WO2014093623A1 - Method and assays for cancer prognosis using jak2 - Google Patents

Abstract

Provided herein are methods and assays for predicting the prognosis of a subject having cancer, comprising measuring the level of JAK2 expression. Also provided herein are methods and assays useful for predicting an adaptive immune response in a tumor (e.g., a malignant tumor), particularly breast cancer.

Description

METHOD AND ASSAYS FOR CANCER PROGNOSIS USING JAK2

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 1 19(e) of U.S. Provisional Application

No. 61/736,330 filed December 12, 2012, the contents of which are incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant nos. P50 CA083636-

S2 and R01 CA135357 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] 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.

BACKGROUND

[0004] 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). Furthermore, 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.

[0005] Studies of mRNA levels in primary breast tumors have been useful for classifying breast cancers into subtypes that correlate with prognosis and drug responsiveness (7-9), for predicting the likelihood of recurrence (10, 1 1), and for delineating gene expression signatures that correlate with prognosis (12-15).

SUMMARY

[0006] 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.

[0007] 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.

[0008] In one embodiment of this aspect and all other aspects provided herein, the method further comprises administering a therapeutic agent with, or without, a JAK2 inhibitor depending upon the JAK2 level.

[0009] In another embodiment of this aspect and all other aspects provided herein, the level of expression of JAK2 is the level of mRNA expression.

[00010] In another embodiment of this aspect and all other aspects provided herein, the cancer is breast cancer.

[00011] In another embodiment of this aspect and all other aspects provided herein, the biological sample is a tumor biopsy.

[00012] In another embodiment of this aspect and all other aspects provided herein, expression of

JAK2 is normalized to expression of a housekeeping gene.

[00013] In another embodiment of this aspect and all other aspects provided herein, JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.

[00014] In another embodiment of this aspect and all other aspects provided herein, JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.

[00015] In another embodiment of this aspect and all other aspects provided herein, the housekeeping gene is hydroxymethylbilane synthase (HMBS).

[00016] 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

wherein the malignant tumor is predicted to have a non-adaptive immune response when the levels of JAK2 expression are low.

[00017] In one embodiment of this aspect and all other aspects provided herein, the method further comprises administering a therapeutic agent with, or without, a JAK2 inhibitor depending upon the JAK2 level. [00018] In another embodiment of this aspect and all other aspects provided herein, the level of expression of JAK2 is the level of mRNA expression.

[00019] In another embodiment of this aspect and all other aspects provided herein, the malignant tumor is breast cancer.

[00020] In another embodiment of this aspect and all other aspects provided herein, expression of

JAK2 is normalized to expression of a housekeeping gene.

[00021] In another embodiment of this aspect and all other aspects provided herein, the housekeeping gene is hydroxymethylbilane synthase (HMBS).

[00022] In another embodiment of this aspect and all other aspects provided herein, wherein

JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.

[00023] In another embodiment of this aspect and all other aspects provided herein, JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.

[00024] Also provided herein are assays for predicting an adaptive immune response in a malignant tumor, the assay 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.

[00025] In one embodiment of this aspect and all other aspects provided herein, the malignant tumor is breast cancer.

[00026] In another embodiment of this aspect and all other aspects provided herein, expression of

JAK2 is normalized to expression of a housekeeping gene.

[00027] In another embodiment of this aspect and all other aspects provided herein, the housekeeping gene is hydroxymethylbilane synthase (HMBS).

[00028] In another embodiment of this aspect and all other aspects provided herein, JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.

[00029] In another embodiment of this aspect and all other aspects provided herein, JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.

[00030] 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.

[00031] Also provided herein are methods for predicting an adaptive immune response in a malignant tumor, the 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 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.

[00032] Also provided herein are assays for predicting an adaptive immune response in a malignant tumor, the assay 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.

BRIEF DESCRIPTION OF THE FIGURES

[00033] 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.

[00034] 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.

[00035] 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.

[00036] FIGs. 4A-4D JAK2 mRNA quantification in primary breast tumors. FIG. 4A

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.

[00037] FIG. 5 Ruxolitinib inhibits the anti-CD3 -dependent production of IFN-γ. Murine splenocytes were stimulated with anti-CD3 in the presence of the indicated concentrations of ruxolitinib or a vehicle control (DMSO). IFN-γ levels in culture supernatants were measured by ELISA. Error bars are the standard deviation of triplicate ELISA determinations.

DETAILED DESCRIPTION

[00038] Provided herein are methods and assays for predicting the prognosis of a subject having cancer, comprising measuring the level of JAK2 expression. Also provided herein are methods and assays useful for predicting an adaptive immune response in a tumor {e.g., a malignant tumor), particularly breast cancer.

Definitions

[00039] As used herein, 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.

[00040] As used herein, the terms "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. In one embodiment, 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. In some embodiments 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.

[00041] As used herein, the term "Janus kinase 2" or "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.

[00042] As used herein, 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.

[00043] As used herein, 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. In other embodiments, 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. Although unlikely, it is also contemplated herein that the subject can also have a reduced risk of cancer metastasis and death as compared to a subject lacking cancer. In some embodiments, "good prognosis" means that a subject is likely to survive for at least 5 years following diagnosis or prognosis.

[00044] As used herein, the term "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. In some embodiments, 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. Similarly, the term "low" when used in reference to JAK2 expression levels (e.g., "when the levels of JAK2 in the biological sample are low") 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.

[00045] As used herein, the phrase " normalized to the expression level of a housekeeping gene" or "normalizing" 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. [00046] As used herein, the terms "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. In some embodiments, 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. Thus, for example, 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.

[00047] As used herein, the term "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.

[00048] 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.

[00049] As used herein, the term "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).

[00050] As used herein, the term "adaptive immune response" refers to a tumor comprising T- cell lymphocytes that actively target cancerous cells in the tumor.

[00051] As used herein, the term "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.

[00052] As used herein, 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. In other embodiments, 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.

[00053] As used herein, 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. [00054] As used herein, 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

embodiment, the reference is obtained from a subject or plurality of subjects having, or diagnosed with having, cancer, such as breast carcinoma.

[00055] As used herein, the term "plurality of subjects" refers to at least two subjects (e.g., 2, 3,

4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 10000 or more subjects).

[00056] 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.

[00057] 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-fold increase or more as compared to a reference level.

[00058] As used herein the term "comprising" or "comprises" is used in reference to

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. [00059] As used herein 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.

[00060] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00061] As used in this specification and the appended claims, the singular forms "a," "an," and

"the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

Cancer

[00062] 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, liposarcoma, fibrosarcoma, synovial sarcoma and osteosarcoma; skin cancer including melanomas, Kaposi's sarcoma, basocellular cancer, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non- seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; transitional cancer and renal cancer including adenocarcinoma and Wilm's tumor.

Biological Samples

[00063] 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. In one embodiment, 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. In one embodiment, the biological sample is a biopsy from a growth or tumor (e.g., a malignant tumor).

[00064] In some embodiments, 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. In addition, 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.

[00065] 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.

[00066] In some embodiments, a biological sample can also be obtained for use as a control sample, and which corresponds to normal tissue having the same origin. For example, one of ordinary skill in the art can take two samples at the time of e.g., biopsy, with one sample corresponding to a tumor or cancer tissue and a second, control sample corresponding to the same tissue in a non-diseased site. In one embodiment, 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. Alternatively, a biological control sample can be a prior biopsy from the same subject to be tested prior to the onset of cancer.

Detection of JAK2

[00067] 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. In one embodiment, the level of JAK2 can be normalized to another protein or its mRNA (e.g., a normalizing gene/protein) such as e.g., a

housekeeping gene or gene product or another gene or gene product determined to lack significant variations in level/ concentration among samples or among relevant disease states. Normalizing genes/proteins and their use are known to those of skill in the art. Protein expression level

[00068] 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.,

immunoblotting, ELISA, immunoprecipitation, lateral flow immunoassay, radioimmunoassay, etc.

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 ABCAM™ (Cambridge, MA), NEW ENGLAND BIOLABS™ (Ipswich, MA), SANTA CRUZ BIOTECHNOLOGIES™ (Santa Cruz, CA), and CELL SIGNALING™ (Danvers, MA), among others.

[00069] 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.

Transcript expression level

[00070] In some embodiments, 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.

[00071] In general, 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. [00072] JAK2 expression levels can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time RT-PCR methods. Methods of RT-PCR and QRT-PCR are known in the art, and are described in more detail below. In one embodiment, 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. During amplification, 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.

[00073] 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

hybridization to a nucleic acid containing a polymorphic region of the invention. Length of the probe used will depend, in part, on the nature of the assay used and the hybridization conditions employed. In one embodiment, probes are labeled with two fluorescent dye molecules to form so-called "molecular beacons" (Tyagi, S. and Kramer, F.R., 1996). Such 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.

[00074] In some embodiments, 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. In some embodiments probes for use in the methods are nucleic acids which hybridize to the region of interest and which are not further extended. The 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. For example, discrete fragments of the DNA can be prepared and cloned using restriction enzymes. Alternatively, 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 BIOSYSTEMS™, etc.). As examples, 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.

Determining high vs. low JAK2 levels in a sample

[00075] The methods and assays described herein employ the use of 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.

[00076] In one embodiment, 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). When expressed in this manner, 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." One will appreciate that any housekeeping gene can be used to determine whether JAK2 levels are high or low.

[00077] In one embodiment, the methods and assays provided herein further comprise a step of measuring the expression of a housekeeping gene, such as HMBS.

Normalizing expression to a control

[00078] In some embodiments, 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. In certain embodiments, 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). One of skill in the art can easily determine if 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. Typically, 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).

[00079] In one embodiment, the normalizing control is the housekeeping gene

hydroxymethylbilane synthase (HMBS). In another embodiment, the methods and assays provided herein further comprise a step of measuring the expression of a housekeeping gene, such as HMBS.

References or Reference Samples

[00080] In one embodiment, 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. In another embodiment, optionally in addition to an internal reference, 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.

[00081] In one embodiment, a biological standard is obtained at an earlier time point

(presumably prior to the onset of cancer) from the same individual that is to be tested or treated as described herein. Alternatively, 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.

[00082] 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. Thus, 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. Generally, 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. The converse is contemplated in cases where 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.

[00083] 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.

[00084] Other standards contemplated for use herein include (i) a sample from a corresponding non-tumor tissue (e.g., a sample obtained from a healthy breast in the same subject or different subjects), or (ii) 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 infiltration

[00085] 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 (APCs) 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.

Following T-cell activation in response to encounter with specific 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. The result of this process is that naive T cells circulate randomly while activated T cells proliferate and home to specific tissue sites.

[00086] Upon trafficking to a tumor, T-lymphocytes localized to a tumor site are referred to as

"tumor-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.

[00087] In cancer, 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. For example, 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.

[00088] In some embodiments, 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. As but one example, 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.

JAK2 inhibitors

[00089] Janus kinase 2 (JAK2) 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.

[00090] Inhibition of JAK2 using JAK2 inhibitors typically occurs by disruption of the JAK-

STAT signaling pathway. JAK2 inhibitors include, but are not limited to, ruxolitinib, baricitinib, CYT387, lestaurtinib, pacritinib, and TG101348. In one embodiment, a JAK2 inhibitor is used herein to treat a subject having cancer. In another embodiment, the JAK2 inhibitor is ruxolitinib.

[00091] The present invention may be as defined in any one of the following numbered paragraphs

1. 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.

2. The method of paragraph 1, wherein the level of expression of JAK2 is the level of mRNA expression.

3. The method of paragraph 1, wherein the cancer is breast cancer.

4. The method of paragraph 1, wherein the biological sample is a tumor biopsy.

5. The method of paragraph 1, wherein expression of JAK2 is normalized to expression of a housekeeping gene. 6. The method of paragraph 1, wherein JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.

7. The method of paragraph 1, wherein JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.

8. The method of paragraph 5, wherein the housekeeping gene is hydroxymethylbilane synthase (HMBS).

9. The method of paragraph 1, wherein the reference is (a) a normalizing factor or (b) a value form a sample (or pool of samples) that have known characteristics.

10. The method of paragraph 1, further comprising administering a therapeutic agent with, or without, a JAK2 inhibitor depending on the JAK2 level.

11. A method for predicting an adaptive immune response in a malignant tumor, the 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

wherein the malignant tumor is predicted to have a non-adaptive immune response when the levels of JAK2 expression are low.

12. The method of paragraph 11, wherein the level of expression of JAK2 is the level of m NA expression.

13. The method of paragraph 11 , wherein the malignant tumor is breast cancer.

14. The method of paragraph 11, wherein expression of JAK2 is normalized to expression of a housekeeping gene.

15. The method of paragraph 14, wherein the housekeeping gene is hydroxymethylbilane synthase (HMBS).

16. The method of paragraph 11, wherein JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.

17. The method of paragraph 11, wherein JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.

18. The method of paragraph 11 , wherein the reference is (a) a normalizing factor or (b) a value form a sample (or pool of samples) that have known characteristics.

19. The method of paragraph 11, further comprising administering a therapeutic agent with, or without, a JAK2 inhibitor depending on the JAK2 level.

20. An assay for predicting an adaptive immune response in a malignant tumor, the assay 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 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.

21. The method of paragraph 20, wherein the malignant tumor is breast cancer.

22. The method of paragraph 20, wherein expression of JAK2 is normalized to expression of a housekeeping gene.

23. The method of paragraph 18, wherein the housekeeping gene is hydroxymethylbilane synthase (HMBS).

24. The method of paragraph 20, wherein JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.

25. The method of paragraph 20, wherein JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.

EXAMPLES

[00092] Summary: Janus Kinase-2 (JAK2) supports breast cancer growth and clinical trials testing inhibitors are underway. 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. Using a case-control design, 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. In the case-control study, increased JAK2 was associated with a decreasing risk of recurrence (multivariate p=0.003, n=223). Similarly, JAK2 was associated with a protective CI (<0.5) in the public cohorts:

NETHERLANDS CI=0.376, n=295; METABRIC CI=0.462, n=l,981; OSLOVAL CI=0.452, n=184. Furthermore, JAK2 strongly correlated with the favorable prognosis LYM metagene signature for infiltrating T cells (r=0.5, p<2xl0-16, n=l,981) and with severe lymphocyte infiltration (p=0.00003, n=156). Moreover, 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.

EXAMPLE 1 : JAK2 Expression is Associated with Tumor-Infiltrating Lymphocytes and

Improved Breast Cancer Outcomes

[00093] Here, the inventors evaluated the association between tumor mRNA levels of JAK2 and clinical outcomes in a novel case-control study and in 3 public cohorts. Outcomes included distant metastatic recurrence in a matched case-control study (n=223); recurrence-free survival in the

Netherlands Cancer Institute cohort that was used to develop the MammaPrint recurrence risk test (n=295) (11); overall and disease-specific survival in METABRIC, currently the largest collection of gene expression and copy number data linked to long term breast cancer outcomes (n=l,981) (14); and overall survival in OSLOVAL, a recent cohort that along with METABRIC formed the basis of the Sage Bionetworks DREAM breast cancer prognosis challenge (n=184) (12, 15).

Case-control study of JAK2 mRNA levels and distant recurrence

[00094] The inventors previously optimized methods for measuring JAK2 mRNA in archival formalin- fixed, paraffin-embedded tumors by quantitative RT-PCR despite the degradation that characterizes RNA extracted from these samples (20). This approach was applied to tumor specimens from 112 women receiving surgery for breast cancer who subsequently experienced a distant metastatic recurrence and 112 women who did not. The clinical characteristics of cases and controls are shown in Table 1.

Table 1. Characteristics of Patients by Recurrence Status

Variable* Value No Recurrence Recurrence P-Value

Recurrence # 112 112

Age at Diagnosis Mean(sd) 51(11) 50(12) 0.47

Year Collected Mean(sd) 1998(5) 1997(5) 0.06

Age>50 Yes 56 (50.0%) 51 (45.5%) 0.59

Ever Pregnant No 24 (21.4%) 25 (22.3%)

Unknown 0 (0.0%) 2 (1.8%)

Yes 88 (78.6%) 85 (75.9%) 0.53

Age at First Pregnancy Mean(sd) 26(6) 27(6) 0.61

Pregnant at Diagnosis Yes 0 (0.0%) 2 (1.8%) 0.50

Ductal Carcinoma In Situ Yes 88 (78.6%) 94 (83.9%) 0.39

Lobular Carcinoma In Situ Yes 17 (15.2%) 10 (8.9%) 0.22

Extra Modal Extensions Yes 33 (29.5%) 41 (36.6%) 0.32

Multifocal Yes 25 (22.3%) 27 (24.1%) 0.87

Histologic Grade Grade 1 5 (4.5%) 0 (0.0%)

Grade 2 19 (17.0%) 21 (18.8%)

Grade 3 84 (75.0%) 83 (74.1%)

Unknown 4 (3.6%) 8 (7.1%) 0.09

Nuclear Grade High 70 (62.5%) 76 (67.9%)

Intermediate 37 (33.0%) 28 (25.0%)

Low 3 (2.7%) 6 (5.4%)

Unknown 2 (1.8%) 2 (1.8%) 0.45

Lymphovascular Invasion No/Unknown 56 (50.0%) 52 (46.4%)

Suspicious 11 (9.8%) 7 (6.2%)

Yes 45 (40.2%) 53 (47.3%) 0.45

Tumor Size <2cm 33 (29.5%) 39 (35.1%)

2 - 5 cm 62 (55.4%) 59 (53.2%)

> 5 cm 17 (15.2%) 13 (11.7%) 0.59

# of Lymph Nodes Examined Mean(sd) 17(8) 18(10) 0.56

# of Positive Lymph Nodes 0 19 (17.0%) 20 (17.9%)

1 to 3 61 (54.5%) 45 (40.2%)

4 to 9 20 (17.9%) 31 (27.7%) Variable* Value No Recurrence Recurrence P-Value

10+ 12 (10.7%) 16 (14.3%) 0.15

Histology Ductal 92 (82.1%) 94 (83.9%)

Lobular 10 (8.9%) 8 (7.1%)

Lobular/Ductal Mixed 9 (8.0%) 7 (6.2%)

Metaplastic

Carcinoma, Not

otherwise specified 1 (0.9%) 3 (2.7%) 0.71

Estrogen Receptor (ER) Negative 24 (21.4%) 34 (30.4%)

Positive 88 (78.6%) 75 (67.0%)

Unknown 0 (0.0%) 3 (2.7%) 0.05

Progesterone Receptor (PR) Negative 43 (38.4%) 44 (39.3%)

Positive 69 (61.6%) 65 (58.0%)

Unknown 0 (0.0%) 3 (2.7%) 0.29

Hormone Receptor (=ER or PR) Negative 23 (20.5%) 29 (25.9%)

Positive 89 (79.5%) 80 (71.4%)

Unknown 0 (0.0%) 3 (2.7%) 0.15

HER2 Negative 56 (50.0%) 51 (45.5%)

Positive 11 (9.8%) 22 (19.6%)

Unknown 45 (40.2%) 39 (34.8%) 0.12

Triple Negative No 90 (80.4%) 87 (77.7%)

Unknown 10 (8.9%) 14 (12.5%)

Yes 12 (10.7%) 11 (9.8%) 0.70

Tamoxifen Given Yes 63 (56.2%) 60 (53.6%) 0.79

Herceptin Given Yes 3 (2.7%) 5 (4.5%) 0.72

Hormone Therapy Yes 81 (73.2%) 71 (63.4%) 0.20

Surgery Type Partial Mastectomy 39 (34.8%) 40 (35.7%)

Total Mastectomy 73 (65.2%) 72 (64.3%) 1.00

Surgical Margins Positive Yes 6 (5.4%) 4 (3.6%) 0.75

Surgical Margins Close/Positive Yes 35 (31.2%) 33 (29.5%) 0.88

Radiation Therapy Yes 78 (69.6%) 68 (60.7%) 0.21

* Variables used in matching are bold

[00095] With the exception of a borderline significant increase in the number of estrogen receptor negative (ER-negative) tumors among recurrences, there were no significant differences between cases and controls. Of note, while ER was one of the variables used in matching, it was not the only variable, which accounts for the residual effect of this strong prognostic factor even after propensity score matching. Sufficient RNA was available for JAK2 mRNA determinations in 223 tumor specimens. The validity of the mRNA measurements was confirmed by 1) the strong correlation in values obtained using probes for both JAK2 exon8/9 and exon23/24; 2) the reproducible mRNA levels across 3 separate tumor specimens for 14 tumors for which this comparison was possible; and 3) the strong concordance in mRNA levels of ESR1, PGR, ERBB2, and the corresponding clinical immunohistochemistry results.

[00096] 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

Correlated Covariates

Continuous Model, All Tissues" JAK2-ex23/24 JAK2-ex8/9

n 223 223

Coefficient -0.4165 -0.3712

Un-adjusted Regression

P-Value 0.0004 0.0007

Coefficient -0.3725 -0.3358

Adjusted Regression

P-Value 0.0032 0.0056

Continuous Model, Primary Tissues Only^b

n 192 192

Coefficient -0.53322 -0.50955

Un-adjusted Regression

P-Value 0.00007 0.00006

Coefficient -0.44692 -0.44983

Adjusted Regression

P-Value 0.00183 0.00175

Dichotomous Median Split Model, All Tissues⁰

n 223 223

Coefficient -0.744 -0.596

Un-adjusted Regression

P-Value 0.006 0.028

Above Median 45 (40.5%) 47 (42.3%)

Recurrence Rates

Below Median 66 (58.9%) 64 (57.1%)

Coefficient -0.653 -0.493

Adjusted Regression

P-Value 0.022 0.097 ^aCoefficients and p-values were obtained using logistic regression with transcript expression as a predictor of recurrence. Coefficients estimate the change in the log of the odds that an individual experienced a recurrence for every two-fold increase in transcript expression. A negative coefficient indicates that increased transcript expression is associated with decreased likelihood of recurrence. ^bCoefficients and p-values were calculated as in (a) using only primary tissue. For individuals with both a primary and node specimen, only the primary tissue was included (n=26). Data from individuals with only a node specimen were excluded (n=31).

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.

[00097] Furthermore, with the exception of the JAK2 exon8/9 probe in the dichotomous model, significance was maintained in multivariate analysis. The association between increasing JAK2 mRNA and decreasing distant recurrence was also significant when analysis was restricted to only primary tumors. Finally, a receiver operator curve revealed that the association between higher JAK2 mRNA and reduced recurrence was maximal when tumor samples with the top 40-50%) of JAK2 expression level were defined as high JAK2 (FIG. IB).

JAK2 mRNA levels and survival outcomes in the NETHERLANDS, METABRIC, and OSLOVAL cohorts

[00098] Next, 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. For example, using disease-specific survival data in METABRIC, 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). Conversely, 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.

[00099] 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. Since METABRIC provided sufficient sample size, we also evaluated the concordance index for JAK2 mRNA in ER- and ER-/PR-/HER2- (triple negative) subtypes; JAK2 mRNA was even more protective for both overall and disease-specific survival in these subtypes.

JAK2 mRNA levels and tumor-infiltrating T cells

[000100] 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). Furthermore, 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). Finally, consistent with a functional role for JAK2 in supporting cytokine receptor signaling during T cell activation, the inventors found that the JAKl/2 inhibitor ruxolitinib markedly inhibited the anti-CD3 -dependent production of IFN-γ, a marker of the differentiation of T-helper cells along the tumor-inhibitory Thl pathway (23). These results indicate that the consistently protective effect of JAK2 mRNA in multiple cohorts is related, at least in part, to levels of infiltrating T cells.

[000101] For the first time, the inventors demonstrate a consistent association between increasing

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. Indeed, 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 reminiscent of the frequent association between single genes and the prognostically significant PCNA and CIN metagene signatures for proliferation and chromosomal instability (12).

[000102] In addition to the present demonstration that the JAKl/2 inhibitor ruxolitinib inhibits

CD3 -dependent Thl differentiation, further support for a functional role of JAK2 in T cells is provided by studies demonstrating that JAK2 is involved in IL-12 and IFN-γ 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).

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EXAMPLE 2: Materials and Methods

Selection of cases and controls

[000130] The research protocol to use a breast cancer research database for case selection, to access institution-archived leftover tumor tissue, and to undertake molecular biology studies of the tissue was approved by the Institutional Review Boards of the Fred Hutchinson Cancer Research Center (File #6643) and Swedish Medical Center (File #4924C-10). Patient consent was not required for

participation. 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. Within each matched pair, the 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).

Quantitative RT-PCR

[000131] RNA was extracted from 4x10 μιη tumor sections using the ABSOLUTELY RNA

FFPE™ system (STRATAGENE™). 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 (INVITROGEN™) and was preamplified for 14 cycles using the TAQMAN™ preamplification system (APPLIED BIOSYSTEMS™). All probes bound to exon junctions to prevent genomic DNA amplification (Table 3).

Table 3. Taqman Assays

Symbol Gene Name Ϊ

Assay ID Amplicon Size

(bp)

ERBB2 human epidermal growth factor receptor 2 Hs01001580 ml 60

(HER-2)

ESR1 estrogen receptor 1 HsOO 174860 ml 62

HMBS hydroxymethylbilane synthase Hs00609297 ml 64

JAK2 janus kinase 2, exon 8-9 Hs01078136 ml 84

JAK2 janus kinase 2, exon 23-24 Hs00234567 ml 101

PGR progesterone receptor Hs01556702 ml 77

Applied Biosystems

[000132] cDNA was diluted and used to seed triplicate real time PCR reactions for each

TAQMAN™ assay using standard cycling conditions for the 7900HT (APPLIED BIOSYSTEMS™). Cycle threshold (Ct) values were determined using Sequence Detection Software (APPLIED

BIOSYSTEMS™). 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.

JAK2 mRNA levels and distant recurrence in the case-control study

[000133] Associations between JAK2 mRNA and the likelihood of recurrence were evaluated by logistic regression. In the continuous model, coefficients were calculated as estimates of the change in the log of the odds that an individual experienced a recurrence for every twofold increase in JAK2 levels, where a negative coefficient indicates that increasing JAK2 levels are associated with a decreasing likelihood of recurrence. Since participants had either one tumor specimen (n=184), two specimens (n=26), or three tumor specimens (n=14), generalized estimating equations were used to account for varying numbers of tissues per individual ("Geepack" package) (18). Logistic regressions were also performed using only primary tumors. For this analysis, among the 26 individuals with both a primary and node specimen, only the primary tissue was included, and data from 31 individuals with only a node specimen were excluded. In the dichotomous model, coefficients were calculated with above-median versus below-median JAK2 levels as a predictor of recurrence, where values for individuals with multiple specimens were averaged to one value per individual. For each model, multivariate analysis was performed to adjust for clinical factors with which JAK2 expression was significantly correlated.

JAK2 mRNA levels and survival outcomes in the NETHERLANDS, METABRIC, and OSLOVAL cohorts

[000134] The inclusion criteria, clinical characteristics, and follow-up of the NETHERLANDS,

METABRIC, and OSLOVAL cohorts were described (11, 12, 14). Data are available via Sage Bionetworks under the following project identifiers: doi: 10.7303/syn4517.1,

doi:10.7303/synl 688369; and doi: 10.7303/synl 688370. In each cohort, the concordance index was determined as described (12, 19).

T cell activation

[000135] Murine splenocytes were collected under a protocol approved by the University of Washington Institutional Animal Care and Use Committee and plated at lxl 0⁶ cells per mL in RPMI, 10% FBS, 50 μΜ beta-mercaptoethanol, 100 units/mL penicillin, 100 μg/mL streptomycin sulfate, and 2.5 μg/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

CHEMICALS™). After 48 hours, interferon-gamma (IFN-γ) levels in culture supernatants were measured using the Ready-Set-Go ELISA™ System (EBIOSCIENCE™).

Claims

1. 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.

2. The method of claim 1, wherein the level of expression of JAK2 is the level of mRNA expression.

3. The method of claim 1, wherein the cancer is breast cancer.

4. The method of claim 1, wherein the biological sample is a tumor biopsy.

5. The method of claim 1, wherein expression of JAK2 is normalized to expression of a housekeeping gene.

6. The method of claim 1, wherein JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.

7. The method of claim 1, wherein JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.

8. The method of claim 5, wherein the housekeeping gene is hydroxymethylbilane synthase (HMBS).

9. The method of claim 1, wherein the reference is (a) a normalizing factor or (b) a value form a sample (or pool of samples) that have known characteristics.

10. The method of claim 1, further comprising administering a therapeutic agent with, or without, a JAK2 inhibitor depending on the JAK2 level.

11. A method for predicting an adaptive immune response in a malignant tumor, the 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

wherein the malignant tumor is predicted to have a non-adaptive immune response when the levels of JAK2 expression are low.

12. The method of claim 11, wherein the level of expression of JAK2 is the level of mRNA expression.

13. The method of claim 11 , wherein the malignant tumor is breast cancer.

14. The method of claim 11, wherein expression of JAK2 is normalized to expression of a housekeeping gene.

15. The method of claim 14, wherein the housekeeping gene is hydroxymethylbilane synthase (HMBS).

16. The method of claim 11, wherein JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.

17. The method of claim 11, wherein JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.

18. The method of paragraph 11 , wherein the reference is (a) a normalizing factor or (b) a value form a sample (or pool of samples) that have known characteristics.

19. The method of paragraph 11, further comprising administering a therapeutic agent with, or without, a JAK2 inhibitor depending on the JAK2 level.

20. An assay for predicting an adaptive immune response in a malignant tumor, the assay 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 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.

21. The method of claim 20, wherein the malignant tumor is breast cancer.

22. The method of claim 20, wherein expression of JAK2 is normalized to expression of a housekeeping gene.

23. The method of claim 22, wherein the housekeeping gene is hydroxymethylbilane synthase (HMBS).

24. The method of claim 20, wherein JAK2 levels are high when the expression level of JAK2 is greater than 10% of the expression level of the housekeeping gene.

25. The method of claim 20, wherein JAK2 expression levels are low when the expression level of JAK2 is less than or equal to the expression level of the housekeeping gene.