaluated whether they are reliably detectable (present call) or not (marginal or absent call) according to Affymetrix manufacturers instructions and known to those skilled in the art. In brief, the Detection algorithm uses probe pair intensities to generate a Detection p-value and assign a Present, Marginal, or Absent call. Each probe pair in a probe set is considered as having a potential vote in determining whether the measured transcript is detected (Present) or not detected (Absent) . The vote is described by a value called the Discrimination score [R] . The score is calculated for each probe pair and is compared to a predefined threshold Tau. Probe pairs with scores higher than Tau vote for the presence of the transcript. Probe pairs with scores lower than Tau vote for the absence of the transcript. The voting result is summarized as a p-value. The greater the number of discrimination scores calculated for a given probe set that are above Tau, the smaller the p-value and the more likely the given transcript is truly Present in the sample. The p-value associated with this test reflects the confidence of the Detection call.

The Discrimination score is a basic property of a probe pair that describes its ability to detect its intended target. It measures the target-specific intensity difference of the probe pair (PM-MM) relative to its overall hybridization intensity (PM+MM) : R = (PM - MM) / (PM + MM) . Further details are described in "GeneChip® Expression Analysis - Data Analysis Fundamentals" available at Affymetrix.

As genes being relevant for said method are particularly present in a specific subtype of breast cancer, genes were grouped according to there frequency of present calls. Importantly, in contrast to standard approaches of data analysis, that filter genes of high prevalence, i.e. with higher percentage of present calls (>40-50%) in a given tumor sample cohort, genes were selected that are only present in <10% of the tumors, 10-25% of the tumors or 25-50% of the tumors (see tables 9 to 11 below) . A substantial number of these genes are not taken into account in standard gene expression analysis. However, the underlying idea was to identify genes that are rarely expressed and particularly linked to the expression of MMP7, thereby further defining the tumor subtype, which has been found to be affected by application of erythropoietin and derivatives thereof as part of this invention.

Table 8: MMP7 Coregulated Genes 0-10%P-Call - Korrelation Value

Table 9: MMP7 Coregulated Genes 10-25% P-CaIl - Correlation Value

Table 10 MMP7 Coregulated Genes 25-50% P-CaIl - Correlation Value

Gene Symbol OMIM RefSeq Transcript UniGene Corr. Coeff.

MMP7 178990 NM_002423 Hs.2256 1

GABRP 602729 NM_014211 Hs.26225 0,73891044

KRT5 148040 NM_000424 Hs.433845 0,64962757

MIA 601340 NM_006533 Hs.279651 0,62844288

KRT17 148069 NM_000422 Hs.2785 0,61626387

SERPINB5 154790 NM_002639 Hs.55279 0,60425866

CDH3 1 14021 NM_001793 Hs.191842 0,59489262

DSC3 600271 NMJ⁾01941 , NM_024423 Hs.41690 0,5858714

RARRES 1 605090 NMJ)02888, NM_206963 Hs.131269 0,58481002

BBOX1 603312 NM_003986 Hs.144845 0,57903713

BCL11A 606557 NM 018014, NM 022893, NM_138553, Hs.370549 0,57575095

NM 138559

KRT6B 148042 NM_005555 Hs.524438 0,5738591

GPM6B 300051 NM 001001994, NM 001001995, Hs.495710 0,55337

NM 001001996, NM 005278

PRKX, PRKY 300083 NM_002760, NM_005044 Hs.390788 0,51488674

LAMB3 150310 NMJ)00228 Hs.497636 0,51479542

C20orf42 607900 NM_017671 Hs.472054 0,50839508

PTX3 602492 NM_002852 Hs.546280 0,50705993

IL27RA 605350 NMJ)04843 Hs.132781 0,5007171

FABP7 602965 NM_001446 Hs.26770 0,48973542

VSNL1 600817 NM_003385 Hs.444212 0,47818118

PSAT1 NM_021154, NM_058179 Hs.494261 0,4750104

QKI NM 006775, NM 206853, NMJ^06854, Hs.510324 0,46944296

NM 206855

KLK5 605643 N MJ) 12427 Hs.50915 0,4679442

EGFR 131550 NM 005228, NM 201282, NM_201283, Hs.488293 0,46774578

NM 201284

FOLR1 136430 NM 000802, NM 016724, NM 016725, Hs.73769 0,4674747

NM 016729, NM 016730, N MJ) 16731

C10orf10 NM_007021 Hs.93675 0,45996296

NFIB 600728 NM_005596 Hs.370359 0,45937616

EN 1 131290 N MJ)01426 Hs.271977 0,45779347

PTPLA N MJ) 14241 Hs.114062 0,44725442

PADI2 607935 NM_007365 Hs.33455 0,44604826

SLC6A14 300444 NMJJ07231 Hs.522109 0,44299024

LAMC2 150292 NMJJ05562, NMJ)18891 Hs.530509 0,43491286

CXCL2 139110 NM_002089 Hs.75765 0,43128723

CXCL1 155730 NMJ)01511 Hs.789 0,4281131

ETV6 600618 N MJ)01987 Hs.504765 0,42688453

ST8SIA1 601 123 NMJJ03034 Hs.408614 0,41741198

DST NM 001723, NM 015548, NM_020388, Hs.485616 0,41263211

NM 183380

MFHAS1 605352 NM_004225 Hs.379414 0,41243863

HLA-DOB 600629 NMJJ02120 Hs.1802 0,40922064

C13orf18 NM_025113 Hs.981 17 0,40284616

KCNK5 603493 NMJJ03740 Hs.444448 0,39959067

CAPN 6 N MJ) 14289 Hs.496593 0,39349055

FAM49A NM 030797 Hs.467769 0,39018506 LIPG 603684 NM _006033 Hs.465102 0,38723004

CRABP 1 180230 NM _004378 Hs.346950 0,38344872

RASSF4 NM _032023, NM J78145 Hs.522895 0,37979156

GAL 137035 NM _015973 Hs.278959 0,37466979

LOC388078 XM _370835 Hs.158949 0,37465245

LCN7 NM _022164 Hs.199368 0,37388504

SAA1 104751 NM _000331 , NM_. J99161 Hs.332053 0,37182701

IL32 606001 NM _004221 Hs.943 0,37109697

FLJ13154 NM _024598 Hs.408702 0,36899638

SPIB 606802 NM _003121 Hs.437905 0,36710572

IFRD1 603502 NM _001007245, NM_001550 Hs.7879 0,36375213

IGHM 147170 Hs.525648 0,36365873

OLFM4 NM _006418 Hs.508113 0,36331367

INDO 147435 NM _002164 Hs.840 0,3608101

PTPRZ1 176891 NM _002851 Hs.489824 0,36046731

CYP39A1 605994 NM _016593 Hs.387367 0,35765034

IGHG1 , XM_372632 Hs.375600 0,35706353

LOC390714

SLC16A1 NM _003051 Hs.75231 0,35227686

IL15RA NM _002189, NM _172200 Hs.524117 0,35096925

IL4R 147781 NM _000418, NM_. _001008699 Hs.513457 0,35042447

PLD1 602382 NM _002662 Hs.478230 0,34841132

MTM R2 603557 NM _016156, NM_. _201278, NM_ 201281 Hs.181326 0,34671074

MME 120520 NM 000902, NM _007287, NM_ 007288, Hs.307734 0,34607881

NM 007289

LAD1 602314 NM _005558 Hs.519035 0,34419352

STRA6 NM _022369 Hs.24553 0,34303415

SLC2A3 138170 NM _006931 Hs.419240 0,3402546

IGM Hs.449011 0,33864814

KIAA1609 NM _020947 Hs.288274 0,33764797

IGLC2 Hs.458262 0,33492953

IgG Hs.449599 0,33346677

KIAA1212 NM _018084 Hs.292925 0,33221185

LCN2 600181 NM _005564 Hs.204238 0,33220989

GLS 138280 NM _014905 Hs.116448 0,32548213

MAGED4 NM _030801 , NM _177535, NM_ 177537 Hs.522650 0,32431769

PTPNS1 602461 NM _080792 Hs.128846 0,3234607

FUT4 104230 NM _002033 Hs.390420 0,31911993

ITGB4 147557 NM 000213, NM _001005619, Hs.370255 0,31797302

NM 001005731

C6orf106 NM _022758, NM_. _024294 Hs.485162 0,31730258

ABTB2 NM _145804 Hs.23361 0,31698745

SRD5A1 184753 NM _001047 Hs.552 0,31652635

CHST11 NM _018413 Hs.546386 0,31598234

BMP1 112264 NM 001199, NM 006128, NM 006129, Hs.1274 0,3158673

NM 006130, NM _006131 , NM_ 006132

CP 117700 NM _000096 Hs.282557 0,31554991

ANGPT1 601667 NM _001146, NM_. _139290 Hs.369675 0,31387395

B3GNT3 605863 NM _014256 Hs.69009 0,31333566

PML 102578 NM 002675, NM 033238, NM 033239, Hs.526464 0,31051713

NM 033240, NM 033242, NM 033244,

NM 033245, NM 033246, NM 033247,

NM 033249, NM 033250

GABRE 300093 NM 004961 , NM 021984, NM 021987, Hs.22785 0,31034768

NM 021990 FOLH1 600934 NM_._004476 Hs.380325 0,31022066

DNMT2 602478 NM 004412, NM 176081, NM 176083, Hs.351665 0,30963284

NM 176084, NM 176085, NM 176086

NFATC1 NM 006162, NM 172387, NM 172388, Hs.534074 0,30747533

NM 172389, NM_. _172390

PTGDS 176803 NM_{. ,}000954 Hs.446429 0,30675441

FLJ12270 NM_. ,030581 Hs.280951 0,3053351

MDFI 604971 NM_. _005586 Hs.520119 0,3052088

RRAGD 608268 NM_. _021244 Hs.485938 0,30451423

MGC8685 NM_. J78012 Hs.300701 0,30423272

ROR1 602336 NM_{. ,}005012 Hs.436456 0,30288291

CD40 109535 NM_. ,001250, NM_. _152854 Hs.472860 0,30285954

IgH Hs.406550, 0,30108827

Hs.525872

CCNE1 123837 NM_. ,001238, NM_. _057182 Hs.244723 0,30083674

PLD2 602384 NM_. ,002663 Hs.104519 0,30069852

KAB NM_{. ,}014812 Hs.533635 0,30042171

ICAM2 146630 NM_. ,000873 Hs.431460 0,30025226

CDC2L6 NM_. ,015076 Hs.193251 0,30023336

LMO4 603129 NM_. ,006769 Hs.436792 0,29955918

CD38 107270 NM_. ,001775 Hs.479214 0,29949397

CD79B 147245 NM_. ,000626, NM_. _021602 Hs.89575 0,29623967

FHOD3 NM_. ,025135 Hs.436636 0,29080468

LIMS2 607908 NM_. ,017980 Hs.469881 0,29068768

CD200 155970 NM 001004196, NM_001004197, Hs.79015 0,2893278

NM 005944

CD79A 112205 NM_. ,001783, NM_. _021601 Hs.79630 0,28932518

STK17B 604727 NM_. ,004226 Hs.88297 0,2890144

MET 164860 NM_. ,000245 Hs.132966 0,28888083

S100A1 176940 NM_{. ,}006271 Hs.515715 0,28875446

NMB 162340 NM_{. ,}021077, NM_. _205858 Hs.386470 0,28821248

OSBPL3 606732 NM 015550, NM 145320, NM 145321, Hs.520259 0,28503752

NM 145322, NM_. J45323, NM_145324

NOLC1 602394 NM_{. ,}004741 Hs.523238 0,28471988

RDX 179410 NM_. ,002906 Hs.263671 0,28452206

FOSL2 NM_. ,005253 Hs.220971 0,28416342

SOCS3 604176 NM_. ,003955 Hs.527973 0,28386813

GZMB 123910 NM_. ,004131 Hs.1051 0,28247213

SYNGR1 603925 NM_{. ,}004711, NM J45731, NM_145738 Hs.216226 0,28080964

MCM5 602696 NM_. ,006739 Hs.517582 0,2805407

SEPT11 NM_. ,018243 Hs.128199 0,27944833

BIN1 601248 NM 004305, NM 139343, NM 139344, Hs.193163 0,27880883

NM 139345, NM 139346, NM 139347,

NM 139348, NM 139349, NM 139350,

NM 139351

BOP1 NM_. ,015201 Hs.535901 0,27867991

EPHA2 176946 NM_. ,004431 Hs.171596 0,27854949

SAA4 104752 NM_{. ,}006512 Hs.512677 0,27764308

CIB2 605564 NM_. ,006383 Hs.129867 0,27763945

COL6A1 120220 NM_. ,001848 Hs.474053 0,27750492

FADS3 606150 NM_{. ,}021727 Hs.21765 0,27530009

MMP14 600754 NM_. ,004995 Hs.2399 0,27499908

CD6 186720 NM_. ,006725 Hs.502710 0,27494496

FLJ10665 NM_. ,018173 Hs.163953 0,27492416

MAP3K14 604655 NM 003954 Hs.404183 0,27455169 FADS1 606148 NM_. _013402 Hs.503546 0,2744469

IL17R 605461 NM_{. ,}014339 Hs.129751 0,273552

BACH1 602751 NM 001011545, NM_001186, Hs.154276 0,27256238

NM 206866

NFKBIE 604548 NM_{. ,}004556 Hs.458276 0,27145654

ST3GAL2 607188 NM_{. ,}006927 Hs.368611 0,27099949

TRA@, TRD@ Hs.74647 0,27065617

EPHB3 601839 NM_. 004443 Hs.2913 0,26838857

APEG1 NM_{. ,}005876 Hs.21639 0,26800662

GEMIN4 606969 NM_{. ,}015721 Hs.499620 0,26787484

RAB23 606144 NM_{. ,}016277, NM_. _183227 Hs.485635 0,26505768

CDC42EP3 606133 NM_{. ,}006449 Hs.369574 0,2642982

NPR3 108962 NM_{. ,}000908 Hs.237028 0,26258355

EM R2 606100 NM 013447, NM 152916, NM 152917, Hs.531619 0,26228088

NM 152918, NM_. J52919, NM_ 152920,

NM 152921

IL8 146930 NM_{. ,}000584 Hs.624 0,26149446

CALD 1 114213 NM 004342, NM 033138, NM 033139, Hs.490203 0,26087511

NM 033140, NM_. _033157

FLJ 12438 NM_{. ,}021933 Hs.8595 0,2603066

KHSRP 603445 NM_{. ,}003685 Hs.91142 0,26022726

SLAMF8 606620 NM_{. ,}020125 Hs.438683 0,26002532

PLEKHM2 XM_ _.290944 Hs.145049 0,25961262

CXorfθ 300120 NM_{. ,}005491 Hs.20136 0,25902784

TGFBR2 190182 NM_{. ,}003242 Hs.82028 0,25899434

ITGB6 147558 NM_{. ,}000888 Hs.470399 0,25835311

LCK 153390 NM_{. ,}005356 Hs.470627 0,25749677

C9orf40 NM_{. ,}017998 Hs.532296 0,25717193

PTPN7 176889 NM_{. ,}002832, NM_. _080588, NM_ 080589 Hs.402773 0,25577092

PRR4 605359 NM_{. ,}007244 Hs.408153 0,25570279

CA9 603179 NM_{. ,}001216 Hs.63287 0,25501394

DPP4 102720 NM_{. ,}001935 Hs.368912 0,25498438

ATP2B4 NM_{. ,}001001396, NM_001684 Hs.343522 0,25474662

PRKAA1 602739 NM_{. ,}006251 , NM _„206907 Hs.43322 0,25440449

KCNJ2 600681 NM_{. ,}000891 Hs.1547 0,25247389

SPRY2 602466 NM_{. ,}005842 Hs.18676 0,25236636

ANGPTL4 605910 NM_{. ,}016109, NM_. J39314 Hs.9613 0,25088108

ANTXR1 606410 NM_{. ,}018153, NM_. _032208, NM_ 053034 Hs.165859 0,24980527

HEM1 141180 NM_{. ,}005337 Hs.182014 0,24886674

ITGA4 192975 NM_{. ,}000885 Hs.440955 0,24860334

PLEKHB1 607651 NM_{. ,}021200 Hs.445489 0,24853754

THEDC1 NM_{. ,}018324 Hs.24309 0,2477231

RUTBC3 NM_{. ,}015705 Hs.474914 0,24722838

PPM1 F NM_{. ,}014634 Hs.112728 0,24719042

TULP3 NM_{. ,}003324 Hs.524187 0,24673349

PRIM2A NM_{. ,}000947 Hs.485640 0,24610269

HRASLS NM_{. ,}020386 Hs.36761 0,24506718

SOX4 184430 0,24478352

HIC NM_{. ,}199072 Hs.546385 0,24451417

BLM 604610 NM_{. ,}000057 Hs.169348 0,24423695

FGF1 131220 NM_{. ,}000800, NM_. _033136, NM_ 033137 Hs.483635 0,24403763

STAG3 608489 NM_{. ,}012447 Hs.521075 0,24399346

GIMAP5 608086 NM_{. ,}018384 Hs.412331 0,24289763

TG M2 190196 NM 004613. NM 198951 Hs.517033 0.24289387 MS4A1 112210 NM _021950, NM _152866, NM_ _152867 Hs.438040 0,24264246

TANK 603893 NM _004180, NM _133484 Hs.516490 0,24248934

RAB3IL1 NM _013401 Hs.13759 0,24200422

DOK2 604997 NM _003974, NM _201349 Hs.71215 0,24000698

MBNL2 607327 NM J44778, NM _207304 Hs.125715 0,23818958

GNAL 139312 NM _002071 , NM _182978 Hs.136295 0,2381376

H2AFY2 NM _018649 Hs.499953 0,23769051

SEPT9 604061 NM _006640 Hs.440932 0,23651183

ALS2CL NM J47129, NM _182774, NM_. J 82775 Hs.517937 0,23640573

SOX11 600898 NM _003108 Hs.432638 0,23627692

KLF6 602053 NM _001008490, NM_001300 Hs.285313 0,23492837

IGHG1 147020 Hs.375600 0,23475569

BM039 NM _018455 Hs.283532 0,23330599

CLEC10A 605999 NM _006344, NM _182906 Hs.54403 0,23322248

PDE4DIP NM 001002810, NM 001002811 , Hs.445881 0,23316765

NM 001002812, NM_014644,

NM 022359

SLA 601099 NM _006748 Hs.75367 0,23299015

BAK 1 600516 NM _001188 Hs.485139 0,23276204

MAP4 157132 NM _002375, NM _030884, NM_ _.030885 Hs.517949 0,23203611

FAM31C NM _024898 Hs.236449 0,23193562

NPAS2 603347 NM _002518 Hs.156832 0,2309894

GTF3C2 604883 NM _001521 Hs.75782 0,23026043

CENPA 117139 NM _001809 Hs.1594 0,22964168

MUF1 NM _006369 Hs.144941 0,22960305

RBM9 NM _014309 Hs.282998 0,22959888

GPR124 606823 NM _032777 Hs.17270 0,2295872

SLC28A3 608269 NM _022127 Hs.149425 0,22878563

SS18 600192 NM _001007559, NM_005637 Hs.404263 0,22848481

CCL18 603757 NM _002988 Hs.143961 0,22843105

PDLIM4 603422 NM _003687 Hs.424312 0,22814375

IL6 147620 NM _000600 Hs.512234 0,22774959

ZNF318 NM _014345 Hs.509718 0,22752988

KIAA0125 NM_014792 Hs.395486 0,22715425

PTPN2 176887 NM _002828, NM _080422, NM_{. .}080423 Hs.123352 0,22680461

C22orf4 NM _014346 Hs.435044 0,22606897

SERPINE1 173360 NM _000602 Hs.414795 0,22573197

KYNU 236800 NM _003937 Hs.470126 0,22450548

LHCGR 152790 NM _000233 Hs.468490 0,22392935

CD86 601020 NM _006889, NM _175862 Hs.171 182 0,223279

PCDHGA10, NM 003735, NM 018913, 0,22316551

PCDHGA11 , NM 018914, NM 018916, NM 018918,

PCDHGA12, NM 018919, NM 032011 , NM 032054,

PCDHGA3, NM 032086, NM 032090, NM 032091 ,

PCDHGA5, NM _032092, NM _032094

PCDHGA6

MRPS6 NM _032476 Hs.302742 0,22290385

DRPLA 607462 NM _001007026, NM_001940 Hs.143766 0,22256058

MCM10 NM _018518, NM _182751 Hs.198363 0,22202879

C16orf5 NM _013399 Hs.7765 0,22128123

XC L2 604828 NM _003175 Hs.458346 0,22100633

IBRDC3 NM _153341 Hs.546478 0,22075397

STX6 603944 NM _005819 Hs.518417 0,21999699

HIC2 607712 NM 015094 Hs.517434 0,21935087 LOC90379 XM_ _.373433 Hs.443636 0,21905839

KIAA0792 XM_ _.375848 Hs.119387 0,2186445

FLJ21924 NM_. _024774 Hs.369368 0,21821481

C6orf68 NM_. _138459 Hs.289008 0,21812248

EHD1 605888 NM_. _006795 Hs.523774 0,21755761

PCOLCE2 607064 NM_{. ,}013363 Hs.8944 0,21728051

PCNX NM_. _014982 Hs.158722 0,21602517

PDLIM7 605903 NM 005451 , NM_{. ,}203352, NM_203353, Hs.533040 0,21556157

NM 213636

EGR2 129010 NM_{. ,}000399 Hs.1395 0,21473932

C1QR1 120577 NM_. _012072 Hs.97199 0,21473044

IL7 146660 NM_{. ,}000880 Hs.536926 0,21466643

FLJ 10357 NM_. _018071 Hs.35125 0,21427947

TAF 1 313650 NM_{. ,}004606, NM_{. ,}138923 Hs.158560 0,21396095

ADAMTS2 604539 NM_. _014244, NM_{. ,}021599 Hs.120330 0,21365327

147570 0,2136451

CALML3 114184 NM_{. ,}005185 Hs.239600 0,21268052

ELN 130160 NM_{. ,}000501 Hs.252418 0,21254057

HSPA4L NM_. _014278 Hs.135554 0,21253169

DDX19, 605812 NM_. _007242, NM_{. ,}018332 Hs.221761 0,21208072

DDX19L

SLC36A1 606561 NM_{. ,}078483 Hs.269004 0,21 150744

CDCA8 NM_. ,018101 Hs.524571 0,21144181

ALOX5 152390 NM_{. ,}000698 Hs.89499 0,21114516

SLCO2B1 604988 NM_{. ,}007256 Hs.7884 0,21097678

POU4F1 601632 NM_{. ,}006237 Hs.211588 0,21038371

ATF5 606398 NM_{. ,}012068 Hs.9754 0,21034312

TGFB2 190220 NM_{. ,}003238 Hs.133379 0,20994902

CCR5 601373 NM_{. ,}000579 Hs.546245 0,20993143

PNAS-4 NM_{. ,}016076 Hs.498317 0,20987713

HIPK3 604424 NM_{. ,}005734 Hs.201918 0,20985919

PRKCI 600539 NM_{. ,}002740 Hs.399873 0,2096312

SSPN 601599 NM_{. ,}005086 Hs.183428 0,20961148

BTN2A1 NM_{. ,}007049, NM_{. ,}078476 Hs.519635 0,20951766

CSGIcA-T 608037 NM_{. ,}019015 Hs.86392 0,20951134

ARID5A NM_{. ,}006673, NM_{. ,}212481 Hs.920 0,20915419

CD96 606037 NM_{. ,}005816, NM_{. ,}198196 Hs.142023 0,20899445

CD1 D 188410 NM_{. ,}001766 Hs.1799 0,20894581

ADARB1 601218 NM_{. ,}001112, NM_{. ,}015833, NM_015834 Hs.474018 0,20840687

SELE 131210 NM_{. ,}000450 Hs.89546 0,20761901

SHB 600314 NM_{. ,}003028 Hs.521482 0,20694989

XDH 607633 NM_{. ,}000379 Hs.250 0,20662272

PLCB4 600810 NM_{. ,}000933, NM_{. ,}182797 Hs.472101 0,20624536

CARD 15 605956 NM_{. ,}022162 Hs.135201 0,20601743

LILRB2, LILRB6 604814 NM_{. ,}005874, NM_{. ,}024318 Hs.534386 0,20496422

CDCA3 607749 NM_{. ,}031299 Hs.524216 0,20485789

SPM 165170 NM_{. ,}003120 Hs.502511 0,20461261

0,20455593

CLEC1A 606782 NM_{. ,}016511 Hs.29549 0,20337689

STAT5A 601511 NM_{. ,}003152 Hs.437058 0,2032969

LOC94431 NM_{. ,}145237 Hs.546468 0,20324272

PACAP NM_{. ,}016459 Hs.409563 0,20274097

EPHB2 600997 NM_{. ,}004442, NM_{. ,}017449 Hs.523329 0,20273918

PRKCB1 176970 NM 002738, NM 212535 Hs.460355 0,20252955 IPO8 605600 NM_006390 Hs.505136 0,2015909

TRMT1 NM_001008568, NM_001008569, Hs.439524 0,20140934

NM_001008570, NM_001008571 , NM_018006

EXTL3 605744 NM_001440 Hs.491354 0,20126313

GNB5 604447 NM_006578, NM_016194 Hs.155090 0,20086384

PEX5 600414 NM_000319 Hs.479944 0,20048004

HCP5 604676 NM_006674 Hs.520017 0,2004233

HFE 235200 — , NM_000410, NM_139002, Hs.233325 0,20032763

NM_139003, NM_139004, NM_139005, NM_139006, NM_139007, NM_139008, NM 139009, NM_139010, NM_139011

Molecular methods

RNA was isolated from formalin-fixed paraffin-embedded ("FFPE") tumor tissue samples employing an experimental method based on proprietary magnetic beads from Siemens Medical Solutions Diagnostics. In short, the FFPE slide were lysed and treated with Proteinase K for 2 hours 55°C with shaking. After adding a binding buffer and the magnetic particles (Siemens Medical Solutions Diagnostic GmbH, Cologne, Germany) nucleic acids were bound to the particles within 15 minutes at room temperature. On a magnetic stand the supernatant was taken away and beads were washed several times with washing buffer. After adding elution buffer and incubating for 10 min at 70⁰C the supernatant was taken away on a magnetic stand without touching the beads. After normal DNAse I treatment for 30 min at 37°C and inactivation of DNAse I the solution was used for reverse transcription- polymerase chain reaction (RT-PCR) .

RT-PCR was run as standard kinetic one-step Reverse Transcriptase TaqMan™ polymerase chain reaction (RT-PCR) analysis on a ABI7900 (Applied Biosystems) PCR system for assessment of mRNA expression. Raw data of the RT-PCR were normalized to one or combinations of the housekeeping genes RPL37A, GAPDH, RPL13, and HPRTl by using the comparative ΔΔCT method, known to those skilled in the art. In brief, a total of 40 cycles of RNA amplification were applied and the cycle threshold (CT) of the target genes was set as being 0.5. CT scores were normalized by subtracting the CT score of the housekeeping gene RPL37A or the mean of the combinations from the CT score of the target gene (Delta CT) . RNA results were then reported as 40-Delta CT or ₂ ^{( (4}°-^{(cτ Target Gene ~ cτ Housekeeping Gerie)M"1)n} (2^Λ(40-(CT Target Gene - CT Housekeeping Gene) * (- 1) ) ) scores, which would correlate proportionally to the mRNA expression level of the target gene. For each gene specific Primer/Probe were designed by Primer Express ® software v2.0 (Applied Biosystems) according to manufacturers instructions . Statistics

The statistical analysis was performed with Graph Pad Prism Version 4 (Graph Pad Prism Software, Inc) and JMP 5.0.1.2 (SAS Institute, Inc) .

The clinical and biological variables were categorised into normal and pathological values according to standard norms. The Chi-square test was used to compare different groups for categorical variables. To examine correlations between different molecular factors, the Spearman rank correlation coefficient test was used.

For univariate analysis, logistic regression models with one covariate were used when looking at categorical outcomes. Survival curves were estimated by the method of Kaplan and Meier, and the curves were compared according to one factor by the log rank or Wilcoxon test. For the estimation of multivariate models, all parameters which were significant at the univariate analysis (p<0.05) were fitted to a Cox regression model using a backward forward stepwise method for the selection of covariates. Confidence intervals (CI) at 95% for hazard rates (HR) were calculated. All the probabilities that were calculated were two-tailed.

An example of the method of the invention is shown in Figs. 1 to 14.

Fig. 1 shows a Kaplan Meyer plot comparing survival of patients in an untested cohort (n=71) . Patients receiving Aranesp® (curve "1") while under taxane-based chemo therapy are compared with patients not receiving Aranesp® (curve "0") while under taxane-based chemotherapy. There is no significant difference (mean survival 207 weeks for Aranesp® + for an observation interval of ca. 280 weeks vs. mean survival 207 weeks for Aranesp® -; log rank p value =

0.3354) . Univariate cox regression analysis revealed no significance in this group of patients (p=0, 33) . Fig. 2 shows a Kaplan Meyer plot comparing survival of patients in a cohort (n=19 which was test as ESRl expression status negative (40-Delta CT < 34) . Patients receiving Aranesp® (curve "1") while under taxane-based chemo therapy are compared with patients not receiving Aranesp® (curve "0") while under taxane-based chemotherapy. There is significantly decreased survival for these patients under Epo administration (mean survival 106 weeks for Aranesp® + for an observation interval of ca. 280 weeks vs. mean survival 280 weeks for Aranesp® -; log rank p value = 0.065) . Univariate cox regression analysis revealed significance in this group of patients (p=0, 02) .

Fig. 3 shows a Kaplan Meyer plot comparing survival of patients in a cohort (n=63 which was test as ESRl expression status positive (40-Delta CT > 34) . Patients receiving Aranesp® (curve "1") while under taxane-based chemotherapy are compared with patients not receiving Aranesp® (curve "0") while under taxane-based chemo therapy. It appears, that ESR positive patients have a small increase in mean survival time, although there is no statistically significant difference for these patients under Epo administration (mean survival 219 weeks for Aranesp® + for an observation interval of ca. 186 weeks vs. mean survival 280 weeks for Aranesp® -; log rank p value = 0.73) . Univariate cox regression analysis revealed no significance in this group of patients (p=0, 74) .

Fig. 4 shows a Kaplan Meyer plot comparing survival of patients in a cohort (n=19 which was tested as MLPH expression status negative (40-Delta CT < 34) . Patients receiving Aranesp® (curve "1") while under taxane-based chemotherapy are compared with patients not receiving Aranesp® (curve "0") while under taxane-based chemotherapy. There is significantly decreased survival for these patients under Epo administration (mean survival 121 weeks for

Aranesp® + for an observation interval of ca. 280 weeks vs. mean survival 280 weeks for Aranesp® -; log rank p value = 0.068) . Univariate cox regression analysis revealed significance in this group of patients (p=0, 03) .

Fig. 5 shows a Kaplan Meyer plot. We have compared survival of patients in a cohort (n=63 which was test as MLPH expression status positive (40-Delta CT > 34) . Patients receiving Aranesp® (curve "1") while under taxane-based chemotherapy are compared with patients not receiving Aranesp® (curve "0") while under taxane-based chemo therapy. There is no statistically significant difference for these patients under Epo administration (mean survival 217 weeks for Aranesp® + for an observation interval of ca. 217 weeks vs. mean survival 186 weeks for Aranesp® -; log rank p value = 0.71) . Univariate cox regression analysis revealed no significance in this group of patients (p=0, 72) .

Fig. 6 shows a diagram of a partitioning test. This test is for the death rate of breast cancer patients (n=160) being treated either with or without Aranesp® ("darbo_B0>=l" vs "darbo_B0<l") based on base line assessment of grading (

"baseLineGrading_Bl<3" refers to Grade 1 and 2 tumors while "baseLineGrading_Bl>=3" refers to Grade 3 tumors) and expression of EpoR. As a cut off the median expression level of EpoR has been chosen. High grade tumors (i.e. grade 3) exhibiting high levels of EpoR expression (40-Delta CT > 32) had a 58% death rate if treated with Aranesp® (7 out of 12), while only having 12% death rate if not treated with Aranesp® (2 out of 17) .

Fig. 7 shows overall Survival of patients with basal like tumors (Arm A and B; +/- Aranesp) ; MMP7 Cut off at the 1st quartile of RT-kPCR expression levels defined by Delta Delta CT Method in relation to RPL37A as housekeeping control, and non-basal like tumors.

Fig. 8 shows overall Survival of patients with non-basal like tumors (Arm A and B; +/- Aranesp) ; MMP7 Cut off at the 1st quartile of RT-kPCR expression levels defined by Delta Delta CT Method in relation to RPL37A as housekeeping control.

Fig. 9 shows overall survival of patients with basal like tumors (Arm A; +/- Aranesp) ; MMP7 cut off at the 2nd tertile of RT-kPCR defined by Delta Delta CT Method in relation to RPL37A as housekeeping control and non-basal like tumors.

Fig. 10 shows overall survival of patients with non-basal like tumors (Arm A; +/- Aranesp) ; MMP7 cut off at the 2nd tertile of RT-kPCR defined by Delta Delta CT Method in relation to RPL37A as housekeeping control and non-basal like tumors .

Fig. 11 shows overall survival of patients with basal like tumors (Arm A; +/- Aranesp) ; MLPH/MMP7 Cut off at upper tertile of RT-kPCR defined by the algorithm 0.642*MMP7 - 0.766 * MLPH. Both MMP7 and MLPH are Delta CT values with respect to the housekeeper expression (RPL37A) .

Fig. 12 shows overall survival of patients with basal like tumors (Arm A; +/- Aranesp) ; MLPH/MMP7 Cut off at the Median of RT-kPCR expression levels of MMP7 and MLPH by simply subtracting the raw values from each other (i.e. two gene ratio without using a house keeping control) .

Fig. 13 shows a ROC Analysis (Arm A; +/- Aranesp) of MLPH cut offs defined by Delta CT method in relation to RPL37A as house keeping control: at three years of follow up a sensitivity of 100% and a Specificity of 42% can be reached on the training set resulting in a Area Under the Curve (AUC) of 0.8120. A the pre-defined cutoff of lower tertile results in a sensitivity of 85.4% and a specificity of 68.5% (diamond) .

Fig. 14 shows a ROC analysis (Arm A; +/- Aranesp) of MMP7 cut offs defined by Delta CT method in relation to RPL37A as house keeping control: at three years of follow up a sensitivity of 100% and a Specificity of 31% can be reached on the training set resulting in a Area Under the Curve (AUC) of 0.7139) . A the pre-defined cutoff of lower tertile results in a sensitivity of 83.5% and a specificity of 42.0%

These data demonstrate that by using the method of invention a group of patients can be identified that is likely to have an adverse response to Epo administration under cytotoxic chemotherapy. They thus demonstrate the utility of the present invention.

Claims

What is claimed is

1. A method for predicting an adverse response of a patient suffering from or at risk of developing a neoplastic disease and undergoing at least one given mode of cytotoxic treatment to the administration of erythropoietin or a functional derivative thereof, said method comprising the steps of:

a) obtaining a biological sample from said patient; b) determining the expression level of at least one gene of interest, said gene being correlated with the expression level status of one of MMP7, MMPl, IGHM, MLPH, ESRl, PGR, RBl, BRCAl, Her-2/neu, EpoR, KRT5, and SPPl in the said sample, c) comparing the expression level determined in (b) with an expression level of at least one reference gene; and d) predicting an adverse response to the administration of erythropoietin or a functional derivative thereof in said patient from the outcome of the comparison in step (C) .

2. Method of claim 1, wherein in step (c) a value of a difference between an expression level value of the at least one gene of interest and the expression level value of the at least one reference gene is determined and a threshold value is defined for said difference, wherein a value of the difference above said threshold value is indicative of a first therapeutic success and a value of the difference below said threshold value is indicative of a second therapeutic success.

3. Method of claim 1 or 2, wherein the gene of interest is a gene selected from the group consisting of MMP7, MMPl, MLPH, IGHM, EPO-R alpha, ESRl, PGR, RBl, BRCAl, EpoR, KRT5, Her-2/neu, and SSPl.

4. Method of any one of the preceding claims wherein the reference gene is a housekeeping gene, in particular RPL37a.

5. Method of any one of the preceding claims, wherein the cancer is breast cancer.

6. Method of any one of the preceding claims, wherein the cytotoxic therapy comprises the administration of a taxane compound, in particular Docetaxel (Taxotere®) or Paclitaxel (Taxol®) .

7. Method of any one of the preceding claims, wherein the adverse response to Epo correlated with a negative Estrogen receptor status in the patient undergoing cytotoxic therapy.

8. The method according to any one of the aforementioned claims, wherein the expression level is determined by

a) a hybridization based method; b) a PCR based method; c) determining the protein level, d) a method based on the electrochemical detection of particular molecules, and/or by e) an array based method.

9. The method according to any one of the aforementioned claims, characterized in that the expression level of at least one of the said genes is determined with rtPCR

(reverse transcriptase polymerase chain reaction) of the gene-related mRNA.

10. The method according to any one of the aforementioned claims, characterized in that the expression level of at least one of the said genes or fragments thereof are detected on protein level.

11. The method according to any one of the aforementioned claims, characterized in that the expression level of at least one of the said ligands of is determined in formalin and/or paraffin fixed tissue samples.

12. The method according to any one of the aforementioned claims, wherein, after lysis, the samples are treated with silica-coated magnetic particles and a chaotropic salt, in order to purify the nucleic acids contained in said sample for further determination.

13. The method according to any one of the aforementioned claims, wherein the gene of interest is a gene correlated with the expression level of MMP7.

14. The method according to claim 13, wherein said gene of interest is a gene selected from the group of genes listed in tables 2, 8, 9, and 10.

15 The method according to claim 14, wherein said gene of interest is selected from genes from the group of genes listed in tables 8, 9, and 10 having a correlation coefficient with MMP7 of at least 0.25, preferably at least 0.3, preferably at least 0.5.

16. A kit useful for carrying out a method according to any one of the aforementioned claims, comprising at least a primer pair and/or a probe each having a sequence sufficiently complementary to at least one gene as set forth in claims 1 or tables 1 to 10.