WO2014093623A1 - Procédé et analyses pour le pronostic du cancer à l'aide de jak2 - Google Patents

Procédé et analyses pour le pronostic du cancer à l'aide de jak2 Download PDF

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WO2014093623A1
WO2014093623A1 PCT/US2013/074669 US2013074669W WO2014093623A1 WO 2014093623 A1 WO2014093623 A1 WO 2014093623A1 US 2013074669 W US2013074669 W US 2013074669W WO 2014093623 A1 WO2014093623 A1 WO 2014093623A1
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jak2
expression
level
levels
cancer
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PCT/US2013/074669
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Christopher P. Miller
J. David BEATTY
Carl Anthony Blau
Nicole Urban
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University Of Washington Through Its Center For Commercialization
Fred Hutchinson Cancer Research Center
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the field of the invention relates to methods and assays for determining the prognosis of a cancer and/or selecting a treatment for such a cancer.
  • JAK2 The tyrosine kinase Janus Kinase 2 (JAK2) is important for the signaling of a variety of cytokine receptors such as erythropoietin receptor signaling during erythropoiesis and prolactin receptor signaling during mammary differentiation (1 , 2).
  • JAK2 has emerged as an important target in myeloproliferative disorders, and increasingly, in solid tumors such as breast cancer. Recent studies have implicated JAK2 in interleukin-6-dependent breast cancer stem cell self-renewal (3), and in both interleukin-6- and interleukin-8-dependent growth of triple negative breast cancers (4).
  • JAK2 signaling has been implicated as a mechanism of escape from other targeted breast cancer therapies (5). Based on these results, JAK2 inhibitors are currently undergoing evaluation in patients with breast cancer (6). JAK2 is also expressed in diverse cell types, including immune cells, and whether overall JAK2 mRNA levels in breast tumors are associated with clinical outcomes has not been evaluated.
  • the methods and assays provided herein are based, in part, on the discovery that JAK2 expression levels can indicate the level of T-cell infiltrate in a tumor (e.g., breast cancer tissue) and can predict outcome or prognosis in cancer patients. Accordingly, provided herein are methods and assays for determining prognosis in a patient having, or suspected of having, cancer (e.g., breast cancer). Also provided herein are methods for predicting the amount of T-cell infiltrate in a tumor, in part, by measuring JAK2 expression levels.
  • One aspect provided herein relates to a method for determining the prognosis of a subject having cancer, the method comprising: (a) measuring the expression of Janus kinase-2 (JAK2) in a biological sample obtained from a subject having or suspected of having cancer, and (b) comparing the level of expression of JAK2 to a reference, wherein the subject is determined to have a good prognosis when the levels of JAK2 in the biological sample are high, and wherein the subject is determined to have a poor prognosis when the level of JAK2 in the biological sample is low.
  • JAK2 Janus kinase-2
  • the method further comprises administering a therapeutic agent with, or without, a JAK2 inhibitor depending upon the JAK2 level.
  • the level of expression of JAK2 is the level of mRNA expression.
  • the cancer is breast cancer.
  • the biological sample is a tumor biopsy.
  • JAK2 is normalized to expression of a housekeeping gene.
  • JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.
  • JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.
  • the housekeeping gene is hydroxymethylbilane synthase (HMBS).
  • Also provided herein are methods for an adaptive immune response in a malignant tumor a method comprising: (a) measuring the expression of JAK2 in a biological sample of a malignant tumor, and (b) comparing the level of expression of JAK2 to a reference, wherein the malignant tumor is predicted to have an adaptive immune response when the levels of JAK2 expression in the biological sample are high, and
  • the malignant tumor is predicted to have a non-adaptive immune response when the levels of JAK2 expression are low.
  • the method further comprises administering a therapeutic agent with, or without, a JAK2 inhibitor depending upon the JAK2 level.
  • a JAK2 inhibitor depending upon the JAK2 level.
  • the level of expression of JAK2 is the level of mRNA expression.
  • the malignant tumor is breast cancer.
  • JAK2 is normalized to expression of a housekeeping gene.
  • the housekeeping gene is hydroxymethylbilane synthase (HMBS).
  • JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.
  • JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.
  • assays for predicting an adaptive immune response in a malignant tumor comprising: (a) isolating mRNA from a malignant tumor sample, (b) contacting the isolated mRNA with a probe or primer specific to JAK2, and (c) assaying the level of mRNA expression of JAK2,wherein the malignant tumor is predicted to have an adaptive immune response when the levels of JAK2 expression are high, and wherein the malignant tumor is predicted to have a non-adaptive immune response when the levels of JAK2 expression are low.
  • the malignant tumor is breast cancer.
  • JAK2 is normalized to expression of a housekeeping gene.
  • the housekeeping gene is hydroxymethylbilane synthase (HMBS).
  • JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.
  • JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.
  • Another aspect provided herein relates to methods for determining the prognosis of a subject having cancer, the method comprising: (a) measuring the expression of JAK2 in a biological sample obtained from a subject having or suspected of having cancer, and (b) comparing the level of expression of JAK2 to a reference, wherein the subject is determined to have a good prognosis when the level of JAK2 in the biological sample is above the level in the reference, and wherein the subject is determined to have a poor prognosis when the level of JAK2 in the biological sample is at or below the level in the reference.
  • Also provided herein are methods for predicting an adaptive immune response in a malignant tumor comprising: (a) measuring the expression of JAK2 in a biological sample of a malignant tumor, and (b) comparing the level of expression of JAK2 to a reference, wherein the malignant tumor is predicted to have an adaptive immune response when the level of JAK2 is increased in the biological sample as compared to the reference, and wherein the malignant tumor is predicted to have a non-adaptive immune response when the level of JAK2 is not changed or is decreased in the biological sample as compared to the reference.
  • assays for predicting an adaptive immune response in a malignant tumor comprising: (a) isolating mRNA from a malignant tumor sample, (b) contacting the isolated mRNA with a probe or primer specific to JAK2, (c) assaying the level of mRNA expression of JAK2, wherein the malignant tumor is predicted to have an adaptive immune response when the level of JAK2 is increased in the biological sample as compared to the reference, and wherein the malignant tumor is predicted to have a non-adaptive immune response when the level of JAK2 is not changed or is decreased in the biological sample as compared to the reference.
  • FIGs. 1A-1B JAK2 mRNA is associated with reduced distant breast cancer recurrence.
  • FIG. 1A JAK2 mRNA was measured by quantitative RT-PCR using RNA samples extracted from 223 breast tumor samples. Values for the JAK2 exon23/24 junction probe are shown normalized to the endogenous control gene HMBS. Box plots depict the distribution of JAK2 mRNA values in quartiles. The circle in the box represents the mean value while the horizontal line represents the median value. Outliers are shown as triangles. The p-value was calculated using the T-test.
  • FIG. IB JAK2 mRNA was modeled in a receiver operator curve as a predictor of reduced recurrence across different thresholds for defining high JAK2.
  • the true positive rate versus false positive rate of distant recurrence for each model versus actual outcomes is plotted for each threshold of defining high JAK2.
  • the percent of samples that are defined as high JAK2 are shown for each model (top %).
  • the p-value for the area under the curve (AUC) was calculated using the Z-test.
  • FIG. 2 JAK2 mRNA is associated with a protective concordance index in the
  • NETHERLANDS, OSLOVAL, and METABRIC cohorts The concordance index in patient rankings based on JAK2 mRNA versus survival outcomes is shown for each indicated cohort. For comparison, the concordance indexes for the least and most protective single genes in METABRIC (CDCA5 and FDG3) are shown.
  • FIGs. 3A-3C JAK2 mRNA correlates with the LYM metagene signature and tumor- infiltrating lymphocytes.
  • FIG. 3A The average expression of the top-ranked genes of the LYM metagene signature (SASH3, CD53, and NCKAP1L) in each tumor sample from METABRIC is shown relative to JAK2 mRNA.
  • FIG. 3B The scatter plot for ESRl, which is restricted to epithelial cells, is shown for comparison.
  • FIG. 3C JAK2 mRNA levels are shown relative to levels of tumor-infiltrating lymphocytes in tumor samples from the favorable prognosis METABRIC Integrative Cluster 4 that is enriched for an adaptive immune response signature. Box plots depict the distribution of JAK2 mRNA values in quartiles. The circle in the box represents the mean value while the horizontal line represents the median value. The p-value was calculated using ANOVA.
  • FIGs. 4A-4D JAK2 mRNA quantification in primary breast tumors.
  • Transcripts for JAK2 (exon 23/24 probe), and the breast cancer genes ESRl, ERBB2, and PGR were measured by quantitative RT-PCR using RNA samples extracted form formalin- fixed, paraffin- embedded breast cancer samples. Values are shown normalized to the endogenous control gene HMBS, and samples are arranged on the x-axis in order of increasing RNA abundance/integrity as measured by HMBS Ct values. Values greater than six standard deviations from the mean (asterisk) are not to scale.
  • FIG. 4B Correlation between intra-transcript measurements using TAQMAN probes for the indicated exon junctions for JAK2.
  • FIG. 4C Transcript levels of JAK2 (exon 23/24 probe) and the breast cancer genes ESRl, ERBB2, and PGR are shown in 14 breast cancer cases in which three separate FFPE tumor samples were available.
  • FIG. 4D Transcript levels of the breast cancer genes ESRl, ERBB2, and PGR2 are shown relative to their respective clinical hormone receptor status. U, unavailable.
  • FIG. 5 Ruxolitinib inhibits the anti-CD3 -dependent production of IFN- ⁇ .
  • Murine splenocytes were stimulated with anti-CD3 in the presence of the indicated concentrations of ruxolitinib or a vehicle control (DMSO).
  • IFN- ⁇ levels in culture supernatants were measured by ELISA. Error bars are the standard deviation of triplicate ELISA determinations.
  • kits for predicting the prognosis of a subject having cancer comprising measuring the level of JAK2 expression.
  • the term "subject" is preferably a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples.
  • sample and “biological sample” are used interchangeably and refer to a sample of biological tissue, cells, or fluid that, in a healthy and/or pathological state, contains cells.
  • the biological sample is a biopsy sample from a site of suspected tumor growth ⁇ e.g., breast tissue or breast cancer tissue).
  • a biopsy sample can include e.g., tissue biopsy, fine needle aspiration, core needle biopsy, vacuum assisted biopsy, open surgical biopsy, among others.
  • a biological sample is taken from a human subject, and in alternative embodiments the biological sample is taken from any mammal, such as rodents, animal models of diseases, commercial animals, companion animals, dogs, cats, sheep, cattle, and pigs, etc.
  • JAK2 Japanese kinase 2
  • JAK2 refers to a mRNA or protein product of the JAK2 gene found on chromosome 9, locus 9p24 (Gene ID: 3717 (human), NCBI database available on the world wide web at ncbi.nlm.nih.gov/sites/gene).
  • the JAK2 protein comprises tyrosine kinase activity and can also be referred to in the literature as JTK10 and THCYT3.
  • the term “poor prognosis” refers to a subject having at least a 2-fold increase in likelihood of cancer metastasis and death compared to a subject without cancer (e.g., a benign tumor). In other embodiments, the subject has at least a 3-fold, at least a 5-fold, at least a 10-fold or at least a 100-fold increase in risk of metastasis and death as compared to a subject without cancer. In one embodiment, “poor prognosis” refers to a subject that is unlikely to survive 5 years from diagnosis and/or prognosis.
  • the term "good prognosis” refers to a subject having less than a 2-fold increase in the likelihood of cancer metastasis, and death as compared to a subject without cancer.
  • the subject with a good prognosis will have less than a 1-fold increase, less than a 50% increase, less than a 25% increase, less than a 10%> increase, less than a 5% increase or even no increase in risk of metastasis and death as compared to a subject without cancer.
  • the subject can also have a reduced risk of cancer metastasis and death as compared to a subject lacking cancer.
  • "good prognosis” means that a subject is likely to survive for at least 5 years following diagnosis or prognosis.
  • high when used in reference to JAK2 expression levels (e.g., when the levels of JAK2 in the biological sample are high) means that the expression levels of JAK2 in a given sample are greater than 10%> of the expression level of a chosen housekeeping gene (e.g., as determined by intensity of a signal using e.g., RT-PCR etc) in the same sample.
  • high JAK2 expression levels means that the expression level of JAK2 in a given sample is greater than at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%>, or more of the expression level of the selected housekeeping gene.
  • the term “low” when used in reference to JAK2 expression levels means that the expression levels of JAK2 in a given sample are ⁇ 10% of the expression level of the selected housekeeping gene in the same sample.
  • normalized to the expression level of a housekeeping gene refers to the conversion of a data value representing the expression level of JAK2 in a sample by dividing it by the expression data value representing the level of a normalizing gene/protein (e.g., hydroxymethylbilane synthase (HMBS)), thereby permitting comparison of normalized marker values among a plurality of samples or to a reference.
  • HMBS hydroxymethylbilane synthase
  • normalizing protein and “normalizing factor” are used interchangeably and refer to expression of mRNA or protein of a control marker against which the amounts of marker or combination of markers of interest are normalized to permit comparison of amounts of the mRNA or protein of interest among different biological samples.
  • a normalizing transcript or protein is constitutively expressed and is not differentially regulated between at least two physiological states or conditions from which samples will be analyzed, e.g., given disease and non-disease states.
  • a normalizing control does not vary substantially outside of a range found in a normal healthy population (e.g., ⁇ 30%, ⁇ 25%, ⁇ 20%, ⁇ 15%, preferably ⁇ 10%>, ⁇ 7%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1% or less) or in the presence and absence of e.g., cancer.
  • housekeeping gene refers to a gene encoding a transcript and/or protein that is constitutively expressed, and is necessary for basic maintenance and essential cellular functions.
  • a housekeeping gene generally is not expressed in a cell- or tissue- dependent manner, most often being expressed by all cells in a given organism.
  • Some examples of housekeeping proteins include e.g., HMBS, actin, tubulin, GAPDH, among others.
  • the term "statistically significant” or “significantly” refers to statistical significance and generally means two standard deviations (2SD) or more above or below normal or a reference. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.
  • malignant tumor refers to a tumor that has been determined to be cancerous (i.e., invasive or metastatic) and/or has been assigned a stage grouping using clinical or pathological staging techniques known to those of skill in the art (e.g., the TNM grading scale or the Stage 0-Stage IV grading scale, e.g., a Stage I or higher grouping refers to a malignant tumor).
  • cancerous i.e., invasive or metastatic
  • stage grouping using clinical or pathological staging techniques known to those of skill in the art (e.g., the TNM grading scale or the Stage 0-Stage IV grading scale, e.g., a Stage I or higher grouping refers to a malignant tumor).
  • adaptive immune response refers to a tumor comprising T- cell lymphocytes that actively target cancerous cells in the tumor.
  • non-adaptive immune response refers to a tumor lacking T- cell lymphocytes or a tumor lacking T-cell lymphocyte activity against cancerous cells in the tumor.
  • the term "high T-cell infiltration” refers to T-cell or T-cell markers in a tumor at a level of at least 20% greater than the level in a tumor sample known to have a poor prognosis or at a level in which T-cells make up greater than 10% of the non-tumor cells in the tumor on a per cell number basis.
  • the T-cell or T-cell markers in a tumor are at a level of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or even 1000- fold greater than the level e.g., in a tumor sample known to have a poor prognosis.
  • the term “low T-cell infiltration” refers to T-cell or T-cell markers in a tumor at a level of less than, or equal to, 10% of the non-tumor cells in the tumor on a per cell number basis.
  • the term “reference” refers to a reference value, or range of values, obtained for JAK2 expression from e.g., at least one subject determined to lack detectable cancer or at least one subject determined to have a cancer.
  • the reference value or range of values can be obtained from a plurality of subjects in a population substantially free of cancer (i.e., cancer is not detectable by typical clinical means) or can be obtained from a plurality of subjects in a population having cancer (e.g., breast cancer).
  • the reference sample can be stored as a value(s) on a computer or PDA device to permit comparison with a value obtained from a subject using the methods described herein.
  • the reference sample can also be obtained from the same subject e.g., at an earlier time point prior to onset of detectable cancer using clinical tests known to those of skill in the art.
  • One of skill in the art can determine an appropriate reference sample for use with the methods described herein. In one
  • the reference is obtained from a subject or plurality of subjects having, or diagnosed with having, cancer, such as breast carcinoma.
  • plural of subjects refers to at least two subjects (e.g., 2, 3,
  • the terms “decrease” , “reduced”, “reduction” , “decrease” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced”, “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference value or reference level, for example a decrease by at least about 20%, or at least about 30%), or at least about 40%, or at least about 50%, or at least about 60%>, or at least about 70%, or at least about 80%o, or at least about 90%> or up to and including a 100% decrease (e.g., absent level or non- detectable level as compared to a reference sample), or any decrease between 10-100%) as compared to a reference level.
  • a 100% decrease e.g., absent level or non- detectable level as compared to a reference sample
  • the terms “increased” /'increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statistically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10%> as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%o, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%o or up to and including a 100% increase or any increase between 10-100%) as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50- fold increase, at least about a 100-fold increase, at least about a 1000
  • compositions, methods, and respective component(s) thereof that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
  • the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the methods and assays provided herein are applicable to determining or predicting the prognosis of a subject having cancer. Essentially any cancer or cancer cell population can be evaluated using the methods and assays described herein. While mammary carcinoma is exemplified, additional epithelial cell cancers are also specifically contemplated, as are non-epithelial cell cancers.
  • Cancers contemplated to be amenable to the methods described herein include, but are not limited to, breast cancer; bladder cancer; brain metastases; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer including colorectal carcinomas; endometrial cancer; esophageal cancer; gastric cancer; head and neck cancer; intraepithelial neoplasms including Bowen's disease and Paget's disease, liver cancer; lung cancer including small cell lung cancer and non-small cell lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; osteosarcomas; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, lip
  • a “biological sample” refers to a sample obtained from a subject that comprises a cell or population of cells or a quantity of tissue or fluid from a subject. Most often, the sample has been removed from a subject, but the term “biological sample” can also refer to cells or tissue analyzed in vivo, i.e. without removal from the subject.
  • a biological sample can be obtained from essentially any tissue suspected of containing cancerous cells. Some non-limiting examples of tissues include e.g., brain, liver, lung, mammary, breast, gut, stomach, fat, muscle, spleen, testes, uterus, ovary, skin, endocrine organ and bone, etc.
  • a biological sample comprises cells including, but not limited to, epithelial, endothelial, neuronal, adipose, cardiac, skeletal muscle, fibroblast, immune cells, hepatic, splenic, lung, circulating blood cells, reproductive cells, gastrointestinal, renal, bone marrow, and pancreatic cells.
  • the biological sample is a biopsy from a growth or tumor (e.g., a malignant tumor).
  • a biological sample is from a resection, biopsy, vacuum assisted biopsy, open surgical biopsy, or core needle biopsy of a primary, secondary or metastatic tumor.
  • fine needle aspirate biological samples are also useful. Samples can be fresh, frozen, fixed or optionally paraffin-embedded, frozen or subjected to other tissue preservation methods.
  • a biological sample can be provided by removing a sample of cells from subject, but archival tissues with an outcome history (e.g., poor prognosis, e.g., death, or good prognosis e.g., 5 year, 10 year, or greater survival) can also be used.
  • the biological sample can be pretreated as necessary for storage or preservation, by dilution in an appropriate buffer solution or concentrated, if desired. Any of a number of standard aqueous buffer solutions, employing one of a variety of buffers, such as phosphate, Tris, or the like, at physiological pH can be used.
  • the biological sample can in certain circumstances be stored for use prior to use in the methods or assays as disclosed herein. Such storage can be at +4°C or frozen, for example at -20°C or -80°C.
  • a biological sample can also be obtained for use as a control sample, and which corresponds to normal tissue having the same origin.
  • a biological sample can be obtained for use as a control sample, and which corresponds to normal tissue having the same origin.
  • the biological sample can be a breast cancer sample and the biological control sample can be breast tissue from another area of the same breast or breast tissue from the other breast.
  • a biological control sample can be a prior biopsy from the same subject to be tested prior to the onset of cancer.
  • JAK2 can be detected by any means of detecting expression of a polypeptide, or fragment thereof, or an mRNA transcript of the polypeptide. These detection methods are known to those skilled in the art and/or are described briefly below.
  • the level of JAK2 can be normalized to another protein or its mRNA (e.g., a normalizing gene/protein) such as e.g., a
  • Protein from a biological sample to be analyzed can be detected or isolated using techniques, including but not limited to immunohistochemistry, Western blot analysis, e.g.,
  • Antibodies directed against JAK2 can be applied for disease diagnostics and prognostics. Such methods can be used to detect abnormalities or differences in the level of expression of JAK2, and/or the tissue, cellular, or subcellular location of the peptide. Generally, however, it will be the amount of JAK2 that is of primary interest. Antibodies to be used for protein analysis are widely available through commercial sources including ABCAMTM (Cambridge, MA), NEW ENGLAND BIOLABSTM (Ipswich, MA), SANTA CRUZ BIOTECHNOLOGIESTM (Santa Cruz, CA), and CELL SIGNALINGTM (Danvers, MA), among others.
  • Antibodies can also be raised against a polypeptide or portion of a polypeptide by methods known to those skilled in the art. Antibodies are readily raised in animals such as rabbits or mice by immunization with the gene product, or a fragment thereof. Immunized mice are particularly useful for providing sources of B cells for the manufacture of hybridomas, which in turn are cultured to produce large quantities of monoclonal antibodies. While both polyclonal and monoclonal antibodies can be used in the methods described herein, it is preferred that a monoclonal antibody is used where conditions require increased specificity for a particular protein. Antibody manufacture methods are described, for example, in Harlow et al., 1988.
  • the antibodies that recognize JAK2 may be any antibody variant, antibody derivative, bispecific molecule, human antibody, humanized antibody, monoclonal antibody, human monoclonal, and variants and antigen-binding fragments thereof. Conventional methods for immunohistochemistry are described in Harlow and Lane, 1988 and Ausbel et al, 1987.
  • expression levels of JAK2 can be determined by measuring the level of messenger RNA (mRNA) expression. Detection of mRNA expression is known by persons skilled in the art, and can comprise, for example PCR procedures, RT-PCR, Northern blot analysis, RNAse protection assay, etc. Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures that are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample.
  • mRNA messenger RNA
  • PCR provides a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for an amplified product of the correct size.
  • the primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, e.g., each primer is specifically designed to be complementary to one strand of the genomic locus to be amplified.
  • JAK2 expression levels can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time RT-PCR methods.
  • RT reverse-transcription
  • QRT-PCR quantitative RT-PCR
  • Methods of RT-PCR and QRT-PCR are known in the art, and are described in more detail below.
  • labeled probes can be used in conjunction with amplification of cDNA. (Holland et al., 1991).
  • U.S. Patent No. 5,210,015 by Gelfand et al. describes fluorescence-based approaches to provide real time measurements of amplification products during PCR.
  • Such approaches have generally either employed intercalating dyes (such as ethidium bromide) to indicate the amount of double-stranded DNA present, or they have employed probes containing fluorescence- quencher pairs (also referred to as the "Taq-Man" approach) where the probe is cleaved during amplification to release a fluorescent molecule whose concentration is proportional to the amount of double-stranded DNA present.
  • the probe is digested by the nuclease activity of a polymerase when hybridized to the target sequence to cause the fluorescent molecule to be separated from the quencher molecule, thereby causing fluorescence from the reporter molecule to appear.
  • the Taq-Man approach uses a probe containing a reporter molecule-quencher molecule pair that specifically anneals to a region of a target polynucleotide.
  • Primers or probes of use in the methods described herein include naturally occurring or recombinant single- or double- stranded nucleic acids or chemically synthesized nucleic acids. They may be labeled by nick translation, Klenow fill-in reaction, PCR or other methods known in the art. Probes useful in the methods described herein, their preparation and/or labeling are described in, for example Sambrook et al. (1989). A probe can be a polynucleotide of any length suitable for selective
  • probes are labeled with two fluorescent dye molecules to form so-called “molecular beacons” (Tyagi, S. and Kramer, F.R., 1996).
  • molecular beacons signal binding to a complementary nucleic acid sequence through relief of intramolecular fluorescence quenching between dyes bound to opposing ends on an oligonucleotide probe.
  • a quenching molecule is useful with a particular fluorophore if it has sufficient spectral overlap to substantially inhibit fluorescence of the fluorophore when the two are held proximal to one another, such as in a molecular beacon, or when attached to the ends of an oligonucleotide probe from about 1 to about 25 nucleotides.
  • primers for use with the methods and assays described herein are nucleic acids which hybridize to a nucleic acid sequence which is adjacent to the region of interest or which covers the region of interest and is extended.
  • a primer can be used alone in a detection method, or a primer can be used together with at least one other primer or probe in a detection method.
  • Primers can also be used to amplify at least a portion of a nucleic acid.
  • probes for use in the methods are nucleic acids which hybridize to the region of interest and which are not further extended.
  • nucleic acids, or fragments thereof, to be used in the methods of the invention can be prepared according to methods known in the art and described, e.g., in Sambrook et al. (1989), supra.
  • discrete fragments of the DNA can be prepared and cloned using restriction enzymes.
  • discrete fragments can be prepared using PCR using primers having an appropriate sequence under the manufacturer's conditions.
  • Oligonucleotides can be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from APPLIED BIOSYSTEMSTM, etc.).
  • phosphorothioate oligonucleotides can be synthesized by the method of Stein et al., 1988, methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988). Reagents and hardware for conducting PCR are commercially available. Primers useful to amplify sequences from a particular gene region are preferably complementary to, and hybridize specifically to sequences in the target region or in its flanking regions. Nucleic acid sequences generated by amplification may be sequenced directly.
  • JAK2 mRNA or protein levels for determining the prognosis of a subject having cancer. In such methods and assays, it is necessary to determine whether the JAK2 levels are "high” or "low.” This can be achieved by any number of methods known to those of skill in the art.
  • the expression value (e.g., intensity of a signal) of JAK2 is compared to the expression value for a given housekeeping gene (e.g., intensity of a signal specific for the housekeeping gene). Since the expression values for housekeeping genes are generally much higher than the expression values of JAK2, one can simply express the expression value of JAK2 in a single sample as a percentage or fraction of expression value for the housekeeping gene in the same sample. For example, the expression value of JAK2 in a given sample can be compared to the expression value for HMBS (Gene ID: 3145) in the same sample; JAK2 levels can be expressed as 20% of the HMBS gene product (e.g., mRNA or protein).
  • HMBS Gene ID: 3145
  • JAK2 levels that are greater than 10% of the levels of the housekeeping gene are considered “high,” whereas JAK2 levels that are less than 10%> of the housekeeping gene are considered “low.”
  • any housekeeping gene can be used to determine whether JAK2 levels are high or low.
  • the methods and assays provided herein further comprise a step of measuring the expression of a housekeeping gene, such as HMBS.
  • the expression levels of JAK2 are normalized to a control, such as a housekeeping gene. This is particularly useful for comparing levels of JAK2 amongst samples (e.g., between individuals etc). Suitable controls for normalizing expression levels of biomarkers are known to those of skill in the art.
  • any gene or gene product can be used as a normalizing control, provided that the mRNA or protein is constitutively expressed, and is not differentially regulated in disease states (e.g., cancer).
  • a gene or gene product can be used as a normalizing marker by comparing the expression levels in samples taken at different time points from one individual, or among a plurality of samples taken from diseased (e.g., cancer) and control populations.
  • diseased e.g., cancer
  • an appropriate normalization control marker will not fluctuate widely (e.g., less than 30%) among time points or among disease populations when assessed using an assay (e.g., a microarray).
  • the normalizing control is the housekeeping gene
  • HMBS hydroxymethylbilane synthase
  • the methods and assays provided herein further comprise a step of measuring the expression of a housekeeping gene, such as HMBS.
  • the level of JAK2 is compared to the level of a housekeeping gene or protein to provide an internal reference. This comparison will also normalize the JAK2 expression to that housekeeping gene or protein level.
  • an external reference is used. This type of reference refers to the level of JAK2 expression or the level of T-cell infiltrate in a known sample against which another sample is compared (e.g., obtained from a subject substantially free of cancer or a cancer sample known to have high, or low, T-cell infiltration).
  • Such a reference or "standard” is useful for determining the amount of JAK2 or the relative increase/ decrease of JAK2 in a biological sample.
  • a standard serves as a reference level for comparison, such that samples can be compared to an appropriate standard in order to infer the presence, absence or extent of cancer in a subject.
  • a biological standard is obtained at an earlier time point
  • a standard can be from the same individual having been taken at a time after the onset or diagnosis of cancer. In such instances, the standard can provide a measure of the efficacy of treatment.
  • a standard can also be drawn from unaffected tissue corresponding to the tumor type from the same individual, e.g., healthy breast tissue of an individual with breast cancer.
  • a standard level can be obtained, for example, from a known biological sample from a different individual (e.g., not the individual being tested) that is substantially free of cancer.
  • a known sample can also be obtained by pooling samples from a plurality of individuals to produce a standard over an averaged population, wherein a standard represents an average level of JAK2 among a population of individuals.
  • the level of JAK2 in a standard obtained in this manner is representative of an average level of this marker in a general population or a diseased population.
  • An individual sample is compared to this population standard by comparing expression of JAK2 from a sample relative to the population standard.
  • an increase in the amount of JAK2 over a standard (e.g., obtained from subjects substantially free of cancer) will indicate that the subject has a good prognosis, while a decrease in the amount of JAK2 will indicate that the subject has a poor prognosis.
  • a standard is obtained from a population of subjects having cancer. It should be noted that there is often variability among individuals in a population, such that some individuals will have higher levels of JAK2 expression, while other individuals have lower levels of expression; for this reason, for JAK2 levels in particular, an internal reference gene e.g., a housekeeping gene expressed in the same biological sample can be particularly appropriate. However, one skilled in the art can make logical inferences on an individual basis regarding the detection and treatment of cancer as described herein.
  • a standard or series of standards can also be synthesized.
  • a known amount of JAK2 (or a series of known amounts) can be prepared within the typical expression range for JAK2 that is observed in a general population.
  • This method has an advantage of being able to compare the extent of disease in two individuals in a mixed population. This method can also be useful for subjects who lack a prior sample to act as a standard or for routine follow-up post-diagnosis. This type of method can also allow standardized tests to be performed among several clinics, institutions, or countries etc.
  • a sample from a corresponding non-tumor tissue e.g., a sample obtained from a healthy breast in the same subject or different subjects
  • a sample from a known or characterized tumor tissue e.g., a known stage of tumor or a known level of T-cell infiltration
  • T-cell trafficking refers to migration of T lymphocytes to a site of immune response activity.
  • Naive T cells recirculate throughout the body, leaving and reentering the lymphoid tissues as they sample their environment for the presence of non-self antigens or "danger" signals.
  • Lymphoid tissues are specially adapted to help promote encounters between antigen-specific T-cell receptors expressed on T cells and their cognate antigens.
  • Specialized antigen-presenting cells concentrate within lymphoid tissues, and are specially adapted to interact with and to present antigens to T cells to initiate an immune response by T cells genetically programmed to recognize a particular antigen.
  • T cells proliferate, undergo differentiation to produce a variety of secreted and cell-associated products, including cytokines, and migrate to tissue sites associated with the antigen.
  • cytokines include cytokines
  • T-lymphocytes localized to a tumor site are referred to as
  • T-infiltrating lymphocytes Infiltrating lymphocytes. Infiltrating lymphocytes are frequently found in tumor tissues, which is indicative of an ongoing host immune response. Clinically, an increase in infiltration of T-cells to the tumor site is closely associated with better prognosis. For example, preventive vaccinations were shown to be effective in inducing the rejection of inoculated tumor cells. Such increases in T-cell infiltration are indicative of an adaptive immune response, as that term is used herein.
  • immunological escape appears to occur when the balance between factors for tumor growth and destruction favor the tumor (e.g., a non-adaptive immune response).
  • Factors that may contribute to immunological escape include tumor kinetics, antigenic modulation, antigen masking and blocking factors.
  • antigenic modulation facilitates escape by removing the target antigens that the immune system's effector cells would recognize. This is known to occur when administering xenogeneic antibodies during immunotherapy.
  • Tumor escape from effector cells may also occur because certain molecules bind to the surfaces of the tumor cell and mask the tumor antigens thereby preventing adhesion of attacking lymphocytes.
  • the presence of infiltrating tumor cells can be confirmed by detecting one or more T-cell markers including, but not limited to, T-cell antigen receptor, CD8, T6, CD4, CD3, TAC, T9, CD2, and VLA-1.
  • T-cell markers including, but not limited to, T-cell antigen receptor, CD8, T6, CD4, CD3, TAC, T9, CD2, and VLA-1.
  • CD3 is generally considered a "pan T-cell" marker, as it is expressed on all CD4+ and CD8+ T-cells.
  • CD3 expression can be evaluated using PCR, for example, or by methods that detect the protein directly, e.g., using anti-CD3 antibodies.
  • Janus kinase 2 is a non-receptor tyrosine kinase in the Janus kinase family.
  • JAK2 is involved in several signaling pathways through receptors including, for example, type II cytokine receptor family (e.g., interferon receptors), the GM-CSF receptor family (e.g., IL-3R, IL-5R and GM-CSF-R), the gpl30 receptor family (e.g., IL-6R), and the single chain receptors (e.g., Epo-R, Tpo-R, GH-R, PRL-R). JAK2 signaling is activated downstream from the prolactin receptor.
  • type II cytokine receptor family e.g., interferon receptors
  • the GM-CSF receptor family e.g., IL-3R, IL-5R and GM-CSF-R
  • the gpl30 receptor family e.g., IL-6R
  • single chain receptors e.g., Epo-R, Tpo-R, GH-R, PRL-R
  • JAK2 inhibitors include, but are not limited to, ruxolitinib, baricitinib, CYT387, lestaurtinib, pacritinib, and TG101348.
  • a JAK2 inhibitor is used herein to treat a subject having cancer.
  • the JAK2 inhibitor is ruxolitinib.
  • a method for determining the prognosis of a subject having cancer comprising:
  • the subject is determined to have a poor prognosis when the level of JAK2 in the biological sample is low.
  • a method for predicting an adaptive immune response in a malignant tumor comprising:
  • the malignant tumor is predicted to have an adaptive immune response when the levels of JAK2 expression in the biological sample are high, and
  • the malignant tumor is predicted to have a non-adaptive immune response when the levels of JAK2 expression are low.
  • HMBS hydroxymethylbilane synthase
  • JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.
  • JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.
  • An assay for predicting an adaptive immune response in a malignant tumor comprising:
  • the malignant tumor is predicted to have an adaptive immune response when the levels of JAK2 expression are high, and
  • the malignant tumor is predicted to have a non-adaptive immune response when the levels of JAK2 expression are low.
  • HMBS hydroxymethylbilane synthase
  • JAK2 Janus Kinase-2
  • JAK2 is also expressed beyond the tumor epithelium, including in immune cells, and whether JAK2 mRNA levels in breast tumors correlate with outcomes has not been evaluated.
  • JAK2 mRNA was measured in 223 archival breast tumors and associations with distant recurrence were evaluated by logistic regression. The frequency of correct pairwise comparisons of patient rankings based on JAK2 levels versus survival outcomes, the concordance index (CI), was evaluated using data from 2,460 patients in 3 cohorts.
  • CI concordance index
  • JAK2 was associated with a protective CI ( ⁇ 0.5) in the public cohorts:
  • the JAK1/2 inhibitor ruxolitinib potently inhibited the anti-CD3 -dependent production of interferon-gamma, a marker of the differentiation of T-helper cells along the tumor- inhibitory Thl pathway. The potential for JAK2 inhibitors to interfere with the anti-tumor capacities of T cells should be evaluated.
  • JAK2 mRNA levels were significantly higher in tumors from women who experienced no distant recurrence compared to those who experienced a distant recurrence (FIG. 1 A).
  • the association between increasing JAK2 mRNA and decreasing distant recurrence was significant for both JAK2 exon8/9 and exon23/24 probes in logistic regression when JAK2 mRNA was treated as a continuous or dichotomous variable (Table 2).
  • Table 2 JAK2 1 Recurrence Associations Before and After Adjusting for Significantly
  • cCoefficients and p-values were obtained using logistic regression with above-median versus below- median transcript expression as a predictor of recurrence. Values for individuals with multiple specimens were averaged to one value per individual.
  • the inventors evaluated the association between JAK2 mRNA levels and outcomes in the NETHERLANDS, METABRIC, and OSLOVAL cohorts.
  • the inventors used the concordance index (19), which provides a convenient numerical measure of the strength and direction of an association between a single gene and outcomes and was used as the metric to score submissions in the Sage Bionetworks DREAM breast cancer prognosis challenge (12).
  • the concordance index is the relative frequency of correct pairwise comparisons of patient rankings based on gene expression levels versus survival outcomes. A concordance index >0.5 indicates that higher expression is associated with shorter survival while a value ⁇ 0.5 indicates that higher expression is associated with longer survival.
  • the single-gene mRNA with the poorest prognosis was previously found to be CDCA5 with a concordance index of 0.651, indicating that if 2 patients were randomly selected, the patient with the higher CDCA5 level will have shorter survival 65.1% of the time (12).
  • the single most protective gene was FGD3 with a concordance index of 0.352, indicating that if 2 patients were randomly selected, the patient with the higher FGD3 level will have the longer survival 64.8% (100%-35.2%) of the time.
  • JAK2 mRNA exhibited a protective concordance index in all 3 datasets (FIG. 2). The strongest effect was observed in the NETHERLANDS cohort, where the concordance index of 0.376 indicates that if 2 patients were randomly selected, the patient with the higher tumor JAK2 mRNA level will have the longer recurrence-free survival 62.4% (100%>-37.6%>) of the time. Similarly, JAK2 mRNA was consistently protective, albeit to a lesser extent, in the METABRIC and OSLOVAL cohorts.
  • JAK2 mRNA was even more protective for both overall and disease-specific survival in these subtypes.
  • JAK2 is expressed in diverse cell types including immune cells, and high levels of tumor-infiltrating lymphocytes, especially T cells, have been associated with a favorable prognosis in breast cancer (14, 21). It was therefore tested whether breast tumor JAK2 mRNA levels correlate with the T cell transcript enriched LYM metagene signature.
  • the top ten genes in the LYM metagene signature are PTPRC (CD45), CD53, LCP2 (SLP-76), LAPTM5, DOCK2, IL10RA, CYBB, CD48, ITGB2 (LFA-1) and EVI2B.
  • the LYM metagene is associated with favorable prognosis in breast cancer, in particular in ER-negative breast cancer and even more so in the absence of multiple positive lymph nodes, and recently formed part of the winning prognostic model in the Sage Bionetworks DREAM breast cancer prognosis challenge (12, 15).
  • the LYM metagene was recently defined with increased accuracy following mining from data sets from multiple cancer types available from The Cancer Genome Atlas (22). Indeed, there was a highly significant correlation between JAK2 mRNA levels and the LYM metagene in tumor samples from METABRIC (FIG. 3A). In contrast, the LYM metagene had an inverse correlation with the breast epithelial associated transcript ESR1 (FIG. 3B).
  • JAK2 mRNA levels correlated strongly with levels of infiltrating lymphocytes as determined by pathologic assessment in a subset of 156 tumors for which these data were available (FIG. 3C). These tumor samples belonged to METABRIC integrative cluster 4, which was previously associated with a favorable prognosis and a strong adaptive immune response signature (14).
  • JAKl/2 inhibitor ruxolitinib markedly inhibited the anti-CD3 -dependent production of IFN- ⁇ , a marker of the differentiation of T-helper cells along the tumor-inhibitory Thl pathway (23).
  • JAK2 mRNA levels and improved breast cancer outcomes This association was the strongest in the case-control study which matched for variables known to influence recurrence. Although the influence of JAK2 mRNA on survival outcomes in the unmatched public cohorts was predictably not as strong, the remarkably consistent association between higher JAK2 mRNA and favorable survival is unexpected in the sense that JAK2 protein collaborates with a variety of cytokine receptors that were recently shown to promote breast cancer growth (3, 4). These results likely reflect a lack of concordance between JAK2 mRNA and levels of phospho-JAK2 protein in breast epithelial cells and/or the presence of additional JAK2-expressing cell types in tumor specimens that are favorable for prognosis.
  • the inventors observed a strong correlation between JAK2 mRNA and levels of tumor-infiltrating lymphocytes and the favorable prognosis LYM metagene signature.
  • the finding that a single gene correlates with a larger biomolecular metagene that is associated with prognosis is pronounced of the frequent association between single genes and the prognostically significant PCNA and CIN metagene signatures for proliferation and chromosomal instability (12).
  • JAK2 is involved in IL-12 and IFN- ⁇ signaling, key regulators of the tumor inhibitory Thl response (23-25). Furthermore, JAK inhibitors have been shown to impair production of these Thl cytokines (26) and to inhibit IFN-y-dependent T cell trafficking in murine preclinical studies (27).
  • JAK2/STAT3 signaling pathway is required for growth of CD44(+)CD24(-) stem cell-like breast cancer cells in human tumors. J Clin Invest. 2011 ;121 :2723-35. [000106] Hartman ZC, Poage GM, den Hollander P, Tsimelzon A, Hill J, Panupinthu N, et al.
  • Pencina MJ DAgostino RB. Overall C as a measure of discrimination in survival analysis: model specific population value and confidence interval estimation. Stat Med. 2004;23:2109- 23.
  • Miller CP Lowe KA, Valliant-Saunders K, Kaiser JF, Mattern D, Urban N, et al.
  • IL-12 induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12. J Exp Med. 1995;181 :399-404.
  • Fridman JS Scherle PA, Collins R, Burn TC, Li Y, Li J, et al. Selective inhibition of
  • JAK1 and JAK2 is efficacious in rodent models of arthritis: preclinical characterization of INCB028050. J Immunol. 2010;184:5298-307.
  • the breast cancer research database at the Swedish Cancer Institute contains patient, tumor, treatment and outcomes data collected prospectively since 1989 for over 12,000 patients.
  • the dataset was reduced to women followed for at least 2 years with invasive carcinoma with Tl-3 primary tumors and treated by partial mastectomy plus breast irradiation or total mastectomy, sentinel node biopsy or axillary dissection, and adjuvant chemotherapy. Patients with multiple primaries, T4 primaries or distant metastases, and those receiving neoadjuvant chemotherapy were excluded.
  • Matching variables included extranodal extension of metastasis, lymphovascular invasion, estrogen receptor (ER) / progesterone receptor (PR) / human epidermal growth factor receptor-2 (HER2) status, T-stage, N-stage, and the interaction between T- and N-stage.
  • the T-N interaction term allowed for the fact that tumor size is more important for women without positive nodes than women with positive nodes.
  • diagnosis dates of the recurring and non-recurring patients were no more than 2 years apart.
  • Propensity scoring was used to match 112 cases of distant recurrence following surgery to 112 nonrecurring controls using the "Optmatch" package (16) for R (17).
  • FFPETM system (STRATAGENETM). The amount of tumor versus normal tissue in each section was greater than 50% for 84% of samples and greater than 90%> tumor tissue for 47% of samples as determined by pathologists' inspection of hematoxylin and eosin stained slides.
  • cDNA was synthesized using random hexamers and Superscript III (INVITROGENTM) and was preamplified for 14 cycles using the TAQMANTM preamplification system (APPLIED BIOSYSTEMSTM). All probes bound to exon junctions to prevent genomic DNA amplification (Table 3).
  • ERBB2 human epidermal growth factor receptor 2 Hs01001580 ml 60
  • BIOSYSTEMSTM BIOSYSTEMSTM Relative quantification was calculated as 2 A -delta Ct, where delta Ct values were calculated by subtracting the indicated control gene mean Ct value from the target gene mean Ct value.
  • Murine splenocytes were collected under a protocol approved by the University of Washington Institutional Animal Care and Use Committee and plated at lxl 0 6 cells per mL in RPMI, 10% FBS, 50 ⁇ beta-mercaptoethanol, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin sulfate, and 2.5 ⁇ g/mL amphotericin-B. Cultures were treated with 25 ng/mL anti-CD3 (purchased from UCSF monoclonal antibody core) plus the indicated concentrations of ruxolitinib (SELLECK
  • IFN- ⁇ interferon-gamma

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Abstract

La présente invention concerne des procédés et des analyses pour la prédiction du pronostic d'un sujet atteint d'un cancer, comportant la mesure du taux d'expression de JAK2. La présente invention concerne également des procédés et des analyses utiles pour la prédiction d'une réponse immunitaire adaptative dans une tumeur (par exemple une tumeur maligne), en particulier le cancer du sein.
PCT/US2013/074669 2012-12-12 2013-12-12 Procédé et analyses pour le pronostic du cancer à l'aide de jak2 WO2014093623A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2007045996A1 (fr) * 2005-10-19 2007-04-26 INSERM (Institut National de la Santé et de la Recherche Médicale) Procede in vitro pour le pronostic de la progression et du resultat d'un cancer chez un patient, et systeme de mise en oeuvre
WO2009048901A1 (fr) * 2007-10-09 2009-04-16 University Of Washington Mesure quantitative/semi-quantitative du récepteur de l'érythropoïétine sur des cellules cancéreuses
US8202881B2 (en) * 2009-09-03 2012-06-19 Bristol-Meyers Squibb Company JAK2 inhibitors and their use for the treatment of myeloproliferative diseases and cancer

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Publication number Priority date Publication date Assignee Title
WO2007045996A1 (fr) * 2005-10-19 2007-04-26 INSERM (Institut National de la Santé et de la Recherche Médicale) Procede in vitro pour le pronostic de la progression et du resultat d'un cancer chez un patient, et systeme de mise en oeuvre
WO2009048901A1 (fr) * 2007-10-09 2009-04-16 University Of Washington Mesure quantitative/semi-quantitative du récepteur de l'érythropoïétine sur des cellules cancéreuses
US8202881B2 (en) * 2009-09-03 2012-06-19 Bristol-Meyers Squibb Company JAK2 inhibitors and their use for the treatment of myeloproliferative diseases and cancer

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MAROTTA ET AL.: "The JAK2/STAT3 signaling pathway is required for growth of CD 44+ CD 24- stem cell -like breast cancer cells in human tumors", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 121, no. 7, 1 July 2011 (2011-07-01), pages 2723 - 2735 *

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