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WO2012056033A1 - Méthode d'analyse d'un échantillon biologique d'un patient - Google Patents

Méthode d'analyse d'un échantillon biologique d'un patient Download PDF

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WO2012056033A1
WO2012056033A1 PCT/EP2011/069064 EP2011069064W WO2012056033A1 WO 2012056033 A1 WO2012056033 A1 WO 2012056033A1 EP 2011069064 W EP2011069064 W EP 2011069064W WO 2012056033 A1 WO2012056033 A1 WO 2012056033A1
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gene
pgr
cancer
sample
malignant
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Axel Niendorf
Klaus Bendrat
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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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to methods for the analysis of a biological sample of a patient suspected of having, developing, or being predisposed to, a malignant or neoplastic disease.
  • Malignant or neoplastic disease especially various types of cancer are a leading causes of death worldwide.
  • simple neoplastic diseases such as benign tumors are self-limited and do not invade or metastasize
  • in cancer i.e., in a malignant disease
  • a group of cells displays uncontrolled growth invasion and sometimes metastasis.
  • cancer is usually treated with a combination of surgery, chemotherapy and radiotherapy.
  • treatments are becoming more specific for different varieties of cancer.
  • Risk assessment is however an important issue.
  • a patient for which a high risk is predicted will have to undergo aggressive medical treatment, including radiotherapy and chemotherapy, which both involve severe side effects, while a patient for which a low risk is predicted can undergo less aggressive treatment, and will thus be saved from severe side effects.
  • multiple 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.
  • endocrine treatment chemotherapy and treatment with targeted therapies.
  • 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.
  • endocrine agents such as tamoxifen with different mode of action and differences in disease outcome exist.
  • the pathological routine diagnostic is still dependent on morphological and clinical parameters, such as tumor type, grade, stage and biomarkers.
  • the Nottingham Prognostic Index combines such parameters into one score and triages tumours into low, medium and high risk of recurrence.
  • This assessment is supplemented by the analysis of molecular markers to add further prognostic information and facilitate therapeutic decisions.
  • EP2036988 describes a method for predicting the response of a tumor in a patient suffering from or at risk of developing recurrent gynecologic cancer towards a chemotherapeutic agent.
  • This approach relies on the predictive power of the several molecular markers such as the estrogen receptor 1 (ESR1) and the progesterone receptor (PGR) and various calculations such as a weighting or scoring of the expression level of the different markers according to their significance.
  • ESR1 estrogen receptor 1
  • PGR progesterone receptor
  • the constants used for weighting or scoring are typically derived as the median expression value of that gene found in samples collected from a representative patient cohort.
  • the expression quotient of the genes HoxB13 and IL17BR is employed for identifying breast cancer patients with early stage estrogen receptor-positive breast cancer that would benefit from treatment with a different agent than tamoxifen (Ma et al. 2004).
  • the gene expression levels of HoxB 13, which is upregulated in recurrence cases that do not benefit from tamoxifen treatment and IL17BR, which is downregulated in recurrence cases that do not benefit from tamoxifen treatment are determined by quantitative real time PCR and the quotient of HoxB13 :IL17BR (H:I) is subsequently calculated for each sample. Since the two genes show opposing expression patterns with regard to clinical outcome this H:I quotient can be used to predict the treatment outcome with tamoxifen, i.e., whether a different treatment might be more successful for a given patient.
  • Fig. 1 A Shows a comparison of estrogen receptor (ESR1) expression levels as determined by protein (immunohistochemistry, IHC) and mRNA (quantitative RT-PCR) detection, respectively.
  • Fig. IB Shows a comparison of progesterone receptor (PGR) expression levels as determined by protein (immunohistochemistry, IHC) and mRNA (quantitative RT-PCR) detection, respectively
  • Fig. 2A Shown is the normalized relative ESR1 expression vs. the normalized relative PGR expression of 218 breast cancer cases ("Hamburg data set").
  • Fig. 2B Shown are the sum of relative ESR1 and PGR expression (S) plotted versus the quotient of relative PGR and ESR1 expression (Q).
  • Fig. 2 C Shown is an identical presentation as in 2B of an external validation using the publicly available data set GSE 3494 [6]
  • a method for the analysis of a biological sample of a patient suspected of having, developing, or being predisposed to, a malignant or neoplastic disease comprising the steps of: a) providing said biological sample,
  • ESR1 estrogen receptor
  • PGR progesterone receptor
  • PGR progesterone receptor
  • ESR1 estrogen receptor
  • biological sample refers to a sample obtained from a patient.
  • the sample may be of any biological tissue, cells or fluid.
  • samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), tissue, nipple aspirate, core or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, urine, peritoneal fluid, and pleural fluid, or cells there from.
  • Biological samples may also include sections of tissues such as frozen or fixed sections taken for histological purposes or microdissected cells or extracellular parts thereof.
  • malignant disease shall relate to a disease with the tendency to become progressively worse and to potentially result in death.
  • neoplastic disease refers in a first instance to disease in which a neoplasm - an abnormal mass of tissue due to abnormal proliferation of cells - is formed. The growth of the cells exceeds, and is uncoordinated with that of the normal tissues around it. The growth persists in the same excessive manner even after cessation of the stimuli.
  • a neoplastic disease does not necessarily have to be malignant.
  • a malignant and neoplastic disease refers to a cancer or a cancerous disease. It includes but is not limited to primary, secondary, or metastatic lesions of cancer patients, and also shall comprise lymph nodes affected by cancer cells or minimal residual disease cells either locally deposited or freely floating throughout the patient's body.
  • cancer is not limited to any stage, grade, histomorphological feature, aggressiveness, or malignancy of an affected tissue or cell aggregation.
  • solid tumors, malignant lymphoma and all other types of cancerous tissue, malignancy and transformations associated therewith, lung cancer, ovarian cancer, cervix cancer, stomach cancer, pancreas cancer, prostate cancer, head and neck cancer, renal cell cancer, colon cancer or breast cancer are included.
  • neoplastic lesion or “neoplastic disease” or “neoplasm” or “cancer” are not limited to any tissue or cell type.
  • determining whether or not the sample is positive for at least one gene means that it is checked whether or not a given gene is expressed in said sample. In a narrower sense, it is meant that the expression of said gene is higher than a certain level.
  • nucleic acid level examples include quantitative PCR, the assessment of the methylation status of a given DNA sequence, the measurement of the number of copies of a given gene as well as analysis of mircoRNAs or siRNAs.
  • level of translated protein examples include immunohistochemistry e.g. with quantitative image analysis, ELISA and mass spectrometry. More preferred options are given in the specification further below.
  • estrogen receptor 1 refers to the gene with the accession numbers NCBI Reference Sequence: NG 008493.1, Entrez Gene ID: 2099 and SEQ ID No. 1.
  • the gene is located on chromosome 6 and encodes a nuclear hormone receptor. Alternative splicing results in several transcript variants, which differ in their 5' UTRs and use different promoters.
  • PGR progesterone receptor
  • the gene is encoding a steroid receptor.
  • gene coexpressed with gene X shall relate to at least two genes, which show the same pattern of expression in response to certain conditions, i.e., which are upregulated or downregulated in a coordinated fashion. This does not mean that both genes are necessarily expressed at the same level, nor that they are involved into the same physiological or developmental processes.
  • co-expression differs from co- regulation in that co-regulation can also refer to a situation where one gene is switched off while the other one is switched on whereas co-expression means that the at least two genes are up- and downregulated at the same time.
  • co-regulation can also refer to a situation where one gene is switched off while the other one is switched on whereas co-expression means that the at least two genes are up- and downregulated at the same time.
  • co-expression means that the at least two genes are up- and downregulated at the same time.
  • a good way to find genes which are coexpressed is to analyze data from a gene chip.
  • Z-ESR1 is the meta-analytical Z- score corresponding to partial coefficient test of the respective gene against ESRl expression.
  • Z-score transformation provides a way of standardizing data across a wide range of experiments and allows the comparison of microarray data independent of the original hybridization intensities. Data normalized by Z score transformation can be used directly in the calculation of significant changes in gene expression between different samples and conditions.
  • the term "gene from the signaling pathway of gene X” shall relate to a gene, which is involved in the signal transduction of gene X.
  • the mitogen-activated protein kinase (MAPK/ERK) pathway and the phosphoinositide 3-kinase (P13K/AKT) pathway are involved in the signaling pathway of estrogen receptors, thus genes encoding for MAPK or ERK would be genes from the signaling pathway of estrogen receptors.
  • Changes in the expression level or expression characteristic e.g. time point, duration etc.
  • the term "expression level of gene X” refers to the amount of gene X mRNA expressed in the biological sample. Moreover, if the levels of expressed mRNA directly correlated with the amount of expressed protein for a given gene X, then the term "expression level of gene X” also refers to the amount of gene X related proteins expressed in the biological sample. This is the case as in such an instance the amount of gene X mRNA expressed in the biological sample can be inferred from the amount of expressed protein.
  • determining the expression level refers to the process of determining whether a gene is expressed and if this is the case assessing to which extend it is expressed. These to assessments are usually carried out in parallel, but of course they can also be carried out after each other. Therefore, the process of determining gene expression may include all necessary preparatory steps know in the art such as protein, mRNA, RNA, DNA and/or cDNA preparation; measurement using techniques such as real time PCR, immunohistochemistry or microarray; basic arithmetic operations such as determining a mean value, if gene expression level for one biological sample is determined using more than one probe since the average of the probes can then be calculated in order to increase the accuracy of the inventive method; etc. .
  • the term "forming a quotient on the basis of said at least two expression levels" refers to the division of one of the determined expression levels by the other one. Thus, the term does not refer to the formation of a quotient using the expression level of the same gene. Moreover, the term excludes weighting or scoring of the determined expression levels for example according to their significance in disease stage or disease progression. It is particularly preferred that for the conclusion in step d), the expression level of the respective genes is determined with immunohistochemical methods or PCR-based methods.
  • the expression level of the respective genes is determined with immunohistochemical methods or PCR-based methods.
  • the inventive method can be used to analyze a biological sample of a patient suspected of having, developing, or being predisposed to, a malignant or neoplastic disease while being considerably less cumbersome than methods known from the art. Especially no weighting or scoring of gene expression levels has to be carried out and the method gives a result independent from a prediction, if a specific treatment e.g. with tamoxifen, benefits a given patient.
  • the gene expression level of a gene co-expressed with ESR1 and the gene expression level of a gene co-expressed with PGR are determined. Then, the quotient of the expression level value of the gene co-expressed with PGR over of the expression level value of the gene co-expressed with ESR1 can be used to predict for a specific patient whether or not that patient falls into a group which usually shows favorable clinical courses.
  • the term "favorable outcome” or “favorable clinical outcome” as used herein refers to the likelihood that given patient will survive or that a malignant or neoplastic disease will not reoccur, if it turns out that that patient indeed has, develops or is predisposed to a malignant or neoplastic disease
  • step d) comprises the step of checking whether or not a given sample is receptor-positive for ESR1 and/or PGR using immunohistochemical methods.
  • cells are considered to be "receptor-positive", if they show distinct nuclear staining with an adequate receptor-specific staining method, e.g., with a receptor specific antibody which is either labelled as such, or is detected by a secondary antibody which is labelled.
  • This embodiment is advantageous since it is in accordance with routine clinical practice and thus the assessment whether or not a given sample is receptor-positive for ESR1 and PGR using immunohistochemical methods is readily available.
  • the method further comprises the step of normalizing at least one of the determined gene expression levels.
  • normalization of the determined expression levels refers to the process of removing error from measured data. Normalization can be carried out against an endogenous unregulated reference gene transcript or against total cellular DNA or RNA content (molecules/g total DNA/RNA and concentrations/g total DNA/RNA). For example when using quantitative real time PCR to determine gene expression level genes, which are largely unregulated are usually assessed in parallel with the target genes. These unregulated genes are termed housekeeping genes.
  • Housekeeping gene refers to genes that usually code for proteins whose activities are essential for the maintenance of cell function. They are thus ubiquitous genes expressed in most organ, tissue and/or cell types of an organism in a mainly unregulated or only weakly regulated fashion, or regulated to a constant gene expression rate. Housekeeping genes include, without limitation, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), Cypl, albumin, actins, e . g .
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • Cypl Cypl
  • albumin actins
  • ⁇ -actin tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), Ribosomal protein L32, 28S, 18S, large ribosomal protein (RPLPO), Transferrin receptor (TFRC) and beta-glucuronidase (GUS).
  • This step is advantageous whenever still other arithmetic operations than formation of a quotient on the basis of said at least two expression levels are to be carried out.
  • step d) comprises the step of checking whether or not the expression level of said gene is higher than a predetermined threshold level.
  • threshold level refers to a level of gene expression above a certain point as determined for example using calibrator samples in a qPCR run.
  • calibrator sample refers to a mixture of cDNAs prepared from different cells, tissues and/or organs, e.g., carcinomas and normal tissues from several organs (breast, lung and colon).
  • a calibrator sample is used as a reference sample for every PCR run and serves two purposes, namely a) to measure the PCR efficiency of each individual run, and b) as a standard for relative quantification, wherein every marker or gene of the study is quantified relative to the calibrator sample.
  • Calibrator samples are obsolete in case a large microarray is being used, as for example an Affymetrix U133 microarray, which covers a large amount of genes (see Fig. 2C).
  • a threshold level is especially advantageous, if gene expression level is inferred from protein expression data. This is the case as the threshold level can be used to asses whether levels of expressed mRNA directly correlated with the amount of expressed protein for a given gene. This is for example the case for ESR1 and PGR as shown in Fig. 1A and Fig. IB.
  • Suitable, and preferred, methods to determine the expression level of a gene as set forth in step d) may be selected from the group consisting of: ⁇ IHC (Immunohistochemistry) combined with quantitative image analysis
  • step d) On the nucleic acid level, other suitable, and preferred, methods to determine the expression level of a gene as set forth in step d) may be selected from the group consisting of:
  • the method further comprises the step of sorting and/or forming a ratio of at least two of the gene expression levels determined. These steps are advantageous in order to determine, which values of the determined expression levels are useful for further analysis.
  • sorting of at least two expression levels determined refers to the process of bringing the expression levels determined for a given gene into an descending order. Therefore, this step is only applicable, if gene expression level is determined in duplicate, triplicate etc.
  • forming a ratio of at least two expression levels determined refers to the process of dividing gene expression levels determined for the same gene through each other. Hence the term ratio differs herein from the term quotient. Whereas the term ratio relates to the dividing expression levels of the same gene the term quotient relates to a division using gene expression levels of two different genes.
  • the method according to the invention further comprises, the step of categorizing at least two of the determined ratios.
  • categorizing refers to the process of classifying gene expression level ratios into quality groups
  • This step is especially advantageous, as it can be employed to further determine, which values of the determined gene expression levels are useful for further analysis.
  • the ratios of the gene expression values Rl and R2 already used for demonstration purposes above are categorized into three quality groups. If the ratio is smaller than 1.15 it is categorized into category 1, when the ratio is between 1.15 and 2 it is categorized into category 2 and if it is above 2 it is categorized into category 3. If the two ratios Rl and R2 of a triplicate measurement fall into the same category, the mean of all three measurements is used for further analysis. If Rl and R2 fall into different categories only the two values closer to each other are combined and averaged.
  • the step of determining whether or not the sample is positive for at least one of the two genes comprises the step of adding at least two of the gene expression levels determined, and checking whether or not the resulting sum is higher than a predetermined threshold level.
  • the inventive method can advantageously be used as basis for stratification for an individual patient.
  • the sum off ESR1 and PGR gene expression levels can be used in combination with the quotient of PGR over ESR1 for risk stratification of a given patient.
  • This is shown in Fig. 2B in an exemplary fashion.
  • the figure depicts the sum of the receptor expression S, here the sum of the gene expression levels of PGR and ESR1 versus the quotient Q (PGR/ESRl).
  • the inventive method surprisingly allows for a fast and simple analysis of a biological sample of a patient suspected of having, developing, or being predisposed to, a malignant or neoplastic disease as well as a prediction whether the clinical outcome of a given patient is favorable or not.
  • this prediction alone does not automatically lead to a specific therapeutic decision, but it can be used to facilitate the therapeutic decision that has to be made for a given patient.
  • the method is characterized in that the formed quotient is a quotient of the gene expression levels of PGR and ESR1.
  • the formed quotient is a quotient of the gene expression levels of PGR and ESR1.
  • said malignant or neoplastic disease is a hormone-dependent malignant or neoplastic disease.
  • hormone-dependent disease shall relate to a disease responsive to presence and/or absence and/or level of one or more hormones. For example some men with advanced prostate cancer, are responsive to withdrawal of the hormone testosterone. In these patients lowering testosterone levels can cause tumors to shrink or slow their growth.
  • the malignant or neoplastic disease is a gynaecological cancer.
  • cancer typically affects two general classes of genes. Cancer-promoting oncogenes are usually activated in cancer cells, giving those cells new properties, such as hyperactive growth and division, protection against programmed cell death, loss of respect for normal tissue boundaries, and the ability to become established in diverse tissue environments. Tumor suppressor genes are inactivated in cancer cells, resulting in the loss of normal functions in those cells, such as accurate DNA replication, control over the cell cycle, orientation and adhesion within tissues, and interaction with protective cells of the immune system.
  • the determination of the expression level of a gene from the ESR1 and/or the PGR signaling pathway might be a tumor suppressor or an oncogene as these are likely to correlate with the predictive power of the ESR1 and/or the PGR gene expression level.
  • glycosaecological cancer shall relate to a group of cancers that affect the tissue and organs of the female reproductive system including the breast tissue. Each type of cancer is named after the organ it originates e.g. the uterus, ovaries, cervix, fallopian tubes, vulva, vagina etc..
  • vaginal cancer • vaginal cancer, and/or
  • breast cancer is particularly preferred.
  • the term "predisposition" refers to an increased risk of having or developing a disease, for example because a given patient has a certain set of alleles.
  • prognosis refers to the likely outcome of said malignant or neoplastic disease.
  • prognostic estimates can be very accurate: for example the statement “45% of patients with severe septic shock will die within 28 days” can be made with some confidence, because previous research found that this proportion of patients died.
  • additional information is needed to determine whether a patient belongs to the 45% who will succumb, or to the 55% who survive.
  • a complete prognosis includes expected time and a description of the disease course such as progressive decline, intermittent crisis, or sudden, unpredictable crisis.
  • the term "prognosis” relates to the risk of a patient developing metastasis, recurrence or death.
  • prediction towards a given cancer treatment relates to an individual assessment of the expected survival rate (DFS, disease free survival) of a patient, if the malignant or neoplastic is treated with a given therapy.
  • DFS expected survival rate
  • RT-PCR stands for reverse transcription PCR and is often erroneously mixed up with the term “real time PCR”. It is a variant of the polymerase chain reaction (PCR), in which an RNA strand, e.g., from an mRNA, is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified and/or detected using traditional or real-time PCR.
  • PCR polymerase chain reaction
  • Quantitative real-time PCR which is also referred to as quantitative PCR (“qPCR”) refers to any type of a PCR method, which allows the quantification of the template in a sample.
  • the setup of the reaction is very similar to conventional end point PCR, but the reaction is carried out in a real-time thermal cycler that allows measurement of fluorescent molecules.
  • probes in qPCR include the following probes: Taqman probes, Molecular Beacons probes, Scorpions probes, and SYBR Green probes. Briefly, Taqman probes, Molecular Beacons, and Scorpion probes each have a fluorophore attached to the 5' end of the probes and a quencher moiety coupled to the 3' end of the probes.
  • the proximity of the fluorophore and the quencher moiety prevents the detection of fluorescent signal from the probe.
  • the 5' to 3' exonuclease activity of the Taq polymerase degrades that proportion of the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore.
  • the exponential increase of the fluorescence is used to determine the threshold cycle, CT, i.e., the number of PCR cycles at which a significant exponential increase in fluorescence is detected, and which is directly correlated with the number of copies of DNA template present in the reaction.
  • CT threshold cycle
  • fluorescence detected in the real-time PCR thermal cycler by Taqman probes, Molecular Beacons, and Scorpion probes is directly proportional to the fluorophore released and the amount of DNA template present in the PCR. This is not necessarily the case when using SYBR Green as this dye will intercatalate with any double stranded nucleic acid, not only with the template. Therefore, when using SYBR Green usually a melt curve analysis is also carried out to ensure that the signals originates from the actual target DNA.
  • nucleic acid microarray (also termed “DNA microarray” or “DNA chip”) relates to a multiplex technology used in molecular biology. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles of a specific DNA sequence, known as probes. This can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample (called target) under high- stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.
  • Examples for isothermal DNA/RNA amplification techniques include LAMP (loop-mediated amplification), RDC (Reaction Deplacement Chimeric, strand displacement) and NASBA (Nucleic Acid Sequence Based Amplification).
  • LAMP loop-mediated amplification
  • RDC Reaction Deplacement Chimeric, strand displacement
  • NASBA Nucleic Acid Sequence Based Amplification
  • hybridization refers to the pairing of complementary nucleic acids.
  • in situ hybridization refers to a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough, in the entire tissue (whole mount ISH). This is distinct from immunohistochemistry, which localizes proteins in tissue sections.
  • immunohistochemistry refers to the process of localizing antigens (e.g. proteins) in cells and/or cells of a tissue section exploiting the principle of antibodies binding specifically to antigens. Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events, such as proliferation or cell death. Visualising an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore thus employing the principles of immunofluorescence.
  • an enzyme such as peroxidase
  • the antibody can also be tagged to a fluorophore thus employing the principles of immunofluorescence.
  • Immunohistochemistry can also be used to evaluate tumor content in the sample on which qPCR is carried out in order to account for the fact that QPCR result will be influenced by the amount of tumor tissue present.
  • the sample is a fresh sample or a frozen sample.
  • the sample is a fixed sample, preferably a formalin fixed paraffin embedded (“FFPE”) sample.
  • FFPE formalin fixed paraffin embedded
  • microslicing refers to the process of cutting extremely thin slices, 0.05 - ⁇ , of material, known as sections. Microslicing is for example carried out using a microtome which allows for the preparation of samples for observation under transmitted light or electron radiation.
  • microdissection refers to the process of cutting apart or separating tissue, cells, chromosomes etc. on mircometer scale. Microdissection is for example carried out using a laser microdissection apparatus for isolating specific cells of interest from microscopic regions of tissues, cells or organisms.
  • needle aspiration refers to a diagnostic procedure used to investigate superficial lumps or masses. In this technique, a thin, hollow needle is inserted into the mass to extract cells that, after being stained, will be examined under a microscope. Fine needle aspiration biopsies are very safe, minor surgical procedures. Often, a major surgical (excisional or open) biopsy can be avoided by performing a needle aspiration biopsy instead.
  • the method according to the invention is carried out using fresh frozen material from breast cancer patients receiving standard therapy.
  • total RNA is extracted using a QIAcube and the miRNeasy mini Kit (QIAGEN, Hilden) and three independent reverse transcription reactions are synthesized for each sample.
  • the three independent cDNA preparations are pooled and PCR analyses of cDNA, in this example for the ESR1 and the PGR, are conducted in triplicates using the QuantiFast SYBR Green PCR Kit (QIAGEN) and a 7500 fast PCR machine (ABI).
  • QIAGEN QuantiFast SYBR Green PCR Kit
  • ABSI fast PCR machine
  • a quotient of PGR over ESR1 is formed that can be used to predict for a specific patient whether or not that patient falls into a group which usually shows favorable clinical course.
  • This can be deducted for example from Fig. 2A, where the said group with favorable clinical courses is situated in the upper right quadrant, i.e., all cases show expression of PGR and ESR1. That this patient group has a favorable clinical courses compared to ESR1 and/or PGR negative patients is known, however, it was surprisingly found that a further subdivision of the ESR1 + /PGR + group can be established by using the PGR/ESRl quotient.
  • the PGR/ESRl quotient is high (Expression level of PGR higher than of ESR1), i.e., the value lies above the diagonal denoting equal expression of ESR1 and PGR the outcome is especially favorable even compared to other ESR1 + /PGR + cases in which ESR1 is expressed to a higher extend than PGR (see Fig. 2A).
  • the first assessment into which outcome group a patient falls, can be further supported by a second arithmetic operation, namely by formation of the sum of ESRl and PGR gene expression levels. This sum is then used in combination with the above described quotient of PGR over ESRl for risk stratification of a given patient.
  • a plot of the sum of receptor expression S (PGR+ESR1) versus the quotient Q (PGR/ESRl) is established on which distinct areas correlate with different outcome groups.
  • This is shown in Fig. 2B in an exemplary fashion.
  • the figure depicts the sum of the receptor expression S, here the sum of the gene expression levels of PGR and ESRl, versus the quotient Q (PGR/ESRl).
  • Threshold values are 7.85 for S and 1.5 for Q. They define 3 distinct areas that correlate with different outcome groups.
  • this low risk group can surprisingly be further divided into a medium risk group in the lower right part (S>7.85 and Q ⁇ 1.5) as well as a low risk group with the best survival properties on the upper right part of the plot (S>7.85 and Q>1.5).
  • Fig. 1A Shows a comparison of estrogen receptor (ESRl) expression levels as determined by protein (immunohistochemistry, IHC) and mRNA (quantitative RT-PCR) detection, respectively. 314 breast cancer specimens were assessed. There were 174 control cases (favourable clinical course; filled diamonds) and 140 events (relapse or death; filled squares). The large symbols of the left curve stand for 36 ESRl -IHC negative tumors (10 controls and 26 events, whereas the small symbols of the right curve stand for 278 ESRl -IHC positive tumors (164 controls, 114 events). The filled squares depict death/relapse cases and the filled diamonds depict non-recurrence cases (controls).
  • ESRl estrogen receptor
  • Fig. IB shows a comparison of progesterone receptor (PGR) expression levels as determined by protein (immunohistochemistry, IHC) and mRNA (quantitative RT-PCR) detection, respectively.
  • PGR progesterone receptor
  • the presentation is the same as in Fig. 1A, i.e., the large symbols of the left curve stand for 67 PGR-IHC negative tumors (23 controls, 44 events), whereas the small symbols of the right curve stand for 247 ESR1-IHC positive tumors (151 controls, 96 events).
  • the filled squares depict death/ relapse cases and the diamonds depict non-recurrence cases (controls).
  • FIG. 2A Shown is the normalized relative ESRl expression vs. the normalized relative PGR expression of 218 breast cancer cases ("Hamburg data set") as investigated with the methods discussed above. Of the 218 cases 147 were controls (filled squares) and 71 were events (crosses). The ESRl(-) PGR(-) double negative cases form a distinct cluster in the lower left quadrant. ESR(+) PGR(-) cases are found further to the right with PGR values ⁇ 1 and ESRl values up to 55. ESR1(+) PGR(+) double positive cases lie in the upper right part of the plot.
  • Fig. 2B Shown are the sum of relative ESRl and PGR expression (S) plotted versus the quotient of relative PGR and ESRl expression (Q) for the same 218 cases as in Fig. 2 A.
  • threshold values were set by correlation of the S- and Q-values with the clinical outcome. It turned out that in cases with values S >7.85 (i.e., on the right of the solid vertical line) the quotient Q is strongly correlated with clinical outcome. Furthermore, it turned out that most favorable outcomes show a (PGR/ESRl) quotients Q > 1.5 (dashed horizontal line). Even more surprisingly, however, the inventors found a second cohort of patients with a favorable clinical outcome. These patients have a high S value, but a very small (PGR/ESRl) quotient Q (i.e., ESR expression is higher than PGR expression).
  • Fig.2C Shown is a similar presentation as in 2B, but with external data from the publicly available data set GSE 3494 [6].
  • the said data have been obtained on Affymetrix U133 A and B arrays.
  • ESR1/PGR (+/+) double positive cases (S>2250) with a high PGR/ESRl quotient (Q>0.33) mostly fall into the favourable clinical outcome group (see also table 2).
  • Fig. 2C the publicly available data set GSE 3494 [6] was used, and, as can be seen, it turned out that the treshold value for S was 2250, compared to 7,85 in Fig. 2A (Hamburg data set).
  • the treshold value for Q was 0.33, compared to 1.5 in Fig. 2 A.
  • the method according to the present invention has to be calibrated individually for a given experimental design (analysis platform primers, buffers etc), with a sample set from a well documented cohort of patients. Once the S- and Q-values have been obtained, the latter will be compared with clinical outcomes of the respective patients, in order to determine the respective treshold values (either mathematically of according to the know how of the respective practitioner), for the assessment of an individual patient. For this reason, the threshold values given herein are exemplary values only, which are applicable for the specific experimental approach, but should by no means be understood as absolute values. 1.
  • RNA isolation and qRT-PCR Total RNAs were extracted automatically from fresh frozen material (three consecutive 5 ⁇ sections) using a QIAcube and the miRNeasy mini Kit (QIAGEN, Hilden) and stored at - 80°C until use. Another section was stained to evaluate tumor content in the sample. Three independent two step reverse transcription reactions (20 ⁇ 1, each containing approx. 300 ng total RNA) were synthesized for each tumor sample according to the manufacturer ' s protocol (miScript Reverse Transcription Kit, QIAGEN). The three independent cDNA preparations were pooled (60 ⁇ ) and diluted 1 : 15 with water resulting in a total volume of 900 ⁇ .
  • PCR analyses of cDNA were conducted in triplicates using the QuantiFast SYBR Green PCR Kit (QIAGEN) and a 7500 fast PCR machine (ABI).
  • a typical 96 well plate contained 30 samples (3x) plus a dilution series (lx, 6x, 36x, 216x, 1296x, etc) of a "calibrator" sample (i.e., a cDNA from a pooled mix of 23 tumor samples) for relative quantification purposes and for determination of individual PCR run efficacy. All samples of one PCR run were set up from one master mix containing primers and reaction mix.
  • Primer sequences for ESR1 and PGR were from Geneglobe (QIAGEN), primer sequences for ERBB2 and the 5 housekeepers (ACTB, GAPDH, GUSB, RPLP0, TFRC) were from reference [1] 1.5 qPCR data processing
  • triplicate ct-values were transformed into triplicate relative mRNA amounts and the PCR efficacy of that individual run was determined.
  • the two ratios were categorized into three quality groups: Q-group 1 if the ratio was smaller than 1.15 (i.e., less than 15% difference between the two values), Q-group 2 if the ratio was between 1.15 and 2 (between 15 and 100% difference) and Q-group 3 if the ratio was above 2 (more than 100% difference). If the two ratios Rl and R2 of a triplicate measurement fell into the same Q- group, the mean of all three measurements was used for further analysis. If Rl and R2 fell into different Q-groups only the two better values were combined and averaged. Relative gene expression values were normalized using the geometric mean of 5 reference genes (ACTB, GAPDH, GUSB, RPLP0, TFRC [1]).
  • Fig. 1A demonstrate the cumulative distribution of 36 IHC negative and 278 positive breast cancer cases for ESR1 expression, demonstrating an excellent correlation between protein and mRNA expression.
  • a second treshold (1.9; solid line) can be defined on the basis of medical criteria.
  • the concordance of IHC and qRT-PCR for progesterone receptor testing is shown in Fig. IB.
  • the PCR-IHC concordance for PGR is lower than for ESR1.
  • a plot of the receptor expression of the remaining 217 cases demonstrates the categories from IHC- diagnostics and the power of ESR1 and PGR (Fig. 2A): ESR1/PGR (-/-) double negative tumors (ESR1 ⁇ 1 and PGR ⁇ 1 : 10 controls, 16 events) form a distinct group in the lower left quadrant. Further to the right with increasing ESR1 expression and a PGR expression ⁇ 1 there is the group of ESRl/PGR (+/-) with 15 controls and 11 events. Only one control and three events fall into the ESRl/PGR (-/+) category.
  • the meaning of the receptor quotient Q is clearer visible in a plot of the sum of receptor expression S (PGR+ESR1) versus the quotient Q (PGR/ESRl) in Fig. 2B.
  • the publicly available data set GSE 3494 was used for external validation. After exclusion of 80 cases with unclear clinical outcome the data set comprised 111 controls and 45 deaths. Although chip data are scaled differently, the same pattern is obvious: the low risk group with the most favourable outcome is characterized by S (PGR+ESR1)>2250 and a quotient Q (PGR/ESR1)>0.33. Data are summarized in table 2.
  • the S/Q quotient In comparison to the NPI and receptor status the S/Q quotient has the best performance in almost every category, i.e., when using the S/Q quotient the prediction corresponds to the actual outcome in the highest number of cases especially with the extremely high negative predictive value (NPV) of 92% (Hamburg data set) and 94% (GSE3494).
  • NPV extremely high negative predictive value
  • Table 1 clinical data of Hamburg breast cancer samples n n recurrent

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Abstract

La présente invention concerne une méthode d'analyse d'un échantillon biologique d'un patient susceptible d'avoir, de développer ou d'être prédisposé à une malignité ou à une maladie néoplasique, ladite méthode comprenant les étapes consistant à : obtenir ledit échantillon biologique, déterminer au moins le niveau d'expression du gène du récepteur des œstrogènes (ESR1), d'au moins un gène coexprimé avec ce dernier et/ou d'au moins un gène issu de la voie de signalisation d'ESR1, déterminer au moins le niveau d'expression du gène du récepteur de la progestérone (PGR), d'au moins un gène coexprimé avec ce dernier et/ou d'au moins un gène issu de la voie de signalisation de PGR, déterminer si l'échantillon est positif ou non pour au moins un des gènes déterminés dans les étapes b) et c) et comparer au moins le niveau d'expression du gène du récepteur de la progestérone (PGR), d'au moins un gène coexprimé avec ce dernier et/ou d'au moins un gène issu de la voie de signalisation de PGR, au niveau d'expression du gène du récepteur des œstrogènes (ESR1), d'au moins un gène coexprimé avec ce dernier et/ou d'au moins un gène issu de la voie de signalisation d'ESR1.
PCT/EP2011/069064 2010-10-29 2011-10-28 Méthode d'analyse d'un échantillon biologique d'un patient Ceased WO2012056033A1 (fr)

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