WO2009071655A2 - Procédés pour un pronostic du cancer du sein - Google Patents

Procédés pour un pronostic du cancer du sein Download PDF

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WO2009071655A2
WO2009071655A2 PCT/EP2008/066868 EP2008066868W WO2009071655A2 WO 2009071655 A2 WO2009071655 A2 WO 2009071655A2 EP 2008066868 W EP2008066868 W EP 2008066868W WO 2009071655 A2 WO2009071655 A2 WO 2009071655A2
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gene
steroid receptor
expression
tumor
regulated
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WO2009071655A3 (fr
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Mathias Gehrmann
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Siemens Healthcare Diagnostics Inc
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Siemens Healthcare Diagnostics Inc
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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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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, kits and systems for the prognosis of the disease outcome of breast cancer in node positive breast cancer patients treated with tamoxifen. More specific, the present invention relates to the prognosis of breast cancer based on measurements of the expression levels of marker genes in tumor samples of breast cancer patients .
  • breast cancer is one of the leading causes of cancer death in women in western countries. More specifically breast cancer claims the lives of approximately 40,000 women and is diagnosed in approximately 200,000 women annually in the United States alone. Over the last few decades, adjuvant systemic therapy has led to markedly improved survival in early breast cancer (EBCTCG, 1998 a+b) . This clinical experience has led to consensus recommendations offering adjuvant systemic therapy for the vast majority of breast cancer patients (Goldhirsch et al . , 2003) . In breast cancer a multitude of treatment options are available which can be applied in addition to the routinely performed surgical removal of the tumor and subsequent radiation of the tumor bed. Three main and conceptually different strategies are endocrine treatment, chemotherapy and treatment with targeted therapies.
  • endocrine agents Prerequisite for treatment with endocrine agents is expression of hormone receptors in the tumor tissue i.e. either estrogen receptor, progesterone receptor or both.
  • hormone receptors i.e. either estrogen receptor, progesterone receptor or both.
  • Tamoxifen has been the mainstay of endocrine treatment for the last three decades. Large clinical trial showed that tamoxifen significantly reduced the risk of tumor recurrence.
  • An additional treatment option is based on aromatase inhibitors which belong to a new endocrine drug class. In contrast to tamoxifen which is a competitive inhibitor of estrogen binding aromatase inhibitors block the production of estrogen itself thereby reducing the growth stimulus for estrogen receptor positive tumor cells.
  • Positive lymph node status is a strong adverse prognostic factor in breast cancer and women with node positive disease are generally considered for chemotherapy.
  • treatment guidelines for women with node positive and hormone receptor positive breast cancer are not well defined by St Gallen or NCI criteria. Neither is chemotherapy definitely recommended nor is a clear advice given regarding the preferred type of endocrine treatment.
  • Gene expression profiling has greatly extended the possibility to analyze the underlying biology of the heterogeneous nature of breast cancer.
  • MSC medullary breast cancer
  • US 2004/0229297-A1 filed 27 January 2004, discloses a method for the prognosis of the breast cancer in a patient said method comprising detecting in human tumor tissues the infiltration of certain immune cells. High infiltration of the tumor with immune cells was associated with poor cancer prognosis. The method, however, does not use information on the nodal status and does not rely on information on the rate of proliferation of the tumor.
  • the present invention fulfills the need for advanced methods for the prognosis of breast cancer on the basis of readily accessible clinical and experimental data. Definitions
  • prediction generally relates to the prediction of an event occurring in the future, e.g. prediction of survival, of prediction of development of metastasis or the like .
  • prognosis generally relates to the course of a disease under a given treatment.
  • prediction of prognosis of a patient receiving a treatment relates to the likelihood that a patient will have either favourable or unfavourable clinical course under a given therapy.
  • prediction of prognosis relates to an individual assessment of the malignancy of a tumor, or to the expected survival rate (DFS, disease free survival) of a patient, if the tumor is treated with a given therapy.
  • prognosis relates to an individual assesment of the malignancy of a tumor, or to the expected survival rate (DFS, disease free survival) of a patient, if the tumor remains untreated.
  • Classification within the meaning of the invention, shall be understood as being the process of classifying objects into one of multiple classes, said classes being defined by certain properties of the objects therein.
  • tumors or patients are classified into one of several "risk classes", said risk classes being defined e.g. by the likelihood of recurrence of cancer in said patients after surgery and treatment with a given drug.
  • a "risk” is the likelihood of the occurrence of a certain event, usually within a given period of time.
  • Sequential classification steps within the meaning of the invention, shall be understood as being multiple classification steps in a sequence of classification steps, wherein the subsequent classification step further classifies a class which was previously classified by a preceding classification step.
  • Sequential classification steps can be sequential classification steps in a "branched" classification scheme, such as the decision tree shown in Figure 1-3. Classification by sequential classification steps is effectively be implemented as a combination of the individual comparison steps, combined by the logical AND operator .
  • carcinomas e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma
  • pre-malignant conditions e.g., adenomas, blood cell neoplasms and neomorphic changes independent of their histological origin.
  • breast cancer relates to any form of cancer of the male or female breast, regardless of its 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.
  • treatment refers to a timely sequential or simultaneous administration of anti-tumor, and/or anti vascular, and/or immune stimulating, and/or blood cell proliferative agents, and/or radiation therapy, and/or hyperthermia, and/or hypothermia and/or hormonal treatment 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.
  • 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. Taxanes enhance stability of microtubules, preventing the separation of chromosomes during anaphase.
  • hormone treatment denotes a treatment which targets hormone signalling, e.g. hormone inhibition, hormone receptor inhibition, use of hormone receptor agonists or antagonists, use scavenger- or orphan receptors, use of hormone derivatives and intereference with hormone production.
  • hormone signalling e.g. hormone inhibition, hormone receptor inhibition, use of hormone receptor agonists or antagonists, use scavenger- or orphan receptors, use of hormone derivatives and intereference with hormone production.
  • hormone signalling e.g. hormone inhibition, hormone receptor inhibition, use of hormone receptor agonists or antagonists, use scavenger- or orphan receptors, use of hormone derivatives and intereference with hormone production.
  • hormone signalling e.g. hormone inhibition, hormone receptor inhibition, use of hormone receptor agonists or antagonists, use scavenger- or orphan receptors, use of hormone derivatives and intereference with hormone production.
  • tamoxifene therapy which modulates signalling of the estrogen receptor
  • aromatase treatment which inter
  • Tamoxifen is an orally active selective estrogen receptor modulator (SERM) that is used in the treatment of breast cancer and is currently the world's largest selling drug for that purpose. Tamoxifen is sold under the trade names Nolvadex, Istubal, and Valodex. However, the drug, even before its patent expiration, was and still is widely referred to by its generic name "tamoxifen.” Tamoxifen and Tamoxifen derivatives competitively bind to estrogen receptors on tumors and other tissue targets, producing a nuclear complex that decreases DNA synthesis and inhibits estrogen effects.
  • SERM selective estrogen receptor modulator
  • Steroid receptors are intracellular receptors (typically cytoplasmic) that perform signal transduction for steroid hormones. Examples include type I Receptors, in particular sex hormone receptors, e.g. androgen receptor, estrogen receptor , progesterone receptor; Glucocorticoid receptor, mineralocorticoid receptor; and type II Receptors, e.g. vitamin A receptor, vitamin D receptor, retinoid receptor, thyroid hormone receptor.
  • expression level refers, e.g., to a determined level of gene expression. In its simplest form this can mean "determining if there is a detectable expression of a gene" (qualitative determination) . This can be done e.g. immunohistochemically by qualitative determination by a pathologist. It can also be done by a quantitative determination wherein the gene is present, if the expression is above a threshold level.
  • 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.
  • 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.
  • 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 .
  • tumor sample or “tumor containing sample”, as used herein, refer to a sample obtained from a patient which sample contains tumor cells.
  • 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, urine, peritoneal fluid, and pleural fluid, liquor cerebrospinalis, tear fluid, or cells isolated therefrom. This may also include sections of tissues such as frozen or fixed sections taken for histological purposes or microdissected cells or extracellular parts thereof.
  • a tumor sample to be analyzed can be 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.
  • 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 comprises tumor cells or tumor cell fragments obtained from the patient.
  • the cells may be found in a cell "smear" collected, for example, by a nipple aspiration, ductal lavage, 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 or “microarray” 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.
  • 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.
  • “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 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 the absence of the given therapy.
  • 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 or “prediction of death after 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 "not sufficiently responding" tumor to the given therapy, or may result in a classification as a tumor with a prolonged disease free post therapy time.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • hybridizing counterparts refers to a nucleic acid molecule that is capable of hybridizing to a nucleic acid molecules under stringent conditions.
  • the present invention is based on the surprising finding that the outcome of cancer, preferably breast cancer in patients which do receive tamoxifen but not chemotherapy, can be accurately predicted from the expression levels of a small number of marker genes. Accordingly, the present invention relates to classification methods for the determination of the outcome of breast cancer in tamoxifen-treated breast cancer patients, using information on the expression of a small number of highly informative marker genes. The method is based on multiple comparisons of determined expression levels with predetermined threshold levels. Depending on the outcome of a first comparison, further comparisons of expression levels with threshold values are performed, as shown in the decision tree in Figure 1. The outcome of the prognostic method is a classification of the tumor under investigation into one of several "risk classes".
  • Risk classes of the invention are preferably a "low risk of recurrence" class, an "intermediate risk of recurrence class” and a "high risk of recurrence class".
  • the outcome of the prognostic methods of the invention provides useful information for the selection of the most suitable treatment regimen for the patient. Accordingly, chemotherapy may be avoided if a patient has a low risk of recurrence, or aggressiveness of chemotherapy may be adjusted to the likelihood of recurrence of the tumor, i.e. a tumor with a intermediate risk might justify a less aggressive treatment compared to a tumor with a high risk of recurrence.
  • Methods of the invention are multi-step classification methods in which consecutive classification steps are performed, the classifiers used in each step (i.e. the marker genes and threshold levels applied) being dependent on the outcome of the previous classification step(s) . It has been found that this classification strategy is in many cases superior to other methods, using a single multi-variate classification step. Without being bound by theory, it is assumed that the superior performance of sequential classification schemes over single-step classification schemes is due to the fact that marker gene expression can be differently correlated with the disease outcome in different subgroups of patients. For example, high expression of a marker gene A may be positively correlated with good disease outcome in patients having high expression of marker gene B, but may be negatively correlated with good disease outcome in patients having low expression of marker gene B. In multi- step classification methods, this can be taken into account by e.g. first classifying on the basis of the expression level of marker gene B, and then classifying on the basis of the correct correlation of marker gene A expression with the disease outcome.
  • the method of the invention is a method of prediction of a prognosis of cancer in a patient treated with Tamoxifen using the classification scheme generally shown in Figures 1 to 3.
  • the present invention provides a method to assess the risk of recurrence of a node positive and hormone receptor positive breast cancer patient when treated with tamoxifen.
  • the method is based on quantitative determination of RNA species isolated from the tumor in order to obtain expression values and subsequent bioinformatic analysis of said determined expression values, as generally depicted in Figs .1 and 2.
  • RNA coding for ESRl and PGR are determined. Based on these expression values the tumor is classified as steroid hormone receptor (SR) present or SR absent. Alternatively, this classification may also be performed on a protein-basis, for example determining the presence or absence of steroid receptor expression by immunohistological staining, as is known in the art.
  • SR steroid hormone receptor
  • RNA species coding for proliferation associated genes are determined. E.g. when expression values of said proliferation associated genes are below a predetermined cut off value said tumor is classified as low proliferation tumor. Suitable genes for making this determination are listed in table 1.
  • expression values of ESRl, PGR or expression values of genes co-regulated with ESRl or PGR are used to determine whether these values are higher then a predetermined cut off value. Tumors with expression values above the cut off value are classified as SR high.
  • expression values of proliferation associated genes are taken into account when determining the SR high class, thereby classifying only those tumors as SR high that do not exhibit a high expression of proliferation associated genes.
  • RNA species suitable to classify the tumor as intermediate or high risk are those indicating the presence of tumor infiltrating T- lymphocytes, in particular RNA molecules coding for genes listed in table 2. Further suitable RNA species are listed in table 2.
  • a combination of genes indicating the presence of T-lymphocyte infiltration and one or more from the genes listed in table 2 is used to classify a tumor as intermediate or high risk tumor.
  • the invention relates to:
  • a Method of classifying a tumor sample for the prediction of prognosis of a breast cancer patient receiving anti-hormonal treatment comprising a) determining in a tumor sample whether steroid receptor expression in a patient sample is present or absent by determining if there is a detectable expression of at least one steroid receptor gene or at least one gene co- regulated with a steroid receptor gene;
  • said sample is classified as low risk if (i) there is a detectable expression of at least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene and
  • said sample is classified as low risk if (i) there is a detectable expression of at least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene,
  • the expression level of said at least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene is above the predetermined first threshold level
  • the expression level of said at least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene is below the predetermined first threshold level
  • the expression level of said at least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene is below the predetermined first threshold level
  • step (b) an expression level of said at least one first marker gene above a third threshold value is indicative of a fast proliferating tumor.
  • At least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene is selected from the group consisting of estrogen receptor 1 (ESRl), progesterone receptor (PGR) or a combination thereof.
  • said anti-hormonal treatment comprises a tamoxifen treatment.
  • the expression level of at least one first marker gene is determined as a proliferation metagene expression value which is constructed by mathematically combining expression values of a plurality of first marker genes using 2, 3, 4, 5, 10, 20, 50, or all of the genes listed in Table 1.
  • a system for classifiying a tumor sample of a breast cancer patient to be selected for receiving anti-hormonal treatment comprising
  • (c) means for comparing said expression level of said first marker gene with a predetermined first threshold value
  • the expression level of said at least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene is above the predetermined second threshold level
  • said sample is classified as intermediate risk if (i) there is a detectable expression of at least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene,
  • the expression level of said at least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene is below the predetermined first threshold level, and (iv) the expression level of the at least one second marker gene in a tumor sample of said patient is above the predetermined second threshold level;
  • said sample is classified as high risk if (i) there is a detectable expression of at least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene,
  • the expression level of said first marker gene indicates that said tumor is fast proliferating, (i ⁇ ) the expression level of said at least one steroid receptor gene or at least one gene co-regulated with a steroid receptor gene is below the predetermined first threshold level, and
  • a prognostic method For a prognostic method to "be based" on a multiple pieces of information (as is the case in the present invention) all individual pieces of information must be taken into consideration for arriving at the prognosis or classification. This means that all individual pieces of information can influence the outcome of the prognosis or classification. It is well understood that a piece of information, such as e.g. the steroid receptor status of a patient, can influence the outcome of the prognosis or classification in that the is only applied when said steroid receptor status is e.g. positive. Likewise, it is understood that a method can "be based" on information relating to the proliferation rate of the tumor, e.g. if fast proliferation is a conditional criterion applied in the course of the method.
  • said classification or prognosis is entirely based on the information that said steroid receptor status is positive and that said tumor is a fast proliferating tumor and on information on the expression level of said second marker gene in said tumor sample.
  • a "fast proliferating tumor” within the meaning of the present invention is a tumor with a gene expression pattern indicative of fast proliferation and not taking into account the actual proliferation rate or rate of tumor growth in terms of mass or size.
  • said classification is an estimation of the likelihood of metastasis fee survival of said patient over a predetermined period of time, e.g. over a period of 5 years.
  • said classification is an estimation of the likelihood of death of disease of said patient over a predetermined period of time, e.g. over a period of 5 years.
  • Death of disease within the meaning of the invention, shall be understood to be the death of a breast cancer patient after recurrence of the disease.
  • Recurrence within the meaning of the invention, shall be understood to be the recurrence of breast cancer in form of metastatic spread of tumor cells, local recurrence, contralateral recurrence or recurrence of breast cancer at any site of the body of the patient.
  • the expression of said first marker gene indicative of fast proliferation of the tumor is a gene selected from Table 1.
  • a single, or 2 to 5, 2 to 10, 2 to 20, 2 to 50 or 2 to 100 first marker genes are used.
  • said first marker gene is TOP2A, UBE2C or a combination of TOP2A and UBE2C.
  • a combination of 2 markers preferably comprises the mathematical combination of the individual signal strengths of each single marker. This may be a direct sum or multiplication product or a weighted sum or product.
  • said first marker gene is a gene co-regulated with TOP2A.
  • Co-regulation of two genes is preferably exemplified by a correlation coefficient between expression levels of said two genes in multiple tissue samples of greater than 0.5, 0.6, 0.7, 0.8, 0.9, or, most preferably greater than 0.9.
  • the statistical accuracy of the determination of said correlation coefficient is preferably +/- 0.1 (absolute standard deviation) .
  • a proliferation metagene expression value is constructed by mathematical combination using at least 2, 3, 4, 5, 10, 20, 50, or all of the genes listed in Table 1.
  • a proliferation metagene expression value is constructed using 2, 3, 4, 5, 6, or all genes from the list of TOP2A, UBE2C, RACGAPl, CENPE, PTTGl, MKI 67, or CCNBl.
  • Proliferation metagene expression value within the meaning of the invention, shall be understood to be a calculated gene expression value representing the proliferative activity of a tumor .
  • a metagene expression value in this context, is to be understood as being the median of the normalized expression of multiple marker genes. Normalization of the expression of multiple marker genes is preferably achieved by dividing the expression level of the individual marker genes to be normalized by the respective individual median expression of these marker genes (per gene normalization) , wherein said median expression is preferably calculated from multiple measurements of the respective gene in a sufficiently large cohort of test individuals.
  • the test cohort preferably comprises at least 3, 10, 100, or 200 individuals.
  • the calculation of the proliferation metagene expression value is performed by: i) determining the gene expression value of at least two, preferably more genes from the list of table 1 ii) "normalizing" the gene expression value of each individual gene by dividing the expression value with a coefficient which is approximately the median expression value of the respective gene in a representative node negative breast cancer cohort iii) calculating the median of the group of normalized gene expression values
  • the present invention further relates to a method as defined above, wherein said second marker gene is a gene expressed in T cells which is specifically expressed in T-cells and not in tumor cells.
  • a gene shall be understood to be specifically expressed in a certain cell type, within the meaning of the invention, if the expression level of said gene in said cell type is at least 2-fold, 5-fold, 10-fold, 100-fold, 1000- fold, or 10000-fold higher than in a reference cell type, or in a mixture of reference cell types.
  • Preferred reference cell types are muscle cells, smooth muscle cells, fibroblast, fat cells, B-cells, macrophages or non-cancerous breast tissue cells.
  • a gene expressed in T cells shall be understood as being a gene selected from Table 2.
  • said second marker gene is selected from Table 2.
  • the claimed methods use the information on the expression of at least one proliferation marker gene (preferably selected from Table 1), and information on the expression of multiple T cell specific genes (preferably selected from Table 2), e.g., an T cell metagene expression is applied.
  • the expression level of multiple first and second marker genes are determined in steps (b) , (c) and/or (d) , and a comparison step between the multiple first and the multiple second marker genes is performed by a "majority voting algorithm".
  • the expression status of a plurality of first marker genes and second marker genes, respectively can also independently be determined by a majority voting algorithm.
  • a suitable threshold level is first determined for each individual first (or each individual second) marker gene used in the method.
  • the suitable threshold level can be determined from measurements of the marker gene expression in multiple individuals from a test cohort. For example, the median expression of the first said marker gene in said multiple expression measurements can be taken as the suitable threshold value for the first said marker gene. And, for example, the third quartile expression of the second said marker gene in said multiple expression measurements is taken as the suitable threshold value for the second said marker gene .
  • the individual marker genes are compared to their respective threshold levels. 2. The number of marker genes, the expression level of which is above their respective threshold level, is determined.
  • a sufficiently large number in this context, means preferably 30%, 50%, 80%, 90%, or 95% of the marker genes used.
  • the claimed methods use the information on the expression of one or two proliferation marker genes (preferably selected from Table 1), but information on the expression of multiple T-cell genes (preferably selected from Table 2) is compared to a threshold level using a majority voting algorithm.
  • a single, or at least 2, 5, 10, 20, 50 or 100 second marker genes are used.
  • the calculation of an T cell metagene is done by
  • determining the gene expression value of at least two, preferably more genes from the list of table 2 2. "normalizing" the gene expression value of each individual gene by dividing the expression value with a coefficient which is approximately the median expression value of the respective gene in a representative node negative breast cancer cohort
  • Fig. 1 Diagram of a decision tree depicting an embodiment of the inventive method
  • Fig. 2 Diagram of a decision tree depicting an embodiment of the inventive method
  • Fig. 3 Diagram of a decision tree depicting an embodiment of the inventive method
  • Fig. 4 Kaplan Meyer Diagram showing the results of a classification according to an embodiment of the inventive method.
  • Fig. 5 Kaplan Meyer Diagram showing the results of a classification according to an embodiment of the inventive method.
  • a preferred method of the invention is a method of prognosis of cancer in a patient using the classification scheme shown in Figures 1 to 3
  • SR present 202225_at Signal > 2000 or 208305_at Signal > 100
  • TOP2AJJBE2C Score (201291_s_at Signal / 350 + 202954_at Signal / 900) / 2
  • ESR1_PGR Score (202225_at Signal / 5000 + 208305_at Signal / 150) / 2
  • T-cell score Median of :
  • Signal refers to the signal strength obtained for the respective Affymetrix probe.
  • HG-U133A array and GeneChip SystemTM was used to quantify the relative transcript abundance in the breast cancer tissues.
  • Starting from 5 ⁇ g total RNA labelled cRNA was prepared using the Roche Microarray cDNA Synthesis, Microarray RNA Target Synthesis (T7) and Microarray Target Purification Kit according to the manufacturer's instruction.
  • T7 Microarray RNA Target Synthesis
  • Microarray Target Purification Kit according to the manufacturer's instruction.
  • synthesis of first strand cDNA was done by a T7-linked oligo- dT primer, followed by second strand synthesis.
  • Double- stranded cDNA product was purified and then used as template for an in vitro transcription reaction (IVT) in the presence of biotinylated UTP.
  • IVTT in vitro transcription reaction
  • Labelled cRNA was hybridized to HG-U133A arrays at 45°C for 16 h in a hybridization oven at a constant rotation (60 r.p.m.) and then washed and stained with a streptavidin-phycoerythrin conjugate using the GeneChip fluidic station.
  • a breast cancer Affymetrix HG-U133A microarray dataset including patient outcome information was downloaded from the
  • NCBI GEO data repository http://www.ncbi.nlm.nih.gov/geo/
  • the data set (GSE2034) represents 180 lymph-node negative relapse free patients and 106 lymph-node negative patients that developed a distant metastasis. None of the patients did receive systemic neoadjuvant or adjuvant therapy.
  • Clinical information was visualized as categorical or continues variable and relative gene expression was visualized on a relative scale from red, indicating high expression, to blue, indicating low expression.
  • Gene groups were defined after manual selection of nodes of the gene dendrogram as suggested by the occurrence of cluster regions within the heatmap.
  • a metagene was calculated as representative of all genes contained within one gene cluster based on the normalized expression values within the respective dataset.
  • the genes contained within the proliferation cluster are listed in Table 1 and the genes contained within the T-cell gene clusters are listed in Table 2.

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Abstract

La présente invention porte sur un procédé d'évaluation du risque de récurrence d'une patiente atteinte d'un cancer du sein positif pour les ganglions lymphatiques et positif pour les récepteurs hormonaux lorsqu'elle est traitée par du tamoxifène. Le procédé est basé sur une détermination quantitative d'espèces d'ARN isolées à partir de la tumeur afin d'obtenir des valeurs d'expression et une analyse bioinformatique ultérieure desdites valeurs d'expression déterminées. Tout d'abord, un état des récepteurs d'hormones stéroïdiennes (SR) est déterminé. Ensuite, une ou plusieurs espèces d'ARN codant pour des gènes associés à la prolifération sont déterminées. Puis une combinaison de gènes indiquant la présence d'une infiltration des lymphocytes T est utilisée pour classifier une tumeur comme étant une tumeur à risque faible, intermédiaire ou élevé.
PCT/EP2008/066868 2007-12-06 2008-12-05 Procédés pour un pronostic du cancer du sein Ceased WO2009071655A2 (fr)

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