WO2019091429A1

WO2019091429A1 - Unique genetic fingerprints for murine tumor model and uses thereof

Info

Publication number: WO2019091429A1
Application number: PCT/CN2018/114595
Authority: WO
Inventors: Jie Cai; Wubin QIAN; Xiaobo Chen; Bin Fan; Sheng Guo; Henry Li
Original assignee: Crown Bioscience Inc Taicang
Current assignee: Crown Bioscience Inc Taicang
Priority date: 2017-11-08
Filing date: 2018-11-08
Publication date: 2019-05-16
Anticipated expiration: 2020-05-08

Abstract

Provided is a method of producing a murine tumor model. In one embodiment, the method comprises transplanting a mouse tumor cell line to a mouse; raising the mouse in a condition that allows the tumor cell line to develop into a tumor; detecting in the tumor the presence of a biomarker that is unique to the mouse tumor cell line; and determining the identification of the mouse tumor cell line.

Description

UNIQUE GENETIC FINGERPRINTS FOR MURINE TUMOR MODEL AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to application PCT/CN2017/109944, filed November 08, 2017, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to cancer diagnosis, prognosis and treatment. In particular, the present invention relates to the identification of murine tumor models.

BACKGROUND

Tumor models are naturally existing or artificially induced systems that share features with human cancers. Experimental systems for studying human cancer include cell lines and animals (e.g., mice) grafted with tumor cells line or tissues, which can be derived from human or non-human animals (e.g., mice) . With the rapid advancement of immune oncology, syngeneic and homograft murine tumor models become valuable experimental tools for preclinical efficacy evaluation of immune therapy and in combination with conventional or targeted cancer therapies. Unlike human tumor xenografts (either patient derived (PDXs) or human cell line derived) , whose genetic identity can readily be tracked based on the well-characterized STR genotypes or HLA tissue types of the individuals from which the tumor grafts are derived, murine tumor models were derived from limited number of laboratory strains of mice and do not have readily and sufficiently diversified common genotyping markers to differentiate individual tumor models. Hence, there is a need to develop markers and methods for identifying individual murine tumor models and for tracking the fidelity in these models.

SUMMARY OF INVENTION

In one aspect, the present disclosure provides a method for producing a non-human tumor model. In one embodiment, the method comprises the steps of transplanting a non-human tumor cell line or a primary tumor tissue to a non-human animal; raising the non-human animal in a condition that allows the non-human tumor cell line or the primary tumor tissue to develop into a tumor; detecting in the tumor the presence of a biomarker that is unique to the non-human tumor cell line or the primary tumor tissue; and determining the identification of the non-human tumor cell line or the primary tumor tissue. In another embodiment, the method comprises the steps of culturing a non-human tumor cell line in vitro; detecting in the non-human tumor cell line the presence of a biomarker that is unique to the non-human tumor cell line; determining the identification of the non-human tumor cell line; transplanting the non-human tumor cell line to a non-human animal; and raising the non-human animal in a condition that allows the non-human tumor cell line to develop into a tumor.

In certain embodiments, the non-human tumor model is a murine tumor model.

In certain embodiments, the non-human tumor cell line is selected from the group consisting of 4T1, A20, B16BL6, B16F0, B16F1, B16F10, C1498, Colon26, CT26, EG7_Ova, EL4, EMT6, H22, Hepa 1-6, J558, JC, KLN205, L1210, L5178-R, LL/2, MBT2, MPC-11, Neuro-2a, P388D1, P815, Renca, S91 and WEHI164.

In certain embodiments, the biomarker is a fusion gene. In certain embodiments, the fusion gene is selected from the fusion genes in Table 1. In certain embodiments, the fusion gene comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-75.

In certain embodiments, the biomarker is detected by an amplification assay, a hybridization assay, a sequencing assay or an array.

In certain embodiments, the biomarker is detected using a primer set capable of detecting a biomarker panel comprising SEQ ID NOs: 1-75.

In certain embodiments, the biomarker is detected using a microarray comprising probes capable of detecting a biomarker panel comprising SEQ ID NOs: 1-75.

In certain embodiments, the non-human tumor model is determined as cell line 4T1 when the biomarker comprises SEQ ID NO: 1.

In certain embodiments, the non-human tumor model is determined as cell line A20 when the biomarker comprises SEQ ID NO: 2 or 3.

In certain embodiments, the non-human tumor model is determined as cell line B16BL6 when the biomarker comprises SEQ ID NO: 4 or 5.

In certain embodiments, the non-human tumor model is determined as cell line B16F0 when the biomarker comprises SEQ ID NO: 6 or 7.

In certain embodiments, the non-human tumor model is determined as cell line B16F1 when the biomarker comprises SEQ ID NO: 8.

In certain embodiments, the non-human tumor model is determined as cell line B16F10 when the biomarker comprises SEQ ID NO: 9.

In certain embodiments, the non-human tumor model is determined as cell line C1498 when the biomarker comprises SEQ ID NO: 10, 11 or 12.

In certain embodiments, the non-human tumor model is determined as cell line Colon26 when the biomarker comprises SEQ ID NO: 13 or 14.

In certain embodiments, the non-human tumor model is determined as cell line CT26 when the biomarker comprises SEQ ID NO: 15 or 16.

In certain embodiments, the non-human tumor model is determined as cell line EG7_Ova when the biomarker comprises SEQ ID NO: 17.

In certain embodiments, the non-human tumor model is determined as cell line EL4 when the biomarker comprises SEQ ID NO: 18, 19 or 20.

In certain embodiments, the non-human tumor model is determined as cell line EMT6 when the biomarker comprises SEQ ID NO: 21, 22 or 23.

In certain embodiments, the non-human tumor model is determined as cell line H22 when the biomarker comprises SEQ ID NO: 24, 25, 26, or 27.

In certain embodiments, the non-human tumor model is determined as cell line Hepa 1-6 when the biomarker comprises SEQ ID NO: 28, 29 or 30.

In certain embodiments, the non-human tumor model is determined as cell line J558 when the biomarker comprises SEQ ID NO: 31 or 32.

In certain embodiments, the non-human tumor model is determined as cell line JC when the biomarker comprises SEQ ID NO: 33 or 34.

In certain embodiments, the non-human tumor model is determined as cell line KLN205 when the biomarker is detected as comprises SEQ ID NO: 35 or 36.

In certain embodiments, the non-human tumor model is determined as cell line L1210 when the biomarker comprises SEQ ID NO: 37, 28, 39 or 40.

In certain embodiments, the non-human tumor model is determined as cell line L5178-R when the biomarker comprises SEQ ID NO: 41, 42, 43, 44, 45, 46, 48, 49 or 50.

In certain embodiments, the non-human tumor model is determined as cell line LL/2 when the biomarker comprises SEQ ID NO: 51, 52 or 53.

In certain embodiments, the non-human tumor model is determined as cell line MBT-2 when the biomarker comprises SEQ ID NO: 54 or 55.

In certain embodiments, the non-human tumor model is determined as cell line MPC-11 when the biomarker comprises SEQ ID NO: 56 or 57.

In certain embodiments, the non-human tumor model is determined as cell line Neuro-2a when the biomarker comprises SEQ ID NO: 58, 59, 60 or 61.

In certain embodiments, the non-human tumor model is determined as cell line P388D1 when the biomarker comprises SEQ ID NO: 62, 63, 64 or 65.

In certain embodiments, the non-human tumor model is determined as cell line P815 when the biomarker comprises SEQ ID NO: 66, 67 or 68.

In certain embodiments, the non-human tumor model is determined as cell line Renca when the biomarker comprises SEQ ID NO: 69 or 70.

In certain embodiments, the non-human tumor model is determined as cell line S91 when the biomarker comprises SEQ ID NO: 71 or 72.

In certain embodiments, the non-human tumor model is determined as cell line WEHI164 when the biomarker comprises SEQ ID NO: 73, 74 or 75.

In certain embodiments, the non-human animal is a rodent. In certain embodiments, the non-human animal is a mouse.

In another aspect, the present disclosure provides a kit for detecting a fusion gene. In certain embodiments, the fusion gene comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-75.

In yet another aspect, the present disclosure provides a hybridization probe for detecting a fusion gene selected from the group consisting of the fusion genes in Table 1. In certain embodiments, the fusion gene comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-75.

In yet another aspect, the present disclosure provides a kit comprising primers for detecting a biomarker panel. In certain embodiments, the biomarder panel comprises SEQ ID NOs: 1-75.

In another aspect, the present disclosure provides a microarray comprising probes for detecting a biomarker panel. In certain embodiments, the biomarder panel comprises SEQ ID NOs: 1-75.

BRIEF DESCRIPTION OF DRAWING

FIGs. 1A-1K illustrate the sequences of fusion genes that are unique to the murine tumor cell lines of the present invention. The junction positions are marked with symbol “|” .

DETAILED DESCRIPTION OF THE INVENTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Definitions

The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over the definition of the term as generally understood in the art.

As used herein, the singular forms “a” , “an” and “the” include plural references unless the context clearly dictates otherwise.

As used herein, the terms “cancer” and “tumor” are interchangeable and refer to any diseases involving an abnormal cell growth and include all stages and all forms of the disease that affects any tissue, organ or cell in the body. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre-and post-metastatic cancers. In general, cancers can be categorized according to the tissue or organ from which the cancer is located or originated and morphology of cancerous tissues and cells. As used herein, cancer types include, without limitation, acute lymphoblastic leukemia (ALL) , acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, childhood cerebellar or cerebral, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, Burkitt's lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, retinoblastoma, gastric (stomach) cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (endocrine pancreas) , Kaposi sarcoma, kidney cancer (renal cell cancer) , laryngeal cancer, leukemia, liver cancer, lung cancer, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer) , retinoblastoma, Ewing family of tumors, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, vaginal cancer.

It is noted that in this disclosure, terms such as “comprises” , “comprised” , “comprising” , “contains” , “containing” and the like have the meaning attributed in United States Patent law; they are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. Terms such as “consisting essentially of” and “consists essentially of”have the meaning attributed in United States Patent law; they allow for the inclusion of additional ingredients or steps that do not materially affect the basic and novel characteristics of the claimed invention. The terms “consists of” and “consisting of” have the meaning ascribed to them in United States Patent law; namely that these terms are close ended.

The term “complementarity” refers to the ability of a nucleic acid to form hydrogen bond (s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%>, 70%>, 80%>, 90%, and 100%complementary) . “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100%over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

The terms “determining, ” “assessing, ” “assaying, ” “measuring” and “detecting” can be used interchangeably and refer to both quantitative and semi-quantitative determinations. Where either a quantitative and semi-quantitative determination is intended, the phrase “determining a level” of a polynucleotide or polypeptide of interest or “detecting” a polynucleotide or polypeptide of interest can be used.

The term “hybridizing” refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. The term “stringent conditions” refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences in a mixed population (e.g., a cell lysate or DNA preparation from a tissue biopsy) . A “stringent hybridization” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization (e.g., as in array, microarray, Southern or northern blot hybridizations) are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I, Ch. 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays, ” (1993) Elsevier, N.Y. Generally, highly stringent hybridization and wash conditions are selected to be about 5℃ lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or northern blot is 42℃. using standard hybridization solutions (see, e.g., Sambrook and Russell Molecular Cloning: A Laboratory Manual (3rd ed. ) Vol. 1-3 (2001) Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY) . An example of highly stringent wash conditions is 0.15 M NaCl at 72℃ for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65℃ for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is l×SSC at 45℃ for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4×SSC to 6×SSC at 40℃ for 15 minutes.

The term “nucleic acid” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

In general, a “protein” is a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds) . Proteins may include moieties other than amino acids (e.g., may be glycoproteins) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence) , or can be a functional portion thereof. Those of ordinary skill will further appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.

Tumor Model

Tumor models, as used herein, refer to cells, tissues or animals used to study the development and progression of cancer, and to test treatments before they are given to human.

In certain embodiments, the tumor models provided herein are non-human animals grafted with tumor cell lines or primary tumor tissues. In certain embodiments, the tumor cell lines used herein include, without limitation, 4T1, A20, B16BL6, B16F0, B16F1, B16F10, C1498, Colon26, CT26, EG7_Ova, EL4, EMT6, H22, Hepa 1-6, J558, JC, KLN205, L1210, L5178-R, LL/2, MBT2, MPC-11, Neuro-2a, P388D1, P815, Renca, S91 and WEHI164.

The 4T1 cell line is a mouse breast tumor cell line that is highly tumorigenic and invasive. The 4T1 cell line can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain and bone. The following properties of 4T1 make it a suitable experimental animal model for human mammary cancer: (1) 4T1 cells are easily transplanted into the mammary gland so that the primary tumor grows in the anatomically correct site; (2) as in human breast cancer, 4T1 metastatic disease develops spontaneously from the primary tumor and the progressive spread of 4T1 metastases to the draining lymph nodes and other organs is very similar to that of human mammary cancer; (3) 4T1 is resistant to 6-thioguanine, hence enabling precise quantitation of metastatic cells, even when they are disseminated and at sub-microscopic levels in distant organ.

The A20 cell line is a BLAB/c B cell lymphoma line derived from a spontaneous reticulum cell neoplasm found in an old BALB/cAnN mouse. The A20 cells express little surface immunoglobulin when grown in Click's medium; however, they express large amounts when grown in RPMI 1640 medium. The A20 cells can present both alloantigens and protein antigens.

The B16BL6 cell line is a murine melanoma cell line derived from B16 cell line. The B16BL6 cells display significantly stronger invasive activity than B16.

The B16F0 cell line is a murine melanoma cell line derived from B16 cell line. It is tumorigenic when being grafted in syngeneic mice but display less invasive activity than B16F10.

The B16F10 cell line is a murine melanoma cell line derived from B16 cell line. The B16F10 cells display significantly stronger invasive activity than B16, B16F0 and B16F1.

The C1498 cell line is a murine AML cell line that arose spontaneously in a C57BL/6 mouse and grows aggressively in syngeneic mice.

The Colon26 cell line is a mouse colon adenocarcinoma derived from BALB/c.

The CT26 cell line is an N-nitroso-N-methylurethan-induced, undifferentiated colon carcinoma cell line. The CT26 cell line has become a preclinical platform for evaluating the potential of drug combinations with immune checkpoint inhibitor antibodies.

The EG7_Ova cell line is a mouse thymoma EL4 cell line derived from C57BL/6. The cell line is stably transfected with the cDNA of chicken ovalbumin (OVA) and thus express OVA epitope as a unique antigen.

The EL4 cell line is a 9, 10-dimethyl-1, 2-benzanthracene induced lymphoma cell line from a C57L mouse.

The EMT6 cell line was established from a transplantable murine mammary carcinoma that arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule. The resulting tumor line (named KHJJ) was propagated in BALB/cKa mice and adapted to tissue culture after the 25th animal passage, and the cell line was named EMT. EMT6 is a clonal isolate of EMT isolated in 1971 at Stanford University.

The H22 cell line is a mouse hepatoma cell line.

The Hepa 1-6 cell line is derived from the BW7756 mouse hepatoma that arose in a C57/L mouse.

The J558 cell line is a mouse B myeloma cell line derived from a BALB/c mouse.

The JC cell line is an epithelial-like cell line derived from a spontaneous primary mammary adenocarcinoma along the milk line.

The KLN205 cell line is a murine lung carcinoma cell line, established form the Nettersheim lung carcinoma.

The L1210 cell line is a mouse lymphocytic leukemia cell line that is derived from the ascetic fluid of an 8-month-old female mouse.

The L5178-R cell line is derived from the L5178 thymic lymphoma induced by methylcholanthrene in a DBA/2 mouse.

The LL/2 cell line is a Lewis lung carcinoma cell line established from the lung of a C57B mouse bearing a tumor resulting from an implantation of primary Lewis lung carcinoma.

The MBT2 cell line is a murine bladder carcinoma cell line.

The MPC11 cell line is a murine myeloma cell line.

The Neuro-2a cell line is a mouse neuroblastoma cell line. It displays a neuronal and amoeboid stem cell morphology.

The P388D1 cell line is a murine macrophage cell line.

The P815 cell line is a mouse mastocytomacell line derived from DBA/2 mice.

The Renca cell line is an adenocarcinoma cell line derived from a tumor that arose spontaneously as a renal cortical adenocarcinoma in Balk/cCr mice.

The S91 cell line is mouse melanoma cell line.

The WEHI164 cell line is a mouse fibrosarcoma cell line induced by subcutaneous injection of 3-methylcholanthrene.

Fusion Gene

In one aspect, the present disclosure provides novel fusion genes in murine tumor models. “Fusion gene” and “gene fusion” are used interchangeably herein and are intended to encompass both DNA and RNA, including but not limited to fusion of two or more separate genes at DNA level (such as genomic DNA or cDNA) , RNA level (such as mRNA) . In certain embodiments, the two genes are fused at genomic DNA level due to chromosome rearrangement, such as a translocation, interstitial deletion, or chromosomal inversion. Fusion gene may be transcribed and/or translated to its gene product, which can be RNA or protein.

The fusion genes provided herein comprise a first encoding sequence for a first gene ( “upstream gene” or “up gene” ) covalently linked to a second encoding sequence for a second gene ( “downstream gene” or “down gene” ) . The upstream gene and the downstream gene as disclosed herein can refer the gene (e.g. the DNA sequence) , the gene transcript (e.g. mRNA) , or the protein product (i.e., the amino acid sequence) , and people skilled in the art can understand the meaning from the context. “Encoding sequence” as used herein refers to the polynucleotide sequence which encodes at least a fragment of a protein product. The encoding sequence can be a DNA sequence such as genomic DNA or cDNA, and can also be a RNA sequence such as mRNA. The fusion of the two encoding sequences can be in frame, such that after being translated into its protein product, a protein fragment of the upstream gene is fused to a protein fragment of the downstream gene.

In some embodiments, the upstream gene and the downstream gene can be found in Table 1. In some embodiments, the first encoding sequence for the upstream gene and the second encoding sequence for the downstream gene can be found in Table 1.

The site where the upstream gene sequence fuses to the downstream gene sequence is referred to as fusion junction. In some embodiments, the fusion genes provided herein comprise a fusion junction as disclosed in Table 1.

In some embodiments, the fusion gene detected in a murine tumor model is unique to the murine tumor model when the fusion gene is not detected in other murine tumor models.

Methods of detecting the fusion gene and/or fusion protein encoded by the fusion gene

Provided herein are also methods of detecting a novel fusion gene in murine tumor models provided herein. In some embodiments, the method comprises: obtaining a nucleic acid containing sample from the murine tumor model, contacting the sample with a detecting agent which specifically detects a target polynucleotide comprising a fusion of a first encoding sequence for a upstream gene and a second encoding sequence for a downstream gene, and detecting the presence of the target polynucleotide.

The nucleic acid-containing sample can be derived from a cell or a tissue of the murine tumor model. “Nucleic acid” as used herein can be a polymer of RNA or a polymer of DNA. The sample may contain isolated nucleic acid such as isolated RNA or cDNA. Alternatively, the sample may contain nucleic acid in its natural or unpurified or unamplified state, for example, the sample may be an isolated cell or tissue, optionally pretreated to release the nucleic acid contained therein. In a further embodiment, the nucleic acid in the sample may be amplified, e.g. by PCR reaction or PCR following reverse transcription.

In certain embodiments, the sample is derived from a non-human animal transplanted with the murine tumor model. The sample can be any suitable biological material collected from the non-human animal, such as body fluid (e.g. blood) and a biopsy sample (e.g. cells or tissues from a disease affected area) . The sample can be treated to extract the nucleic acid.

In the detecting method, the sample is contacted with an oligonucleotide which specifically detects a target polynucleotide comprising the fusion gene. The target polynucleotide can be cDNA, DNA, or mRNA, depending on the type of nucleic acid contained in the sample. The target polynucleotide can comprise any of the fusion genes as provided herein.

The target polynucleotide can be detected based on any suitable methods known in the art, for example but not limited to, hybridization-based methods and amplification-based methods. Hybridization-based methods usually involve using a probe to hybridize and detect the target sequence. Examples of hybridization-based methods include, Northern blot, DNA microarray, whole genome sequencing, RNA sequencing (RNA-seq) , quantitative real time PCR (qRT-PCR) , digital multiplexed gene expression analysis method (see, e.g., Kulkarni MM, Curr Protoc Mol Biol. 2011 Apr, Chapter 25: Unit25B. 10. ) , FISH method (Fluorescence In Situ Hybridization) , CISH (Chromogenic In Situ Hybridization) method, SISH (silver in situ hybridization) methods, and the like. Amplification-based methods usually involve using primers, polymerase and mixture of nucleotide monomers to synthesize nascent polynucleotide chain based on the base sequence of the target template polynucleotide. Examples of amplification-based methods include, PCR (polymerase chain reaction) , LCR (Ligase chain reaction) , SDA (Strand displacement amplification) , isothermal and chimeric primer-initiated amplification of nucleic acids) , loop-mediated isothermal amplification, transcription-mediated amplification and the like.

In certain embodiments, the detecting step involves an amplification step. In such case, the detecting agent comprises at least a pair of primers which can hybridize to the target polynucleotide and amplify a target region encompassing the fusion junction in the presence of a polymerase. In one embodiment, the detecting agent comprises a first primer directed to the first encoding sequence for the upstream gene, and a second primer directed to the second encoding sequence for the downstream gene. As used herein, a primer or a probe “directed to” a sequence, means that the primer or the probe has sufficient identity with or complementarity to at least a portion of the sequence such that the primer or the probe can specifically hybridize to the sequence or to its complementary strand. “Specifically hybridize” as used herein means the primer or probe can hybridize to the intended sequence under stringent conditions. “Stringent condition” as used herein refers to hybridizing at 42 ℃ in a solution consisting of 5× SSPE, 5× Denhardt’s solution, 0.5%SDS, and 100 ug/mL denatured salmon sperm DNA, and then washing at 42 ℃ with a solution comprising 0.5×SSC and 0.1%SDS.

In another embodiment, the detecting agent comprises a junction primer directed to a fragment containing the fusion junction, and a non-junction primer directed to the first or the second encoding sequence. The junction primer would specifically hybridize to the fusion junction, thereby specifically enabling the amplification when the target polynucleotide is present. Otherwise, if the nucleic acid in the sample does not contain the target polynucleotide, the junction primer would not specifically hybridize to its target sequence, and cannot effectuate a meaningful amplification.

After amplification by a suitable nucleic acid amplification method such as PCR, the amplification product is detected. In certain embodiments, the amplification product has a length of 100bp-1500bp (e.g. 100bp-1000bp, 100bp-900bp, 100bp-800bp, 100bp-700bp, 100bp-600bp, 100bp-500bp, 100bp-400bp, 100bp-350bp, 100bp-300bp, 200bp-1000bp, 200bp-900bp, 200bp-800bp, 200bp-700bp, 200bp-600bp, 200bp-500bp, 200bp-400bp, 200bp-350bp, 200bp-300bp, etc. ) . In certain embodiments, the presence of the amplification product would be indicative of the presence of the target polynucleotide. In certain embodiments, the molecular weight or size or sequence of the amplification product is further detected, and a desired size or sequence of the amplification product indicates presence of the target polynucleotide.

When the target polynucleotide is RNA, the amplification step may optionally further comprises a reverse transcription step to produce cDNA of the RNA in the sample. The cDNA is then amplified using the primers to allow detection of presence of the fusion junction.

In certain embodiments, the first primer or the second primer is directed to a region at least 80bp upstream or downstream of the fusion junction of the fusion gene. In certain embodiments, the fusion gene comprises a fusion junction as disclosed in Table 1. In certain embodiments, the first primer and the second primer are useful of amplifying an amplicon having a length of about 200bp to 400bp.

The non-junction primer can be designed based on the desired length of the amplification product, once the junction primer is determined. For example, when it is desired to have a 300bp amplification product, then the non-junction primer can be designed to be complementary to the target polynucleotide about 300bp 5’ upstream the fusion junction or 3’ downstream of the fusion junction.

In certain embodiments, the detecting step involves a hybridization step. Probes can be designed to specifically hybridize to the target polynucleotide, thereby allowing its detection. Probes provided herein can have a suitable length, for example, about 20-200bp (e.g. 20-190bp, 20-150bp, 20-120bp, 20-100bp, 20-90bp, 20-80bp, 20-70bp, 20-60bp, 20-50bp, 20-40bp, and etc. ) .

In certain embodiments, the detecting agent comprises a first probe directed to the first encoding sequence for the upstream gene, and a second probe directed to the second encoding sequence for the downstream gene. In an illustrative example, one of the first and the second probes can be a capture probe which further comprises an immobilizing moiety capable of associating with a substrate through a covalent or a non-covalent bond, and the other probe can be a detecting probe which further comprises a detectable label. The capture probe can be first contacted with the sample to allow hybridization with the nucleic acid, then the complex is immobilized on a substrate via the immobilizing moiety on the capture probe, and the unbound molecules are removed. The detecting probe is then added to the immobilized complexes to allow hybridization to occur. After washing away the excess probe, the detectable label immobilized on the substrate is detected. Illustrative examples of the immobilizing moiety include, but are not limited to, biotin, streptavidin, antigen, antibody, protein A, protein G, oligonucleotide, etc. The detectable label on the detecting probe can be, for example, fluorescent dye, radioisotope, antibody, enzyme, and oligonucleotide (e.g. an oligonucleotide barcode) . In another illustrative example, one of the first and the second probes further comprises a fluorescent dye and the other probe comprises a quencher. After both probes are bound to the target polynucleotide, the two probes are in proximity to each other such that the quencher on one probe quenches the fluorescent signal of the dye on the other probe. Illustrative examples of fluorescent dye include, but are not limited to fluorescein isothiocyanate (FITC) , Alexa 488, Alexa 532, cy3, cy5, 6-joe, EDANS; rhodamine 6G (P6G) and its derivatives (tetramethyirhodamine (TMR) , tetramethylrhodamine isothiocyanate (TMRITC) , x-rhodamine, Texas red, "BODJPY FL" (trade name, product of Molecular Probes, Inc. (Eugene, Oregon, U. S. A. ) , "BODIPY FL/C3" (trade name, product of Molecular Probes, Inc. ) , "BODIPY EL/C6" (trade name, product of Molecular Probes, Inc. ) , "BODIPY 5-FAM" (trade name, product of Molecular Probes, Inc. ) , "BODIPY TMR" (trade name, product of Molecular Probes, Inc. ) , and derivatives thereof (for example, "BODIPY TR" (trade name, product of Molecular Probes, Inc. ) , "BODIPY R6G" (trade name, product of Molecular Probes, Inc. ) , "BODIPY 564" (trade name, product of Molecular Probes, Inc. ) , and "BODIPY 581" (trade name, product of MolecularProbes, Inc. ) ) . Illustrative examples of the quencher include, but are not limited to, Dabcyl, "QSY7" (Molecular Probes) , "QSY33" (Molecular Probes) , Ferrocene and its derivatives, methyl viologen, and N, N'-dimethyl-2, 9-diazopyrenium and the like.

In certain embodiments, the detecting agent comprises a junction probe directed to a fragment containing the fusion junction. The junction probe may further comprise a detectable label. In an illustrative example, the nucleic acid in the sample may be immobilized on a substrate, and then contacted with the probe which recognizes the fusion junction. After washing away the unreacted probes, the substrate can be detected for presence of the probe, which can indicate the presence of the fusion junction of the fusion gene. In another illustrative example, the junction probe can comprise both a fluorescent dye and a quencher, such that the quencher quenches the fluorescence of the dye when the probe is intact. The probe can be used in an amplification method in which a target region encompassing the fusion junction is to be amplified using a polymerase having 5’ -3’ exonuclease activity (such as Taq polymerase) . During the amplification, the probe which hybridizes to the fusion junction can be degraded by the polymerase as it proceeds along the target polynucleotide, thereby separating the fluorescent dye and the quencher on the probe, and allow the fluorescent dye to emit its signal to be detected.

In another aspect, the present disclosure further provides methods of detecting the fusion gene provided herein in a protein-containing sample, comprising contacting the sample with a detecting agent which specifically detects a fusion protein encoded by the fusion gene, and detecting the presence of the fusion protein.

The presence and level of the fusion protein encoded by the fusion gene can be detected. For example, the sample may be contacted with an antibody specific for the fusion protein, and formation of a complex between the antibody and the fusion protein can be detected using methods known in the art, such as, for example, an immunoassay, Western blot method, ELISA, ELIFA, fluorescence immunoassay method, radioimmunoassay method, enzymatic immunoassay method, double antibodies sandwich method, and etc.

Kits

The primer set or probe sets or junction probe as provided herein are useful in detecting the novel fusion genes in the murine tumor models disclosed herein. Therefore, another aspect of the present disclosure relates to kits comprising the primer sets, or the probe sets, or the junction probe described herein.

In certain embodiments, the kits comprise a first primer directed to a first encoding sequence for a first gene, and a second primer directed to a second encoding sequence for a second gene. In certain embodiments, the kits comprise a junction primer directed to a fragment containing the fusion junction of the first gene and the second gene, and a non-junction primer directed to the first encoding sequence for the first gene or the second encoding sequence for the second gene.

In certain embodiments, the kits comprise a first probe directed to a first encoding sequence for the first gene, and a second probe directed to a second encoding sequence for the second gene. In certain embodiments, the kits comprise a junction probe directed to a fragment containing the fusion junction.

The kits provided herein may further comprise one or more components useful for the detection, for example, polymerase, a buffer useful for amplification, and/or a buffer useful for probe hybridization.

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.

EXAMPLE 1

This example shows the identification of unique gene signature (genetic fingerprints) of individual murine tumor homografts derived from murine cell lines or murine primary tumors.

Whole transcriptome sequencing (RNAseq) or other forms of NGS, e.g. WGS or WES, was performed on syngeneic cell lines or tumors derived from the syngeneic cell lines, or on murine primary tumors. The unique gene features with respect to a given syngeneic cell line or tumor sample were obtained through bioinformatics analysis of the NGS data sets. Once identified by bioinformatics analysis, the unique gene features (e.g. gene fusions or mutations) were validated by RT-PCR or PCR followed by Sanger sequencing. Once validated, the unique gene features of a given cell line or murine tumor can be used as a genetic fingerprint to track the passage of murine cell lines or murine tumors of the same lineage.

Syngeneic and murine primary cancer model establishment

For syngeneic tumor model establishment, a predetermined number of cells suspended in 0.1ml PBS were inoculated within the right flank of immunocompetent mice (C57BL/6, BALB/c, or C3H) . For murine primary tumor model establishment, spontaneous tumors from GEMM models were surgically removed from GEMM model and subsequently implanted within the right flank of immunocompetent mice of the same genetic background. Tumor bearing mice were monitored twice a week and tumors were collected when the mean tumor size reached 250～350mm ³.

RNA extraction and transcriptome sequencing

Untreated tumors at 250-350mm ³ were collected for RNAseq. Mutational and genetic analysis was carried out on basal tumor samples. RNA was extracted with RNeasy Mini Kit (Qiagen) , and the library for RNAseq was prepared using

TruSeq ^TM RNA Sample Preparation Kit. The transcriptome sequencing was performed using Illumina

system.

Data analysis and results description

By comparing the RNAseq data of all available syngeneic cell lines and murine primary tumor samples, gene fusions or mutations that are unique to each sample (i. e. these features are not present in other sample data) was extracted. Table 1 below displayed unique gene fusion in selected murine syngeneic cell lines and tumors.

Table 1. Fusion genes in selected murine syngeneic cell lines

Cell Line	Fused gene	Up Gene	Dw Gene	SEQ ID NO
4T1	Kntc1-Snrnp35	Kntc1	Snrnp35	1
A20	Cyfip1-A230056P14Rik	Cyfip1	A230056P14Rik	2
A20	Prcp-Rab30	Prcp	Rab30	3
B16BL6	Atp1b3-Rnf7	Atp1b3	Rnf7	4
B16BL6	Prkaca-Sgip1	Prkaca	Sgip1	5
B16F0	Atp7a-Cox7b	Atp7a	Cox7b	6
B16F0	Rpl17-BC031181	Rpl17	BC031181	7
B16F1	Piezo1-Ankrd11	Piezo1	Ankrd11	8
B16F10	Nadk-Cfap74	Nadk	Cfap74	9
C1498	Lpin2-Nr3c1	Lpin2	Nr3c1	10
C1498	Noa1-Spink2	Noa1	Spink2	11
C1498	Nup205-Cntnap2	Nup205	Cntnap2	12
Colon26	Kdm4c-Slc24a2	Kdm4c	Slc24a2	13
Colon26	Ncor2-1700013M08Rik	Ncor2	1700013M08Rik	14
CT26	Cdh13-Hsbp1	Cdh13	Hsbp1	15
CT26	Synpo-Smim3	Synpo	Smim3	16
EG7_Ova	Pds5a-Cd82	Pds5a	Cd82	17
EL4	Alkbh1-Ism2	Alkbh1	Ism2	18
EL4	Ccnt1-Rpap3	Ccnt1	Rpap3	19
EL4	Ikzf2-Erbb4	Ikzf2	Erbb4	20
EMT6	Pls3-Gm14715	Pls3	Gm14715	21
EMT6	Med13-Ints2	Med13	Ints2	22
EMT6	Susd6-Exd2	Susd6	Exd2	23
H22	Arhgap21-Gm13376	Arhgap21	Gm13376	24
H22	Edem1-Arl8b	Edem1	Arl8b	25
H22	Kifap3-Cpne7	Kifap3	Cpne7	26
H22	Rps6kb1-Nkain2	Rps6kb1	Nkain2	27
Hepa 1-6	Csnk1e-Tmem184b	Csnk1e	Tmem184b	28
Hepa 1-6	Hnrnph1-Cby3	Hnrnph1	Cby3	29
Hepa 1-6	Smg1-Pdilt	Smg1	Pdilt	30
J558	Gm12865-Gm12863	Gm12865	Gm12863	31
J558	Tnfrsf17-Snx29	Tnfrsf17	Snx29	32
JC	Bdp1-Plpp1	Bdp1	Plpp1	33
JC	St7-Copg2	St7	Copg2	34
KLN205	AI846148-Rtn3	AI846148	Rtn3	35
KLN205	Caap1-Gm12655	Caap1	Gm12655	36
L1210	Git1-Ssh2	Git1	Ssh2	37
L1210	Gtf2i-Wbscr16	Gtf2i	Wbscr16	38
L1210	Lnx2-Tyw1	Lnx2	Tyw1	39

L1210	Plcg2-mip	Plcg2	Cmip	40
L5178-R	Acly-Alg1	Acly	Alg1	41
L5178-R	Dennd4a-Megf11	Dennd4a	Megf11	42
L5178-R	Epb41-Fgr	Epb41	Fgr	43
L5178-R	Fbxw7-D930015E06Rik	Fbxw7	D930015E06Rik	44
L5178-R	Gba-Thbs3	Gba	Thbs3	45
L5178-R	Jmy-Fam172a	Jmy	Fam172a	46
L5178-R	Ly6k-D730001G18Rik	Ly6k	D730001G18Rik	47
L5178-R	Papd7-Jmy	Papd7	Jmy	48
L5178-R	Rnf2-Swt1	Rnf2	Swt1	49
L5178-R	Tmem208-Plekhg4	Tmem208	Plekhg4	50
LL/2	Cby1-Sh3bp1	Cby1	Sh3bp1	51
LL/2	Cfdp1-Chst5	Cfdp1	Chst5	52
LL/2	Ppat-Igfbp7	Ppat	Igfbp7	53
MBT-2	Dpep1-Spata33	Dpep1	Spata33	54
MBT-2	Lmo1-Ric3	Lmo1	Ric3	55
MPC-11	Cnot2 (116549033) -Kcnmb4	Cnot2 (116549033)	Kcnmb4	56
MPC-11	Gm43041-Tmem106b	Gm43041	Tmem106b	57
Neuro-2a	Arid1a-Rps6ka1	Arid1a	Rps6ka1	58
Neuro-2a	C920009B18Rik-H60b	C920009B18Rik	H60b	59
Neuro-2a	Poldip3-Pacsin2	Poldip3	Pacsin2	60
Neuro-2a	Ppp2ca-Cdkl3	Ppp2ca	Cdkl3	61
P388D1	2810402E24Rik-Adamts13	2810402E24Rik	Adamts13	62
P388D1	Gm42566-Chd2	Gm42566	Chd2	63
P388D1	Tial1-Fmo1	Tial1	Fmo1	64
P388D1	Tial1-Sgip1	Tial1	Sgip1	65
P815	Capzb-Emc1	Capzb	Emc1	66
P815	Gramd4-Tbc1d22a	Gramd4	Tbc1d22a	67
P815	Runx1-Erg	Runx1	Erg	68
Renca	Fbxo31-1700018B08Rik	Fbxo31	1700018B08Rik	69
Renca	Neat1-Scyl1	Neat1	Scyl1	70
S91	Atad1-Inadl	Atad1	Inadl	71
S91	Naca-Prim1	Naca	Prim1	72
WEHI164	Ago2-Slc45a4	Ago2	Slc45a4	73
WEHI164	Rps6kb1-Vmp1	Rps6kb1	Vmp1	74
WEHI164	Tulp4-Synj2	Tulp4	Synj2	75

While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments) , it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

Claims

A method for producing a non-human tumor model, the method comprising:

transplanting a non-human tumor cell line or a primary tumor tissue to a non-human animal;

raising the non-human animal in a condition that allows the non-human tumor cell line or the primary tumor tissue to develop into a tumor;

detecting in the tumor the presence of a biomarker that is unique to the non-human tumor cell line or the primary tumor tissue; and

determining the identification of the non-human tumor cell line or the primary tumor tissue.
A method of producing a non-human tumor model, the method comprising:

culturing a non-human tumor cell line in vitro;

detecting in the non-human tumor cell line the presence of a biomarker that is unique to the non-human tumor cell line;

determining the identification of the non-human tumor cell line;

transplanting the non-human tumor cell line to a non-human animal; and

raising the non-human animal in a condition that allows the non-human tumor cell line to develop into a tumor.
The method of claim 1 or 2, wherein the non-human tumor model is a murine tumor model.
The method of claim 3, wherein the non-human tumor cell line is selected from the group consisting of 4T1, A20, B16BL6, B16F0, B16F1, B16F10, C1498, Colon26, CT26, EG7_Ova, EL4, EMT6, H22, Hepa 1-6, J558, JC, KLN205, L1210, L5178-R, LL/2, MBT2, MPC-11, Neuro-2a, P388D1, P815, Renca, S91 and WEHI164.
The method of claim 1 or 2, wherein the biomarker is a fusion gene.
The method of claim 5, wherein the fusion gene comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-75.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line 4T1 when the biomarker comprises SEQ ID NO: 1.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line A20 when the biomarker comprises SEQ ID NO: 2 or 3.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line B16BL6 when the biomarker comprises SEQ ID NO: 4 or 5.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line B16F0 when the biomarker comprises SEQ ID NO: 6 or 7.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line B16F1 when the biomarker comprises SEQ ID NO: 8.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line B16F10 when the biomarker comprises SEQ ID NO: 9.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line C1498 when the biomarker comprises SEQ ID NO: 10, 11 or 12.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line Colon26 when the biomarker comprises SEQ ID NO: 13 or 14.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line CT26 when the biomarker comprises SEQ ID NO: 15 or 16.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line EG7_Ova when the biomarker comprises SEQ ID NO: 17.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line EL4 when the biomarker comprises SEQ ID NO: 18, 19 or 20.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line EMT6 when the biomarker comprises SEQ ID NO: 21, 22 or 23.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line H22 when the biomarker comprises SEQ ID NO: 24, 25, 26, or 27.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line Hepa 1-6 when the biomarker comprises SEQ ID NO: 28, 29 or 30.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line J558 when the biomarker comprises SEQ ID NO: 31 or 32.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line JC when the biomarker comprises SEQ ID NO: 33 or 34.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line KLN205 when the biomarker comprises SEQ ID NO: 35 or 36.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line L1210 when the biomarker comprises SEQ ID NO: 37, 28, 39 or 40.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line L5178-R when the biomarker comprises SEQ ID NO: 41, 42, 43, 44, 45, 46, 48, 49 or 50.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line LL/2 when the biomarker comprises SEQ ID NO: 51, 52 or 53.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line MBT-2 when the biomarker comprises SEQ ID NO: 54 or 55.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line MPC-11 when the biomarker comprises SEQ ID NO: 56 or 57.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line Neuro-2a when the biomarker comprises SEQ ID NO: 58, 59, 60 or 61.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line P388D1 when the biomarker comprises SEQ ID NO: 62, 63, 64 or 65.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line P815 when the biomarker comprises SEQ ID NO: 66, 67 or 68.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line Renca when the biomarker comprises SEQ ID NO: 69 or 70.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line S91 when the biomarker comprises SEQ ID NO: 71 or 72.
The method of claim 6, wherein the non-human tumor cell line is determined as cell line WEHI164 when the biomarker comprises SEQ ID NO: 73, 74 or 75.
The method of claim 1 or 2, wherein the biomarker is detected by an amplification assay, a hybridization assay, a sequencing assay or an array.
The method of claim 1 or 2, wherein the biomarker is detected using a primer set capable of detecting a biomarker panel comprising SEQ ID NOs: 1-75.
The method of claim 1 or 2, wherein the biomarker is detected using a microarray comprising probes capable of detecting a biomarker panel comprising SEQ ID NOs: 1-75.
The method of claim 1 or 2, wherein the non-human animal is a rodent.
The method of claim 38, wherein the non-human animal is a mouse.
A kit comprising primers for detecting a fusion gene comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-75.
A hybridization probe for detecting a fusion gene comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-75.
A kit comprising primers for detecting a biomarker panel, wherein the biomarker comprises SEQ ID NOs: 1-75.
A microarray comprising probes for detecting a biomarker panel, wherein the biomarker panel comprises SEQ ID NOs: 1-75.