WO2016057852A1 - Markers for hematological cancers - Google Patents

Abstract

Described herein are markers for hematological cancers, such as lymphoma, and uses of such markers in methods, kits, and devices. Th invention provides methods for identifying mutations associated with certain types of cancers. Mutations are identified in cancerous B cell or T cells, e.g. somatic markers, but not in matched normal B cells or T cells from the same individual, in accordance with the invention. In some aspects, the invention provides a method, comprising analyzing genomic DNA from a subject for the presence of a mutation in a gene selected from the group. In some embodiments of any one of the methods provided, the genomic DNA is obtained from white blood cells of the subject.

Description

MARKERS FOR HEMATOLOGICAL CANCERS

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application number 62/061,211, filed October 8, 2014, the contents of which are

incorporated by reference herein in their entirety.

BACKGROUND OF INVENTION

Several types of human and canine malignancies arise from cells that belong to the hematologic system, including peripheral blood cells, bone marrow, lymph nodes and lymphatic and blood vasculature. Lymphomas refer to a group of blood cell tumors that develop from lymphocytes, such as B-cell lymphoma and T-cell lymphoma. Identification of mutations associated with these diseases is helpful for developing diagnostics and treatments.

SUMMARY OF THE INVENTION

The invention provides methods for identifying mutations associated with certain types of cancers. Mutations are identified in cancerous B cell or T cells, e.g. somatic markers, but not in matched normal B cells or T cells from the same individual, in accordance with the invention.

The invention is premised, in part, on mutations in genes identified by sequencing of paired tumor and normal DNA obtained from canines having B-cell or T-cell lymphomas. Exemplary significantly mutated genes identified in B-cell or T-cell lymphomas included but were not limited to POT1, FBXW7, TRAF3, PSMA1, COX8A, PTEN, SATB1, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRC1, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPL11, EEF1A1, KLRK1, GRIFIN, FAM90A1, ZNF486 and ZFN706.

In some aspects, the invention provides a method, comprising analyzing genomic DNA from a subject for the presence of a mutation in a gene selected from the group including but not limited to POT1, FBXW7, TRAF3, PSMA1, COX8A, PTEN, SATB1, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRC1, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPL11, EEF1A1, KLRK1, GRIFIN, FAM90A1, ZNF486 and ZFN706; and identifying a subject having the mutation as a subject having a hematological cancer. In some embodiments, the gene is selected from a group consisting oiPOTl, FBXW7, TRAF3, PSMA1, PTEN, SATB1, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP 53, cMYC, MAP3K14, KPNA2, NLRP14, EEF1A1, KLRKl, GRIFIN, FAM90A1, and ZNF486. In some embodiments, the gene is KCND2 or mTOR.

In some embodiments, the gene is selected from a group consisting oiSATBl, TBC1D26, PSMA1, COX8A, PTEN, LTA4H, and a potential TMC4 antisense. In some embodiments, the gene is SATB1, TBC1D26, PSMA1, PTEN, or a potential TMC4 antisense. The hematological cancer may involve T cells, such as a T cell lymphoma.

In some embodiments, the gene is selected from a group consisting of FBXW7, TRAF3, MAP3K14, POT1, TP 53, SETD2 and DDX3X. The hematological cancer may involve B cells, such as a B cell lymphoma.

In some embodiments of any one of the methods provided, the genomic DNA is obtained from white blood cells of the subject. In some embodiments of any one of the methods provided, the genomic DNA is analyzed using a single nucleotide polymorphism (SNP) array. In some embodiments of any one of the methods provided, the genomic DNA is analyzed using a bead array. In some embodiments of any one of the methods provided, the genomic DNA is analyzed using a nucleic acid sequencing assay.

In some embodiments of any one of the methods provided, the subject is a human subject. In some embodiments of any one of the methods provided, the subject is a canine subject. In some embodiments of any one of the methods provided, the cancer is a lymphoma. In some embodiments of any one of the methods provided, the cancer is a B cell lymphoma. In some embodiments of any one of the methods provided, the cancer is a T cell lymphoma.

In some embodiments of any one of the methods provided, the mutation is two or more mutations. In some embodiments of any one of the methods provided, the gene is two or more genes.

In other aspects, the invention provides a method, comprising measuring, in a cancer cell or population of cancer cells in the absence of at least one agent, a first expression level of one or more genes selected from the group consisting oiPOTl, FBXW7, TRAF3, PSMAl, COX8A, PTEN, SATBl, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRC1, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPL11, EEFlAl, KLRKl, GRIFIN, FAM90A1, ZNF486, and ZFN706; and measuring, in a cancer cell or population of cancer cells in the presence of the at least one agent, a second expression level of one or more genes selected from the group consisting oiPOTl, FBXW7, TRAF3, PSMAl, COX8A, PTEN, SATBl, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP 53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRC1, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPL11, EEFlAl, KLRKl, GRIFIN, FAM90A1, ZNF486, and ZFN706; and identifying the at least one agent as capable of modulating expression of the one or more genes in the cancer cell or population of cancer cells if (i) the second expression level of the one or more genes is up- regulated compared to the first expression level or (ii) the second expression level of the one or more genes is down-regulated compared to the first expression level.

In some embodiments, the first expression level is the expression level of one or more genes selected from a group consisting oiPOTl, FBXW7, TRAF3, PSMAl, PTEN, SATBl, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, KPNA2, NLRP14, EEFlAl, KLRKl, GRIFIN, FAM90A1, and ZNF486. In some embodiments, the second expression level is the expression level of one or more genes selected from a group consisting oiPOTl, FBXW7, TRAF3, PSMAl, PTEN, SATBl, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, KPNA2, NLRP14, EEFlAl, KLRKl, GRIFIN, FAM90A1, and ZNF486. In some embodiments, the first and second expression levels are expression levels oiKCND2 or mTOR.

In some embodiments, the at least one agent is identified as suitable for treating a hematological cancer if the agent is identified as capable of modulating expression of the one or more genes.

In some embodiments of any one of the methods provided, the cancer cell or population of cancer cells is a lymphoma cell or a population of lymphoma cells. In some embodiments of any one of the methods provided, the cancer cell or population of cancer cells comprises a mutation in the one or more genes. In some embodiments of any one of the methods provided, the cancer cell or population of cancer cells is a canine cancer cell or a population of canine cancer cells. In some embodiments of any one of the methods provided, the cancer cell or population of cancer cells are a human cancer cell or a population of human cancer cells.

In some embodiments of any one of the methods provided, the one or more genes is selected from the group consisting ofNLRP5, NLRP14, TBC1D26 and GRIFIN. In some embodiments of any one of the methods provided, the one or more genes is selected from the group consisting ofNLRPI4, TBC1D26 and GRIFIN.

In some embodiments of any one of the methods provided, the one or more genes is selected from the group consisting of FBXW7, TRAF3, MAP SKI 4, POTI, TP53, PNRCI, FKBP3, SOCS2, SETD2, MITF, and DDX3X. In some embodiments of any one of the methods provided, the one or more genes is selected from the group consisting of FBXW7, TRAF3, MAP3KI4, POTI, TP '53, SETD2 and DDX3X.

In some embodiments of any one of the methods provided, the one or more genes is selected from the group consisting of PSMA1, COX8A, LTA4H, TBC1D26, ZNF706, ATP5H, NLRP5, GLUD2, SATBI, PTEN, MAP2KI, NLRPI4, KCND2, mTOR, and a potential TMC4 antisense. In some embodiments of any one of the methods provided, the one or more genes is selected from the group consisting ofPSMAI, TBCID26, SATBI, PTEN, NLRPI4, KCND2, mTOR, and a potential TMC4 antisense.

The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGs. 1A and B show the project outline including sample numbers.

FIGs. 2A and B provide a graph showing the average tumor mutation load and relative distribution of mutations in the samples analyzed. Cocker B = Cocker spaniel B-cell lymphoma, Golden B = Golden retriever B-cell lymphoma, Boxer T = Boxer T-cell lymphoma, Golden T = Golden retriever T-cell lymphoma, Cocker + Golden B = combined data for Cocker spaniel B-cell lymphoma and Golden retriever B-cell lymphoma, Boxer + Golden T = combined data for Boxer T-cell lymphoma and Golden retriever T cell lymphoma. The correlation between the data in the top and bottom panels of FIG. 2B is clearly apparent in the color rendering. The top panel is comprised of 6 rectangles, 3 on the left and 3 on the right. The bottom panel is comprised of 4 different 3-D bar graphs, each corresponding to a different dog breeds. The mutations associated with each of the 6 top panel rectangles are shown in each of the 3-D graphs in essentially the same arrangement as in the rectangles.

FIGs. 3A-C are a series of Venn diagrams showing overlap in significantly mutated genes detected in B-cell lymphomas and T-cell lymphoma in different dog breeds, exemplifying the overlap between significantly mutated genes in the two breeds predisposed to B-cell lymphoma and the difference between the breeds predisposed to T-cell lymphoma, as well as the difference between B- and T-cell lymphoma.

FIG. 4 is a diagram of a TumorPortal analysis showing FBXW7 recurrently mutated at a specific position. It visualizes that the position commonly seen mutated in canine lymphoma, particularly in Golden retriever B-cell lymphoma, is the same amino acid as one of the two positions recurrently mutated in human cancer. The human data come from TumorPortal (Lawrence et al., Nature 2014, 505:495-501). The canine data show that of the two positions known to be recurrently mutated in partially overlapping, partially different human cancers (including human lymphoma and leukemia), one (R465 in human) is recurrently mutated in canine lymphoma.

FIG. 5 is a table illustrating the most commonly mutated genes and selected interaction partners observed in B- and T-cell lymphoma. For B-cell lymphoma, the three top significantly mutated genes and two pathways are highlighted. For T-cell lymphoma, three genes, and one pathway are listed. For all entries, the fraction of individuals in a group with at least one mutation in that gene or pathway is indicated. Most of the entries were significantly differently mutated between B-cell lymphomas and T-cell lymphomas, as calculated by Fisher's exact test (right-most column).

FIG. 6 A is a table of genes commonly and significantly mutated in B- or T-cell lymphoma. None of these genes has been previously implicated in human lymphoma, although they have been observed in other human cancers. These genes may be used for identifying chemotherapy targets used in other cancers, which could also help lymphoma patients.

FIG. 6B is a table of genes not previously mentioned with human cancer. These genes are highly relevant for further studies to identify new chemotherapy targets.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based in part on the discovery of somatic markers associated with particular cancers in canine subjects. The two canine cancer types studied were B-cell lymphoma and T-cell lymphoma. DNA from tumor cells and matched normal cells from canine subjects was sequenced, and differences between the tumor and normal samples were identified. A variety of somatic mutations were discovered in tumor cells that were not present in normal cells.

The invention therefore provides diagnostic and treatment methods that involve detecting one or more of markers, e.g., somatic markers, in canine subjects in order to identify subjects having a hematological cancer, and optionally determining one or more methods of treatment based on the presence or absence of a mutated gene or mutated pathway.

Where a drug or treatment regimen is known to modulate a disclosed gene or pathway now shown herein to be mutated in association with a B cell or T cell lymphoma, the invention provides methods of treating the respective disease with that drug or treatment regimen. Where a disclosed gene or pathway has not been previously associated with a B cell or T cell lymphoma, the invention provides methods of screening for drugs that modulate an affected pathway for use in treatment of the respective disease.

In addition, in view of the clinical and histological similarity between canine and human lymphomas, the markers identified by the invention may also be markers and/or mediators of disease progression in these human cancers as well. Accordingly, the invention provides diagnostic and treatment methods for use in canines, human subjects, as well as others.

The invention refers to the somatic markers described herein as cancer-associated markers to convey that the presence of these various markers has been shown to be associated with the occurrence of certain cancer types in accordance with the invention. These various marker types will be discussed in greater detail herein.

Hematological cancers

In some embodiments, aspects of the invention relate to methods of identifying a subject has having cancer (e.g., a hematological cancer) based on the presence of one or more cancer associated markers described herein. This may include identify a subject as having a malignant cancer and not a benign cancer based on the presence of one or more cancer associated markers described herein. Hematological cancers include cancers of the blood, lymph nodes, and bone marrow. Exemplary hematological cancers include leukemias, myelomas, and lymphomas. In some embodiments of any one of the methods provided herein, the hematological cancer is lymphoma.

Lymphomas are a group of blood cell cancers that develop from lymphocytes. In humans, lymphomas may be classified as Hodgkin's lymphoma or non-Hodgkin's lymphoma. Lymphomas may also be classified by cell type, e.g., T-cell lymphoma or B-cell lymphoma. Canines also develop lymphomas, including T-cell and B-cell lymphomas.

Exemplary lymphomas include precursor T-cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, Mantle cell lymphoma, B-cell chronic lymphocytic lymphoma, MALT lymphoma, Burkitt's lymphoma, Mycosis fungoides, Peripheral T-cell lymphoma- Not-Otherwise-Specified, Nodular sclerosis form of Hodgkin's lymphoma, and Mixed- cellularity subtype of Hodgkin's lymphoma.

Lymphomas may be diagnosed, e.g., using a lymph node biopsy, bone marrow aspiration, lumbar puncture and biopsy or other method known in the art. In some embodiments, other tests may be used to diagnose lymphoma, e.g., blood counts, blood viscosity test, immunophenotyping, beta-2-microglobulin test, flow cytometry, and/or FISH (fluorescent in situ hybridization) testing.

In some embodiments, any one of the methods described herein comprises measuring a mutation as described herein in combination with a known diagnostic method (e.g., lymph node biopsy, bone marrow aspiration and biopsy, lumbar puncture, blood counts, blood viscosity test, immunophenotyping, flow cytometry, beta-2 -microglobulin test, and/or FISH).

Somatic markers

The invention is based in part on the discovery of various somatic mutations present in tumors that are not present in normal cells from the same subject. Somatic mutations were identified by performing a genome-wide sequencing of tumor cells and normal cells from dogs with T-cell or B-cell lymphoma. The markers demonstrating at least one mutation include, but are not limited to, POT1, FBXW7, TRAF3, PSMA1, COX8A, PTEN, SATB1, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRC1, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPL11, EEF1A1, KLRK1, GRIFIN, FAM90A1, ZNF486, and ZFN706. In some embodiments, the markers demonstrating at least one mutation include POT I, FBXW7, TRAF3, PSMA1, PTEN, SATB1, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, KPNA2, NLRP14, EEF1A1, KLRK1, GRIFIN, FAM90A1, and ZNF486. In some embodiments, the marker demonstrating at least one mutation is KCND2 or mTOR.

The invention therefore provides, in part, methods for detecting the presence of a mutation in one or more of such genes and identifying a subject as having at least one cancer based on the presence of such mutation(s). Other aspects of the invention relate to methods of treatment involving detection of the presence of a mutation in one or more such genes. A gene may include regulatory sequences (e.g., promoters, enhancers, or suppressors, either adjacent to or far from the coding sequence) and coding sequences. As used herein, a coding sequence includes the first DNA nucleotide to the last DNA nucleotide that is transcribed into an mRNA that includes the untranslated regions (UTRs), exons, and introns. The coding sequence for each gene can be obtained using the Ensembl database by entering the Ensembl gene IDs provided in Table 1, or by other methods known in the art. In some embodiments, the mutation is within or near (e.g., within 100 kb of) the coding sequence of a gene. Thus, it is to be understood that this disclosure provides for detecting mutations within or near "genes" or within or near a coding sequence. In some embodiments, the mutation is within 5000 kb, 2500 kb, 1000 kb, 900 kb, 800 kb, 700 kb, 600 kb, 500 kb, 400 kb, 300 kb, 200 kb, 150 kb, 100 kb, 50 kb, 25 kb, 10 kb, or 5 kb of a gene or of the coding sequence of the gene, as described herein. In some embodiments, all mutations described herein are non- synonymous, coding mutations, e.g., mutations that alter the amino acid sequence of any protein produced by a gene described herein.

In some instances, the invention provides methods for detecting the presence of a mutation in one or more genes selected from POT1, FBXW7, TRAF3, PSMA1, COX8A, PTEN, SATB1, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRC1, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPL11, EEF1A1, KLRKl, GRIFIN, FAM90A1, ZNF486, and ZFN706, and identifying a subject as having cancer or as having a malignant cancer and not a benign cancer based on the presence of such mutation(s). The subject may be a canine subject or a human subject, although it is not so limited. In some aspects, the cancer can be treated with compounds known to modulate at least one of the mutated genes, or that is developed as a modulator of at least of the mutated genes described herein. In some embodiments, the methods detect a mutation in one or more genes the gene is selected from a group consisting oiPOTl, FBXW7, TRAF3, PSMAl, PTEN, SATBl, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, KPNA2, NLRP14, EEF1A1, KLRK1, GRIFIN, FAM90A1, and ZNF486. In some embodiments, the methods detect a mutation in KCND2 or mTOR.

Table 1 lists the Ensembl numbers for several of these markers in the canine genome and in the human genome. Nucleic acid sequences may be obtained by entering the Ensembl numbers into the Ensembl database (Ensembl release 77). In some instances, a human orthologue of the locus has not yet been identified. In those instances, the invention contemplates that the human orthologue possesses at least 60% homology, or at least 70% homology, or at least 75% homology to the canine sequence and the methods described herein can be based on an analysis of loci in the human genome that share these degrees of homology.

Table 1: List of top significantly mutated genes in lymphoma tumors in canines

EEF1A1 ENSCAFG00000006937 ENSG00000196205

ENSCAFG00000024406 ENSG00000156508

ENSCAFG00000024474

ENSCAFG00000007877

FBXW7 ENSCAFG00000008141 ENSG00000109670

RPL11 ENSCAFG00000013232 ENSG00000142676

SETD2 ENSCAFG00000013392 ENSG00000181555

ATP5H ENSCAFG00000013551 ENSG00000167863

DDX3X ENSCAFG00000014251 ENSG00000215301

PTPN6 ENSCAFG00000014463 ENSG00000111679

PTEN ENSCAFG00000015670 ENSG00000171862

P0T1 ENSCAFGOOOOOOl 6403 ENSG00000275572

ENSCAFGOOOOOOOl 754 ENSG00000128513

TP53 ENSCAFGOOOOOOl 6714 ENSG00000141510

MAP2K1 ENSCAFGOOOOOOl 7298 ENSG00000169032

TRAF3 ENSCAFG00000018075 ENSG00000131323

KCND2 ENSCAFG00000024216 ENSG00000184408

C0X8A ENSCAFG00000024399 ENSG00000176340

TBC1D26 ENSCAFG00000025100 ENSG00000214946

ENSCAFG00000023521

FAM90A1 ENSCAFG00000025123 ENSG00000171847

FAM90A1 ENSCAFG00000030674 ENSG00000171847

ENSCAFG00000025286

PNRC1 ENSCAFG00000030839 ENSG00000146278

KLRK1 ENSCAFG00000013453, ENSG00000255819

ENSCAFG00000014042

ENSCAFG00000028587

KPNA2 ENSCAFG00000011598 ENSG00000182481

MAP3K14 ENSCAFG00000013774 ENSG00000006062

ZNF486 ENSCAFG00000031827 ENSG00000256229

antisense in ENSCAFG00000031638 antisense against part of TMC4 ENSG00000167608

The Ensembl gene IDs for mTor are ENSCAFGOOOOOOl 6648 (canine) and ENSGOOOOO 198793 (human). Mutations

Aspects of the invention relate to a (i.e., at least one) mutation in a gene described herein (e.g., a gene identified as containing somatic mutations) and uses and detection thereof in various methods. As used herein, a mutation is one or more changes in the nucleotide sequence of the genome of the subject. As used herein, mutations include, but are not limited to, point mutations (e.g., SNPs), insertions, deletions, rearrangements, inversions and duplications. Mutations also include, but are not limited to, silent mutations, missense mutations, and nonsense mutations.

In some embodiments, the mutation is a somatic mutation. A germ-line mutation is generally found in the majority, if not all, of the cells in a subject. Germ-line mutations are generally inherited from one or both parents of the subject (i.e., were present in the germ cells of one or both parents). Germ-line mutations as used herein also include de novo germ-line mutations, which are spontaneous mutations that occur at single-cell stage level during development. A somatic mutation occurs after the single-cell stage during development. Somatic mutations are considered to be spontaneous mutations. Somatic mutations generally originate in a single cell or subset of cells in the subject.

A mutation as described herein may be found within a gene described herein or within a region encompassing such a gene (e.g., a region that encompasses the gene as well as 100 kb or more upstream and 100 kb or more downstream of the gene). In some embodiments, all genes described as mutated have non-synonymous mutations in their coding sequence, where the boundaries of coding sequence come from the CanFam3.1 canine genome assembly.

In some embodiments, the mutation is at R470 or R465 in FBXW7. In some embodiments, the mutation is at R470 in canine FBXW7 or at R465 in human FBXW7. In some embodiments, the mutation is at R470 in canine FBXW7.

Screening methods

Aspects of the invention relate to methods of screening agents that may be suitable for treatment of a hematological cancer based on the mutations, genes, and pathways described herein. In some embodiments, the invention provides a method for screening a molecular or chemical library, including for example a combinatorial library, to identify an agent or combination of agents that modulate a gene or pathway described herein.

The agents may be biological (e.g., protein, peptide, nucleic acid, antibody, etc.) in nature or they may be chemical in nature (e.g., a small molecule, small organic molecule, etc.). They may be present in a library such as a molecular library, a combinatorial library, a chemical library, and the like. It is to be understood that the agent may be of virtually any nature.

In some embodiments, the method comprises exposing a cell or a population of cells (such as cell line or a primary tumor sample) to an agent and measuring expression of one or more markers provided herein in the cell or cell population before and after exposure to the agent. The method may comprise measuring one or more markers as described herein such as measuring an expression level of a gene selected from POT1, FBXW7, TRAF3, PSMA1, COX8A, PTEN, SATB1, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRC1, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPL11, EEF1A1, KLRK1, GRIFIN, FAM90A1, ZNF486, and ZFN706. In some embodiments, the expression level is of a gene selected from the group POT1, FBXW7, TRAF3, PSMA1, PTEN, SATB1, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, KPNA2, NLRP14, EEF1A1, KLRKl, GRIFIN, FAM90A1, and ZNF486. In some embodiments, the expression level is expression level of KCND2 or mTOR.

In some embodiments, the method comprises measuring a first expression level of one or more markers in a cancer cell or population of cancer cells, and measuring a second expression level of the same one or more markers in a cancer cell or population of cancer cells in the presence of at least one agent, wherein a difference between the first and second expression level is used to determine whether the at least one agent modulates the level of the marker including its expression level and/or its activity level.

The expression and activity may be measured using any method known in the art. The art is familiar with assays for mRNA expression levels, protein expression levels, and activity levels of the one or more markers (see, e.g., Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, (Current Edition); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (Current Edition)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (Current Edition) ANTIBODIES, A LABORATORY MANUAL and

ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)). DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition);

Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.). Examples of mRNA- based assays include but are not limited to oligonucleotide microarray assays, quantitative RT-PCR, Northern analysis, and multiplex bead-based assays. Protein levels may be measured using protein-based assays such as but not limited to immunoassays, Western blots, Western immunoblotting, multiplex bead-based assays, and assays involving aptamers (such as SOMAmer™ technology) and related affinity agents. Other examples of protein detection and quantitation methods include multiplexed immunoassays as described for example in US Patent Nos. 6939720 and 8148171, and published US Patent Application No. 2008/0255766, and protein microarrays as described for example in published US Patent Application No. 2009/0088329.

In some embodiments, the cell or population of cells is a lymphoma cell or population of such cells such as a lymphoma cell line or lymphoma cells derived from a subject having lymphoma. It is to be understood that these screening methods may be used to identify agents that might treat other cancers containing markers described herein.

In some embodiments, the cancer cell or population of cancer cells is a canine cancer cell or a population of canine cancer cells. In some embodiments, the cancer cell or population of cancer cells is a human cancer cell or a population of human cancer cells. In some embodiments, the cancer cell or population of cancer cells comprises a mutation in one or more genes selected from POT1, FBXW7, TRAF3, PSMA1, COX8A, PTEN, SATB1, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRC1, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPL11, EEF1A1, KLRK1, GRIFIN, FAM90A1, ZNF486, and ZFN706. In some embodiments, the cancer cell or population of cancer cells comprises a mutation in one or more genes selected from POT1, FBXW7, TRAF3, PSMA1, PTEN, SATB1, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, KPNA2, NLRP14, EEF1A1, KLRKl, GRIFIN, FAM90A1, and ZNF486. In some embodiments, the cancer cell or population of cancer cells comprises a mutation in KCND2 or mTOR.

In some embodiments, the agent is a member of a molecular library. Accordingly, a method of screening may be a high-throughput screen. The molecular library may be, for example, recombinantly produced or chemically produced. Non-limiting examples of molecular libraries include a small molecule library (e.g., a small organic or inorganic molecule library), a chemical library, a peptide library, an siRNA library, an shRNA library, an anti-sense oligonucleotide library, or an open-reading frame library. In general, a molecular library contains from two to 10¹² molecules, and any integer number therebetween. Methods for preparing libraries of molecules and screening such molecules are well known in the art.

Genome analysis methods

Methods of genetic analysis are known in the art. Examples of genetic analysis methods and commercially available tools are described below.

Affymetrix: The Affymetrix SNP 6.0 array contains over 1.8 million SNP and copy number probes on a single array. The method utilizes at a simple restriction enzyme digestion of 250 ng of genomic DNA, followed by linker-ligation of a common adaptor sequence to every fragment, a tactic that allows multiple loci to be amplified using a single primer complementary to this adaptor. Standard PCR then amplifies a predictable size range of fragments, which converts the genomic DNA into a sample of reduced complexity as well as increases the concentration of the fragments that reside within this predicted size range. The target is fragmented, labeled with biotin, hybridized to microarrays, stained with streptavidin- phycoerythrin and scanned. To support this method, Affymetrix Fluidics Stations and integrated GS-3000 Scanners can be used.

Illumina Infinium: Examples of commercially available Infinium array options include the 660W-Quad (>660,000 probes), the lMDuo (over 1 million probes), and the custom iSelect (up to 200,000 SNPs selected by user). Samples begin the process with a whole genome amplification step, then 200 ng is transferred to a plate to be denatured and neutralized, and finally plates are incubated overnight to amplify. After amplification the samples are enzymatically fragmented using end-point fragmentation. Precipitation and resuspension clean up the DNA before hybridization onto the chips. The fragmented, resuspended DNA samples are then dispensed onto the appropriate BeadChips and placed in the hybridization oven to incubate overnight. After hybridization the chips are washed and labeled nucleotides are added to extend the primers by one base. The chips are immediately stained and coated for protection before scanning. Scanning is done with one of the two Illumina iScan™ Readers, which use a laser to excite the fluorophore of the single-base extension product on the beads. The scanner records high-resolution images of the light emitted from the fluorophores. All plates and chips are barcoded and tracked with an internally derived laboratory information management system. The data from these images are analyzed to determine SNP genotypes using Illumina's BeadStudio. To support this process, Biomek F/X, three Tecan Freedom Evos, and two Tecan Genesis Workstation 150s can be used to automate all liquid handling steps throughout the sample and chip prep process.

Illumina BeadArrav: The Illumina Bead Lab system is a multiplexed array-based format. Illumina's BeadArray Technology is based on 3-micron silica beads that self- assemble in microwells on either of two substrates: fiber optic bundles or planar silica slides. When randomly assembled on one of these two substrates, the beads have a uniform spacing of -5.7 microns. Each bead is covered with hundreds of thousands of copies of a specific oligonucleotide that act as the capture sequences in one of Illumina's assays. BeadArray technology is utilized in Illumina's iScan System. Sequenom: During pre-PCR, either of two Packard Multiprobes is used to pool oligonucleotides, and a Tomtec Quadra 384 is used to transfer DNA. A Cartesian nanodispenser is used for small-volume transfer in pre-PCR, and another in post-PCR. Beckman Multimeks, equipped with either a 96-tip head or a 384-tip head, are used for more substantial liquid handling of mixes. Two Sequenom pin-tool are used to dispense nanoliter volumes of analytes onto target chips for detection by mass spectrometry. Sequenom Compact mass spectrometers can be used for genotype detection.

Sequencing methods

Methods of genome sequencing are known in the art. Examples of genome sequencing methods and commercially available tools are described below.

Illumina Sequencing: 89 GAIIx Sequencers are used for sequencing of samples. Library construction is supported with 6 Agilent Bravo plate-based automation, Stratagene MX3005p qPCR machines, Matrix 2-D barcode scanners on all automation decks and 2 Multimek Automated Pipettors for library normalization.

454 Sequencing: Roche® 454 FLX-Titanium instruments are used for sequencing of samples. Library construction capacity is supported by Agilent Bravo automation deck, Biomek FX and Janus PCR normalization.

SOLiD Sequencing: SOLiD v3.0 instruments are used for sequencing of samples. Sequencing set-up is supported by a Stratagene MX3005p qPCR machine and a Beckman SC Quanter for bead counting.

ABI Prism® 3730 XL Sequencing: ABI Prism® 3730 XL machines are used for sequencing samples. Automated Sequencing reaction set-up is supported by 2 Multimek Automated Pipettors and 2 Deerac Fluidics - Equator systems. PCR is performed on 60 Thermo-Hybaid 384-well systems.

Ion Torrent: Ion PGM™ or Ion Proton™ machines are used for sequencing samples. Ion library kits (Invitrogen) can be used to prepare samples for sequencing. Other Technologies: Examples of other commercially available platforms include Helicos Heliscope Single-Molecule Sequencer, Polonator G.007, and Raindance RDT 1000 Rainstorm.

Controls

Some of the methods provided herein involve measuring a level of a marker in a biological sample and then comparing that level to a control in order to identify a subject having a cancer such as a hematological cancer. The control may be a control level that is a level of the same marker in a control tissue (such as normal, non-tumor tissue or blood from the same subject from which the biological sample is derived), control subject, or a population of control subjects.

In some embodiments, the control is non-tumor tissue or non-tumor cells derived from the same subject from which the biological sample was obtained.

In some embodiments, the control may be (or may be derived from) a normal subject (or normal subjects). Normal subjects, as used herein, refer to subjects that are apparently healthy and show no tumor manifestation. The control population may therefore be a population of normal subjects.

In other instances, the control may be (or may be derived from) a subject (a) having a similar tumor to that of the subject being tested and (b) who is negative for the cancer- associated allele.

It is to be understood that the methods provided herein do not require that a control level be measured every time a subject is tested. Rather, it is contemplated that control levels of markers are obtained and recorded and that any test level is compared to such a predetermined level (or threshold).

Samples

The methods provided herein detect and sometimes measure (and thus analyze) levels or particular markers in biological samples. Biological samples, as used herein, refer to samples taken or derived from a subject. These samples may be tissue samples or they may be fluid samples (e.g., bodily fluid). Examples of biological fluid samples are whole blood, plasma, serum, urine, sputum, phlegm, saliva, tears, and other bodily fluids. In some embodiments, the biological sample is a whole blood sample, or a sample of white blood cells from a subject. In some embodiments, the biological sample is a tumor, a fragment of a tumor, or a tumor cell(s). The sample may be taken from the mouth of a subject using a swab or it may be obtained from other mucosal tissue in the subject.

Subjects

Certain methods of the invention are intended for canine subjects, including for example golden retrievers, boxers, and cocker spaniels. Other methods of the invention may be used in a variety of subjects including but not limited to humans and canine subjects. In some embodiments, a subject (e.g., a subject identified in a method herein) has a

hematological cancer such as a T-cell or B-cell lymphoma.

It is to be understood that methods of the invention may be used in a variety of other subjects including but not limited to mammals such as humans, canines, felines, mice, rats, rabbits, and apes.

Computational analysis

Methods of computation analysis of sequencing data and somatic mutation data are known in the art. Examples of available computational programs are: Genome Analysis Toolkit (GATK, Broad Institute, Cambridge, MA), muTect (Broad Institute, Cambridge, MA), and Genome MuSiC (Dees et al. 2012. MuSiC: Identifying mutational significance in cancer genomes. Genome Research 22:1589-1598.). .

Devices and Kits

Any of the methods provided herein can be performed on a device, e.g., an array. Suitable arrays are described herein and known in the art. Accordingly, a device, e.g., an array, for detecting any of the mutations or markers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations or markers, or at least 10, at least 20, at least 30, at least 40, at least 50, or more mutations or markers, or up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 75 or up to 100 mutations or markers) described herein is also contemplated.

Reagents for use in any of the methods provided herein can be in the form of a kit. Accordingly, a kit for detecting any of the mutations or markers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations or markers, or at least 10, at least 20, at least 30, at least 40, at least 50, or more mutations or markers, or up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 75 or up to 100 mutations) described herein is also contemplated. In some embodiments, the kit comprises reagents for detecting any of the mutations or markers described herein, e.g., reagents for use in a method described herein. Suitable reagents are described herein and art known in the art.

Breeding programs

Other aspects of the invention relate to use of the diagnostic methods in connection with a breeding program. A breeding program is a planned, intentional breeding of a group of animals to reduce detrimental or undesirable traits and/or increase beneficial or desirable traits in offspring of the animals. Thus, a subject identified using the methods described herein as not having a cancer-associated marker of the invention may be included in a breeding program to reduce the risk of developing hematological cancer in the offspring of said subject. Alternatively, a subject identified using the methods described herein as having a cancer-associated marker of the invention may be excluded from a breeding program.

Treatment

Other aspects of the invention relate to methods of treatment or to diagnostic methods that comprise a treatment step (also referred to as "theranostic" methods due to the inclusion of the treatment step). Any treatment for a hematological cancer, such as T-cell or B-cell lymphoma, is contemplated herein. In some embodiments, treatment comprises one or more of surgery, chemotherapy, immunotherapy, and radiation. In some embodiments, treatment comprises a bone-marrow transplant. Examples of treatment of hematological cancers include rituximab, Obinutuzumab, Ofatumumab, Ibritumomab tiuxetan, Alemtuzumab, Brentuximab vedotin, Interferon, cyclophosphamide, Chlorambucil, Bendamustine,

Ifosfamide, Dexamethasone, Cisplatin, Carboplatin, Oxaliplatin, doxorubicin, vincristine, Mitoxantrone, Etoposide, Bleomycin Cytarabine, Gemcitabine, Methotrexat, Pralatrexate, and/or prednisone.

In some embodiments, the treatment is a treatment that modulates a gene disclosed herein or a pathway disclosed herein, such as a treatment known to modulate a gene disclosed herein or a pathway disclosed herein. In some embodiments, the treatment is a treatment that is identified using a screening method provided herein, e.g., an agent that is identified as modulating a gene or pathway disclosed herein.

In some embodiments, a subject identified as having a hematological cancer is treated. In some embodiments, the method comprises selecting a subject for treatment on the basis of the presence of one or more cancer-associated markers as described herein. In some embodiments, the method comprises treating a subject with a hematological cancer characterized by the presence of one or more cancer-associated markers as defined herein.

Administration of a treatment may be accomplished by any method known in the art (see, e.g., Harrison's Principle of Internal Medicine, McGraw Hill Inc.). Administration may be local or systemic. Administration may be parenteral (e.g., intravenous, subcutaneous, or intradermal) or oral. Compositions for different routes of administration are well known in the art (see, e.g., Remington's Pharmaceutical Sciences by E. W. Martin). Dosage will depend on the subject and the route of administration. Dosage can be determined by the skilled artisan.

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. EXAMPLES

Example 1. Tumor/normal sequencing in dogs identifies known and novel lymphoma genes

There are 400+ dog breeds world-wide, having extraordinary diversity between breeds. Because of inbreeding within breeds, there is an enrichment of disease genes within certain breeds, predisposing these breeds to certain diseases. As a result, simple and complex traits can be mapped using breeds that are predisposed to develop certain diseases.

In addition, the limited genetic diversity of each breed can help identify which typical somatic mutations happen depending on the background gene expression, or help subdivide canine lymphomas into molecular subtypes reflecting different human lymphoma subtypes.

This study sought to identify somatic mutations in lymphomas in certain canine breeds. Canine lymphomas can be well diagnosed and treatment is the same or similar to human lymphoma, highlighting the similarly between human and canine lymphomas. The most common type of canine B-cell lymphoma is equivalent to human Diffuse Large B-cell lymphoma (DLBCL), while T-cell lymphoma in canines is heterogeneous. Canines presenting with high-grade lymphoma have poor prognosis, with 40-45% living about one year with treatment and only 4-6 weeks if untreated.

In this study, tumor and normal paired samples were collected from 3 canine breeds: Golden retriever, Boxer, and Cocker spaniel. The breeds were picked for their differential predisposition to B- and T-cell lymphoma. Golden retrievers have a 13% chance of developing either B-cell or T-cell lymphoma during their lifetime. Boxers have a 20% chance of developing lymphoma during their lifetime and mostly develop T-cell lymphomas while Cocker spaniels have a 7% chance of developing lymphoma with a high prevalence for B-cell lymphoma during their lifetime.

The number of paired samples collected for each breed are summarized in the table below. Breed B-cell pairs T-cell pairs

Boxer - 16

Cocker 10 - Spaniel

Golden 54 25

Retriever

Each sample was subjected to Exome Capture (Roche/Nimblegen) using lug DNA from the sample. The processed samples were then sequenced using Illumina Sequencing. Base calling values for normal (N) and tumor (T) samples were preset at >30x for the normal sample and >60x for the tumor sample. After standard quality control filtering, the sequence information was aligned to CanFam3.1. Variant calling in tumor and normal, followed by selection of somatic variants was accomplished using muTect (Cibulskis et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature Biotechnology 31 :213— 219 (2013)). Significantly mutated genes were then identified in each sample group using Genome MuSiC (Mutational Significance in Cancer, Dees et al. MuSiC: Identifying mutational significance in cancer genomes. Genome Research 22:1589- 1598. (2012)).

The overall tumor mutation load identified by breed is shown in the tables below (average number of total mutations and non-synonymous mutations, respectively, per individual).

Total tumor mutations, average

Non-synonymous mutations, average

As shown in the table below, it was found that the number of samples to some extent correlated with the number of significant genes detected.

There was overlap in the genes identified in the different breeds that are predisposed to B-cell lymphoma (FIG. 3A). However, there was little overlap between the breeds that are predisposed to T-cell lymphoma (FIG. 3B), and little overlap between B-cell lymphoma and T-cell lymphoma (FIG. 3C). FIG. 3C shows that most genes that are significantly mutated are specific for either subtype of lymphoma. Genes that did overlap included PSMA1, TBC1D26, FAM90A1, and potential TMC4 antisense. It was found that the top genes identified in B-cell lymphomas were similar, whereas the top genes identified in T-cell lymphomas were not similar. Due to the very different number of samples and hence different number of significantly mutated genes, comparison of all significantly mutated genes is not entirely representative. Rather, the top most significantly mutated genes were compared. Of all genes significantly mutated in Cocker spaniel B-cell lymphoma (the smaller sample group for B-cell lymphoma, 8 significantly mutated genes), TRAF3, POTl, FAM90A1, PSMA1, and DDX3X were among the top 15 significantly mutated genes in Golden retriever B-cell lymphomas. The shared genes include genes known to be involved in human lymphoma, genes implicated in other human cancers, and genes not previously described in human cancer. Of all significantly mutated genes in Boxer T-cell lymphomas (the smaller sample group for T-cell lymphoma, 6 significantly mutated genes) none is among the top 10 significantly mutated genes in Golden retriever T-cell lymphoma but one (SATB1) is significantly mutated in the entire list of significantly mutated genes in Golden retriever T-cell lymphoma.

The key pathways/genes identified in B-cell lymphomas included FBXWl-cMyc, TRAF3-MAP3K14, POT1 and DDX3X and are summarized in the table below and in FIG. 5. FBXW7 was the most commonly mutated gene in Golden retriever B-cell lymphomas (28%) but was rarely mutated in Cocker spaniel B-cell lymphomas (10%). It was found that the position commonly seen mutated in canine lymphoma, particularly in Golden retriever B-cell lymphoma, R470, was at a homologous position to one of the two positions recurrently mutated in human cancer, R465 (FIG. 4). However the ubiquitination and half-life of cMyc, a well-known oncogene in human tumors, is regulated by FBXW7 (PMID 23791182), and together, FBXW7 and/or cMyc were mutated in almost a third of all B-cell lymphoma samples regardless of breed. TRAF3 was the second most commonly mutated gene in Golden retriever B-cell lymphoma and, together with two other genes, the most commonly mutated gene in Cocker spaniel B-cell lymphoma tumors. TRAF3 signals through MAP3K14 in the alternative NF-kB signaling pathway. These two genes are commonly mutated in classical Hodgkin's lymphoma in humans (PMID 22469134). A mutation either of these two genes was seen in half of all Cocker spaniel B-cell lymphoma tumors. TRAF2 is also part of the same complex as TRAF3 and MAP3K14. TRAF2 was also observed to be mutated in more than one sample, but did not reach significance in any group. Two additional examples of genes typically mutated in B-cell lymphoma but not seen mutated in any T-cell lymphoma sample were POT1 (protection of telomeres 1) and DDX3X, an ATP-dependent RNA helicase.

KLRK1 was observed to contain both mutations and somatic copy number alterations. KLRK1 was significantly mutated in Golden B cell lymphoma, and the combined T cell group (but neither Golden T nor Boxer T alone).

FBXW7 (SEL-10) encodes an E3 ubiquitin ligase and is involved in cyclin E degradation and cMyc stability (see above). TRAF3 encodes TNF receptor-associated factor 3, which is involved in the CD40 signaling cascade and cell death via NF-KB.

The key pathways/genes identified in T-cell lymphomas included SATB1, PTEN, and LTA4H. PTEN signals via mTOR and PI3K and this pathway appears to be highly specific to Boxer T-cell lymphomas. The fraction of individuals with at least one mutation in a gene or pathway are summarized in the table above and in FIG. 5.

SATB1, which was found to be mutated mostly in Boxer T-cell lymphoma samples but also in Golden retriever T-cell lymphomas, encodes a protein that binds the nuclear matrix and is involved in chromatin remodeling. SATB1 also recruits chromatin remodeling factors and controls genome organization and gene regulation. The PTEN-mTOR-PI3K pathway, where mutations were found mostly in Boxer T-cell lymphoma samples, is involved in tumor suppression and cell cycle regulation. In particular, PTEN dephosphorylates PIP3, thereby inhibiting the AKT pathway. LTA4H encodes a leukotriene A4 hydrolase involved in metabolism, and is the most significantly mutated gene among Golden retriever T-cell lymphomas that was never seen mutated in the Boxer T-cell lymphomas in this study.

Of the genes identified, some are associated with human lymphomas or other cancers, showing that canine and human lymphomas share similarities. A summary of the associations are shown in FIG. 6. In particular, genes seen in other cancers but not human lymphoma included PSMA1, KPNA2, and GLUD2. Novel genes include two NLRP genes implicated in innate immunity, TBC1D26 which has not been much studied although the involvement of other TBC1 family members in the cell cycle could suggest a similar role for this gene's product, and GRIFIN, encoding a galectin-like protein.

In conclusion, these results show that the typical somatic mutations in B-cell lymphomas from the two compared breeds are similar, whereas in T-cell lymphoma, the genetic background may affect somatic mutations or predispose to a certain T-cell lymphoma subtype. Additionally, the novel genes and the genes identified that have been associated with other cancers may help in development of new treatment options for lymphoma patients.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any

combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

What is claimed is: CLAIMS

1. A method, comprising:

analyzing genomic DNA from a subject for the presence of a mutation in a gene selected from the group consisting of POTl, FBXW7, TRAF3, PSMA1, COX8A, PTEN, SATB1, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRCl, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPLll, EEFlAl, KLRKl, GRIFIN, FAM90A1, ZNF486, ZFN706, KCND2, and mTOR; and

identifying a subject having the mutation as a subject having a hematological cancer.

2. The method of claim 1, wherein the gene is selected from the group consisting of POTl, FBXW7, TRAF3, PSMA1, PTEN, SATB1, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP53, cMYC, MAP3K14, KPNA2, NLRP14, EEFlAl, KLRKl, GRIFIN, FAM90A1, and ZNF486.

3. The method of claim 1, wherein the gene is selected from the group consisting of NLRP5, NLRP14, TBC1D26, and GRIFIN.

4. The method of claim 1, wherein the gene is selected from the group consisting of NLRP14, TBC1D26, and GRIFIN.

5. The method of claim 1, wherein the gene is selected from the group consisting of FBXW7, TRAF3, MAP3K14, POTl, TP53, PNRCl, FKBP3, SOCS2, SETD2, MITF, and DDX3X .

6. The method of claim 1, wherein the gene is selected from the group consisting of FBXW7, TRAF3, MAP3K14, POTl, TP53, SETD2, and DDX3X.

7. The method of claim 1, wherein the gene is selected from the group consisting of PSMA1, COX8A, LTA4H, TBC1D26, ZNF706, ATP5H, NLRP5, GLUD2, SATB1, PTEN, MAP2K1, NLRP14, KCND2, mTOR, and a potential TMC4 antisense.

8. The method of claim 1, wherein the gene is selected from the group consisting of PSMA1, TBC1D26, SATB1, PTEN, NLRP14, KCND2, mTOR, and a potential TMC4 antisense.

9. The method of any one of claims 1 to 8, wherein the genomic DNA is obtained from white blood cells of the subject.

10. The method of any one of claims 1 to 9, wherein the genomic DNA is analyzed using a single nucleotide polymorphism (SNP) array.

11. The method of any one of claims 1 to 9, wherein the genomic DNA is analyzed using a bead array.

12. The method of any one of claims 1 to 9, wherein the genomic DNA is analyzed using a nucleic acid sequencing assay.

13. The method of any one of claims 1 to 12, wherein the subject is a human subject.

14. The method of any one of claims 1 to 12, wherein the subject is a canine subject.

15. The method of any one of claims 1 to 14, wherein the cancer is a lymphoma.

16. The method of claim 1, 2, 5 or 6, wherein the cancer is a B cell lymphoma.

17. The method of claim 1, 2, 7 or 8, wherein the cancer is a T cell lymphoma.

18. The method of any one of claims 1 to 17, wherein the mutation is two or more mutations.

19. The method of any one of claims 1 to 18, wherein the gene is two or more genes.

20. A method, comprising:

measuring, in a cancer cell or population of cancer cells in the absence of at least one agent, a first expression level of one or more genes selected from the group consisting of POT1, FBXW7, TRAF3, PSMAl, COX8A, PTEN, SATBl, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP 53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRC1, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPL11, EEF1A1, KLRK1, GRIFIN, FAM90A1, ZNF486, ZFN706, KCND2, and mTOR; and

measuring, in a cancer cell or population of cancer cells in the presence of the at least one agent, a second expression level of one or more genes selected from the group consisting oiPOTl, FBXW7, TRAF3, PSMAl, COX8A, PTEN, SATBl, LTA4H, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP 53, cMYC, MAP3K14, MAP2K1, KPNA2, NLRP5, NLRP14, MITF, PNRC1, FKBP3, SOCS2, ATP5H, PTPN6, GLUD2, RPL11, EEF1A1, KLRK1, GRIFIN, FAM90A1, ZNF486, ZFN706, KCND2, and mTOR; and

identifying the at least one agent as capable of modulating expression of the one or more genes in the cancer cell or population of cancer cells if

(i) the second expression level of the one or more genes is up-regulated compared to the first expression level or

(ii) the second expression level of the one or more genes is down-regulated compared to the first expression level.

21. The method of claim 20, wherein the first and second expression levels are expression levels of one or more genes selected from the group consisting oiPOTl, FBXW7, TRAF3, PSMAl, PTEN, SATBl, DDX3X, TBC1D26, a potential TMC4 antisense, SETD2, TP 53, cMYC, MAP3K14, KPNA2, NLRP14, EEFlAl, KLRKl, GRIFIN, FAM90A1, and ZNF486.

22. The method of claim 20 or 21, wherein the at least one agent is identified as suitable for treating a hematological cancer if the agent is identified as capable of modulating expression of the one or more genes.

23. The method of claim 20, 21 or 22, wherein the cancer cell or population of cancer cells is a lymphoma cell or a population of lymphoma cells.

24. The method of any one of claims 20-23, wherein the cancer cell or population of cancer cells comprises a mutation in the one or more genes.

25. The method of any one of claims 20-24, wherein the cancer cell or population of cancer cells is a canine cancer cell or a population of canine cancer cells.

26. The method of any one of claims 20-25, wherein the cancer cell or population of cancer cells are a human cancer cell or a population of human cancer cells.

27. The method of any one of claims 20-26, wherein the one or more genes is selected from the group consisting oiNLRP5, NLRP14, TBC1D26, and GRIFIN.

28. The method of any one of claims 20-26, wherein the one or more genes is selected from the group consisting oiNLRP14, TBC1D26, and GRIFIN.

29. The method of any one of claims 20-26, wherein the one or more genes is selected from the group consisting of FBXW7, TRAF3, MAP3K14, POTl, TP53, PNRCl, FKBP3, SOCS2, SETD2, MITF, and DDX3X.

30. The method of any one of claims 20-26, wherein the one or more genes is selected from the group consisting of FBXW7, TRAF3, MAP3K14, POTl, TP53, SETD2, and DDX3X.

31. The method of any one of claims 20-26, wherein the one or more genes is selected from the group consisting oiPSMAl, COX8A, LTA4H, TBC1D26, ZNF706, ATP5H, NLRP5, GLUD2, SATBl, PTEN, MAP2K1, NLRP14, KCND2, mTOR, and a potential TMC4

antisense.

32. The method of any one of claims 20-26, wherein the one or more genes is selected from the group consisting oiPSMAl, TBC1D26, SATBl, PTEN, NLRP14, KCND2, mTOR, and a potential TMC4 antisense.