WO2010003772A1 - Procédé permettant de prévoir une réaction indésirable à l'érythropoïétine dans le cadre du traitement d'un cancer du sein - Google Patents

Procédé permettant de prévoir une réaction indésirable à l'érythropoïétine dans le cadre du traitement d'un cancer du sein Download PDF

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WO2010003772A1
WO2010003772A1 PCT/EP2009/057425 EP2009057425W WO2010003772A1 WO 2010003772 A1 WO2010003772 A1 WO 2010003772A1 EP 2009057425 W EP2009057425 W EP 2009057425W WO 2010003772 A1 WO2010003772 A1 WO 2010003772A1
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gene
expression level
genes
mmp7
expression
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PCT/EP2009/057425
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English (en)
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Michael Untch
Ralph Markus Wirtz
Christian VON TÖRNE
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Siemens Medical Solutions Diagnostics Gmbh
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Publication of WO2010003772A1 publication Critical patent/WO2010003772A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to methods for prediction of the therapeutic success of cancer therapy.
  • 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.
  • pCR pathological complete remission
  • 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.
  • 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) .
  • ADRs severe side 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.
  • 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.
  • PREPARE Preoperative Epirubicin Paclitaxel Aranesp Studie
  • the patients are treated with or without Darbepoietin alfa (Aranesp®) , as a class member of the erythropoietin derivatives.
  • Adbepoietin alfa Adbepoietin alfa
  • the interim results were published by the investigator and the sponsoring company Amgen showing numerically more deaths in the AranespTM 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
  • RNA analysis of selected candidate genes from paraffin embedded tissue biopsies to identify the patients being harmed by an EPO treatment.
  • prediction relates to the likelihood that a patient will respond either favourably or unfavourably to a given therapy.
  • prediction 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.
  • 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.
  • 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.
  • DFS Disease Free Survival
  • OFS Overall Survival
  • MSS Metastasis Specific Survival
  • the term "clinical response" of a patient 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
  • DFRS distant recurrence-free survival
  • 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.
  • abnormal 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.
  • 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 .
  • carcinoma 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 .
  • carcinoma 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, endo
  • carcinomas e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma
  • pre-malignant conditions adenomas, blood cell neoplasms and neomorphic changes independent of their histological origin.
  • carcinomas e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma
  • pre-malignant conditions adenomas, blood cell neoplasms and neomorphic changes independent of their histological origin.
  • cancer is not limited to any stage, grade, histomorphological feature, invasiveness, aggressiveness or malignancy of an affected tissue or cell aggregation.
  • 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.
  • 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.
  • tumor 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.
  • 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.
  • neoplastic cells of the invention may be cells of a benign neoplasm or may be cells of a malignant neoplasm.
  • 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.
  • TNM Malignant Tumours
  • 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.
  • various combinations of various drugs and/or physical methods, and various schedules are under investigation.
  • 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.
  • 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.
  • EPO Erythropoietin
  • erythrocyte red blood cell
  • hematopoietin erythrocyte (red blood cell) precursors in the bone marrow.
  • hematopoietin erythrocyte (red blood cell) precursors in the bone marrow.
  • hematopoietin erythrocyte (red blood cell) precursors in the bone marrow.
  • 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.
  • Epo epoietin
  • Procrit also known as Eprex and Epogen
  • NeoRecormon darbepoietin
  • PDpoietin PDpoietin
  • 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.
  • 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.
  • expression level refers, e.g., to a determined level of gene expression.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • said genes can be used without comparison to a reference pattern or after normalization to a reference gene or multiple reference genes.
  • biological sample refers to a sample obtained from a patient.
  • the sample may be of any biological tissue or fluid.
  • 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.
  • 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.
  • 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 .
  • 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.
  • oligonucleotide arrays wherein the probes are oligonucleotides are referred to as "oligonucleotide arrays" or “oligonucleotide chips.”
  • the regions in a microarray have typical dimensions, e.g., diameters, in the range of between about 10-250 ⁇ m, and are separated from other regions in the array by about the same distance.
  • 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.
  • 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.
  • “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.
  • 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.
  • the individual “arrayed probes” can be identified by their respective position on the solid support, e.g., on a "chip”.
  • response 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.
  • 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 .
  • 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 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.
  • Bioactivity 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.
  • a bioactivity can be altered by modulating the level of the polypeptide, such as by modulating expression of the corresponding gene.
  • marker 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.
  • marker gene 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 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.
  • receptor 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.
  • 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.
  • 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".
  • intercellular hormones and growth factors
  • intracellular second messengers
  • small molecule 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.
  • 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.
  • hybridization is used in reference to the pairing of complementary nucleic acids.
  • hybridization based method 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.
  • 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.
  • 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.
  • a PCR based method refers to methods comprising a polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • This is an approach for exponentially amplifying nucleic acids, like DNA or RNA, via enzymatic replication, without using a living organism.
  • 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.
  • 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) .
  • rtPCR reverse transcriptase PCR
  • 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.
  • the probe is modified at its 5'- or 3 ' -end with a thiol group which binds to the electrode comprising a gold surface.
  • target nucleic acid molecules may carry, for example, an enzyme label, like horseradish peroxidase (HRP) or alkaline phosphatase.
  • HRP horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • a substrate is then added (e.g., ⁇ -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.
  • 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.
  • 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) .
  • Tm thermal melting point
  • 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.
  • 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” .
  • 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.
  • 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.
  • derivative 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
  • hybridizing counterparts refers to a nucleic acid molecule that is capable of hybridizing to a nucleic acid molecules under stringent conditions.
  • 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.
  • 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.
  • a erythropoietin derivative caused increased death rates in cancer patients treated with standard regimen.
  • 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.
  • 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.
  • 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 .
  • the genes of interest comprise MMPl.
  • This marker has been shown to give a good prediction of the adverse response to EPO.
  • 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.
  • 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.
  • cytotoxic therapy comprises the administration of anthracycline, cyclophosphamide, methotrexate, fluoruracil, Docetaxel (Taxotere®) or Paclitaxel (Taxol®) .
  • 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.
  • said gene of interest is a gene selected from the group of genes listed in tables 2, 8, 9, and 10.
  • 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.
  • a deviating expression level of either of the aformentioned genes can have different reasons, these being
  • the adverse response to Epo correlated with a negative hormone receptor status in the patient undergoing cytotoxic therapy.
  • said hormone receptor is Estrogen receptor 1.
  • 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.
  • 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.
  • 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.
  • 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.
  • the expression level of at least one of the said gene/s is determined in formalin and/or paraffin fixed tissue samples.
  • the expression level of at least one of the said gene/s is determined in serum, plasma or whole blood samples.
  • 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.
  • the expression levels relate to protein fragments of said factors (e.g. EpoR extracellular parts) .
  • 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
  • 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.
  • RNA samples are taken as biopsies form a patient and undergo diagnostic procedures.
  • 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) .
  • RNAses ubiquitous RNA-digesting enzymes
  • 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 (SiO 2 ) •
  • 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 said approach is particularly useful for the purification of mRNA out of formaline and/or paraffine fixed tissue samples.
  • 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.
  • 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.
  • RNA level 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%) .
  • 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.
  • 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.
  • these motifs may be selected from the group comprising at least
  • MMP Matrix Metallo Proteinases
  • 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.
  • PGR Progesteron receptor
  • 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.
  • basal markers are derived from the appearance of the respective cells, which is similar to basal cells.
  • 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 ESRl, PGR, MLPH, MMP7, KRT5 , MMPl, Her-2/neu, and IGHM.
  • 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.
  • 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
  • BCNU Carmustin
  • Cardubris® Busulfan
  • Myleran® Bleomycin
  • Carboplatin Carboplat®
  • Chlorambucil Leukeran®
  • Cis-Platin Cis-Platin
  • Platinex Platinumiblastin®
  • dacarbazin DTIC®; Detimedac®
  • Docetaxel Taxotere®
  • Epirubicin Flumorubicin®
  • Etoposid Etoposid
  • Vepesid® 5-Fluorouracil
  • Fluorouracil Fluorouracil
  • Mitomycin C Mitomycin®
  • Mitoxantron Novantron®
  • Oxaliplatin Eloxatine®
  • Paclitaxel Tetraphalol
  • Vinblastin Vinblastin
  • Vincristin Vincristin®
  • Vindesin Eldisine®
  • Vinorelbin Vinorelbin
  • cytotoxic therapy may comprise the administration of Docetaxel (Taxotere®) or Paclitaxel (Taxol®) .
  • 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 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.
  • 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.
  • standard e.g. Arm A of the PREPARE trial
  • more-harsh dose intensified regimen e.g. Arm A of the PREPARE trial
  • 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.
  • 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.
  • 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) .
  • KLK5, KLK7 extracellular matrix genes
  • Laminins extracellular matrix genes
  • GASl cell cycle genes
  • 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 SystemTM was used to quantify the relative transcript abundance in the breast cancer tissues.
  • 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.
  • T7 Microarray RNA Target Synthesis
  • Microarray Target Purification Kit Microarray Target Purification Kit
  • 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.
  • 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 p-value associated with this test reflects the confidence of the Detection call.
  • 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.
  • OSBPL3 606732 NM 015550, NM 145320, NM 145321, Hs.520259 0,28503752
  • FGF1 131220 NM . , 000800, NM . _033136, NM_ 033137 Hs.483635 0,24403763
  • ARID5A NM . , 006673, NM . , 212481 Hs.920 0,20915419
  • LILRB2 LILRB6 604814 NM . , 005874, NM . , 024318 Hs.534386 0,20496422
  • HFE 235200 — NM_000410, NM_139002, Hs.233325 0,20032763
  • FFPE formalin-fixed paraffin-embedded
  • the FFPE slide were lysed and treated with Proteinase K for 2 hours 55°C with shaking.
  • 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.
  • the supernatant was taken away and beads were washed several times with washing buffer.
  • RT-PCR reverse transcription- polymerase chain reaction
  • RT-PCR was run as standard kinetic one-step Reverse Transcriptase TaqManTM 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 ⁇ CT method, known to those skilled in the art.
  • CT cycle threshold
  • 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 °- (c ⁇ Target Gene ⁇ c ⁇ Housekeeping Gerie)M"1)n (2 ⁇ (40-(CT Target Gene - CT Housekeeping Gene) * (- 1) ) ) scores, which would correlate proportionally to the mRNA expression level of the target gene.
  • Primer/Probe were designed by Primer Express ® software v2.0 (Applied Biosystems) according to manufacturers instructions .
  • 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.
  • the Spearman rank correlation coefficient test was used.
  • FIGs. 1 to 14 An example of the method of the invention is shown in Figs. 1 to 14.
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • High grade tumors i.e. grade 3
  • high levels of EpoR expression 40-Delta CT > 32
  • 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.
  • AUC Area Under the Curve
  • AUC Area Under the Curve
  • 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) .
  • AUC Area Under the Curve
  • AUC Area Under the Curve

Abstract

La présente invention concerne des procédés permettant de prévoir une réaction indésirable à l'érythropoïétine dans le cadre du traitement d'un cancer, lesdits procédés comprenant la détermination du niveau d'expression d'au moins un gène d'intérêt, ledit gène étant corrélé au statut du niveau d'expression de MMP7, MMP1, IGHM, MLPH, ESR1, PGR, RB1, BRCA1, Her-2/neu, EpoR, KRT5 ou SPP1.
PCT/EP2009/057425 2008-06-16 2009-06-16 Procédé permettant de prévoir une réaction indésirable à l'érythropoïétine dans le cadre du traitement d'un cancer du sein WO2010003772A1 (fr)

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AU2015249113B2 (en) * 2008-09-09 2017-09-07 Somalogic Operating Co., Inc. Lung cancer biomarkers and uses thereof
WO2010136192A1 (fr) * 2009-05-28 2010-12-02 Alepor Gmbh & Co. Kg Nouveau récepteur de l'érythropoïétine protecteur des tissus (nepor) et procédés d'utilisation
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CN113061655A (zh) * 2021-03-25 2021-07-02 中国科学院合肥物质科学研究院 一组用于预测乳腺癌新辅助化疗敏感性的基因标签及应用
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