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WO2011080373A1 - Empreinte génomique utilisée en tant que prédicteur de réponse à un traitement - Google Patents

Empreinte génomique utilisée en tant que prédicteur de réponse à un traitement Download PDF

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WO2011080373A1
WO2011080373A1 PCT/ES2010/070874 ES2010070874W WO2011080373A1 WO 2011080373 A1 WO2011080373 A1 WO 2011080373A1 ES 2010070874 W ES2010070874 W ES 2010070874W WO 2011080373 A1 WO2011080373 A1 WO 2011080373A1
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genes
treatment
response
expression
breast cancer
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Ramón GARCÍA ESCUDERO
Jesús María PARAMIO GONZÁLEZ
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Centro de Investigaciones Energeticas Medioambientales y Tecnologicas CIEMAT
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Centro de Investigaciones Energeticas Medioambientales y Tecnologicas CIEMAT
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to in vitro methods for predicting the response of a subject with breast cancer to the treatment of a taxane plus chemotherapy with an antimetabolite, an intercalating agent and an alkylating agent.
  • breast cancer is the second most common type of cancer (10.4%; after lung cancer) and the fifth most common cause of cancer death (after lung cancer, stomach cancer, cancer of liver, and colon cancer).
  • breast cancer is the most common cause of cancer death.
  • breast cancer produced 502,000 deaths worldwide (7% of cancer deaths; almost 1% of all deaths).
  • the number of global cases has increased significantly since the 1970s, a phenomenon partially blamed on modern lifestyles in the western world.
  • Women in North America have the highest incidence of breast cancer in the world. Because the breast is composed of identical tissues in men and women, breast cancer also occurs in men. The incidence of breast cancer in men is approximately 100 times lower than in women, although men with breast cancer are considered to have statistically the same survival rates as women.
  • TNM Breast cancer is classified in phases according to the TNM system.
  • the prognosis is closely linked to the results of the phase classification, and the phase classification is also used to assign patients to treatments both in clinical trials and in medical practice.
  • the different stages can be classified following the TNM system developed by the AJCC (American Joint Committee on Cancer) in collaboration with the UICC (International Union against Cancer):
  • TX The primary tumor cannot be evaluated.
  • T0 There is no evidence of tumor.
  • Tis Carcinoma in situ, not invasion.
  • TI The tumor is 2 cm or smaller.
  • T2 The tumor is larger than 2 cm but smaller than 5 cm.
  • T3 The tumor is larger than 5 cm.
  • T4 Tumor of any size that grows on the chest wall or skin, or inflammatory breast cancer.
  • NX Nearby lymph nodes cannot be evaluated.
  • NO The cancer has not spread to regional lymph nodes.
  • NI The cancer has spread to 1 to 3 lymph nodes in the armpit or to one internal breast.
  • N2 The cancer has spread to 4 to 9 lymph nodes in the armpit or to multiple internal mammary nodes.
  • N3 One of the following applies:
  • the cancer has spread to 10 or more lymph nodes in the armpit, or the cancer has spread to the lymph nodes under the collarbone, or the cancer has spread to the lymph nodes above the collarbone or the cancer affects the lymph nodes in the armpit and has spread to the internal mammary lymph nodes, or the cancer affects 4 or more lymph nodes in the armpit, and minimal amounts of cancer are found in the internal mammary nodes or in sentinel lymph node biopsy .
  • MX The presence of distant extension (metastasis) cannot be evaluated. M0: There is no distant metastasis. MI: Extension to distant organs has occurred, which does not include the supraclavicular lymph node.
  • HA grade T0, TI, NI, M0 or T2, NO, M0
  • Neoadjuvant chemotherapy is the administration of chemotherapy before breast cancer surgery, with the aim of reducing the extent of surgery or preventing surgery altogether.
  • the response to neoadjuvant chemotherapy is usually dichotomized as a complete pathological response (RCp) or residual disease (ER).
  • RCp pathological response
  • ER residual disease
  • RCp is a significant clinical endpoint to predict, since these patients have overall survival and prolonged disease-free survival when compared to patients with lesser response.
  • neoadjuvant or adjuvant therapy with trastuzumab is effective in breast cancer patients with overexpression of the HER-2 gene along with chemotherapy, this combined regimen is currently recommended for patients positive for HER-2.
  • Hess et al. (Hess et al, 2006. J. Clin. Oncol. 24: 4236-44) describe the development of a multigenic "predictor" of complete Pathological Response (RCp) of patients with breast cancer for paclitaxel / fluorouracil, adriamycin and cyclophosphamide (T / FAC) and determined its predictive accuracy in independent cases.
  • This study included 99 patients with normal levels of HER-2. In this way, they identified a predictor with 30 probes that predicted the RCp to T / FAC chemotherapy.
  • genomic breast cancer tests capable of identifying tumors with poor baseline prognosis in response to chemotherapy have been described known in the prior art include 21-gene tests (Paik et al, 2004, N Engl J Med. 351: 2817- 2826), intrinsic subtypes (Parker et al, 2009, J. Clin. Oncol. 27: 1160- 1167) or Genomic Grade Index (Liedtke et al, 2009, J. Clin. Oncol. 27: 3185-3191).
  • 21-gene tests Pieric et al, 2004, N Engl J Med. 351: 2817- 2826
  • intrinsic subtypes Parker et al, 2009, J. Clin. Oncol. 27: 1160- 116
  • Genomic Grade Index Liedtke et al, 2009, J. Clin. Oncol. 27: 3185-3191
  • these genomic tests do not have high sensitivity or specificity.
  • the present invention relates to an in vitro method for predicting the response of a subject diagnosed with breast cancer to treatment with a taxane and chemotherapy with an antimetabolite, an intercalating agent and a DNA alkylating agent, hereinafter the method of the invention, which comprises determining the expression levels of the genes identified in Table 1 and in Table 2 in a sample of tumor tissue from said subject, wherein an increase in the expression of the genes identified in the Table 1 and a decrease in the expression of the genes identified in Table 2 with respect to a reference value is indicative of a better response to treatment.
  • Figure 1 shows the relationship between the 40 gene test and the response to chemotherapy T / FAC.
  • A Risk value derived from the test 40 genes versus probability of RCp. The Y axis on the right shows the proportion of RCp patients (0) versus ER patients (1).
  • B Risk value derived from the test 40 genes versus probability of survival free of metastasis distant to 5 years in the Loi dataseis (GSE6532 and GSE9195). The Y axis on the right shows the proportion of patients who developed metastases distant at 5 years (0) versus those patients who did not (1).
  • C ROC curve analysis of logistic regression models for RCp to T / FAC chemotherapy.
  • the authors of the present invention have selected a signature of genes that hybridize with probes and whose expression correlates with the prediction of the response of a subject diagnosed with breast cancer upon treatment with a taxane, a pyrimidine analog antimetabolite, an intercalating agent and a DNA alkylating agent.
  • the invention relates to an in vitro method for predicting the response of a subject diagnosed with breast cancer to treatment with a taxane and chemotherapy with an antimetabolite, an intercalating agent and a DNA alkylating agent, hereinafter method of the invention, which comprises determining the levels of expression of the genes identified in Table 1 and Table 2 in a sample of tumor tissue from said subject, wherein an increase in the expression of the genes identified in Table 1 and a decrease in the expression of the genes identified in Table 2 with respect to a reference value is indicative of a better response to treatment.
  • "Predicting the response” means, in the context of the present invention, the determination of the likelihood of the patient responding favorably or unfavorably to a given therapy.
  • the term "prediction”, as used herein, refers to an individual evaluation of any parameter that may be useful in determining the evolution of a patient.
  • the prediction of the clinical response to tyrosine kinase treatment does not need to be correct for 100% of the subjects to be diagnosed or evaluated.
  • the term requires that a statistically significant part of the subjects can be identified as having an increased probability of having a positive response.
  • the person skilled in the art can easily determine if a subject is statistically significant using several well-known statistical evaluation tools, for example, determination of confidence intervals, determination of p-values, Student's t-test, Mann Whitney test, etc. .
  • Preferred confidence intervals are at least 50%>, at least 60%>, at least 70%>, at less than 80%>, at least 90%), at least 95%>.
  • P values are preferably 0.2, 0.1 or 0.05.
  • the prediction of the clinical response can be made using any assessment criteria used in oncology and known to the person skilled in the art. Valuation parameters useful to describe the evolution of a disease include:
  • Tumor control which, as used in the present invention, refers to the proportion of people treated in which a complete response, partial response, minor response or stable disease> 6 months is observed.
  • DFS disease-free survival
  • PFS6 rate refers to the percentage of people who are free of progression in the first six months after the start of therapy
  • MS median survival
  • subject refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, for example, humans, nonhuman primates, cows , horses, pigs, sheep, goats, dogs, cats or rodents.
  • the patient is a human being male or female of any age or race.
  • breast cancer refers to a breast tumor and includes any histological subtype that typically appears in breast cancer such as ductal carcinoma, lobular carcinoma, hemangioma, sarcomas, etc. any clinical subtype such as superficial cancer, muscle invader or metastatic disease and any TMN phase including Tis, TI, T2, T3 or T4 that depends on the presence or absence of cancer Invasive, invasive cancer dimensions, and the presence or absence of invasion outside the breast, NO, NI, N2 or N3 that depends on the number size and location of breast cancer cell deposits in the lymph nodes and M0 or MI that depends on the presence or absence of breast cancer cells in locations other than the breast and lymph nodes (the so-called distant metastases, for example, bone, brain, lung).
  • any histological subtype that typically appears in breast cancer such as ductal carcinoma, lobular carcinoma, hemangioma, sarcomas, etc.
  • any clinical subtype such as superficial cancer, muscle invader or metastatic disease and any TMN
  • taxanes are a set of antineoplastic drugs that prevent cell growth by preventing cell division. They are therefore used for the treatment of cancer. They are also called antimicrotubules, antimitotic and mitotic inhibitors. These are terpenes produced by plants of the genus Taxus, such as yew, hence its generic name. They are taxanes, for example, paclitaxel and docetaxel. Taxanes are used to perform chemotherapy treatment in cancer patients. The main mechanism of the taxanes is the inhibition of the function of the microtubule, which is carried out by stabilizing the guanosindifosphate (GDP) attached to the tubulin, preventing the normal functioning of the microtubules. Microtubules are essential for cell division and, therefore, taxanes stop cell mitosis. In a preferred embodiment, the taxane is paclitaxel.
  • antimetabolite is meant a substance that replaces, inhibits or competes with a specific chemical compound inside a cell, whose function is therefore to destabilize. Because its structure resembles that of the compound, it is absorbed by the cell but does not react in the same way as with the enzyme that acts on the ordinary substance. It can inhibit the enzyme or be converted into an atypical chemical agent. Many antimetabolites are useful in the treatment of various types of cancer.
  • folic acid analogs such as methotrexate
  • analogs of pyrimididnas where compounds such as 5-fluorouracil, widely used in colon, breast and ovarian cancer, would be included, without limitation
  • cytarabine which is used in the treatment of leukemia or gemcitabine, which is used in metastatic non-small cell lung cancer, in pancreatic carcinoma and in bladder cancer, or they may also be purine analogs, such as cladribine, mercaptopurine or fludarabine
  • the antimetabolite is an analogue of pyrimidine and in particular 5-fluorouracil or 5-FU.
  • 5-FU fluorouracil
  • thymidylate synthase acts in several ways, but mainly as an inhibitor of thymidylate synthase. Disrupting the action of this enzyme blocks the synthesis of pyrimidine thymidine, which is a nucleotide required for DNA replication. Thymidylate synthase methylates deoxyuracil monophosphate (dUMP) to deoxythymidine monophosphate (dTMP).
  • dUMP deoxyuracil monophosphate
  • dTMP deoxythymidine monophosphate
  • a pyrimidine analog As a pyrimidine analog, it is transformed into several cytotoxic metabolites inside the cell that are then incorporated into DNA and RNA, eventually inducing cell cycle arrest and apoptosis by inhibiting the ability of the cell to synthesize DNA. It is a specific S phase drug and active only during certain cell cycles. In addition to being incorporated into DNA and RNA, the drug has been shown to inhibit the activity of the exosome complex, an exoribonuclease complex, the activity of which is essential for cell survival.
  • Intercalating agent means a molecule that is capable of reversibly inserting itself between two strands of DNA.
  • the intercalating agent is an anthracycline.
  • Anthracyclines are tumor antibiotics used for different types of cancer, derived from the Streptomyces bacteria, specifically from Streptomyces peucetius var. Caesius The first anthracycline discovered was daunorubicin (trade name daunomycin), produced naturally by Streptomyces peucetius, an actinobacterium. Doxorubicin (adriamycin) was developed later and other related compounds subsequently developed.
  • anthracyclines include, without limitation, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin.
  • the adriamycin intercalating agent examples include, without limitation, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin.
  • the adriamycin intercalating agent include, without limitation, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin.
  • the adriamycin intercalating agent examples include, without limitation, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pixantrone, valrubicin.
  • DNA alkylating agents are known agents used for the treatment of cancer that bind an alkyl group (CnH2n + l) to DNA.
  • the alkyl group joins the DNA guanine, specifically the nitrogen atom number 7 of the imidazole ring.
  • alkylating agents can be used to treat various types of cancer. Examples of alkylating agents are cyclophosphamide, iofosfamide, melphalan, mechlorethamine or mustine, uramustine, chlorambucil, carmustine, lomustine, streptozocin and busulfan.
  • the alkylating agent of DNA is cyclophosphamide.
  • Cyclophosphamide also known as the phosphate cycle, is a nitrogen mustard alkylating agent, from the oxazoforin group. It is a "prodrug"; It becomes the liver in the active forms that have chemotherapeutic activity. Cyclophosphamide is converted in the liver by mixed-function oxidases enzymes into active metabolites. The main active metabolite is 4-hydroxycyclophosphamide. 4- Hydroxy-cyclophosphamide exists in equilibrium with its tautomer, aldophosphamide.
  • aldophosphamide is oxidized by the enzyme aldehyde dehydrogenase (ALDH) to produce carboxyphosphamide.
  • a small proportion of aldophosphamide is converted into phosphoramide and acrolein mustard.
  • Acrolein is toxic to the bladder epithelium and can lead to hemorrhagic cystitis. This can be prevented by the use of aggressive hydration and / or Mesna.
  • sample refers to any sample that can be obtained from a patient.
  • the present method can be applied to any type of biological sample of a patient, such as a biopsy sample, tissue, cell or fluid (serum, saliva, semen, sputum, cerebrospinal fluid (CSF), tears, mucus, sweat, milk , brain extracts and the like).
  • said sample is a sample of tumor tissue or a part thereof.
  • said tumor tissue sample is a sample of breast tumor tissue from a patient suffering from breast cancer.
  • Said sample can be obtained by conventional methods, for example, biopsy, using methods well known to those skilled in related medical techniques.
  • Methods to obtain a biopsy sample include partitioning into large pieces of a dough, or microdissection or other cell separation methods known in the art.
  • Tumor cells can be obtained additionally by aspiration cytology with a fine needle.
  • they can be fixed in formalin and embedded in paraffin or frozen first and then embedded in a cryosolidifiable medium, such as OCT compound, by immersion in a highly cryogenic medium that allows rapid freezing.
  • the taxane of the method of the invention is paclitaxel.
  • the antimetabolite is fluorouracil.
  • anthracycline is adriamycin.
  • the alkylating agent of DNA is the phosphamide cycle.
  • the invention relates to an in vitro method for predicting the response of a subject diagnosed with breast cancer to treatment with paclitaxel and chemotherapy with fluorouracil, adriamycin and cyclophosphamide, which comprises determining the levels of expression of the genes identified in Table 1 and Table 2 in a sample of tumor tissue from said subject, where an increase in the expression of the genes identified in Table 1 and a decrease in the expression of the genes identified in Table 2 with respect to a reference value is indicative of a better response to treatment.
  • the determination of the expression levels of the genes according to the method of the invention needs to be correlated with the reference values corresponding to the median value of the expression levels of said genes measured in a collection of tumor tissues in biopsy samples of cancer patients, before neoadjuvant chemotherapy treatment. Once this median value has been established, the level of this marker expressed in tumor tissues of patients with this median value can be compared, and thus be assigned to the level of "low", "normal” or "high".
  • the collection of samples from which the reference level is derived will preferably consist of patients suffering from the same type of cancer.
  • a reference value used to determine whether the expression of a gene sample is "increased” or “decreased” corresponds to the median value of the levels of BRCA expression measured in an RNA sample obtained by gathering equal amounts of RNA from each tumor sample obtained by biopsy of cancer patients before neoadjuvant chemotherapeutic treatment. Once this median value has been established, the level of this marker expressed in tumor tissues of patients with this median value can be compared, and thus assigned to the level of "increased” or “decreased.” Due to the variability between subjects (for example, aspects related to age, race, etc.) it is very difficult (if not practically impossible) to establish absolute reference values for BRCAl.
  • the reference values for "increased” or “decreased” BRCA expression are determined by calculating the percentiles by conventional means that involves the testing of a group of samples isolated from normal subjects (ie , people without breast cancer diagnosis) for the expression levels of the BRCAl gene.
  • the "increased” levels can then be assigned, preferably, to samples where the expression levels of the BRCAl genes is equal to or exceed the 50th percentile in the normal population, including, for example, expression levels equal to or in excess of the percentile.
  • the expression "increase in expression”, as used herein, refers to a change in the expression levels of a given gene with respect to the expression levels in the reference sample of at least 5%, in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%>, at least 45%, at least 50%>, at least 55%, at least 60%>, at least 65%, at least 70%, at least 75%, at at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more.
  • control RNAs are mRNAs derived from maintenance genes and encoding proteins that are constitutively expressed and that perform essential cellular functions.
  • Preferred maintenance genes for use in the present invention include ⁇ -2-microglobulin, ubiquitin, 18-S ribosomal protein, philine cycle, GAPDH and actin.
  • the control RNA is the BETA-actin mRNA.
  • the quantification of the relative gene expression is calculated according to the comparative method Ct using ⁇ -actin as endogenous control and commercial RNA controls as calibrators.
  • the final results are determined according to formula 2- (ACt of the sample-ACt of the calibrator), where the ACT values of the calibrator and the sample are determined by subtracting the CT value of the target gene from the value of the ⁇ -actin gene.
  • the method of the invention predicts the response of a subject diagnosed with breast cancer, which also has normal levels of HER-2.
  • normal levels of HER-2 is considered when the HER-2 detection test is negative, that is, there is no amplification or expression of HER-2.
  • the person skilled in the art has the necessary knowledge to determine the levels of HER-2. They can be measured, for example by:
  • IHC test ImmunoHistoChemistry
  • FISH Test Fluorescence In Situ Hybridization
  • the result of this test can be positive (more than 2 copies are amplified) or negative (2 or less copies, there is no amplification).
  • the method of the invention predicts the response of a subject diagnosed with advanced breast cancer.
  • advanced breast cancer refers to the fact that breast cancer has spread beyond the breast and it has reached the lymph nodes and distal tissues, that is, it would correspond to the NI, N2, N3 and M phases of the TNM classification or to grades IB, IIIA, IIIB or IV according to the classification of the "American Joint Committee on Cancer Staging. " Most cases of advanced breast cancer are found in women over 50 years. Currently, there is a growing number of options for the treatment of advanced breast cancer. Traditional methods of local control include surgery (procedures such as lumpectomy or mastectomy) and radiotherapy (applied at the cancer site). Systemic treatments include chemotherapy and hormonal therapy.
  • said treatment is a neoadjuvant treatment, that is, it is a treatment that is administered as a first step to reduce the size of the tumor before the main treatment that generally consists of surgery.
  • the main benefit that is obtained with neoadjuvant therapy is that it decreases the size of the tumor, which occurs in 80-90% of cases, and that allows a mastectomy to be avoided in many cases.
  • said method further comprises determining the level of expression of the genes identified in Table 1 and Table 2, also determining at least one clinical parameter selected from the subject's age, state of the estrogen receptor (ER) and tumor grade, where at a younger age of the subject, negative subjects for estrogen receptor and more advanced tumor grade, is indicative of a better response to treatment.
  • ER estrogen receptor
  • subjects under 50 or 50 years respond better to treatment.
  • Estrogen receptors are overexpressed in about 70% of breast cancer cases and are referred to as "ER positive.”
  • subjects with ER-negative tumors that is to say that they do not have estrogen receptors, would respond better to treatment than subjects with ER-positive tumors.
  • genes with more advanced breast cancer respond better to treatment, specifically, subjects diagnosed with grade III cancer respond better than subjects diagnosed with grade I or II cancer.
  • said genes are the genes whose nucleotide sequences hybridize with the probes identified in Tables 3 and 4.
  • the genes identified for the genetic signature or genomic test of 40 genes correspond to those of the Table 1 and Table 2.
  • the quantification of the expression levels of the genes identified in Table 1 and Table 2 can be performed from the RNA resulting from the transcription of said genes (mRNA) or, alternatively, from the complementary DNA (cDNA) of said genes.
  • the quantification of the expression levels of the genes identified in Table 1 and Table 2 comprises the quantification of the messenger RNA (mRNA) of said genes, or a fragment of said mRNA, the DNA complementary (cDNA) of said genes, or a fragment of said cDNA, or mixtures thereof.
  • the method of the invention may include performing an extraction step in order to obtain total RNA, which can be performed by conventional techniques (Chomczynski et al., Anal.
  • any conventional method can be used within the framework of the invention to detect and quantify mRNA levels encoded by genes whose nucleotide sequences hybridize with the probes of Tables 3 and 4 or their corresponding cDNA.
  • the levels of mRNA encoded by said genes can be quantified by the use of conventional methods, for example, methods comprising amplification of mRNA and quantification of the product of amplification of said mRNA, such as electrophoresis.
  • the levels of the cDNA corresponding to said mRNAs encoded by the genes of Tables 1 and 2 can also be quantified by the use of conventional techniques; in this case, the method of the invention includes a step of synthesis of the corresponding cDNA by reverse transcription (RT) of the corresponding mRNA followed by amplification and quantification of the amplification product of said cDNA.
  • RT reverse transcription
  • the quantification of the expression levels of the genes identified in Tables 1 and 2 is performed by a multiplex quantitative polymerase chain reaction (PCR).
  • the determination of the expression levels of the genes identified in Table 1 and Table 2 is carried out by means of a DNA array comprising the probes identified in Tables 3 and 4.
  • said array comprises at least one set of 11 probes per gene to determine the expression levels of each of the genes of Tables 1 and 2.
  • the expression levels of said probes will be taken into account.
  • the method of the invention comprises the determination of the expression levels of said genes of Tables 1 and 2 with respect to a reference value.
  • said reference value is the gene expression value of said genes from Tables 1 and 2 in a sample of primary tumors of patients who do not develop metastases.
  • genes will be considered to have an increased expression when the expression ratio of a gene is at least 1.5 times with respect to a reference value, preferably greater than 2 times, more preferably greater than, 3, 4, 5 and 10 times.
  • genes will be considered to have a diminished expression with respect to a reference value, when the expression ratio of a gene is at least 1.5 times lower with respect to the value reference.
  • the method of the invention comprises performing a proportional hazards regression analysis based on said expression values of the genes identified in Tables 1 and 2.
  • the determination of said prognosis comprises a regression analysis of proportional risks of said prognosis based on the expression levels of the genes identified in Table 1 and Table 2.
  • said proportional hazard regression analysis is a Cox analysis.
  • the inventors have used a Cox-type proportional hazards regression analysis to predict the response to treatment of a subject diagnosed with breast cancer.
  • Said Cox analysis assigns a regression coefficient for each gene, so that the gene whose expression is directly correlated with the prognostic variable, for example with the complete pathological response (RCp), is greater than 0, and if its expression is inversely related to said variable is less than 0.
  • the complete pathological response is established as a prognostic variable.
  • the inventors have demonstrated that by means of the method of the invention it is possible to determine the prognosis of the response of a patient with breast cancer and with normal levels of HER-2 with a high sensitivity, specifically 90%.
  • the inventors From the gene expression values of the genes of Tables 1 and 2 as described above, and the value of the Wald statistic of the proportional hazards regression analysis, the inventors have shown that it is possible to determine said prognosis. applying the following formula:
  • s ⁇ is the Wald statistic value of the Cox-type regression analysis for each of these genes identified in Tables 1 and 2, where if the value obtained is greater than zero then it is indicative of a better response to neoadjuvant treatment with a taxane and chemotherapy with a pyrimidine analog antimetabolite, an anthracycline and a DNA alkylating agent in a patient with breast cancer.
  • the value of the Wald statistic is a value commonly used by the person skilled in the art to know if the variables that are introduced in the statistical analysis are relevant or not. Such value can be calculated as described in Wald A. (1943) (Transactions of the American Mathematical Society. 1943; 54: 426-482) and Silvey (1959) (Silvey SD. Annals of Mathematical Statistics. 1959; 30: 389 -407).
  • the level of expression of the proteins encoded by said genes can also be quantified.
  • the quantification of the levels of the proteins encoded by the genes identified in Tables 1 and 2 comprises the quantification of said proteins or variables thereof.
  • protein refers to a molecular chain of amino acids, linked by covalent or non-covalent bonds.
  • the term also includes all forms of physiologically relevant post-translational chemical modifications, for example, glycosylation, phosphorylation or acetylation, etc.
  • variant is understood as a protein whose amino acid sequence is substantially homologous to the amino acid sequence of a particular protein.
  • An amino acid sequence is substantially homologous to a given amino acid sequence when it has a degree of identity of at least 70%, advantageously of at least 75%, typically at least 80%), preferably of at least 85%, more preferably of at least 90%), even more preferably of at least 95%, 97%, 98% or 99%, with respect to said determined amino acid sequence.
  • the degree of identity between two Amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as, for example, BLAST [Altschul SF et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215 (3): 403-10].
  • fragment refers to a peptide comprising a portion of a protein.
  • the level of expression of the proteins encoded by the genes identified in Tables 1 and 2 can be quantified by any conventional method that allows detecting and quantifying said proteins in a sample of a subject.
  • the levels of said proteins can be quantified, for example, by the use of antibodies capable of binding to said proteins (or fragments thereof containing an antigenic determinant) and the subsequent quantification of the complexes formed.
  • the antibodies used in these assays may or may not be labeled.
  • markers that can be used include radioactive isotopes, enzymes, fluorophores, luminescent chemo reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, etc.
  • the quantification of the protein levels encoded by the genes identified in Tables 1 and 2 is performed by western blot, ELISA, immunohistochemistry or an array of proteins.
  • the following example illustrates the invention and should not be considered as limiting its scope.
  • the genomic test of 40 genes is based on the determination of the expression levels of the genes in Tables 1 and 2 from 40 Affymetrix probes (Tables 3 and 4). From the levels of these probes, a "risk value" (VR) of each tumor is obtained by a formula. This formula assigns a numerical value to each sample (VR), based on the sum of the products of the expression values of each probe and the values of the Wald statistic of each gene according to the Cox model.
  • the CEL files were taken to extract the signal strength values using the RMAExpress program.
  • the RLE and NUSE charts allowed us to identify some tumor microarrays that did not pass the criteria of optimal normalization with RMA.
  • the corresponding CEL files were removed from subsequent analyzes.
  • a Cox proportional hazard regression analysis was performed using 5-year distal metastasis (MD) for the 707 U133A probes (and also present in U133Plus 2.0) corresponding to the 427 human genes mapped from the differential expression analysis of tumor tumors. mouse, using the survival utility implemented on the GEPAS website (www.gepas.org) (Vaquerizas JM. et al. Nucleic Acids Res 2005; 33 (Web Server issue): W616-620).
  • Cox analysis assigns a Cox regression coefficient for each probe, so that a probe whose expression is directly correlated with the appearance of MD is greater than 0, and if its expression is inversely related to MD it is less than 0
  • the Wald test (Wald A. Transactions of the American Mathematical Society 1943; 54: 426-482; Silvey SD. Annals of Mathematical Statistics 1959; 30: 389-407) is applied to analyze the null hypothesis that the coefficient be 0 (not related to MD), each probe being assigned a Wald statistic value, its corresponding P value, and P value corrected by the FDR method. Probes with Wald statistic values> 3 or ⁇ -3 were chosen for further analysis. These analyzes aim to verify the ability to predict MD at 5 and 10 years of human breast cancer.
  • Loi breast cancer dataseis (GSE6532 and GSE9195) were used to demonstrate the relationship between VR and the likelihood of developing 5-year distal metastases.
  • Table 5 shows the genes of the signature of the invention, and the corresponding values of si.
  • the inventors have managed to determine whether a patient would belong to the group with the best response to the treatment or the worst response to said treatment.
  • This method is based on the formula for the calculation of the risk value (VR), and on the ROC curve of MD at 5 years of the group of tumors Desmed ⁇ of 142 samples ( Figure 1, panel C). According to the COR curves, the predictor would have 90% sensitivity.
  • Figure 1C shows the operator-receiver (COR) curves for the different predictive models.
  • the formal statistical comparison of the three ROC curves showed no significant differences between the three curves.
  • models that include genomic data have a substantially higher sensitivity (90%) compared to the clinical model (70%>) (Table 7).
  • the likelihood ratio test to compare between the clinical model and the complete models demonstrates that the 40-gene test significantly improves the prediction performance, both as a continuous and dichotomized variable (Table 6).
  • the complete predictive model exhibits greater sensitivity (90% vs. 70%) than a model based on clinical variables.
  • the positive predictive value (PPV) of the complete model (56%; 95% confidence interval, 38% to 73%) indicates that you can define a patient population that is more likely to achieve CRP than the general population, since the overall observed rate of RCp is 26%, below the lower limit of the 95% confidence interval.
  • the negative predictive value (NPV) of the test is also high (97%, 95% confidence interval, 89% to 99%), indicating that less than 3% of patients who test negative (predicted to have residual disease) achieve RCp .

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Abstract

L'invention concerne un procédé de prédiction de la réponse au traitement à l'aide d'un taxane associé à une chimiothérapie avec un antimétabolite, un agent intercalant et un agent alkylant de l'ADN, chez des patients atteints d'un cancer du sein. Concrètement, les inventeurs ont développé un modèle par régression logistique dans lequel le test génomique de 40 gènes, combiné à des paramètres cliniques, permet de prédire avec précision la réponse pathologique complète (RCp) au traitement néoadjuvant de paclitaxel associé à une chimiothérapie avec du fluorouracyle, de l'adriamicine et du cyclophosphamide (T/FAC) chez des patients atteints de cancer du sein avec une expression normale du gène HER-2. Ledit modèle présente un meilleur rendement comparé aux variables cliniques utilisées ordinairement.
PCT/ES2010/070874 2009-12-31 2010-12-30 Empreinte génomique utilisée en tant que prédicteur de réponse à un traitement Ceased WO2011080373A1 (fr)

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WO2014080060A1 (fr) * 2012-11-23 2014-05-30 Servicio Andaluz De Salud Méthode destinée à prédire la réponse au traitement chimiothérapeutique de patients atteints de cancer
CN104024851A (zh) * 2011-11-28 2014-09-03 加拿大国家研究委员会 针对癌症的紫杉醇反应标志物

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EP1209612A2 (fr) * 2000-11-21 2002-05-29 Affymetrix, Inc. Procédés et produits logiciels d'ordinateur pour la prédiction de l'affinité d'hybridation d'acides nucléides
EP1644858A2 (fr) * 2003-07-10 2006-04-12 Genomic Health, Inc. Algorithme de profile d'expression et test du pronostic du cancer
EP1844334A2 (fr) * 2004-11-30 2007-10-17 Brystol-Myers Squibb Company Biomarqueurs et méthodes permettant de déterminer la sensibilité d'agents de stabilisation de microtubules
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104024851A (zh) * 2011-11-28 2014-09-03 加拿大国家研究委员会 针对癌症的紫杉醇反应标志物
WO2014080060A1 (fr) * 2012-11-23 2014-05-30 Servicio Andaluz De Salud Méthode destinée à prédire la réponse au traitement chimiothérapeutique de patients atteints de cancer

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