Method for classifying patients with solid tumors - Patents.com

Abstract

The present invention relates to a method for classifying patients suffering from solid tumors, in particular for prognosticating the survival time of patients suffering from solid tumors and / or for testing the responsiveness of patients suffering from solid tumors to antitumor treatment. The method is based on quantifying multiple immune response markers and comparing them to a reference distribution to determine the percentile of the distribution to which the values ​​correspond. The mean or median of the determined percentiles for the different markers is calculated, and this value is compared to the mean or median percentile reference value, with the result correlating to survival or response.

Description

[Technical field]

[0001] The present invention relates to a method for classifying patients suffering from solid tumors, in particular for the prognosis of survival time of patients suffering from solid tumors and / or for testing the responsiveness of patients suffering from solid tumors to antitumor treatment. [Background technology]

[0002] As detailed by Galon et al. (Cancer Classification Using Immunoscore: A Global Task Force J Transl Med. 2012; 10: 205), prediction of clinical outcome in cancer is usually achieved by histopathological examination of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC / UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N), and signs of metastasis (M). However, it is now recognized that clinical outcomes can vary significantly between patients with the same stage. Current classifications provide limited prognostic information and do not predict response to treatment. Recent literature suggests the importance of the host immune system in controlling tumor progression. And evidence supports the idea of ​​including immunological biomarkers as tools to predict prognosis and response to treatment. Accumulating data collected from large cohorts of human cancers have shown the impact of immunoclassification with prognostic value that may add to the significance of AJCC / UICC TNM classification. It is therefore essential to introduce the "Immunoscore" into traditional classification and begin to provide an essential prognostic and powerful predictive tool. Introducing this parameter as a biomarker to classify cancers as part of the routine diagnostic and prognostic evaluation of tumors will facilitate clinical decision making, including rational stratification of patient treatment.

[0003] Numerous patent applications describe methods for prognosticating the survival of patients with solid tumors and / or for assessing the response of patients with solid tumors to antitumor treatment by measuring immunological biomarkers. For example, WO2015007625, WO2014023706, WO2014009535, WO2013186374, WO2013107907, WO2013107900, WO2012095448, WO2012072750 and WO2007045996 can be mentioned. All these methods provide good results. As explained in the review by Galon et al., Immunity 39, 2013, 11-26, prognostic and predictive immune signatures overlap widely. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2015007625 [Patent Document 2] WO2014023706 [Patent Document 3] WO2014009535 [Patent Document 4] WO2013186374 [Patent Document 5] WO2013107907 [Patent Document 6] WO2013107900 [Patent Document 7] WO2012095448 [Patent Document 8] WO2012072750 [Patent Document 9] WO2007045996 [Non-patent literature]

[0005] [Non-Patent Document 1] J Transl Med. 2012;10:205 [Non-Patent Document 2] Galon et al., Immunity 39, 2013, 11-26 Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have further improved the accuracy of methods for prognosticating survival time in patients with solid tumors and for assessing the response of patients with solid tumors to antitumor treatment. [Means for solving the problem]

[0007] The present invention relates to an in vitro method for prognosing survival of a patient suffering from a solid tumor, said method comprising the steps of: a) quantifying two or more biological markers indicative of the state of the patient's immune response to the cancer, wherein each biological marker indicative of the state of the immune response is quantified in a tumor sample obtained from the patient; b) comparing each of the values ​​obtained in step a) for said two or more biological markers with a distribution of values ​​obtained for each of said two or more biological markers from a reference group of patients affected by said cancer; c) determining for each of the values ​​obtained in step a) for said two or more biological markers a percentile of the distribution corresponding to the value obtained in step a); d) calculating the arithmetic mean or median value of the percentiles; and e) comparing a predetermined reference arithmetic mean or a predetermined median value of the percentiles with the arithmetic mean or median value of the percentiles obtained in step d), said reference value correlating with survival time.

[0008] Typically, survival is disease-free survival (DFS), progression-free survival (PFS), disease-specific survival (DSS) or overall survival (OS).

[0009] The present invention also relates to an in vitro method for assessing the response of a patient suffering from a solid tumor to an antitumor treatment, said method comprising the steps of: a) quantifying two or more biological markers indicative of the state of the patient's immune response to the cancer, wherein each biological marker indicative of the state of the immune response is quantified in a tumor sample obtained from the patient; b) comparing each of the values ​​obtained in step a) for said two or more biological markers with a distribution of values ​​obtained for each of said two or more biological markers from a reference group of patients affected by said cancer; c) determining for each of the values ​​obtained in step a) for said two or more biological markers a percentile of the distribution corresponding to the value obtained in step a); d) calculating the arithmetic mean or median value of the percentiles; and e) comparing a predetermined reference arithmetic mean or a predetermined median value of a percentile of a predetermined reference value with the arithmetic mean or median value of the percentile of step d), wherein said reference value correlates to said anti-tumor treatment.

[0010] The term "arithmetic mean" should be understood broadly and includes the "classical" arithmetic mean of a sample, where the sum of the sample values ​​is divided by the number of elements in the sample, as well as weighted arithmetic means.

number

[0011] The weights Wi may indicate a measure for the reliability of the influence of each value on the average.

[0012] Median values ​​are particularly applicable when many (typically 5, 10, 15, 200 or more) biological markers are quantified.

[0013] The method of the present invention has unique and multiple advantages.

[0014] The biological markers considered in these methods have important and specific properties, which make the methods extremely useful and have a strong discriminatory power.

[0015] The biomarkers considered are immune biomarkers. In particular, the biomarkers are adaptive immune biomarkers. One of the essential properties is that these immune biomarkers are associated with the time to relapse, the time before death, the time before treatment response, the amplification of the treatment response, and the extended period during which the patient responds to treatment.

[0016] One advantage of the methods of the present invention is that the level of intensity, expression, concentration, or amount of each biomarker correlates with length, survival, extended survival, and extended response to treatment.

[0017] The methods of the present invention are applicable because the higher or lower the level of the biomarker, the longer or shorter the survival time and the better or worse the response to treatment.

[0018] One important novel element related to the great feasibility of the method is that it is independent of the patient's clinical data and follow-up: mean percentiles can be calculated based on the raw and normalized biomarker data, independent of the patient's outcome.

[0019] A globally applicable immune response is characterized by a set of biomarkers that, when enriched together, improve stratification of patients into categories of extended survival and better response to treatment.

[0020] The percentile average of all biomarkers, weighted by each individual biomarker, allows for highly accurate stratification of patients into risk categories.

[0021] Typically, said reference group of patients affected by cancer comprises at least: 100, 200, 300, 400, 500, 1000 or 2000 patients.

[0022] Typically, the methods of the present invention are applied to various tissues of cancer origin (e.g., breast cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, bladder cancer, ovarian cancer, prostate cancer), as well as various types of cancer cells (adenocarcinoma, squamous cell carcinoma, large cell carcinoma, melanoma, etc.).

[0023] Typically, the patient subjected to the above method may suffer from a solid cancer selected from the group consisting of adrenocortical carcinoma, anal carcinoma, bile duct carcinoma (e.g., peripheral carcinoma, distal bile duct carcinoma, intrahepatic cholangiocarcinoma), bladder carcinoma, osteosarcoma (e.g., osteoblastoma, chondrosarcoma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, multiple myeloma), brain and central nervous system cancer (e.g., , meningioma, astrocytoma, oligodendroglioma, ependymoma, glioma, medulloblastoma, glioma, schwannoma, germinoma, craniopharyngioma), breast cancer (e.g., ductal carcinoma in situ, invasive ductal carcinoma, invasive lobular carcinoma, noninvasive carcinoma) lobular carcinoma, gynecomastia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenoacanthomas, papillary serous adenocarcinoma, clear cell), esophageal cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoma tumors (e.g. choriomas, destructive chorioadenomas), Kaposi's sarcoma, kidney cancer (e.g. renal cell carcinoma), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatocellular adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung carcinoma, non-small cell lung carcinoma), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. nasal neuroblastoma, midline granuloma), nasopharyngeal carcinoma, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g., embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g., melanoma, non-melanoma skin cancer), stomach cancer, testicular cancer (e.g., seminoma, non-seminomatous germ cell carcinoma), thymus cancer, thyroid cancer (e.g., follicular adenocarcinoma, undifferentiated carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g., uterine leiomyosarcoma).

[0024] In a preferred embodiment, the cancer is colorectal cancer.

[0025] As used herein, "tumor tissue sample" refers to a tumor sample of any tissue derived from a patient. The tissue sample is obtained for the purpose of in vitro evaluation. In some embodiments, the tumor sample may be obtained from a tumor excised from a patient. In some embodiments, the tumor sample may be obtained from a biopsy performed on the patient's primary tumor or on a metastatic sample distant from the patient's primary tumor. For example, an endoscopic biopsy performed on the intestine of a patient suffering from colorectal cancer. Typically, the tumor tissue sample is fixed in formalin, embedded in a robust fixative such as paraffin (wax) or epoxy, and placed in a mold and later hardened to produce a block that can be easily cut. Thin slices of the material can then be prepared using a microtome, placed on glass slides, and subjected to, for example, immunoanatomical analysis (using an IHC automated device such as BenchMark® XT to obtain stained slides). The tumor tissue sample can be used in a microarray called a tissue microarray (TMA). TMA consists of a paraffin block in which up to 1000 isolated tissue cores are assembled into an array, allowing multiplexed histological analysis. This technique allows rapid identification of molecular targets at either the DNA, RNA or protein level in a tissue sample at once. TMA technology has been described in WO2004000992, US8068988, Olli et al 2001 Human Molecular Genetics, Tzankov et al 2005, Elsevier, Kononen et al 1198; Nature Medicine.

[0026] In several embodiments, tumor tissue samples include: (i) a spherical primary tumor (as a whole); (ii) tissue samples from the center of the tumor; (iii) tissue samples from tissue immediately surrounding the tumor, which may more specifically be termed the "invasive margin" of the tumor; (iv) lymphatic islets in close proximity to the tumor; (v) lymph nodes located in close proximity to the tumor; (vi) tumor tissue samples collected prior to surgery (e.g., for patient follow-up after treatment); and (vii) metastases to distant sites.

[0027] As used herein, the term "invasive margin" has its usual meaning in the art and refers to the cellular environment surrounding a tumor. In some embodiments, the tumor tissue sample includes a block or section of tissue removed from the tumor center or the invasive margin surrounding the tumor, including following surgical tumor resection or collection of tissue samples for biopsy, further quantification of one or more biological markers, particularly through anatomical or immunocytochemical methods, through flow cytometry methods, and through gene or protein expression analysis methods, including genomics and proteomic analysis, whether from the tumor center, the invasive margin of the tumor, or a nearby lymph node. Of course, the tumor tissue sample can be subjected to various known post-collection preparation and preservation techniques (e.g., fixation, storage, freezing, etc.). The sample can be fresh, frozen, fixed (e.g., formalin-fixed) or embedded (e.g., paraffin-embedded). In some embodiments, when the quantification of multiple tumor-draining lymph nodes is performed on a resectioned tumor, the tumor tissue sample is derived from the resectioned tumor, including the tumor center and optionally the tumor's invasive edge. In said embodiments, the quantification of the markers of immune adaptive response is typically performed by immunoanatomy (IHC) as described below. In some embodiments, when the quantification of multiple tumor-draining lymph nodes is measured by imaging, the tumor tissue sample may be the result of a biopsy. In said embodiments, the quantification of the markers of immune adaptive response may typically be performed by measuring the expression level of at least one gene.

[0028] Typically, the tumor tissue sample may be selected from the group consisting of: (i) a spherical primary tumor (as a whole); (ii) a tissue sample from the center of the tumor; (iii) a tissue sample from the tissue immediately surrounding the tumor, which may more specifically be termed the "invasive margin" of the tumor; (iv) lymphatic islets in close proximity to the tumor, or tertiary lymphatic structures derived by the tumor; (v) a tumor biopsy taken at any time, and typically prior to surgery; and (vii) a metastasis to a distant site.

[0029] In a preferred embodiment, two or more biological markers are quantified in the center of the tumor and / or the invasive margin of the tumor.

[0030] In a preferred embodiment, two or more biological markers are quantified in the center of the tumor and / or the invasive margin of the tumor.

[0031] The sample can be fresh, frozen, fixed (e.g., formalin-fixed) or embedded (e.g., paraffin-embedded). In a preferred embodiment, the tumor sample is the result of a biopsy performed on the patient's tumor.

[0032] An example is an endoscopic biopsy performed on the abdomen of a patient suffering from or suspected of suffering from colorectal cancer.

[0033] As used herein, a "biological marker" consists of any detectable, measurable and quantifiable parameter that is indicative of the status of a cancer patient's immune response against the tumor.

[0034] Biological markers include the presence, or number or concentration, of cells derived from the immune system at the tumor site.

[0035] Biological markers also include the presence or amount of proteins specifically produced by cells from the immune system at the tumor site.

[0036] Biological markers also include the presence or amount of any biological substance at the tumor site that is indicative of the expression level of genes associated with eliciting a specific immune response in the host. Thus, biological markers also include the presence or amount of messenger RNA (mRNA) transcribed from genomic DNA that encodes a protein specifically produced by cells from the immune system at the tumor site.

[0037] Thus, biological markers include surface antigens specifically expressed by cells from the immune system, including B lymphocytes, T lymphocytes, monocytes / macrophages dendritic cells, NK cells, NKT cells, and NK-DC cells, that are recruited into tumor tissue or to nearby sites, including within the invasive margin of the tumor and nearby lymph nodes, or mRNA encoding such surface antigens.

[0038] By way of example, desirable surface antigens for use as biological markers include CD3, CD4, CD8 and CD45RO expressed by T cells or T cell subsets.

[0039] For example, if the expression of the CD3 antigen, or the expression of its mRNA, is used as a biological marker, the quantification of this biological marker in step a) of the method of the invention is indicative of the level of the patient's adaptive immune response with respect to total T lymphocytes and NKT cells.

[0040] For example, if the expression of the CD8 antigen, or the expression of its mRNA, is used as a biological marker, the quantification of this biological marker in step a) of the method of the invention is indicative of the level of the patient's adaptive immune response with respect to cytotoxic T lymphocytes.

[0041] For example, if expression of the CD45RO antigen, or expression of its mRNA, is used as a biological marker, the quantification of this biological marker in step a) of the method of the invention is indicative of the level of the patient's adaptive immune response with respect to memory T lymphocytes or memory effector T cells.

[0042] Further by way of example, proteins used as biological markers also include cytotoxic proteins specifically produced by cells from the immune system, such as perforin, granulysin and also granzyme-B.

[0043] Numerous patent applications describe numerous biological markers that are indicative of immune response status that may be used in the methods of the present invention.

[0044] Typically, biological markers indicative of immune response status, as described in WO2015007625, WO2014023706, WO2014009535, WO2013186374, WO2013107907, WO2013107900, WO2012095448, WO2012072750 and WO2007045996 (all of which are incorporated by reference), can be used.

[0045] Typically, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 individual biological markers may be quantified, preferably a combination of 2, 3, 4, 5, 6, 7, 8, 9 or 10 biological markers, more preferably a combination of 2, 3, 4, 5 or 6 biological markers may be quantified.

[0046] In a preferred embodiment, the biological markers indicative of the immune response status are those described in WO2007045996.

[0047] Typically, a biological marker that may be used is the cell concentration of cells derived from the immune system.

[0048] In a preferred embodiment, the two or more biological markers comprise CD3+ cell concentration, CD8+ cell concentration, CD45RO+ cell concentration, GZM-B+ cell concentration and / or B cell concentration.

[0049] In a most preferred embodiment, the two or more biological markers comprise CD3+ cell concentration and CD8+ cell concentration, CD3+ cell concentration and CD45RO+ cell concentration, CD3+ cell concentration and GZM-B+ cell concentration, CD8+ cell concentration and CD45RO+ cell concentration, CD8+ cell concentration and GZM-B+ cell concentration, or CD45RO+ cell concentration and GZM-B+ cell concentration.

[0050] B cell concentrations may also be measured (see WO2013107900 and WO2013107907). DC cell concentrations may also be measured (see WO2013107907).

[0051] In a preferred embodiment, the concentration of cells from the immune system is quantified in the center of the tumor and / or at the tumor's invasive margin.

[0052] In a most preferred embodiment, the two or more biological markers comprise CD3+ cell concentration in the tumor center, CD8+ cell concentration in the tumor center, CD3+ cell concentration at the invading edge, and CD8+ cell concentration at the invading edge.

[0053] The concentration may be measured at the "cold spot", i.e. the area of ​​the tumor sample with the lowest concentration, or at 2, 3, 4, 5, 6, 7, 8, 9, 10 "cold spots" corresponding to 2 to 10 areas with the lowest concentration.

[0054] The concentration may also be measured at the "hot spots", i.e. the areas of highest concentration, or at 2, 3, 4, 5, 6, 7, 8, 9, 10 "hot spots", corresponding to 2 to 10 areas having the highest concentration.

[0055] The average concentration across the tumor sample may be determined.

[0056] Typically, the methods described in WO2013186374 may be used to quantify immune cells in tumor samples.

[0057] As used herein, a "marker" consists of a detectable, measurable, or quantifiable parameter that is indicative of the state of the adaptive immune response of a subject. A marker becomes a "biological marker" for the purposes of carrying out the methods of the present invention if a good statistical correlation is found between (i) an increase or decrease in the quantitative value for said marker, and (ii) the survival time actually observed in the patient. Any statistical method known to those skilled in the art may be used to calculate the correlation value for each marker tested and determine the statistical relevance of said marker as a "biological marker" of the present invention. Illustratively, statistical methods using Kaplan-Meier curves and / or log-rank-test and / or Cox proportional hazards model may be used, as shown in the examples herein. Univariate and multivariate analyses (e.g., log-rank-test and Cox test, respectively) should provide a correlation value of less than 0.5, more preferably less than 10. -3 , 10 -4 , 10 -5 , 10 -6 or 10 -7Any marker for which a P-value of less than 0.001 is measured comprises a "biological marker" that can be used in the cancer prognosis method of the present invention. In some embodiments, the marker comprises the presence, number or concentration of cells from the immune system. In some embodiments, the marker comprises the presence or amount of a protein specifically produced by cells from the immune system. In some embodiments, the marker comprises the presence or amount of a biological substance indicative of the level of a gene associated with eliciting a specific immune response in a host. Thus, in some embodiments, the marker comprises the presence or amount of a messenger RNA (mRNA) transcribed by genomic DNA that encodes a protein specifically produced by cells from the immune system. In some embodiments, the marker comprises a surface antigen specifically expressed by cells from the immune system, including B lymphocytes, T lymphocytes, monocyte / macrophage dendritic cells, NK cells, NKT cells, and NK-DC cells, or an mRNA encoding such a surface antigen. When carrying out the method of the present invention with more than one biological marker, the number of specific biological markers quantified in step a) will usually be less than 100 specific markers, and in most embodiments, less than 50 specific markers. The number of specific biological markers required for accurate and reliable prognosis using the method of the present invention may vary significantly depending on the type of technique for quantification. Exemplarily, when the method of the present invention is carried out by in situ immunoanatomical detection of the desired protein markers, especially when the individual quantification of said markers is carried out both at the tumor center (CT) and at the invasion margin (IM), high statistical significance is found with a combination of a small number of biological markers. Exemplarily, high statistical significance was obtained with only one marker or with a combination of two markers, as described in the Examples. Further exemplarily, when the method of the present invention is carried out by gene expression analysis of the desired gene markers, high statistical significance was found even with a small number of biological markers. Without intending to be limited to any particular theory, it is believed that by using gene expression analysis for biological marker quantification and by using a combination of 10 specific biological markers, more preferably a combination of 15 specific biological markers, and most preferably a combination of 20 specific biological markers, high statistical associations (10 -3The inventors believe that the level of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 markers is measured. As used herein, the name of each of the various biological markers of interest refers to the internationally recognized name of the corresponding gene as found in internationally recognized gene and protein sequence databases, including those from the HUGO Gene Nomenclature Committee. In this specification, the name of each of the various biological markers of interest may refer to the internationally recognized name of the corresponding gene as found in the internationally recognized gene and protein sequence database Genbank. Through these internationally recognized sequence databases, a person skilled in the art may search for the nucleic acid and amino acid sequences corresponding to each of the various biological markers of interest described herein.

[0058] Biological markers indicative of the state of the immune response may include the expression level of one or more of the genes or corresponding proteins listed in Table 9 of WO2007045996: 18s, ACE, ACTB, AGTR1, AGTR2, APC, APOA1, ARF1, AXIN1, BAX, BCL2, BCL2L1, CXCR5, BMP2, BRCA1, BTLA, C3, CASP3, CASP9, CCL1, CCL11, CCL13, CCL16, CCL17, CCL18, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL29, CCL30, CCL31, CCL32, CCL33, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39, CCL40, CCL41, CCL42, CCL43, CCL44, CCL45, CCL46, CCL47, CCL48, CCL49, CCL49, CCL50, CCL51, CCL52, CCL53, CCL54, CCL55, CCL56, CCL57, CCL58, CCL59, CCL60, CCL61, CCL62, CCL63, CCL64, CCL65, CCL66, CCL67, CCL68, CCL69, CCL69, CCL70, CCL71, CCL72, CCL73, CCL74, CCL75, CCL76, CCL77, CCL78, CCL79, CCL70, CCL71, CCL72, CCL73, CCL74, CCL75, CCL76, CCL77, CCL78 CL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL5, CCL7, CCL8, CCNB1, CCND1, CCNE1, CCR1, CC R10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, CD154, CD19, CD1a, CD2, CD226, CD244, PDCD1LG1, CD28, CD34, CD36, CD3 8, CD3E, CD3G, CD3Z, CD4, CD40LG, CD5, CD54, CD6, CD68, CD69, CLIP, CD80, CD83, SLAMF5, CD86, CD8A, CDH1, CDH7, CDK2, CDK4, CDKN1 A, CDKN1B, CDKN2A, CDKN2B, CEACAM1, COL4A5, CREBBP, CRLF2, CSF1, CSF2, CSF3, CTLA4, CTNNB1, CTSC, CX3CL1, CX3CR1, CXCL1, CXCL1 0, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL2, CXCL3, CXCL5, CXCL6, CXCL9, CXCR3, CXCR4, CXCR6, CYP1A2, CYP7A1, DCC, DCN, DE FA6, DICER1, DKK1, Dok-1, Dok-2, DOK6, DVL1, E2F4, EBI3, ECE1, ECGF1, EDN1, EGF, EGFR, EIF4E, CD105, ENPEP, ERBB2, EREG, FCGR3A,, CGR3B, FN1, FOXP3, FYN, FZD1, GAPD, GLI2, GNLY, GOLPH4, GRB2, GSK3B, GSTP1, GUSB, GZMA, GZMB, GZMH, GZMK, HLA-B, HLA-C, HLA-, MA, HLA-DMB, HLA-DOA, HLA-DOB, HL A-DPA1, HLA-DQA2, HLA-DRA, HLX1, HMOX1, HRAS, HSPB3, HUWE1, ICAM1, ICAM-2, ICOS, ID1, ifna1, ifna17, ifna2, ifna5, ifna6, ifna8, IFNAR1, IFNAR2, IFNG, IFNGR1, IFNGR2, IGF1, IHH, IKBKB, IL10, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA2, IL15, IL15RA, IL17, IL17R, IL17RB, IL18, IL1A, IL1B, IL1R1, IL2, IL21, IL21R, IL23A, IL23R, IL24, IL27, IL2RA, IL2RB, IL2RG, IL3, IL31RA, IL4, IL4RA, IL5, IL6, IL7, IL7RA, IL8, CXCR1, CXCR2, IL9, IL9R, IRF1, ISGF3G, ITGA4, ITGA7, integrin alpha E (antigen CD103, human mucosal lymphocyte, antigen 1; alpha polypeptide), genes hCG33203, ITGB3, JAK2, JAK3, KLRB1, KLRC4, KLRF1, KLRG1, KRAS, LAG3, LAIR2, LEF1, LGALS9, LILRB3, LRP2, LTA, SLAMF3, MADCAM1, MADH3, MADH7,MAF, MAP2K1, MDM2, MICA, MICB, MKI67, MMP12, MMP9, MTA1, MTSS1, MYC, MYD88, MYH6, NCAM1, NFATC1, NKG7, NLK, NOS2A, P.S 2X7, PDCD1, PECAM, CXCL4, PGK1, PIAS1, PIAS2, PIAS3, PIAS4, PLAT, PML, PP1A, CXCL7, PPP2CA, PRF1, PROM1, PSMB5, ​​PTC H, PTGS2, PTP4A3, PTPN6, PTPRC, RAB23, RAC / RHO, RAC2, RAF, RB1, RBL1, REN, Drosha, SELE, SELL, SELP, SERPINE1, SFRP 1, SIRPベータ1, SKI, SLAMF1, SLAMF6, SLAMF7, SLAMF8, SMAD2, SMAD4, SMO, SMOH, SMURF1, SOCS1, SOCS2, SOCS3, SOCS4, SOCS 5. SOCS6, SOCS7, SOD1, SOD2, SOD3, SOS1, SOX17, CD43, ST14, STAM, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6 STK36, TAP1, TAP2, TBX21, TCF7, TERT, TFRC, TGFA, TGFB1, TGFBR1, TGFBR2, TIM-3, TLR1, TLR10, TLR2, TLR3, TLR4, TLR5 TLR6, TLR7, TLR8, TLR9, TNF, TNFRSF10A, TNFRSF11A, TNFRSF18, TNFRSF1A, TNFRSF1B, OX-40, TNFRSF5, TNFRSF6, TNFR SF7, TNFRSF8, TNFRSF9, TNFSF10, TNFSF6, TOB1, TP53, TSLP, VCAM1, VEGF, WIF1, WNT1, WNT4, XCL1, XCR1, ZAP70, and ZIC2.

[0059] As used herein, the name of each desired gene refers to the internationally recognized name of the corresponding gene as found in internationally recognized gene and protein sequence databases, including those from the HUGO Gene Nomenclature Committee. As used herein, the name of each desired gene may refer to the internationally recognized name of the corresponding gene as found in the internationally recognized gene and protein sequence database Genbank. Through these internationally recognized sequence databases, one skilled in the art may search for the nucleic acid for each desired gene described herein.

[0060] In a preferred embodiment, the biological markers indicative of the state of the immune response are those described in WO2014023706 (incorporated by reference).

[0061] Under this embodiment, the expression levels EL1 of a single gene indicative of a human adaptive immune response and a single gene (pair of genes) indicative of a human immunosuppressive response are assessed with the method of the present invention. Preferably, 1 to 3 genes of each are used, more preferably 1 or 2 genes of each. A limited number of genes of each species gives good and reliable results and is easy to implement. In particular, a single reference value for each of both genes is sufficient. With a higher number of genes, the reference value becomes more refined. An example of the measurement of the reference value is described below.

[0062] The use of more than one or two pairs of genes is more difficult to implement, more expensive, and more time consuming, but offers other advantages: if the expression level of one gene is erroneously assessed, the rest of the gene of the same species (human adaptive immune response or immunosuppressive response) can compensate for the overall result.

[0063] As used herein, the expression "genes indicative of adaptive immune response" refers to any gene expressed by cells that are effectors of adaptive immune response in tumors or contribute to the stabilization of adaptive immune response in tumors. Adaptive immune response is also called "acquired immune response" and includes antigen-dependent stimulation of T cell subtypes, B cell activation, and antibody production. For example, cells of adaptive immune response include, but are not limited to, cytotoxic T cells, memory T cells, Th1 and Th2 cells, adaptive macrophages and adaptive dendritic cells, NK cells, and NKT cells. Thus, genes indicative of adaptive immune response may typically be selected from a cluster of co-regulated genes for Th1 adaptive immunity, cytotoxic response, or memory response, and may code for a Th1 cell phenotype marker, an interleukin (or interleukin receptor) or a chemokine (or chemokine receptor).

[0064] In certain embodiments, the genes indicative of an adaptive immune response are selected from the following group: - a family of chemokines and chemokine receptors selected from the group consisting of CXCL13, CXCL9, CCL5, CCR2, CXCL10, CXCL11, CXCR3, CCL2 and CX3CL1, -A family of cytokines consisting of IL15, - the TH1 family consisting of IFNG, IRF1, STAT1, STAT4 and TBX21; - a family of lymphocyte membrane receptors consisting of ITGAE, CD3D, CD3E, CD3G, CD8A, CD247, CD69 and ICOS, - a family of cytotoxic molecules consisting of GNLY, GZMH, GZMA, GZMB, GZMK, GZMM and PRF1, as well as the kinase LTK.

[0065] Such preferred genes and corresponding proteins are reported in Table 1 below, as they give the best results in terms of patient response to treatment, as shown in Table 5 below. [Table 1]

[0066] As used herein, the expression "genes indicative of an immunosuppressive response" refers to any gene expressed by a cell that is an effector of, or contributes to the stabilization of, an immunosuppressive response in a tumor. For example, an immunosuppressive response includes: -co-inhibition of antigen-dependent stimulation of T cell subtypes: genes CD276, CTLA4, PDCD1, CD274, TIM-3 or VTCN1 (B7H4), - inactivation of macrophages and dendritic cells, and inactivation of NK cells: genes TSLP, CD1A or VEGFA, - Expression of cancer stem cell markers, differentiation, and / or tumorigenesis: PROM1, IHH, -Expression of immunosuppressive proteins produced in the tumor environment: genes PF4, REN, VEGFA.

[0067] For example, cells of the immunosuppressive response include immature dendritic cells (CD1A), regulatory T cells (Treg cells), and Th17 cells that express the IL17A gene.

[0068] Thus, genes indicative of an adaptive immune response may be selected from a group of adaptive immune genes that are typically co-regulated, while immunosuppressive genes may be indicative of inactivation of immune cells (e.g., dendritic cells) and contribute to the induction of an immunosuppressive response.

[0069] In certain embodiments, the gene or corresponding protein indicative of an immunosuppressive response is selected from the group consisting of the genes reported in Table 2 below. [Table 2]

[0070] Said genes preferably provide the best outcome for the patient's response to treatment.

[0071] Under preferred conditions in the context of the invention, the genes indicative of an adaptive immune response are selected from the group consisting of GNLY, CXCL13, CX3CL1, CXCL9, ITGAE, CCL5, GZMH, IFNG, CCR2, CD3D, CD3E, CD3G, CD8A, CXCL10, CXCL11, GZMA, GZMB, GZMK, GZMM, IL15, IRF1, LTK, PRF1, STAT1, CD69, CD247, ICOS, CXCR3, STAT4, CCL2 and TBX21, and the genes indicative of an immunosuppressive response are selected from the group consisting of PF4, REN, VEGFA, TSLP, IL17A, PROM1, IHH, CD1A, CTLA4, PDCD1, CD276, CD274, TIM-3 and VTCN1 (B7H4).

[0072] Since multiple genes are more frequently found when combining one adaptive gene and one immunosuppressive gene, the most preferred genes are: - Genes indicative of adaptive immune responses: CD3G, CD8A, CCR2 and GZMA - Genes indicative of an immunosuppressive response: REN, IL17A, CTLA4 and PDCD1.

[0073] Under further preferred conditions in accordance with the present invention, the genes indicative of an adaptive immune response and the genes indicative of an immunosuppressive response are selected from the group consisting of the genes of Tables 1 and 2 above, respectively.

[0074] The preferred two-gene pair combinations (four genes total) are: - CCR2, CD3G, IL17A and REN, and -CD8A, CCR2, REN and PDCD1.

[0075] The exact choice of genes selected for use in the methods of the invention may depend on the type of treatment contemplated for the patient. For example, if treatment with a drug such as a monoclonal antibody that works by activating the immune system, such as Ipilimumab, marketed as Yervoy® and also known as MDX-010 or MDX-101, is considered for the patient, genes selected from the group consisting of CX3CL1 IL15, CD247, CD3G, CD8A, PRF1, CCL5 and TBX21 for an immunosuppressive response, preferably CX3CL1 and IL15, and the gene CTLA4 for an adaptive suppressive response, would be preferred.

[0076] If treatment such as antibodies that inhibit vascular endothelial growth factor A (VEGF-A), such as bevacizumab, marketed as Avastin®, are considered for the patient, genes selected from the group consisting of IL-15 and GZMA for an adaptive immune response, and the gene VEGFA for an immunosuppressive response, would be preferred.

[0077] Similar considerations apply, for example, to the gene pair GZMA - PDCD1 (also called CD279), when a treatment such as an antibody targeting PD-1, such as BMS-936558, is considered for a patient.

[0078] In a preferred embodiment, the biological markers indicative of the state of the immune response are those described in WO2014009535 (incorporated by reference).

[0079] Biological markers indicative of the state of the immune response may include the expression level of one or more genes selected from the group consisting of CCR2, CD3D, CD3E, CD3G, CD8A, CXCL10, CXCL11, GZMA, GZMB, GZMK, GZMM, IL15, IRF1, PRF1, STAT1, CD69, ICOS, CXCR3, STAT4, CCL2 and TBX21.

[0080] In a preferred embodiment, the biological markers indicative of the state of the immune response are those described in WO2012095448 (incorporated by reference).

[0081] Biological markers indicative of the state of the immune response may include the expression level of one or more genes selected from the group consisting of GZMH, IFNG, CXCL13, GNLY, LAG3, ITGAE, CCL5, CXCL9, PF4, IL17A, TSLP, REN, IHH, PROM1 and VEGFA.

[0082] In a preferred embodiment, the biological marker indicative of the state of the immune response is one described in WO2012072750 (incorporated by reference). The biological marker indicative of the state of the immune response may comprise the expression level of a miRNA cluster comprising miR.609, miR.518c, miR.520f, miR.220a, miR.362, miR.29a, miR.660, miR.603, miR.558, miR519b, miR.494, miR.130a or miR.639.

[0083] Common methods for quantifying biological markers Any method known to those skilled in the art for quantifying the cell-type, protein-type, or nucleic acid-type biological markers encompassed herein may be used to perform the present cancer prognosis method. Thus, any standard and non-standard (emerging) techniques known to those skilled in the art for detecting and quantifying proteins or nucleic acids in a sample can be readily applied.

[0084] Expression of the biological markers of the invention may be assessed by any of a wide variety of known methods for detecting expression of transcribed nucleic acids or proteins, non-limiting examples of which include immunological methods for expression of secreted, cell surface, cytoplasmic or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

[0085] In a preferred embodiment, expression of the markers is assessed using antibodies (e.g., radiolabeled, chromophore-labeled, fluorescently labeled, polymer-chained, or enzyme-labeled antibodies), antibody derivatives (e.g., antibodies conjugated to a substrate or to a protein or ligand of a protein-ligand pair (e.g., biotin-streptavidin)), or antibody fragments (e.g., single chain antibodies, isolated antibody hypervariable domains, etc.) that specifically bind to a marker protein or a fragment thereof, including a marker protein that has carried all or some of its usual post-translational modifications.

[0086] In certain embodiments, the biological marker, or set of biological markers, may be quantified by any of the known immunohistological methods.

[0087] Typically, for further analysis, one of the tumor thin sections is first incubated with a labeled antibody against one of the desired biological markers. After washing, the labeled antibody bound to said desired biological marker is revealed by an appropriate technique, depending on the type of label carried by the labeled antibody, e.g., radioactive, fluorescent or enzymatic label. Multiple labelings can be carried out simultaneously.

[0088] Immunohistology typically involves the following steps: i) fixing a tumor tissue sample with formalin, ii) embedding the tumor tissue sample in paraffin, iii) cutting the tumor tissue sample into sections for staining, iv) incubating the sections with a binding partner specific for the desired immune checkpoint protein, v) rinsing the sections, vi) incubating the sections with a secondary antibody, typically biotinylated, and vii) revealing the antigen-antibody complex, typically with an avidin-biotin-peroxidase complex. Thus, the tumor tissue sample is first incubated with a binding protein for the desired immune checkpoint protein. After washing, the labeled antibody bound to the desired immune checkpoint protein is revealed by an appropriate technique, depending on the type of label carried by the labeled antibody, e.g., radioactive, fluorescent or enzymatic label. Multiple labelings can be performed simultaneously. Alternatively, the method of the present invention may use an amplification system (to enhance the staining signal) and a secondary antibody linked to an enzymatic molecule. Such linked secondary antibodies are commercially available, e.g., from Dako, EnVision systems. Counterstaining may be done, e.g., with hematoxylin and eosin, DAPI, Hoechst. Other staining methods may be performed using any suitable method or system apparent to one of skill in the art, including automated, semi-automated or manual systems.

[0089] For example, one or more labels can be attached to the antibody, thereby allowing detection of the target protein (i.e., biological marker). Exemplary labels include radioisotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof. Non-limiting examples of labels that can be conjugated to primary and / or secondary affinity ligands include fluorescent dyes or metals (e.g., fluorescein, rhodamine, phycoerythrin, fluoroscamine), chromophore dyes (e.g., rhodopsin), chemiluminescent compounds (e.g., luminal, imidazole), and bioluminescent proteins (e.g., luciferin, luciferase), heptanes (e.g., biotin). A variety of other useful fluorescent dyes and chromophores are described in Stryer L (1968) Science 162:526-533, and Brand L and Gohlke JR (1972) Annu. Rev. Biochem. 41:843-868. Affinity ligands also include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g., 3 H, 14 C. 32 P, 35 S or 125I) and particles (e.g., gold). Different types of labels can be conjugated to the affinity ligand using various chemical reactions, e.g., amine or thiol reactions. However, other reactive groups besides amines and thiols, e.g., aldehydes, carboxylic acids, and glutamines, can be used. Various enzymatic staining methods are known to those skilled in the art to detect the desired protein. For example, different enzymes, such as peroxidase, alkaline phosphatase, or different chromogens, such as DAB, AEC, or Fast Red, can be used to visualize the enzymatic reaction. In some embodiments, the label is a quantum dot. For example, quantum dots (Q dots) are becoming useful in a growing list of applications, including immunohistochemistry, flow cytometry, and plate-based assays, and therefore would be useful in conjunction with the present invention. Q dot nanocrystals have unique optical properties, including extremely bright signals for sensitivity and quantification; high optical stability for imaging and analysis; and no single excitation light source is required. The increased range of conjugates makes them useful in a wide range of cell-based applications. Q-dot bioconjugates are characterized by quantum yields comparable to the brightest conventional commercial stains. Furthermore, these quantum dot-based fluorophores absorb light 10 to 1000 times higher than conventional stains. The emission potential of Q-dot quantum dots is narrow and symmetrical, which means that there is minimal overlap with other colors, minimizing bleeding through adjacent detection channels and weakening crosstalk, despite the fact that many colors can be used simultaneously. In other examples, antibodies can be conjugated to peptides or proteins that can be detected via a labeled binding partner or antibody. In indirect IHC assays, a secondary antibody or secondary binding partner is required to detect binding of the primary binding partner because it is unlabeled.

[0090] In some embodiments, the resulting stained specimens are imaged using a system for detecting detectable signals and obtaining images, such as digital images of the staining. Methods for image acquisition are known to those skilled in the art. For example, once the sample is stained, any optical or non-optical imaging device can be used to detect the stain or biomarker label, such as, for example, an upright or inverted optical microscope, a scanning confocal microscope, a camera, a scanning or tunneling electron microscope, a canning probe microscope, and an infrared image detector. In some examples, the image can be digitally captured. The image obtained can then be used to quantitatively or semi-quantitatively measure the amount of immune checkpoint protein in the sample, the absolute number of cells positive for a desired marker, or the cell surface positive for a desired marker. A variety of automated sample processing, scanning, and analysis systems suitable for use with IHC are available to those skilled in the art. Such systems can include automated staining and microscope scanning, computerized image analysis, serial section comparison (to control for variations in sample orientation and size), digital record generators, and archiving and tracking of samples (such as slides on which tissue sections are placed). Cellular imaging systems are commercially available that combine conventional optical microscopes with image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples. See, for example, the CAS-200 system (Becton, Dickinson & Co.). In particular, detection can be performed manually or by image processing techniques that include computer processors and software. Such software can be used to modify, calibrate, standardize, and / or evaluate images based on factors including, for example, staining quality or staining intensity, using procedures known to those skilled in the art (see, for example, those published as U.S. Patent No. US20100136549). Images can be quantitatively or semi-quantitatively analyzed and scored based on the staining intensity of the sample.Quantitative or semi-quantitative histochemistry refers to the scanning and scoring of samples on which histochemistry is performed to identify and quantify the presence of a specified biomarker (i.e., immune checkpoint protein). Quantitative or semi-quantitative methods can use imaging software to detect staining density or amount, or can use human visual staining detection methods where a trained operator ranks the results numerically. For example, images can be quantitatively analyzed using pixel counting algorithms and tissue recognition patterns (e.g., Aperio Spectral software, automated quantitative analysis platform (AQUA® platform), or Tribvn with Ilastic and Calopix software) and other standard methods to measure or quantify or semi-quantify staining intensity; see, e.g., U.S. Pat. No. 8,023,714, U.S. Pat. No. 7,257,268, U.S. Pat. No. 7,219,016, U.S. Pat. No. 7,646,905, U.S. Patent Publication Nos. US20100136549 and 20110111435; Camp et al. (2002) Nature Medicine, 8:1323-1327; Bacus et al. (1997) Analyt Quant Cytol Histol, 19:316-328. The ratio of strong positive reproductives (such as brown staining) to the total of the total stained area can be calculated and scored. The amount of detected biomarkers (i.e. immune checkpoint proteins) can be quantified and shown as a percentage of positive pixels and / or score. For example, the amount can be quantified as a percentage of positive pixels. In some examples, the amount is quantified as a percentage of stained area, for example, a percentage of positive pixels.For example, a sample can have at least, or about at least, or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more positive pixels relative to the total stained area. The amount can be quantified, for example, as an absolute number of cells positive for the desired marker. In some embodiments, a numerical representation of the intensity or amount of tissue staining of the sample is presented for the sample, indicating the amount of target biomarker (e.g., immune checkpoint protein) present in the sample. Optical density or percentage range values ​​can be presented, for example, as a measurement score in an integer measurement.

[0091] Thus, in some embodiments, the method of the present invention comprises the steps of: i) providing one or more immunostained sections of a tissue section obtained by an automated slide staining system by using a binding partner capable of selectively interacting with a biological marker, ii) performing digitization of the slide of step i) by high resolution scan capture, iii) detecting slices of the tissue section on the digital image, iv) providing a size reference grid having uniformly distributed units with the same surface, said grid being adjusted to the size of the tissue section to be analyzed, and v) detecting, quantifying and measuring the intensity or absolute number of stained cells in each unit.

[0092] Multiple tissue analysis techniques are particularly useful for quantifying multiple immune checkpoint proteins in tumor tissue samples. Such techniques would allow for at least 5, or at least 10, or more biomarkers to be measured from a single tumor tissue sample. Additionally, it would be beneficial for the technique to preserve the localization of the biomarkers and be able to distinguish between the presence of the biomarkers in cancer cells and non-cancerous cells. Such methods include, for example, multi-layer immunohistology (L-IHC), multi-layer expression scanning (LES) or multiple tissue immunoblotting (MTI) as taught in U.S. Patent Nos. 6,602,661, 6,969,615, 7,214,477, 7,838,222; U.S. Patent Publication No. 2011 / 0306514 (incorporated herein by reference); and Chung & Hewitt, Meth Mol Biol, Prot Blotting Detect, Kurlen & Scofield, eds. 536: 139-148, 2009, each of which teaches that up to eight, nine, ten, eleven or more images of tissue sections, such as multi-layers and blotted membranes, papers, filters, etc., can be used. Coating membranes useful for carrying out the L-IHC / MTI process are commercially available from 20 / 20 GeneSystems, Inc. (Rockville, MD).

[0093] In some embodiments, the L-IHC method can be performed on a variety of tissue samples, either fresh or preserved. Tissue samples include core needle biopsies routinely fixed in 10% normal buffered formalin and processed in the pathology department. Standard 5 μm thick tissue sections are cut from tissue blocks onto charged slides used for L-IHC. L-IHC then allows for the examination of multiple markers in obtaining copies of molecules transferred from tissue sections to multiple bioaffinity coated membranes, essentially producing a copy of the tissue "image". In the case of paraffin sections, the tissue sections are deparaffinized in known manner, for example by exposing the sections to xylene or a xylene substitute such as NEO-CLEAR®. Sections can be treated with proteinases such as papain, trypsin, proteinase K, and the like. A membrane matrix laminate, including a plurality of 10 μm thick sheets of coated polymer backbones with 0.4 μm diameter holes that introduce tissue molecules, such as proteins, through the laminate, is then provided and placed on the tissue section. The transfer of liquid and tissue molecules is designed to be essentially perpendicular to the membrane surface. The sandwich of section, membrane, spacer paper, absorbent paper, weight, etc. can be exposed to heat to facilitate transfer of molecules from the tissue to the membrane laminate. A portion of the tissue proteins is captured on each of the bioaffinity coated membranes (commercially available from 20 / 20 GeneSystems, Inc., Rockville, MD) of the laminate. Each membrane then contains a copy of the tissue and can be probed for different biomarkers using standard immunoblot techniques, allowing for open-ended development of marker profiles performed on a single tissue section. For example, since there may be lower amounts of protein in the more distal membranes in a stack from tissue, as may occur for different amounts of molecules in tissue samples, different mobilities of molecules released from tissue samples, different binding affinities of molecules to membranes, length of transfer, etc., normalization of values, running controls, evaluation of transfer levels of tissue molecules, etc., may be included in the calibration procedure for changes occurring within and between membranes, allowing direct comparison of information within and between membranes. Thus, for example, protein Total protein per membrane can be measured using any means known in the art, such as by exposing the membrane to labeled avidin or streptavidin; a protein stain, such as fastStain, Ponceau Red, Brilliant Blue stain, etc., to reveal bound biotin, using standard reagents and methods for quantifying protein, such as biotinylated commercially available molecules such as protein.

[0094] In some embodiments, the methods of the present invention utilize multiple tissue imprinting (MTI) technology to measure biomarkers, which allows for multiple biomarkers, in some cases at least six biomarkers, thereby preserving valuable biopsy tissue.

[0095] In some embodiments, there are other multiple tissue analysis systems that may be used as part of the present invention. One such technology is the mass spectrometry-based Selected Reaction Monitoring (SRM) Assay System (available as "Liquid Tissue" from OncoPlexDx (Rockville, MD)). This technology is described in U.S. Patent No. 7,473,532.

[0096] In some embodiments, the methods of the present invention utilize multiplex IHC technology developed by GE Global Research (Niskayuna, NY), which is described in U.S. Patent Publication Nos. 2008 / 0118916 and 2008 / 0118934, in which a sequential analysis is performed on a biological sample containing multiple targets, including binding a fluorescent probe to the sample, followed by signal detection, then inactivating the probe, followed by binding, detection and inactivation of the probe to another target, and continuing this process until all targets are detected.

[0097] In some embodiments, composite tissue imaging can be performed when using fluorescence (e.g., fluorophores or quantum dots) whose signals can be measured by a multispectral imaging system. Multispectral imaging is a technique that accumulates spectroscopic information at each pixel of an image and analyzes the resulting data with spectral image processing software. For example, the system can take a series of images at different wavelengths that can be electronically selected in succession and then utilize an analysis program designed to handle the data. Thus, the system can simultaneously obtain quantitative information from multiple dyes, even if the dyes' spectra are highly overlapping or they are co-localized or occur at the same point in the sample, but with different spectral curves. Many biological materials are autofluorescent or emit lower energy light when excited by higher energy light. This signal can reduce the contrast of the image and data. High sensitivity cameras that do not have composite spectrum imaging capabilities only increase the autofluorescence signal along with the fluorescence signal. Multispectral imaging separates the autofluorescence from the tissue without mixing it, thereby improving the achievable signal-to-noise ratio. Briefly, quantification can be performed by the following steps: i) obtaining a tumor tissue microarray (TMA) from a patient, ii) then staining the TMA sample with an anti-antibody with specificity for the desired immune checkpoint protein, iii) further staining the TMA slide with an epithelial cell marker to aid in automated segmentation of the tumor and stroma, iv) then scanning the TMA slide using a multispectral imaging system, v) processing the scanned image using automated image analysis software (e.g., Perkin Elmer Technology) that allows for detection, quantification, and segmentation of specific tissues via powerful pattern recognition algorithms. Typically, an automated learning algorithm is trained to segment the tumor from the stroma and identify labeled cells.

[0098] Measuring gene expression levels in tumor samples obtained from patients can be carried out by techniques known to those of skill in the art.

[0099] Typically, the expression level of a gene is assessed by measuring the amount of mRNA produced by the gene.

[0100] Techniques for measuring the amount of mRNA are known to those skilled in the art. For example, the nucleic acid contained in a sample (e.g., cells or tissues prepared from a patient) is first extracted by standard methods, for example, using lytic enzymes or chemical solutions, or extracted by nucleic acid binding resins according to the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and / or amplification (e.g., RT-PCR). Quantitative or semi-quantitative RT-PCR is preferably used. Quantitative or semi-quantitative RT-PCR is preferably used.

[0101] Other amplification methods include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA), next-generation quantitative sequencing (NGS) of RNA.

[0102] Nucleic acids that contain at least 10 nucleotides and present a sequence that is complementary or homologous to the desired mRNA herein are useful as hybridization probes or amplification primers. Such nucleic acids do not need to be completely identical, but are typically at least about 80% identical, more preferably 85% identical, and even more preferably 90 to 95% identical to the homologous region of corresponding size. In some embodiments, it will be beneficial to use nucleic acids in combination with a suitable means, such as a detectable label, to detect hybridization. A wide range of suitable indicators are known to those skilled in the art, including fluorescent, radioactive, enzymatic or other ligands (e.g., avidin / biotin).

[0103] Typically, a probe comprises a single-stranded nucleic acid of 10 to 1000 nucleotides in length, e.g., 10 to 800, more preferably 15 to 700, typically 20 to 500 nucleotides in length. Primers are typically shorter single-stranded nucleic acids of 10 to 25 nucleotides in length designed to match perfectly or nearly perfectly to the desired nucleic acid to be amplified. Probes and primers are "specific" for the nucleic acids to which they hybridize, i.e., they preferably hybridize under highly stringent conditions (at the highest melting temperature Tm, e.g., 50% formamide, 5x or 6x SCC (SCC is 0.15M NaCl, 0.015M Na citrate)).

[0104] Nucleic acids that may be used as primers or probes in the above amplification and detection methods may be combined into a kit. Such kits include consensus primers and molecular probes. Preferred kits also include components necessary to measure whether amplification has occurred. The kits may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting specific sequences.

[0105] In certain embodiments, expression of a biological marker of the present invention may be assessed by tagging the biomarker (in its DNA, RNA or protein counterpart) with a digital oligonucleotide barcode and measuring or counting the number of barcodes.

[0106] In a particular embodiment, the method of the invention comprises the step of providing total RNA extracted from cumulus cells and subjecting said RNA to amplification and hybridization to specific probes, more preferably by quantitative or semi-quantitative RT-PCR.

[0107] The probes used in the disclosed methods can be used for nucleic acid detection, such as in situ hybridization (ISH) procedures (e.g., fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver staining in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

[0108] In situ hybridization involves contacting a sample containing a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe capable of specifically hybridizing to or specific for the target nucleic acid sequence (e.g., a genomic target nucleic acid sequence). The slide may optionally be pretreated, for example to remove paraffin or other substances that may inhibit uniform hybridization. Both the sample and the probe are treated, for example by heating to denature double-stranded nucleic acids. The probe and sample (formulated in an appropriate hybridization buffer) are combined under conditions and for a sufficient time to allow hybridization to occur (typically until equilibrium is reached). The chromosome preparation is washed to remove excess probe, and detection of the specific target of the chromosomal target is performed using standard techniques.

[0109] For example, fluorescently labeled avidin or avidin-alkaline phosphatase can be used to detect biotinylated probes. For fluorescent dye detection, fluorescent dyes can be detected directly or samples can be incubated with, for example, fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of FITC signal can be enabled, if necessary, by incubation with biotin-conjugated goat anti-avidin antibody, washing, and a second incubation with FITC-conjugated avidin. For detection by enzyme activity, samples can be incubated with, for example, streptavidin, washed, incubated with biotin-conjugated phosphatase, washed again, and pre-equilibrated (for example in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization, see U.S. Pat. No. 4,888,278.

[0110] Numerous procedures for FISH, CISH and SISH are known to those skilled in the art. For example, procedures for performing FISH are described in U.S. Patent Nos. 5,447,841, 5,472,842 and 5,427,932; and for example, Pinkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described, for example, in Tanner et al., Am. J. Pathol. 157:1467-1472, 2000 and U.S. Patent No. 6,942,970. Further detection methods are described in US Pat. No. 6,280,929.

[0111] A myriad of reagents and detection schemes can be combined with FISH, CISH, and SISH to improve sensitivity, resolution, or other desired properties. As discussed above, probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be optionally detected directly when performing FISH. Alternatively, probes can be labeled with haptens, ligands, or other indirectly detectable moieties (such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenone, coumarins, coumarin-based compounds, podophyllotoxins, podophyllotoxin-based compounds, and combinations thereof). Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., a cell or tissue sample to which the probe binds) with a labeled detection agent, such as an antibody (or receptor, or other specific binding partner) specific for the selected hapten or ligand. The detection agent can be labeled with a fluorophore (e.g., a QUANTUM DOT® or other indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., a secondary antibody or a specific antibody) that can be labeled with a fluorophore.

[0112] In another example, the probe or specific binding agent (such as an antibody, e.g., a primary antibody, a receptor, or other binding agent) is labeled with an enzyme that can convert a fluorescent or staining composition into a detectable fluorescent, staining, or other detectable signal (e.g., in the accumulation of metal particles detectable in SISH). As described above, the enzyme can be attached to the associated probe or detection agent directly or indirectly via a linker. Examples of suitable agents (e.g., binding agents) and chemicals (e.g., linkers and attachment chemicals) are described in U.S. Patent Application Nos. 2006 / 0246524; 2006 / 0246523, and 2007 / 0117153.

[0113] Those skilled in the art will appreciate that by appropriately selecting labeled probe-specific binding agent pairs, multiple detection schemes can be generated to facilitate the detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample, or on more than one cell or tissue sample). For example, a first probe corresponding to a first target sequence can be labeled with a first hapten, such as biotin, while a second probe corresponding to a second target sequence can be labeled with a second hapten, such as DNP. After exposing the sample to the probe, the bound probe can be detected by contacting the sample with a first specific binding agent (in this case, avidin labeled with a first fluorophore, e.g., a first spectrally distinct QUANTUM DOT® that emits at 585 nm), as well as a second specific binding agent (in this case, an anti-DNP antibody or antibody fragment labeled with a second fluorophore, e.g., a second spectrally distinct QUANTUM DOT® that emits at 705 nm). Additional probes / binding agents can be added to multiplex detection schemes using other spectrally distinct fluorophores. A myriad of direct or indirect (one-step, two-step or more) variations can be envisioned, all of which are appropriate in the context of the probes and assays described.

[0114] Typically, a probe comprises a single-stranded nucleic acid of 10 to 100 nucleotides in length, e.g., 10 to 800, more preferably 15 to 700, typically 20 to 500. Primers are typically shorter single-stranded nucleic acids of 10 to 25 nucleotides in length that are designed to match perfectly or nearly perfectly to the desired nucleic acid to be amplified. Probes and primers are "specific" for the nucleic acid to which they hybridize, i.e., they preferably hybridize under highly stringent conditions (at the highest melting temperature Tm, e.g., 50% formamide, 5x or 6x SCC (SCC is 0.15M NaCl, 0.015M Na citrate)).

[0115] The nucleic acid primers or probes used in the above amplification and detection methods may be combined into a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes components necessary to measure whether amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting specific sequences.

[0116] In a particular embodiment, the method of the invention comprises the step of providing total RNA extracted from cumulus cells and subjecting said RNA to amplification and hybridization to specific probes, more preferably by quantitative or semi-quantitative RT-PCR.

[0117] In another preferred embodiment, the expression level is measured by DNA chip analysis. Such DNA chips or nucleic acid microarrays consist of different nucleic acid probes chemically attached to a substrate, which may be a microchip, a glass slide or a microsphere-sized bead. The microchip may be composed of polymer, plastic, resin, polysaccharide, silica or silica-based material, carbon, metal, inorganic glass, or nitrocellulose. The probes include nucleic acids such as cDNA or oligonucleotides, which may be about 10 to about 60 base pairs. To measure the expression level, a sample from a test subject, which may be first subjected to reverse transcription, is labeled and contacted with the microarray under hybridization conditions to form a complex between the target nucleic acid that is complementary to the probe sequence attached to the microarray surface. The labeled and hybridized complexes can then be detected and quantified or semi-quantified. Labeling may be achieved by various methods, for example by using radioactive or fluorescent labels. A wide variety of microarray hybridization techniques are available to those of skill in the art (see, for example, review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

[0118] The expression level of gene can be expressed as absolute expression level or normalized expression level.The method of the present invention uses both types of values.When quantitative PCR is used as expression evaluation method, it is preferred to express as normalized expression level, because a small difference at the beginning of experiment can cause huge difference after several cycles.

[0119] Typically, expression levels are normalized by comparing the expression of a gene with that of a gene that is not relevant for measuring the cancer stage of a patient, such as a constitutively expressed housekeeping gene, to calibrate the absolute expression level of the gene. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, the ribosomal 18S gene, GUSB, PGK1, and TFRC. This normalization allows the expression level of one sample, such as a patient sample, to be compared with that of another sample, or to compare groups of samples from different sources.

[0120] The name of each desired gene herein refers to the internationally recognized name of the corresponding gene as found in internationally recognized gene and protein sequence databases, including those from the HUGO Gene Nomenclature Committee. The name of each desired gene herein may refer to the internationally recognized name of the corresponding gene as found in the internationally recognized gene and protein sequence database Genbank. Through these internationally recognized sequence databases, a person skilled in the art may search for a nucleic acid corresponding to each desired gene described herein.

[0121] When the method for evaluating cancer prognosis of the present invention comprises a combination of at least one gene that indicates adaptive immune response and at least one gene that indicates immunosuppressive response, the combination of genes can be carried out.The number of genes that can be used in the method of the present invention is limited only by the number of different biological genes that are actually available when carrying out the method.Therefore, in one embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 different gene combinations are quantified, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 combinations, more preferably 2, 3, 4, 5 or 6 combinations. However, there is a high statistical correlation (10 -3 The number of combined genes required to reach a lower P value will depend on the technique used to quantify the gene combination. The number of genes used as genes indicative of an adaptive immune response and the number of genes used as genes indicative of an immunosuppressive response may be the same or different.

[0122] Under preferred conditions in the context of the present invention, an approximately balanced number of genes of each type (adaptive immune response and immunosuppressive response) are used, for example two of each, or three of each, or five in one species and five in the other.

[0123] Measuring gene expression levels in a tumor sample obtained from a patient may be performed by techniques known to those of skill in the art.

[0124] Typically, the expression level of a gene is assessed by measuring the amount of mRNA produced by this gene. A subject of the present invention is therefore a method for screening patients with a cancer as defined above, comprising a step of measuring the expression level ELA of one or more genes indicative of a human adaptive immune response, or the expression level ELI of one or more genes indicative of a human immunosuppressive response, by measuring the amount of mRNA corresponding to said genes.

[0125] The method of measuring the amount of mRNA is known to those skilled in the art. For example, the nucleic acid contained in the sample (e.g., cells or tissues prepared from a patient) is first extracted by standard methods, for example, using lytic enzymes or chemical solutions, or extracted by nucleic acid binding resin according to the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and / or amplification (e.g., RT-PCR). Quantitative or semi-quantitative RT-PCR is preferred. Real-time or semi-quantitative RT-PCR is particularly advantageous.

[0126] Other amplification methods include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA), next-generation quantitative sequencing (NGS) of RNA.

[0127] Nucleic acids that contain at least 10 nucleotides and present a sequence that is complementary or homologous to the desired mRNA herein are useful as hybridization probes or amplification primers. Such nucleic acids do not need to be completely identical, but are typically at least about 80% identical, more preferably 85% identical, and even more preferably 90 to 95% identical to the homologous region of corresponding size. In some embodiments, it will be beneficial to use nucleic acids in combination with a suitable means, such as a detectable label, to detect hybridization. A wide range of suitable indicators are known to those skilled in the art, including fluorescent, radioactive, enzymatic or other ligands (e.g., avidin / biotin).

[0128] Typically, a probe comprises a single-stranded nucleic acid of 10 to 1000 nucleotides in length, e.g., 10 to 800, more preferably 15 to 700, typically 20 to 500 nucleotides in length. Primers are typically shorter single-stranded nucleic acids of 10 to 25 nucleotides in length designed to match perfectly or nearly perfectly to the desired nucleic acid to be amplified. Probes and primers are "specific" for the nucleic acids to which they hybridize, i.e., they preferably hybridize under highly stringent conditions (at the highest melting temperature Tm, e.g., 50% formamide, 5x or 6x SCC (SCC is 0.15M NaCl, 0.015M Na citrate)).

[0129] Nucleic acids that may be used as primers or probes in the above amplification and detection methods may be combined into a kit. Such kits include consensus primers and molecular probes. Preferred kits also include components necessary to measure whether amplification has occurred. The kits may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting specific sequences.

[0130] In a particular embodiment, the method of the invention comprises the step of providing total RNA extracted from cumulus cells and subjecting said RNA to amplification and hybridization to specific probes, more preferably by quantitative or semi-quantitative RT-PCR.

[0131] The probes used in the disclosed methods can be used for nucleic acid detection, such as in situ hybridization (ISH) procedures (e.g., fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver staining in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

[0132] In situ hybridization involves contacting a sample containing a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe capable of specifically hybridizing to or specific for the target nucleic acid sequence (e.g., a genomic target nucleic acid sequence). The slide may optionally be pretreated, for example to remove paraffin or other substances that may inhibit uniform hybridization. Both the sample and the probe are treated, for example by heating to denature double-stranded nucleic acids. The probe and sample (formulated in an appropriate hybridization buffer) are combined under conditions and for a sufficient time to allow hybridization to occur (typically until equilibrium is reached). The chromosome preparation is washed to remove excess probe, and detection of the specific target of the chromosomal target is performed using standard techniques.

[0133] For example, fluorescently labeled avidin or avidin-alkaline phosphatase can be used to detect biotinylated probes. For fluorescent dye detection, fluorescent dyes can be detected directly or samples can be incubated with, for example, fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of FITC signal can be enabled, if necessary, by incubation with biotin-conjugated goat anti-avidin antibody, washing, and a second incubation with FITC-conjugated avidin. For detection by enzyme activity, samples can be incubated with, for example, streptavidin, washed, incubated with biotin-conjugated phosphatase, washed again, and pre-equilibrated (for example in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization, see U.S. Pat. No. 4,888,278.

[0134] Numerous procedures for FISH, CISH and SISH are known to those skilled in the art. For example, procedures for performing FISH are described in U.S. Patent Nos. 5,447,841, 5,472,842 and 5,427,932; and for example, Pinkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described, for example, in Tanner et al., Am. J. Pathol. 157:1467-1472, 2000 and U.S. Patent No. 6,942,970. Further detection methods are described in US Pat. No. 6,280,929.

[0135] A myriad of reagents and detection schemes can be combined with FISH, CISH, and SISH to improve sensitivity, resolution, or other desired properties. As discussed above, probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be optionally detected directly when performing FISH. Alternatively, probes can be labeled with haptens, ligands, or other indirectly detectable moieties (such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenone, coumarins, coumarin-based compounds, podophyllotoxins, podophyllotoxin-based compounds, and combinations thereof). Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., a cell or tissue sample to which the probe binds) with a labeled detection agent, such as an antibody (or receptor, or other specific binding partner) specific for the selected hapten or ligand. The detection agent can be labeled with a fluorophore (e.g., a QUANTUM DOT® or other indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., a secondary antibody or a specific antibody) that can be labeled with a fluorophore.

[0136] In another example, the probe or specific binding agent (such as an antibody, e.g., a primary antibody, a receptor, or other binding agent) is labeled with an enzyme that can convert a fluorescent or staining composition into a detectable fluorescent, staining, or other detectable signal (e.g., in the accumulation of metal particles detectable in SISH). As described above, the enzyme can be attached to the associated probe or detection agent directly or indirectly via a linker. Examples of suitable agents (e.g., binding agents) and chemicals (e.g., linkers and attachment chemicals) are described in U.S. Patent Application Nos. 2006 / 0246524; 2006 / 0246523, and 2007 / 0117153.

[0137] Those skilled in the art will appreciate that by appropriately selecting labeled probe-specific binding agent pairs, multiple detection schemes can be generated to facilitate the detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample, or on more than one cell or tissue sample). For example, a first probe corresponding to a first target sequence can be labeled with a first hapten, such as biotin, while a second probe corresponding to a second target sequence can be labeled with a second hapten, such as DNP. After exposing the sample to the probe, the bound probe can be detected by contacting the sample with a first specific binding agent (in this case, avidin labeled with a first fluorophore, e.g., a first spectrally distinct QUANTUM DOT® that emits at 585 nm), as well as a second specific binding agent (in this case, an anti-DNP antibody or antibody fragment labeled with a second fluorophore, e.g., a second spectrally distinct QUANTUM DOT® that emits at 705 nm). Additional probes / binding agents can be added to multiplex detection schemes using other spectrally distinct fluorophores. A myriad of direct or indirect (one-step, two-step or more) variations can be envisioned, all of which are appropriate in the context of the probes and assays described.

[0138] Typically, a probe comprises a single-stranded nucleic acid of 10 to 100 nucleotides in length, e.g., 10 to 800, more preferably 15 to 700, typically 20 to 500. Primers are typically shorter single-stranded nucleic acids of 10 to 25 nucleotides in length that are designed to match perfectly or nearly perfectly to the desired nucleic acid to be amplified. Probes and primers are "specific" for the nucleic acid to which they hybridize, i.e., they preferably hybridize under highly stringent conditions (at the highest melting temperature Tm, e.g., 50% formamide, 5x or 6x SCC (SCC is 0.15M NaCl, 0.015M Na citrate)).

[0139] The nucleic acid primers or probes used in the above amplification and detection methods may be combined into a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes components necessary to measure whether amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting specific sequences.

[0140] In a particular embodiment, the method of the invention comprises the step of providing total RNA extracted from cumulus cells and subjecting said RNA to amplification and hybridization to specific probes, more preferably by quantitative or semi-quantitative RT-PCR.

[0141] In another preferred embodiment, the expression level is measured by DNA chip analysis. Such DNA chips or nucleic acid microarrays consist of different nucleic acid probes chemically attached to a substrate, which may be a microchip, a glass slide or a microsphere-sized bead. The microchip may be composed of polymer, plastic, resin, polysaccharide, silica or silica-based material, carbon, metal, inorganic glass, or nitrocellulose. The probes include nucleic acids such as cDNA or oligonucleotides, which may be about 10 to about 60 base pairs. To measure the expression level, a sample from a test subject, which may be first subjected to reverse transcription, is labeled and contacted with the microarray under hybridization conditions to form a complex between the target nucleic acid that is complementary to the probe sequence attached to the microarray surface. The labeled and hybridized complexes can then be detected and quantified or semi-quantified. Labeling may be achieved by various methods, for example by using radioactive or fluorescent labels. A wide variety of microarray hybridization techniques are available to those of skill in the art (see, for example, review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

[0142] The expression level of gene can be expressed as absolute expression level or normalized expression level.The method of the present invention uses both types of values.When quantitative PCR is used as expression evaluation method, it is preferred to express as normalized expression level, because a small difference at the beginning of experiment can cause huge difference after several cycles.

[0143] Typically, expression levels are normalized by comparing the expression of a gene with that of a gene that is not relevant for measuring the cancer stage of a patient, such as a constitutively expressed housekeeping gene, to calibrate the absolute expression level of the gene. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, the ribosomal 18S gene, GUSB, PGK1, and TFRC. This normalization allows the expression level of one sample, such as a patient sample, to be compared with that of another sample, or to compare groups of samples from different sources.

[0144] Antitumor Treatment The method of the invention therefore makes it possible to define previously unidentified and thus new patient groups, ie patient groups whose cancer will be successfully treated by anti-cancer therapy.

[0145] The anti-cancer treatment may consist of radiotherapy, chemotherapy or immunotherapy. The treatment may consist of adjuvant therapy (i.e. treatment after surgery of the primary tumor) or neoadjuvant therapy (i.e. treatment before surgery of the primary tumor).

[0146] The present invention therefore relates to a chemotherapeutic, radiotherapeutic or immunotherapeutic agent, preferably the latter, for use in the treatment of stage I to III cancer patients who are considered good responders to antitumor treatment according to the above-mentioned method of the invention.

[0147] The term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, metuledopa and uredopa; ethylenimines and methylamelanamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophophoramide and trimethylolomelanamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including the synthetic analogs adozelesin, carzelesin and bizeresin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictiin; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estranustine, isosphamide, mechlorethamine, meclomethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; notorosourea, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin 111 and calicheamicin 211, e.g., Agnew See Chem Intl. Ed. Engl. 33:183-186 (1994); the dynemicins, including dynemicin A; the esperamicins; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores;Aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, canninomycin, carcinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including deoxydoxorubicin), epirubicin, esorubicin, idanurubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, and queramycin. , lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, ptropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; ancitabine, azacitidine, 6-azauridine, carmophor, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5 -Pyrimidine analogues such as FU;androgens such as calcineurone, dromostanol propionate, epithiostanol, mepitostanol, testolactone;antiadrenergics such as aminoglutethimide, mitotane, trilostane;folic acid supplements such as folinic acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;amsacrine;bestrabulil;bisantrene;edatraxate;defofamine;demecolcine;diaziquone;eflornithine;elpitinium acetate;epothilone;etoglucide;gallium nitrate;hydrochloride Xyurea;Lentinan;Lonidamine;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidamol;Nitracrine;Pentostatin;Fenamet;Pirarubicin;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK (registered trademark);Razoxane;Rhizoxane;Sizofiran;Spirogenanium;Tenuazonic acid;Triaziquone;2,2',2"-Trichlorotriethylamine;Tricothecenes (especially T-2 toxin, veracrine A, roridin A and anguidine);Urethane;Vindesine;Decarbazine; mannommustine; mitobromtol; mitolactol; pipobroman; gasytosin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxides such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N) and doxorubicin (TAXOTERE®, Rhone-Poulenc Rorer, Ant; ony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included within this definition are: antihormonal agents which act to regulate or inhibit hormone action on tumors, such as tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY117018, onapristone, and toremifene (Fareston), and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0148] The term "immunotherapeutic agent" as used herein refers to a compound, composition, or treatment that indirectly or directly enhances, stimulates, or increases the body's immune response to cancer cells and / or reduces the side effects of other anti-cancer treatments. Thus, immunotherapy is a treatment that indirectly or directly stimulates or increases the immune system's response to cancer cells and / or reduces the side effects that may be caused by other anti-cancer agents. Immunotherapies are also referred to by those skilled in the art as immunological therapy, biological therapy, biological response modification therapy, and biotherapy. Examples of common immunotherapeutic agents known to those skilled in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies, and non-cytokine adjuvants. Alternatively, immunotherapeutic treatment may consist of administering a certain amount of immune cells (T cells, NK cells, dendritic cells, B cells, etc.) to the patient.

[0149] Immunotherapeutic agents can be non-specific, i.e., they generally boost the immune system, making the body more effective at fighting the growth and / or spread of cancer cells, or they can be specific, i.e., they target the cancer cells themselves, and immunotherapeutic regimens may be combined with the use of non-specific or specific immunotherapeutic agents.

[0150] Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Non-specific immunotherapeutic agents are used alone as the main treatment for cancer treatment, or even in addition to the main treatment, where the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other treatments (e.g., cancer vaccines). In the latter context, non-specific immunotherapeutic agents can also act to reduce the side effects of other treatments, such as bone marrow suppression induced by certain chemotherapeutic agents. Non-specific immunotherapeutic agents can act on key immune system cells to induce secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agent may itself contain cytokines. Non-specific immunotherapeutic agents are generally classified as cytokine or non-cytokine adjuvants.

[0151] Members of the cytokine family find application in cancer therapy, either as general non-specific immunotherapy designed to boost the immune system or as adjuvants provided with other treatments. Suitable cytokines include, but are not limited to, interferons, interleukins, and colony stimulating factors.

[0152] Interferon (IFN) as understood in the present invention includes the general types of IFN, IFN-alpha (IFN-α), IFN-beta (IFN-β) and IFN-gamma (IFN-γ). IFN can act directly on cancer cells, for example by slowing their growth, promoting their growth into cells with more normal behavior, and / or increasing their antigen production, making them more susceptible to recognition and destruction by the immune system. IFN can also act indirectly on cancer cells, for example by slowing down angiogenesis, boosting the immune system, and / or stimulating natural killer (NK) cells, T cells and macrophages. Recombinant IFN-alpha is commercially available as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation). The use of IFN-alpha, alone or in combination with other immunotherapies or chemotherapy, has been shown to be effective in the treatment of a variety of cancers, including melanoma (including metastatic melanoma), kidney cancer (including metastatic kidney cancer), breast cancer, prostate cancer, and cervical cancer (including metastatic cervical cancer).

[0153] Interleukins as understood in the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Wash.) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combination of the present invention. Interleukins alone or in combination with other immunotherapies or chemotherapy have been shown to be effective in the treatment of a variety of cancers, including renal cancer (including metastatic renal cancer), melanoma (including metastatic melanoma), ovarian cancer (including recurrent ovarian cancer), cervical cancer (including metastatic cervical cancer), breast cancer, breast cancer, colorectal cancer, lung cancer, brain cancer and prostate cancer.

[0154] Interleukins have also shown good activity in combination with IFN-alpha in the treatment of various cancers (Negrier et al., Ann Oncol. 2002 13(9):1460-8; Touranietal, J. Clin. Oncol. 2003 21(21):398794).

[0155] Colony stimulating factors (CSFs) contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramoutim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help stimulate new production of blood cells in patients undergoing traditional chemotherapy. Thus, treatment with CSFs may be useful in reducing side effects associated with chemotherapy and allows for higher doses of chemotherapy to be used. Various recombinant colony stimulating factors are commercially available, such as Neupogen® (G-CSF; Amgen), Neulasta (perfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), and Arnesp (erythropoietin). Colony stimulating factors have been shown to be effective in the treatment of cancers, including melanoma, colorectal cancer (including metastatic colorectal cancer), and lung cancer.

[0156] Non-cytokine adjuvants suitable for use in the combinations of the invention include, but are not limited to, levamisol, alum hydroxide (alum), Bacillus Calmette-Guerin (ACG), incomplete Freund's adjuvant (IFA), QS-21, DETOX, keyhole limpet homocyanin (KLH) and dinitrophenyl phosphate (DNP). Non-cytokine adjuvants in combination with other immunological and / or chemotherapy treatments have shown efficacy against a variety of cancers including, for example, colon and colorectal cancer (levamisol); melanoma (BCG and QS-21); kidney and bladder cancer (BCG).

[0157] In addition to specific or non-specific targeting, immunotherapeutic agents can be active, i.e., stimulate the body's own immune response, or they can be inactive, i.e., contain immune system components produced outside the body.

[0158] Inactive specific immunotherapy typically involves the use of one or more monoclonal antibodies that are specific to a particular antigen found on the surface of cancer cells or that are specific to a particular cell growth factor.Monoclonal antibodies can be used in the treatment of cancer in a number of ways, for example, by enhancing the subject's immune response to a specific type of cancer, by targeting a specific cell growth factor, such as those involved in angiogenesis, or by enhancing the delivery of other anti-cancer drugs to cancer cells when linked or conjugated to drugs such as chemotherapeutic agents, radioactive particles or toxins.

[0159] Monoclonal antibodies currently used as cancer immunotherapeutics suitable for inclusion in combination with the present invention include, but are not limited to, rituximab (Rituxan®), trastuzumab (Herceptin®), ibritumomab tiuexan (Zevalin®), tositumomab (Bexar®), cetuximab (C-225, Erbitux®), bevacizumab (Avastin®), gemtuzumab ozogamicin (Mylotarg®), alemtuzumab (Campas®), and BL22. Monoclonal antibodies are used in the treatment of a wide range of cancers, including breast cancer (including advanced metastatic breast cancer), colorectal cancer (including advanced and / or metastatic colorectal cancer), ovarian cancer, lung cancer, prostate cancer, cervical cancer, melanoma, and brain cancer. Other examples include anti-CTLA4 antibodies (e.g., iprimumab), anti-PD1 antibodies, anti-PDL1 antibodies, anti-TIM3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies.

[0160] Monoclonal antibodies can be used alone or in combination with other immunotherapeutic or chemotherapeutic agents.

[0161] Active specific immunotherapy is typically related to the use of cancer vaccines.Cancer vaccines have been developed that contain whole cancer cells, parts of cancer cells, or one or more antigens derived from cancer cells.Cancer vaccines, alone or in combination with one or more immune or chemotherapeutic agents, have been studied in the treatment of multiple types of cancer, including melanoma, renal cancer, breast cancer, colorectal cancer, and lung cancer.Non-specific immunotherapy is useful in combination with cancer vaccines to enhance the body's immune response.

[0162] Immunotherapeutic treatment may consist of adaptive immunotherapy, as described by Nicholas P. Restifo, Mark E. Dudley and Steven A. Rosenberg in "Adoptive immunotherapy for cancer: harnessing the T cell response" (Nature Reviews Immunology, Volume 12, April 2012). In adaptive immunotherapy, the patient's circulating or tumor-infiltrating lymphocytes are isolated in vitro, activated with lymphokines such as IL-2 or induced with genes for tumor necrosis, and re-administered (Rosenberg et al., 1988; 1989). The activated lymphocytes are most preferably the patient's own cells that have been previously isolated from blood or tumor samples and activated ("expanded") in vitro. This form of immunotherapy has produced several cases of regression of melanoma and renal cancer.

[0163] As used herein, the term "radiotherapeutic agent" is intended to refer to, without limitation, any radiotherapeutic agent known to those skilled in the art to be effective for treating or ameliorating cancer. For example, the radiotherapeutic agent may be an agent such as those administered in brachytherapy or radionuclide therapy. Such methods may optionally further include administration of one or more additional cancer treatments, such as, but not limited to, chemotherapy and / or other radiation therapies.

[0164] In a preferred embodiment, the anti-tumor treatment is treatment with a checkpoint inhibitor cancer immunotherapeutic agent.

[0165] As used herein, the expressions "checkpoint inhibitor cancer immunotherapeutic agent" or "immune checkpoint inhibitor" (although both will be used interchangeably) have their general meaning to those skilled in the art and refer to any compound that inhibits the function of an immune checkpoint inhibitor protein. Inhibition includes reduction and complete blocking of function. A preferred immune checkpoint inhibitor is an antibody that specifically recognizes an immune checkpoint protein. Several immune checkpoint inhibitors are known, and in analogy with these known immune checkpoint inhibitors, other immune checkpoint inhibitors may be developed in the (near) future. Immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules, and small molecules. In particular, the immune checkpoint inhibitors of the present invention are administered to enhance the proliferation, migration, persistence, and / or cytotoxic activity of CD8+ T cells in a subject, and tumor infiltration of CD8+ T cells in a subject. As used herein, "CD8+ T cells" have their general meaning to those skilled in the art and refer to a subset of T cells that express CD8 on their surface. They are MHC class I restricted and function as cytotoxic T cells. "CD8+ T cells" are also called cytotoxic T lymphocytes (CTL), T killer cells, cytotoxic T cells, CD8+ T cells or killer T cells. The CD8 antigen is a member of the immunoglobulin supergene family and is a binding recognition factor in major histocompatibility complex class I restricted interactions. The ability of immune checkpoint inhibitors to enhance the killing activity of CD8 T cells may be measured by any assay known to those skilled in the art. Typically, said assay is an in vitro assay in which CD8+ T cells are brought into contact with target cells (e.g., target cells that are recognized and / or lysed by CD8+ T cells). For example, immune checkpoint inhibitors of the invention can be selected for the ability to increase specific lysis by CD8+ T cells by more than about 20%, preferably more than about 30%, at least about 40%, at least about 50%, or more, of the specific lysis obtained with CD8+ T cells or CD8 T cells contacted with an immune checkpoint inhibitor of the invention at the same effector:target cell ratio. Classical cytotoxic activity The example protocol for is conventional.

[0166] Typically, checkpoint blockade cancer immunotherapeutics are agents that block immunoinhibitory receptors expressed by activated T cells, such as cytotoxic T-cell associated protein (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, such as various members of the killer cell immunoglobulin-like receptor (KIR) family, or the primary ligands for these receptors, such as the PD-1 ligand CD274 (best known as PD-L1 or B7-H1).

[0167] Typically, checkpoint blockade cancer immunotherapeutics are antibodies.

[0168] In embodiments, the checkpoint block cancer immunotherapeutic is an antibody selected from the group consisting of: anti-CTLA4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-IDO1 antibody, anti-TIGIT antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-BTLA antibody, and anti-B7H6 antibody.

[0169] Examples of anti-CTLA-4 antibodies are described in U.S. Patent Nos. 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; ​​6,682,736; 6,984,720; and 7,605,238. One anti-CTLA-4 antibody is tremelimumab (ticilimumab, CP-675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-D010), which is a fully human monoclonal IgG antibody that binds to CTLA-4.

[0170] Examples of PD-1 and PD-L1 antibodies are described in U.S. Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149; and PCT Patent Publication Nos. WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699. In some embodiments, the PD-1 blocking agent comprises an anti-PD-L1 antibody. In certain other embodiments, D-1 blocking agents include anti-PD-1 antibodies and similar binding proteins such as nivolumab (MDX 1106, BMS 936558, ONO 4538), a fully human IgG4 antibody that binds to PD-1 and blocks activation by its ligands PD-L1 and PD-L2; lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-1; CT-011, a humanized antibody that binds to PD-1; AMP-224, a fusion protein of B7-DC; antibody Fc portions; and BMS-936559 (MDX-1105-01) for PD-L1 (B7-H1) blocking.

[0171] Other immune checkpoint inhibitors include lymphocyte-activation gene 3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211).

[0172] Other immune checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors, including in particular the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834).

[0173] Also included are TIM3 (T cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94). As used herein, the term "TIM-3" has its general meaning in the art and refers to T cell immunoglobulin domain and mucin domain-containing molecule 3. The natural ligand of TIM-3 is galectin 9 (Gal9). Thus, as used herein, the term "TIM-3 inhibitor" refers to a compound, substance, or composition that can inhibit the function of TIM-3. For example, the composition can inhibit the expression or activity of TIM-3, regulate or block the TIM-3 signaling pathway, and / or block the binding of TIM-3 to galectin-9. Antibodies with specificity for TIM-3 are known to those skilled in the art, typically those described in International Publication Nos. WO2011155607, WO2013006490 and WO2010117057. In some embodiments, the immune checkpoint inhibitor is an indoleamine 2,3-dioxygenase (IDO) inhibitor, preferably an IDO1 inhibitor. Examples of IDO inhibitors are described in International Publication No. WO2014150677.Examples of IDO inhibitors include, without limitation, 1-methyl-tryptophan (IMT), β-(3-benzofuranyl)-alanine, β-(3-benzo(b)thienyl)-alanine, 6-nitro-tryptophan, 6-fluoro-tryptophan, 4-methyl-tryptophan, 5-methyl-tryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5-hydroxy-tryptophan, indole 3-carbonyl, 3, The IDO inhibitors include 3'-diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5-bromo-indoxyl diacetate, 3-amino-naphtholic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole, brassinin derivatives, thiohydantonin derivatives, β-carboline derivatives or brassilexin derivatives.Preferably, the IDO inhibitor is selected from 1-methyl-tryptophan, β-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-amino-naphtholic acid and β-[3-benzo(b)thienyl]-alanine or derivatives or prodrugs thereof.

[0174] In some embodiments, the immune checkpoint inhibitor is an anti-TIGIT (T cell immunoglobulin and ITIM domain) antibody.

[0175] In a preferred embodiment, the checkpoint blockade cancer immunotherapeutic is a CTLA4 blocking antibody, such as ipilimumab, a PD-1 blocking antibody, such as nivolumab or pembrolizumab, or a combination thereof.

[0176] The present invention will be further illustrated by the following figures and examples, which should not be construed as limiting the scope of the present invention in any way. [Brief description of the drawings]

[0177] [Figure 1] 1 is a schematic illustration of a system for carrying out the present invention. [Diagram 2] 1 is a flow diagram showing an example of a procedure related to the present invention. [Diagram 3] Kaplan Meir curves of five pre-determined reference arithmetic mean percentiles are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0178] Working Example A reference group of stage I / II / III colorectal cancer patients (n=539) was analyzed by the algorithm score method. Whole tumor slides were stained with CD3 and CD8 antibodies. Digital pathology and image analysis software was used to measure tumor invasion margin (IM) and core (CT) tumor. CD3 and CD8 concentrations in the CT and IM regions were quantified (cells / mm 2 ). The distribution of all concentrations or all markers is plotted and reported in a table. The corresponding percentiles (i.e., 5% interval steps) were derived. In the example shown in the table below, for each biomarker, the mean concentration biomarker value above 4 was calculated with a weight of 1. Kaplan Meier curves for each cohort are shown in Figure 3 with five group categories (I0, I1, I2, I3, I4) corresponding to the mean percentile values ​​of 0-10%, 10-25%, 25-75%, 75-95%, 95-100%, respectively.

[0179] In the table below, an example of the calculation of percentile arithmetic mean values ​​for a hypothetical patient is shown.

[0180] [Table 3]

[0181] The percentile arithmetic mean for the hypothetical patient would be approximately 47.5. The hypothetical patient would be classified as belonging to group I2.

[0182] The same type of experiment was performed with 21 biological markers, which were the expression levels of 21 genes from the group consisting of CCR2, CD3D, CD3E, CD3G, CD8A, CXCL10, CXCL11, GZMA, GZMB, GZMK, GZMM, IL15, IRF1, PRF1, STAT1, CD69, ICOS, CXCR3, STAT4, CCL2 and TBX21, and with 15 biological markers, which were the expression levels of 15 genes from the group consisting of GZMH, IFNG, CXCL13, GNLY, LAG3, ITGAE, CCL5, CXCL9, PF4, IL17A, TSLP, REN, IHH, PROM1 and VEGFA.

[0183] Calculating the percentile average gave very accurate results.

[0184] References Throughout this application, various references are cited which describe the state of the art to which this invention pertains. The disclosures of these references are incorporated by reference into this disclosure.

Claims

1. 1. An in vitro method for prognosticating survival of a patient suffering from colorectal cancer, comprising: a) detecting and quantifying a biological marker indicative of the state of the patient's immune response against the cancer as defined in (i) or (ii) below, wherein each of the biological markers indicative of the state of the immune response is detected and quantified in vitro in a tumor sample obtained from the patient, and the biological markers indicative of the state of the immune response are (i) the expression levels of all of the following genes: CCR2, CD3D, CD3E, CD3G, CD8A, CXCL10, CXCL11, GZMA, GZMB, GZMK, GZMM, IL15, IRF1, PRF1, STAT1, CD69, ICOS, CXCR3, STAT4, CCL2, and TBX21; or (ii) Expression levels of all of the genes GZMH, IFNG, CXCL13, GNLY, LAG3, ITGAE, CCL5, CXCL9, PF4, IL17A, TSLP, REN, IHH, PROM1, and VEGFA. a process comprising: b) comparing each of the values ​​obtained in step a) for said biological markers with the distribution of values ​​obtained for each of said biological markers from a reference group of patients affected by said cancer; c) determining for each of the values ​​obtained in step a) for said biological marker the percentile of the distribution corresponding to the value obtained in step a); d) calculating the arithmetic mean or median value of the percentiles of the biological markers determined in step c); and e) comparing a predetermined reference arithmetic mean or a predetermined median value of the percentiles with the arithmetic mean or median value of the percentiles obtained in step d), said predetermined reference value correlating with survival time. The method includes:

2. The method of claim 1 , wherein each of the biological markers is a nucleic acid.

3. The method of claim 1 or 2, wherein each of the biological markers is detected by mRNA hybridization and / or amplification.

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