WO2010151133A1 - Procédés pour établir et prédire la résistance à une endocrinothérapie en utilisant un profil d'arnmi - Google Patents

Procédés pour établir et prédire la résistance à une endocrinothérapie en utilisant un profil d'arnmi Download PDF

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WO2010151133A1
WO2010151133A1 PCT/NL2010/050401 NL2010050401W WO2010151133A1 WO 2010151133 A1 WO2010151133 A1 WO 2010151133A1 NL 2010050401 W NL2010050401 W NL 2010050401W WO 2010151133 A1 WO2010151133 A1 WO 2010151133A1
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hsa
mir
tumor
breast cancer
mirna
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Johannes Wilhelmus Maria Martens
Johannes Albert Foekens
Anita Maria Sieuwerts
Francisco Germán RODRIQUEZ-GONZALEZ
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Erasmus University Medical Center Rotterdam
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • 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
    • C12Q2600/16—Primer sets for multiplex assays
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates to the field of medical diagnostics, more specifically to the field of cancer diagnostics, especially breast cancer.
  • ER+ breast cancer Resistance to anti-estrogens is one of the major challenges in the treatment of breast cancer.
  • the golden standard for the endocrine treatment of all stages of estrogen receptor-positive (ER+) breast cancer has been tamoxifen or more recently an aromatase inhibitor.
  • ER+ breast tumors Approximately 30-50% of ER+ breast tumors do not respond to tamoxifen treatment (de novo resistance), and those that do respond often eventually progress to a state in which tumor cell proliferation is no longer inhibited, and may even be stimulated, by tamoxifen treatment (acquired resistance).
  • response rates in patients with ER- ⁇ negative primary tumors are very low. Comparable but slightly superior figures are seen for aromatase inhibitors.
  • Aromatase inhibitors may be more effective than tamoxifen at treating primary breast cancer, and offer a very promising alternative.
  • many Tam-resistant tumors retain sensitivity to steroidal anti- estrogens such as ICI 182,780 (ICI) (Fulvestrant® or Faslodex®), and this compound is approved as a second line therapy for patients who relapse while undergoing tamoxifen treatment.
  • ICI 182,780 ICI 182,780
  • Faslodex® Faslodex®
  • biomarkers are needed to identify patients who will not respond to tamoxifen or an aromatase inhibitor and to select patients who are likely to benefit therefrom.
  • the present invention provides in a first aspect a method of predicting or establishing the resistance of a breast cancer tumor to endocrine therapy, comprising the steps of: a. determining the expression profile essentially consisting of miRNAs hsa-miR-7, hsa-let-7g, hsa-miR-17-5p, hsa-miR-126, hsa-miR-126#, hsa-miR-141, hsa-miR-145, hsa-miR-335, hsa-miR-365, hsa-miR-489, hsa- miR-22, hsa-miR-30a-3p, hsa-miR-30c, and hsa-miR-182, preferably the expression profile essentially consisting of miRNAs hsa-miR-7, hsa-let-7g, hsa-miR-30a-3p,
  • Step a) suitable comprises the step of providing a breast cancer tumor sample.
  • a breast cancer tumor sample This may be in the form of a tumor biopsie, wherein a small part of the tumor is sampled and taken as a representative sample for the tumor itself.
  • Step b), determining the expression profile, in all aspects of the invention relates to the bonding of specific binding partners to the nucleic acid molecules of the miRNAs to be detected, for instance in the form of sequence-specific hybridization probes or sequence specific amplification primers. Such bonding may involve the liberation and isolation of nucleic acid molecules from the tumor tissue sample and hence disruption of tumor tissue in order to assess the presence and expression level therein of the of the miRNAs.
  • the invention further provides in another aspect a method of predicting or establishing the resistance of a breast cancer tumor to endocrine therapy, comprising the steps of: a.
  • diagnostic methods Methods of predicting or establishing the resistance of a breast cancer tumor to endocrine therapy as indicated herein are diagnostic methods. However, emphasis is placed on the analytical part of the diagnosis involving the actual biochemical test. Hence, the present invention in diagnostic aspects refers to methods of analysis of biological samples for the presence of biomarkers that have a predictive, diagnostic and/or prognostic value, wherein said biomarker may be a miRNA profile or a single miRNA expression level.
  • the preferred miRNAs have high statistical correlation with progression free survival and/ or clinical benefit from tamoxifen therapy
  • said at least one miRNA is selected from hsa-miR- 30a-3p, hsa-miR-30c and hsa-miR-182.
  • step a) is performed on a sample which is to be exposed to said estrogen antagonist (anti-estrogen) or an aromatase inhibitor prior to determining said level.
  • said estrogen antagonist anti-estrogen
  • an aromatase inhibitor prior to determining said level.
  • said difference between the level of said at least one miRNA and said reference value or a said level of a control sample is greater than one-fold.
  • said endocrine therapy comprises the administration of an anti-estrogen (most preferably tamoxifen) or an aromatase inhibitor.
  • said breast cancer tumor is a recurring tumor, preferably an ER+ tumor.
  • the invention further provides the use of a marker selected from miRNAs miRNAs hsa-miR-7, hsa-let-7g, hsa-miR-17-5p, hsa- miR-126, hsa-miR-126#, hsa-miR-141, hsa-miR-145, hsa-miR-335, hsa-miR- 365, hsa-miR-489, hsa-miR-22, hsa-miR-30a-3p, hsa-miR-30c, and hsa-miR- 182, preferably hsa-miR-30a-3p, hsa-miR-30c, and hsa-miR-182 for diagnostic use in a method for predicting or establishing the resistance of a breast cancer tumor.
  • a marker selected from miRNAs miRNAs hsa-miR-7
  • the invention further provides a kit of parts for performing a method of predicting or establishing the resistance of a breast cancer tumor to endocrine therapy comprising RT primers and/or probes specific for the detection of at least one miRNA selected from the group consisting of hsa-miR-7, hsa-let-7g, hsa-miR-17-5p, hsa-miR-30c, hsa-miR- 30a-3p, hsa-miR-126, hsa-miR-126#, hsa-miR-141, hsa-miR-145, hsa-miR- 182, hsa-miR-335, hsa-miR-365, hsa-miR-489, hsa-miR-22, hsa-miR-30a-3p, hsa-miR-30c, and hsa-miR-18
  • Figure 1 Kaplan-Meier PFS curves of 246 primary breast cancer patients as a function of the levels of hsa-miR-30a-3p (a), hsa-miR-30c (b), and hsa-miR-182 (c).
  • Samples are divided in quartiles based on the levels of the respective hsa-miRNAs. Patients at risk at 12-month time intervals are indicated at the bottom of each graph.
  • HR hazard ratio.
  • Figure 2 Pathways associated with tamoxifen response and most relevant hsa-miRNAs.
  • a pathway plot [generated by the Global Test program ] shows the genes annotated to the indicated pathway in Biocarta with their association with tumors showing a low versus high expression of the indicated miRNA.
  • Each bar represents a gene in the pathway. The height of the bar indicates the contribution (influence) of each individual gene to the significance of the pathway.
  • Horizontal markers in a bar indicate 1 SD away from the reference point, and two or more horizontal lines in a bar indicate a statistically significant association of the corresponding gene with the subgroups identified by a specific hsa- miRNA. Dark bars indicate positive and light bars indicate negative associations with the tumor subgroup having high versus low expression of a particular hsa-miRNA.
  • breast cancer refers to the erratic growth and proliferation of cells that originate in the breast tissue.
  • a group of rapidly dividing cells may form a lump or mass of extra tissue. These masses are called tumors.
  • Tumors can either be cancerous (malignant) or non-cancerous (benign). Malignant tumors penetrate and destroy healthy body tissues.
  • a group of cells within a tumor may also break away and spread to other parts of the body. Cells that spread from one region of the body into another are called metastases.
  • breast cancer refers to a malignant tumor that has developed from cells in the breast.
  • the teachings of the present invention could be extended to anti-estrogen and aromatase inhibitor-resistant of other tumor types.
  • predict refers to identifying or forecasting whether a breast cancer tumor will exhibit resistance or develop resistance against endocrine therapy.
  • resistance refers the fact that the normal therapeutic efficacy of the drug is not attained and that, for instance, a tumor continues to grow.
  • a tumor is classified as being resistant if the patient from whom said tumor was derived is classified as being a non-responder according to, for instance, the criteria used or set forth by the European Organisation for Research and Treatment of Cancer (EORTC) or the Union International Contre Ie Cancer (UICC).
  • EORTC European Organisation for Research and Treatment of Cancer
  • UICC Union International Contre Ie Cancer
  • control sample refers to a reference sample that is a sample from a tumor which is resistant or that is a sample from a tumor that is not resistant to endocrine therapy or that is a sample from a normal breast tissue, and which is used for comparison purpose with a test sample, that is, in order to classify the test sample — or more specifically the levels of certain miRNAs therein, as being indicative of a resistant of non- resistant tumor.
  • said control sample is from a resistant tumor.
  • the level of a miRNA of a control sample as described herein or an expression profile of miRNAs as described herein is compared to the level or profile in a suspected tumor sample. If there is no significant difference between said levels or profiles, then the suspected tumor sample is determined as having the same indication as that control sample.
  • a control sample may be from a single individual or from multiple individuals.
  • reference profile refers to a collection of expression levels for a multitude of given miRNAs, which expression levels in combination and/or relative to each other provide specific information which can be used for the purpose of comparison with other profiles.
  • the collection in aspects of this invention is indicative for a resistant or non-resistant phenotype of the tumor and can be used as a reference for comparison with the test profile.
  • reference profile is based on samples of one or more non-resistant breast tumors.
  • the reference profile may be based on samples of one or more resistant breast tumors.
  • the skilled person is well aware of methods for comparing data collections such as test and reference profiles of gene expression data, and such methods can suitably be used in aspects of the present invention.
  • anti-estrogen or an aromatase inhibitor-resistant tumor refers to a tumor, including the individual cells therein, that is or becomes refractory to treatment by an anti-estrogen or an aromatase inhibitor.
  • the anti-estrogen or aromatase inhibitor- resistant tumor becomes resistant to anti-estrogen or aromatase inhibitor treatment after initiation of the treatment and may occur during the treatment.
  • the resistance to anti-estrogen or aromatase inhibitor manifests at about 2-24 months while the patient is receiving hormonal therapy. In de novo resistance, the patient does not respond to initial therapy. Acquired resistance is where the patient develops metastatic disease during therapy.
  • Acquired resistance to hormone therapy such as anti-estrogen or aromatase inhibitor is well-known in the art.
  • hormone therapy such as anti-estrogen or aromatase inhibitor
  • breast cancer patients while undergoing treatment with anti-estrogen or aromatase inhibitor have recurrence of the disease.
  • the disease metastasizes during therapy with anti-estrogen or aromatase inhibitor, which results in resistant metastases.
  • the present invention predicts the occurrence of potential metastases, provides information concerning present metastases and/or prevents additional mestastases by identifying tumors susceptible to becoming resistant or being resistant.
  • anti-estrogen or an aromatase inhibitor-sensitive tumor refers to a tumor, including the individual cells therein, that is treatable with anti-estrogen or an aromatase inhibitor.
  • the anti-estrogen or an aromatase inhibitor-sensitive tumor remains sensitive during the treatment.
  • the anti-estrogen or an aromatase inhibitor- sensitive tumor is still sensitive up to at least about seven to ten years.
  • a tumor is classified as being sensitive if the patient from whom said tumor was derived is classified as being a responder as can be classified using EORTC or UICC criteria.
  • said responder is classified using EORTC or UICC criteria.
  • the term "different” as used herein with reference to the comparison of expression profiles or expression levels refers to a degree of difference which is statistically significantly increased or decreased compared to a reference or a control.
  • the term “significantly” or “statistically significant” refers to statistical significance and generally means that values differ two standard deviations (SD).
  • SD standard deviations
  • said difference is classified as statistically significant if the expression level is at least a 20 percent increased or decreased compared to expression level of the same expression product in control individuals.
  • the increase or decrease is at least 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or 250 percent. Most preferably, said increase or decrease is at least 100 percent (herein also referred to as "one fold").
  • level refers to the measurable absolute level or a measurable relative level compared to the level of another miRNA. If the expression level is determined of a miRNA from more than one individual (as a control), usually the median or mean expression level of these individuals is used for comparison.
  • miRNA refers to MicroRNAs (miRs) which are small RNA molecules encoded in the genomes of plants and animals. They are present within introns of protein-coding genes, in polycistronic transcripts encoding multiple miRNAs and in individual miRNA genes.
  • RNAs with a length of approximately 21-23 nucleotides regulate the expression of genes by binding to the 3'-untranslated regions (3'- UTRs) of specific mRNAs.
  • Each miRNA is thought to regulate multiple genes, and since hundreds of miRNA genes are predicted to be present in higher eukaryotes the potential regulatory circuitry afforded by miRNA is enormous.
  • MiRNAs are believed to act as key regulators of processes as diverse as early development, cell proliferation and cell death, apoptosis and fat metabolism, and cell differentiation. There is speculation that in higher eukaryotes, the role of miRNAs in regulating gene expression could be as important as that of transcription factors.
  • Over 540 human miRNAs have been validated to date; however, computer models suggest there may be thousands more. It is believed that up to 30% of human genes are regulated by miRNAs.
  • Pri- miRNAs are processed in the cell nucleus to shorter, 70-100 nucleotide stem- loop structures known as pre-miRNAs. This processing is performed in animals by the RNase III endonuclease Drosha. Pre-miRNAs are subsequently transported into the cytoplasm, where they are processed to double- stranded miRNAs with a length of 21-23 nucleotides by a second RNase III endonuclease, DICER.
  • aromatase inhibitor relates to a compound which inhibits the estrogen production, i.e., the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively.
  • the term includes, but is not limited to, steroids, especially atamestane, exemestane and formestane; and, in particular, non-steroids, especially aminoglutethimide, roglethimide, pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole, fadrozole, anastrozole and letrozole.
  • Exemestane is marketed as AROMASIN; formestane as LENTARON; fadrozole as AFEMA; anastrozole as ARIMIDEX; letrozole as FEMARA or FEMAR; and aminoglutethimide as ORIMETEN.
  • An aromatase inhibitor is particularly useful for the treatment of hormone receptor positive tumors, e.g., breast tumors.
  • anti-estrogen relates to a compound which antagonizes the effect of estrogens at the estrogen receptor level.
  • the term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride.
  • Tamoxifen can be administered in the form as it is marketed, e.g., NOLVADEX; and raloxifene hydrochloride as EVISTA.
  • Fulvestrant can be formulated as disclosed in U.S. Pat. No. 4,659,516 and is marketed as FASLODEX.
  • a combination of the invention comprising a chemotherapeutic agent which is an anti-estrogen is particularly useful for the treatment of estrogen receptor positive tumors, e.g., breast tumors.
  • Tamoxifen breast cancer treatment is widely used as an endocrine therapy in patients with recurrent disease in ER- ⁇ and/or PgR positive tumors, and objective response (CR or PR) to tamoxifen approximately is present in 60—70% in women with positive receptors tumors.
  • CR or PR objective response
  • Tissue specific miRNAs expression is commonly deregulated in human cancers. Down regulated hsa-miR-125a and hsa-miR-125b in breast cancer, hsa-miR-let-7 in lung cancer and hsa-miR-143 and hsa-miR-145 in several different cancer types suggest that their regulation plays a significant role in tumor biology.
  • miRNAs in primary human cancers may be used for tumor diagnosis and prognosis and it's potential involvement in cancer treatment response in general has been implicated, in breast cancer tumors, this has not been suggested in relation to tamoxifen or an aromatase inhibitor therapy and patient behaviour.
  • miRNA expression levels of hsa-miR-30a-3p, hsa- miR-30c, and hsa-miR-182 in 246 ER- ⁇ positive breast cancer patients treated with tamoxifen for recurrent disease.
  • MAPKK mitogen-activated protein kinase kinase
  • MKK4 mitogen-activated protein kinase kinase
  • Hsa-miR-30c has now been found to be correlated to "Rac 1 cell motility signalling pathway" as well as hsa-miR-30a-3p. It suggests that both miRNAs have a collaborative participation on different pathways and conferring better tamoxifen response.
  • the present invention is based on the discovery of a connection between expression levels of hsa-miR-30a-3p, hsa-miR-30c, and hsa-miR-182 and their association with relevant genes included in pathways that plays an important role on tamoxifen or an aromatase inhibitor treatment response.
  • the determination of the miRNAs expression levels on primary tumors can help clinicians to choose a better treatment for recurrent breast cancer on ER+ tumors and predict the individual clinical benefits for individual patients.
  • the present invention concerns the prediction or the detection of a tumor of being resistant to cancer therapy, in particular hormone therapy in breast cancer patients, using miRNA expression profiling.
  • Information obtained from the present invention will assist a health care provider in determining whether or not a tumor (including cells therein) will become resistant to the hormone therapy or whether or not a tumor is resistant to the hormone therapy prior to the start of said therapy.
  • the present invention will provide direction whether or not to continue with hormone therapy, or whether to start at all.
  • tamoxifen or an aromatase inhibitor is the exemplary embodiment described for illustrative purposes only, and a skilled artisan recognizes that the invention can be utilized for other chemotherapeutic drugs also, including other hormone therapy drugs, in particular estrogen inhibitors or estrogen receptor antagonists.
  • the present invention predicts the occurrence or recurrence of metastases due to therapy resistance, provides information concerning present metastases and/or helps to prevent mestastases by early intervention by identifying tumors susceptible to becoming resistant or being resistant and taking necessary actions based thereon.
  • the present invention identifies specific miRNAs which are differentially expressed in a tumor that is resistant compared to a tumor that is not resistant. More specifically, tumors with higher expression levels of hsa-miR-30a-3p, hsa-miR-30c, and hsa-miR-182 than a control were shown to be associated with a better response to treatment with tamoxifen or an aromatase inhibitor.
  • the miRNAs as described herein are useful as biomarkers to avoid hormonal therapies, such as antiestrogens, including tamoxifen or an aromatase inhibitor, raloxifene (Evista), and/or fulvestrant, for example, in case there is a likelihood of developing resistance.
  • hormonal therapies such as antiestrogens, including tamoxifen or an aromatase inhibitor, raloxifene (Evista), and/or fulvestrant, for example, in case there is a likelihood of developing resistance.
  • the present invention employs miRNA expression profiling to compare biological phenotypes of tumors and evaluating the outcome thereof in the light of the chance that the tumor is resistant.
  • the test sample can be compared to a positive (resistant) control.
  • the test sample can be compared to a negative/susceptible control.
  • Certain miRNA's can be used as internal controls for normalizing expression levels.
  • miRNA levels in tumors sensitive to endocrine therapy are compared to miRNA level in tumors resistant to endocrine therapy to establish significance levels for the diagnostic test.
  • said tumor comprises a representative part of said tumor.
  • said tumor is classified as ER- or PgR- positive, preferably using E. O. R. T. C. or UICC criteria.
  • the cut-point which is used to classify a tumor as ER- or PgR- positive is 10 fmol/mg cytosolic protein.
  • said ER or PgR- positive tumor comprises at least 30% nuclei evidently of epithelial tumor origin. More preferably, said at least 30% of the nuclei are distributed uniformly over at least 70% of the section area.
  • said tumor sensitive to endocrine therapy is a tumor which has not progressed after about 2 years following initial diagnosis.
  • said resistant tumor is a tumor that has pregressed within less than 6 months, preferably during ongoing endocrine therapy treatment.
  • the invention exploits the biological characteristic of primary, circulating or metastatic tumor cells of endocrine therapy sensitive patients being molecularly different from the primary or metastatic, circulating or metastatic tumor cells of endocrine therapy refractory patient.
  • the difference between said level of said at least one miRNA and said reference value or said level of a control sample is at least greater than 1, 1.5, 2, 2.5, 3 or 3.5.
  • said endocrine therapy comprises an estrogen antagonist.
  • said estrogen antagonist comprises tamoxifen or an aromatase inhibitor, most preferably tamoxifen.
  • CMF cyclophosphamide, methotrexate, 5-fluorouracil
  • Relevant clinicopathologic characteristics of the patients and their primary tumor are given in Table 1.
  • Ml-patients primary surgery
  • These 30 patients and the 216 patients who developed a recurrence during follow-up [26 patients with local-regional relapse (LRR), 209 patients with distant metastasis (DM)] were treated with first-line tamoxifen (40 mg daily).
  • the median time between primary surgery and start of therapy was 29 months (range, 2-134 months).
  • the median age of the patients was 60 years (range, 26-89 years) and at start of tamoxifen therapy for recurrent disease 62 years (range, 29-90 years).
  • Response to tamoxifen therapy was defined by standard Union International Contre Ie Cancer (UICC) criteria criteria (1).
  • UICC Union International Contre Ie Cancer
  • Objective response was observed in 45 patients (12 complete remission, CR, and 33 partial remission, PR), and 78 patients had a tumor progression of 25% or more, or showed new tumor lesions within 3 months (progressive disease, PD).
  • the median progression free survival (PFS) was for CR: 41 months, PR: 14 months, SD: 14 months, NC ⁇ 6 months: 6 months, and for PD: 3 months. Because the patients with SD had a PFS similar to patients with PR, we classified these patients as responders to tamoxifen as advised by the E.O.R.T.C. Therefore, clinical benefit was defined in our study as CR + PR + SD. Only patients with measurable disease were evaluated.
  • ER and progesterone receptor PgR
  • Tumor cytosols were prepared and processed as recommended by the European Organization for Research and Treatment of Cancer.
  • the cut-point used to classify tumors as ER- or PgR-positive was 10 fmol/mg cytosolic protein.
  • the remainder of the tumor tissue was stored in our liquid nitrogen tumor bank at the Erasmus MC.
  • RNA isolation Before, during, and after cutting the sections for RNA isolation, 5 ⁇ m sections were cut for H&E staining to assess the amount of tumor cells relative to the amount of surrounding stromal cells. In this study, only ER-positive specimens with at least 30% of the nuclei evidently of epithelial tumor cell origin and distributed uniformly over at least 70% of the section area were included.
  • RT samples were diluted in nuclease-free ddH2O and amplified in a final volume of 20 ⁇ l containing cDNA synthesized from 0.5 ng of total RNA, 6 ⁇ l of TaqMan universal PCR master mix without UNG (ABI), 0.6 ⁇ l of the individual TaqMan MicroRNA primer and probe assays and 8.4 ⁇ l of nuclease-free dH2O.
  • Real-time PCR was performed using the Stratagene Mx3000P QPCR System (Agilent Technologies, Waldbronn, Germany), with cycling conditions according to the manufacturer's instructions.
  • a minus RT reaction was incorporated, which proved to be negative for all assays.
  • PCR efficiency, linearity, and the upper and lower detection limits of each of the individual miRNA assays were validated with the standard curve constructed from the simultaneously run serially diluted pool of RNA.
  • the performance of all assays were validated with this standard curve set of samples in uniplex RT reactions.
  • RNA input integrity, and yield of amplifiable cDNA, we proceeded as previously reported.
  • STATA statistical package 10.0 STATA Corp., College Station, TX
  • Differences in levels were assessed with the Mann- Whitney U test or Kruskal-Wallis test, including a Wilcoxon-type test for trend, when appropriate.
  • patient and tumor characteristics were used as grouping variables.
  • the strengths of the associations between continuous variables were tested with the Spearman rank correlation (Rs).
  • Rs Spearman rank correlation
  • To reduce the skewness most variables were log- transformed or Box- Cox transformed. Four equal parts were used to categorize the variable to low, low-intermediate, intermediate-high and high. Survival curves were generated using the method of Kaplan and Meier (1958) and the logrank test was used to test for differences.
  • the Cox proportional hazard model was used to calculate the hazard ratio (HR) and 95% confidence interval (CI) in the analyses of time to progression free survival (PFS), with the starting point of PFS being defined as the start of the first-line of systemic treatment with tamoxifen for recurrence and the endpoint as the first detection of progression of the disease.
  • Logistic regression analysis was used to examine the relation of miRNA levels with clinical benefit of tamoxifen therapy and for the calculation of the Odds Ratio (OR) and its 95% CI. A two-sided P-value of ⁇ .05 was considered statistically significant.
  • Affymetrix microarray gene expression data (HG-U133A chips) previously deposited in the NCBI/GEO database (entries GSE2034 and GSE5327) were combined with gene expression data from HG-U133-plus2 chips. In total, 425 ER-positive samples were available for analysis. Data were preprocessed as previously described (Campbell et al. J
  • a sample was labeled 'high' or 'low' for a miRNA expression. Taken in to account the top 20 or bottom 20 of all samples, according to the expression of that particular miRNA. The 20 samples with the highest hsa-mir-30c expression were compared with the 20 samples with the lowest hsa-mir-30c expression, and likewise for each miRNA of interest.
  • the Global Test program was used (version 4.4.0) to associate Biocarta pathways (http://www.biocarta.com/) to samples expressing high or low amounts of a particular miRNA, irrespective of their association with response to therapy. All P-values were corrected for multiple testing and checked by re-sampling if an equally sized, randomly chosen group of genes is also significant (1,000 samplings). Pathways were considered of interest if the P-value of the Global Test, after correcting for multiple testing and the resampling P-value were either at or below 0.05. Pathway P-values mentioned in the text are two-sided P-values corrected for multiple testing, except where stated otherwise. The contribution of individual genes in a pathway was evaluated using z- scores calculated by the Global Test program. Genes with z-scores>1.96 were considered significant contributors to the pathway. R version 2.4.1 (http://www.cran.r-project.org) was used to run the Global Test package.
  • Tumour miRNA expression levels were categorized in quartiles to find out its possible effect in patient's response to tamoxifen therapy (Table 2).
  • FIG. 1 shows progression-free survival as finction of the categorized miRNA level in all 246 patients.
  • hsa-miR-30c also was related to "ERK 1.
  • Bcl-2 and MAP2K4 were overexpressed in tumors having high versus those having low hsa-miR-30a-3p expression.
  • Several studies show a positive correlation between Bcl-2 and ER- ⁇ expression levels imply that Bcl-2 is a significant favourable prognostic factor for breast cancer treated with chemotherapy and endocrine therapy (35-37).
  • Spearman Rank test showed a significant negative correlation between hsa-miR-30c and EGFR, PDGFRA, CHNl, hsa-miR-30a- 3p only with CHNl and has-miR-182 with PDGFRA mRNA levels.
  • Example 1 we measured using real-time PCR levels over various candidate microRNAs (Hsa-miR-7, hsa-miR-210, hsa-miR-373 and hsa-miR-10b, hsa- miR-205, hsa-miR-98, hsa-miR-374, hsa-miR-335, hsa-miR-22, hsa-miR-214, hsa-miR-212, hsa-miR-21, hsa-miR-187, hsa-miR-17-5p, hsa-miR-132, hsa- miR-125b, hsa-let-7g, hsa-miR-365, hsa-miR-518b, hsa-miR-126*, hsa-
  • Hsa-miR-7, hsa-miR-210, hsa-miR-373 and hsa-miR-lOb, hsa- miR-205 were selected because us and others have shown that these microRNAs are associated with aggressiveness. For this reason these microRNAs for their role in endocrine resistance in breast cancer.
  • microRNAs hsa-miR-98, hsa-miR-374, hsa-miR- 335, hsa-miR-22, hsa-miR-214, hsa-miR-212, hsa-miR-21, hsa-miR-187, hsa- miR-17-5p, hsa-miR-132, hsa-miR-125b, hsa-let-7g, hsa-miR-365, hsa-miR- 518b, hsa-miR-126*, hsa-miR-182, hsa-miR-34a, hsa-miR-432, hsa-miR-141.
  • hsa-miR-126, hsa-miR-422a, hsa-miR-489, hsa-miR-520c were included because they were predicted targets for factors involved in tamoxifen resistence such as ER, PgR, Her-2, ERBB3, ERBB4, EGFR, EZH2, GRB7, SIAH2, BCARl, TNC (and other extracellular matrix genes), AKT3, NCOl, TFFl or other work had suggested a role in therapy resistance (hsa-miR-212).
  • Biological factors were separately introduced as log-transformed continuous variable to the base multivariate model that included the factors menopausal status, dominant site of relapse, disease-free interval, and ER-a and PgR mRNA levels as log-transformed continuous variables 2At start of first-line therapy for recurrent disease Table 3.
  • Biological factors were separately introduced as log-transformed continuous variable to the base multivariate model that included the factors menopausal status, dominant site of relapse, disease-free interval, and ER-a and PgR mRNA levels as log-transformed continuous variables 2At start of first-line therapy for recurrent disease Table 3. List of the most significant pathways associated with hsa-miR-30a-3p, hsa-miR-30c and hsa-miR-182
  • HIVI Nef. negative effector of Fas and TNF 48 36 19.999 6.5573 2.6118 6.38E-04 1.64E-02 0.062 Telomeres,Telomerase.
  • Cellular aging and immortality 16 12 37.646 9.5416 5.6001 1.45E-03 2.21E-02 0.066 Keratinocyte differentiation 33 26 30.007 8.9835 4.0308 8.80E-04 1.64E-02 0.072 hsa-miR-30c
  • variable able 4 (continued).

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Abstract

La présente invention concerne un procédé pour prédire ou établir la résistance d'une tumeur de cancer du sein à une endocrinothérapie, comprenant les étapes de détermination du profil d'expression, composé essentiellement des éléments suivants, les ARNmi de hsa-miR-7, hsa-let-7g, hsa-miR-17-5p, hsa-miR-126, hsa-miR-126#, hsa-miR-141, hsa-miR-145, hsa-miR-335, hsa-miR-365, hsa-miR-489, hsa-miR-22, hsa-miR-30a-3p, hsa-miR-30c, et hsa-miR-182, de préférence hsa-miR-30a-3p, hsa-miR-30c, et hsa-miR-182 dans un échantillon d'une tumeur de cancer du sein ; de comparaison dudit profil à un profil de référence ; et de prédiction ou d'établissement de la résistance dudit cancer du sein à ladite endocrinothérapie sur base de ladite comparaison.
PCT/NL2010/050401 2009-06-25 2010-06-25 Procédés pour établir et prédire la résistance à une endocrinothérapie en utilisant un profil d'arnmi WO2010151133A1 (fr)

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WO2013121380A1 (fr) * 2012-02-15 2013-08-22 Basf Se Moyens et procédés pour l'évaluation d'une maladie endocrinienne ou d'un trouble endocrinien
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EP2940151A1 (fr) * 2014-05-02 2015-11-04 Ruprecht-Karls-Universität Heidelberg Circulation de microRNA en tant que marqueur de détection précoce et marqueur pronostique
CN105441449A (zh) * 2015-12-01 2016-03-30 中国人民解放军第四军医大学 一种与他莫昔芬耐药性相关的microRNA分子MiR-141及其应用
CN106222270A (zh) * 2016-08-02 2016-12-14 滨州医学院 一种乳腺癌发生或转移的诊断试剂盒
WO2017207623A1 (fr) * 2016-05-31 2017-12-07 Université de Lausanne Miarn utilisés en tant que biomarqueurs et régulateurs de cellules souches cancéreuses

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013121380A1 (fr) * 2012-02-15 2013-08-22 Basf Se Moyens et procédés pour l'évaluation d'une maladie endocrinienne ou d'un trouble endocrinien
JP2014117282A (ja) * 2012-12-18 2014-06-30 Samsung Electronics Co Ltd 小胞内のポリヌクレオチドを含む乳がん診断用組成物及びキット、並びにそれを利用した乳がん診断方法
EP2940151A1 (fr) * 2014-05-02 2015-11-04 Ruprecht-Karls-Universität Heidelberg Circulation de microRNA en tant que marqueur de détection précoce et marqueur pronostique
US20150315659A1 (en) * 2014-05-02 2015-11-05 Ruprecht-Karls-Universitat CIRCULATING miRNAs AS EARLY DETECTION MARKER AND PROGNOSTIC MARKER
US9683264B2 (en) 2014-05-02 2017-06-20 Deutsches Krebsforschungszentrum Circulating miRNAs as early detection marker and prognostic marker
CN105441449A (zh) * 2015-12-01 2016-03-30 中国人民解放军第四军医大学 一种与他莫昔芬耐药性相关的microRNA分子MiR-141及其应用
WO2017207623A1 (fr) * 2016-05-31 2017-12-07 Université de Lausanne Miarn utilisés en tant que biomarqueurs et régulateurs de cellules souches cancéreuses
CN106222270A (zh) * 2016-08-02 2016-12-14 滨州医学院 一种乳腺癌发生或转移的诊断试剂盒

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