USE OF FGFR MUTANT GENE PANELS IN IDENTIFYING CANCER PATIENTS THAT WILL BE RESPONSIVE TO TREATMENT WITH AN FGFR INHIBITOR

Abstract

Disclosed herein are methods of identifying a cancer patient that will be responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor and methods of treating cancer patients. The methods involve evaluating a biological sample from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel. Kits and primers for identifying the presence of one or more FGFR mutant genes in a biological sample are also disclosed herein.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/056,159, filed Sep. 26, 2014, the disclosure of which hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 6, 2015, is named 103693.000781_SL.txt and is 64,689 bytes in size.

TECHNICAL FIELD

[0003] Provided herein are methods of identifying a cancer patient that will be responsive to treatment with a fibroblast growth factor receptor inhibitor and methods of treating the same.

BACKGROUND

[0004] The identification of genetic abnormalities can be useful in selecting the appropriate therapeutic(s) for cancer patients. This is also useful for cancer patients failing the main therapeutic option (front-line therapy) for that cancer type, particularly if there is no accepted standard of care for second and subsequent-line therapy. Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases involved in regulating cell survival, proliferation, migration and differentiation. FGFR alterations have been observed in some cancers. To date, there are no approved therapies that are efficacious in patients with FGFR alterations.

SUMMARY

[0005] Disclosed herein are methods of identifying a cancer patient that will be responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor comprising: evaluating a biological sample from the patient for a FGFR mutant from a FGFR mutant gene panel, wherein the FGFR mutant is a FGFR fusion gene or a FGFR single nucleotide polymorphism, and wherein said evaluating comprises amplifying cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel; and determining whether the one or more FGFR mutants from the gene panel are present in the sample, wherein the presence of the one or more FGFR mutants indicates that the patient will be responsive to treatment with the FGFR inhibitor.

[0006] Also disclosed are methods of treating cancer in a patient comprising: evaluating a biological sample from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel; and treating the patient with an FGFR inhibitor if one or more FGFR mutants are present in the sample.

[0007] Kits and primers for identifying the presence of one or more FGFR mutant genes in a biological sample are further provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed methods, kits, and primers, there are shown in the drawings exemplary embodiments of the methods, kits, and primers; however, the methods, kits, and primers are not limited to the specific embodiments disclosed. In the drawings:

[0009] FIG. 1 is an illustration of exemplary FGFR fusion genes, the presence of at least one of which indicates that a patient will be responsive to treatment with an FGFR inhibitor. Also illustrated (small arrows) are exemplary primer locations for amplifying the fusion genes.

[0010] FIG. 2, comprising FIGS. 2A-2I, represents Sanger sequencing results from FFPET samples positive for: A) FGFR3:TACC3 v1; B) FGFR3:TACC3 v3; C) FGFR3:TACC3 Intron; D) FGFR3:BAIAP2L1; E) FGFR2:AFF3; F) FGFR2:BICC1; G) FGFR2:CASP7; H) FGFR2:CCDC6; and I) FGFR2:OFD1.

[0011] FIG. 3 illustrates an exemplary strategy for SNP-specific qRT-PCR using a 3' dideoxy wild type (WT) blocker oligonucleotide.

[0012] FIG. 4 illustrates an exemplary analytical validation strategy for detecting FGFR SNPs. Experiments were performed on engineered RK3E cell lines expressing the FGFR fusions and diluted into a wild type cell line harboring no FGFR3/FGFR2 fusions.

[0013] FIG. 5, comprising FIGS. 5A-5D, illustrates SNP-specific PCR with dideoxy WT blocker for (a) G370C, (B) Y373C, (C) S249C, and (D) R248C.

[0014] FIG. 6, comprising FIGS. 6A-6I, represents efficiency standard curves for the FGFR fusion gene assays: A) FGFR3:TACC3 v1; B) FGFR3:TACC3 v3; C) FGFR3:TACC3 Intron; D) FGFR3:BAIAP2L1; E) FGFR2:AFF3; F) FGFR2:BICC1; G) FGFR2:CASP7; H) FGFR2:CCDC6; and I) FGFR2:OFD1.

[0015] FIG. 7 is an exemplary representation of FGFR fusion gene status in bladder (primary and metastatic), NSCLC (adenocarcinoma and squamous), ovarian, esophageal (primary and metastatic), head and neck (H&N; primary and metastatic), endometrial (metastatic), breast, and prostate cancer.

[0016] FIG. 8 is an exemplary representation of FGFR fusion gene and mutation status in NSCLC adenocarcinoma and squamous cell carcinoma.

[0017] FIG. 9, comprising FIGS. 9A-9D, represents exemplary results from phase I patient samples. Assays were performed using synthetic template assay control (ST), primers for GAPDH (quality control sample), or primers specific for: A) FGFR2:BICC1 fusions; B) FGFR3:TACC3 (exon 18:exon 1) fusions; C) FGFR2:CCDC6 fusions; or D) FGFR3:TACC3 v1, FGFR3:TACC3 v3, or FGFR2:CCDC6 fusions. Patient samples are as follows: A--urothelial carcinoma; B--bladder cancer; C--cholangiocarcinoma; and D--adrenal carcinoma.

[0018] FIG. 10 represents an exemplary Phase I Study design for a First-In-Human Study of JNJ-42756493 in patients with advanced solid tumor.

[0019] FIG. 11 represents the maximal inhibitory percentage reduction of sum of the diameters of targeted lesions from baseline with dose level greater than or equal to 6 mg. Solid tumor patients were treated with the FGFR inhibitor JNJ-42756493 at different doses administered either as a daily regimen or as an intermittent dosing regimen (7 days on-7 days off). Doses and tumor types are indicated. Reduction in tumor was measured as per the RECIST criteria. Patients whose tumors harbor FGFR gene translocations and mutations appear to be more sensitive to the FGFR inhibitor JNJ-42756493.

[0020] FIG. 12 illustrates the expression of various FGFR fusions in RK3E cells stably transfected with the indicated FGFR fusion.

[0021] FIG. 13, comprising FIGS. 13A-13B, illustrates colony formation assays in RK3E cells stably transfected with the indicated FGFR fusion. (A) 0.1% cresyl crystal violet stained 6-well chambers and (B) bar graph illustrating the number of colonies/100 cells plated. Results are representative of two independent experiments.

[0022] FIG. 14, comprising FIGS. 14A-14H, illustrates the expression of exemplary downstream targets in RK3E cells stably transfected with the indicated FGFR fusion.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0023] The disclosed methods, kits, and primers may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods, kits, and primers are not limited to the specific methods, kits, and primers described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods, kits, and primers.

[0024] Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Further, reference to values stated in ranges include each and every value within that range. All ranges are inclusive and combinable.

[0025] It is to be appreciated that certain features of the disclosed methods, kits, and primers which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods, kits, and primers that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0026] As used herein, the singular forms "a," "an," and "the" include the plural.

[0027] The following abbreviations are used throughout the specification: FGFR (fibroblast growth factor receptor); LLOQ (lower limit of quantitation); FGFR3:TACC3 (fusion between genes encoding FGFR3 and transforming acidic coiled-coil containing protein 3); FGFR3:BAIAP2L1 (fusion between genes encoding FGFR3 and brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 1); FGFR2:AFF3 (fusion between genes encoding FGFR2 and AF4/FMR2 family, member 3); FGFR2:BICC1 (fusion between genes encoding FGFR2 and bicaudal C homolog 1); FGFR2: CASP7 (fusion between genes encoding FGFR2 and caspase 7); FGFR2:CCDC6 (fusion between genes encoding FGFR2 and coiled-coil domain containing 6); FGFR2:OFD1 (fusion between genes encoding FGFR2 and oral-facial-digital syndrome 1); FFPET (Formalin-Fixed Paraffin-Embedded Tissue); SNP (single nucleotide polymorphism); NSCLC (Non-small-cell lung cancer), ct (cycle threshold).

[0028] As used herein, "treating" and like terms refer to reducing the severity and/or frequency of cancer symptoms, eliminating cancer symptoms and/or the underlying cause of said symptoms, reducing the frequency or likelihood of cancer symptoms and/or their underlying cause, and improving or remediating damage caused, directly or indirectly, by cancer.

[0029] "Biological samples" refers to any sample from a patient in which cancerous cells can be obtained and RNA can be isolated. Suitable biological samples include, but are not limited to, blood, lymph fluid, bone marrow, a solid tumor sample, or any combination thereof. In some embodiments, the biological sample can be FFPET.

[0030] As used herein, "pre-amplification" refers to a PCR procedure that is performed prior to the amplifying step in order to increase the quantity of template cDNA for the amplification step. A pre-amplification step can be performed, for example, using the TaqMan® PreAmp Master Mix (Life Technologies/Applied Biosystems® product #4391128).

[0031] As used herein, "amplifying," "amplify," and like terms refer to the generation of numerous identical copies of a nucleic acid sample. Suitable techniques for amplifying a nucleic acid sample include, but are not limited to, polymerase chain reaction (PCR) and real-time polymerase chain reaction (RT-PCR). In some embodiments, the amplifying step comprises RT-PCR.

FGFR Mutants

[0032] As used herein, the phrase "FGFR mutant" refers to a FGFR fusion gene, a FGFR single nucleotide polymorphism, or both.

[0033] "FGFR fusion" or "FGFR fusion gene" refers to a gene encoding FGFR (e.g., FGRF2 or FGFR3), or a portion thereof, and one of the herein disclosed fusion partners, or portion thereof, created by a translocation between the two genes. The presence of one or more of the following FGFR fusion genes in a biological sample from a patient can be determined using the disclosed methods: FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, or any combination thereof. Table 1 provides the FGFR fusion genes and the FGFR and fusion partner exons that are fused. FIG. 1 provides an illustration of the various FGFR fusion genes. The sequences of the individual FGFR fusion genes are disclosed in Table 16.

TABLE-US-00001 TABLE 1 Fusion Gene FGFR Exon Partner Exon FGFR3:TACC3 v1 18 11 FCFR3:TACC3 v3 18 10 FGFR3:TACC3 Intron 18 4 FGFR3:BAIAP2L1 18 2 FGFR2:AFF3 19 8 FGFR2:BICC1 19 3 FGFR2:CASP7 19 4 FGFR2:CCDC6 19 2 FGFR2:OFD1 19 3

[0034] "FGFR single nucleotide polymorphism" (SNP) refers to a FGFR2 or FGFR3 gene in which a single nucleotide differs among individuals. In particular, FGFR single nucleotide polymorphism" (SNP) refers to a FGFR3 gene in which a single nucleotide differs among individuals. The presence of one or more of the following FGFR SNPs in a biological sample from a patient can be determined using the disclosed methods: FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, FGFR3 Y373C, or any combination thereof. The sequences of the FGFR SNPs are provided in Table 2.

TABLE-US-00002 TABLE 2 FGFR3 mutant Sequence FGFR3 TCGGACCGCGGCAACTACACCTGCGTCGTGGAGAAC R248C AAGTTTGGCAGCATCCGGCAGACGTACACGCTGGAC GTGCTGGAG(T)GCTCCCCGCACCGGCCCATCCTGC AGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGG GCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTG ACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGG AGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC CCTACGTTACCGTGCTCA (SEQ ID NO: 1) FGFR3 GACCGCGGCAACTACACCTGCGTCGTGGAGAACAAG S249C TTTGGCAGCATCCGGCAGACGTACACGCTGGACGTG CTGGGTGAGGGCCCTGGGGCGGCGCGGGGGTGGGGG CGGCAGTGGCGGTGGTGGTGAGGGAGGGGGTGGCCC CTGAGCGTCATCTGCCCCCACAGAGCGCT(G)CCCG CACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAAC CAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCAC TGCAAGGTGTACAGTGACGCACAGCCCCACATCCAG TGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTG GGCCCGGACGGCACACCCTACGTTACCGTGCTCAAG GTGGGCCACCGTGTGCACGT (SEQ ID NO: 2) FGFR3 GCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCG G370C TGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTG GAGGCTGACGAGGCG(T)GCAGTGTGTATGCAGGCA TCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCC TGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCA GCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGC ACAAGATCTCCCGCTTCCCG (SEQ ID NO: 3) FGFR3 CTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAG Y373C* GACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCT ATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTG CTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAG GCGGGCAGTGTGT(G)TGCAGGCATCCTCAGCTACG GGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGG CTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGA AAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCC GCTTCCCGCTCAAGC (SEQ ID NO: 4) Sequences correspond to nucleotides 920-1510 of FGFR3 (Genebank ID # NM_000142.4). Nucleotides in bold underline represent the SNP. *Sometimes mistakenly referred to as Y375C in the literature.

[0035] As used herein, "FGFR mutant gene panel" includes one or more of the above listed FGFR mutants. In some embodiments, the FGFR mutant gene panel is dependent upon the patient's cancer type.

[0036] The FGFR mutant panel that is used in the evaluating step of the disclosed methods is based, in part, on the patient's cancer type. For patients with bladder cancer, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0037] For patients with metastatic bladder cancer, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0038] For patients with ovarian cancer, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGER3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0039] For patients with head and neck cancer, a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0040] For patients with metastatic head and neck cancer, a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof.

[0041] For patients with esophageal cancer, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0042] For patients with metastatic esophageal cancer, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.

[0043] For patients with non-small-cell lung adenocarcinoma, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0044] For patients with non-small cell lung squamous cell carcinoma, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0045] For patients with metastatic endometrial cancer, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof.

[0046] For patients with breast cancer, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.

Primers for Amplifying FGFR Mutants

[0047] One skilled in the art knows that amplification of nucleic acid requires primers that are complementary, and bind to, a 5' and 3' region of the nucleic acid strand that flanks the region sought to be amplified. As used herein, "pair of primers" refers to the forward and reverse primers used in an amplifying step. Pairs of primers suitable for performing the disclosed methods are listed in Table 3.

TABLE-US-00003 TABLE 3 Reverse Forward Primer Target Primer 5'-3' FGFR3:TACC3 V1 GACCTGGACCGTGTC CTTCCCCAGTTCCAG CTTACC GTTCTT (SEQ ID NO: 5) (SEQ ID NO: 6) FGFR3:TACC3 V3 AGGACCTGGACCGTG TATAGGTCCGGTGGA TCCTT CAGGG (SEQ ID NO: 7) (SEQ ID NO: 8) FGFR3:TACC3 GGCCATCCTGCCCCC GAGCAGTCCAGGTCA Intron (SEQ ID NO: 9) GCCAG (SEQ ID NO: 10) FGFR3:BAIAP2L1 CTGGACCGTGTCCTT GCAGCCCAGGATTGA ACCGT ACTGT (SEQ ID NO: 11) (SEQ ID NO: 12) FGFR2:BICC1 TGGATCGAATTCTCA GCCAAGCAATCTGCG CTCTCACA TATTTG (SEQ ID NO: 13) (SEQ ID NO: 14) FGFR2:AFF3 TGGTAGAAGACTTGG TCTCCCGGATTATTT ATCGAATTCT CTTCAACA (SEQ ID NO: 15) (SEQ ID NO: 16) FGFR2:CASP7 GCTCTTCAATACAGC ACTTGGATCGAATTC CCTGATCA TCACTCTCA (SEQ ID NO: 17) (SEQ ID NO: 18) FGFR2:CCDC6 TGGATCGAATTCTCA GCAAAGCCTGAATTT CTCTCACA TCTTGAATAA (SEQ ID NO: 19) (SEQ ID NO: 20) FGFR2:OFD1 AGGGTGCATCAACTC ACTTGGATCGAATTC ATGAATTAG TCACTCTCA (SEQ ID NO: 21) (SEQ ID NO: 22) FGFR3 R248C GCATCCGGCAGACGT CCCCGCCTGCAGGAT ACA (SEQ ID NO: 24) (SEQ ID NO: 23) FGFR3 S249C GCATCCGGCAGACGT CCCCGCCTGCAGGAT ACA (SEQ ID NO: 26) (SEQ ID NO: 25) FGFR3 G370C AGGAGCTGGTGGAGG CCGTAGCTGAGGATG CTGA CCTG (SEQ ID NO: 27) (SEQ ID NO: 28) FGFR3 Y373C CTGGTGGAGGCTGAC AGCCCACCCCGTAGC GAG T (SEQ ID NO: 29) (SEQ ID NO: 30) FGFR3 R248C GTCGTGGAGAACAAG GTCTGGTTGGCCGGC TTTGGC AG (SEQ ID NO: 31) (SEQ ID NO: 32) FGFR3 S249C GTCGTGGAGAACAAG GTCTGGTTGGCCGGC TTTGGC AG (SEQ ID NO: 33) (SEQ ID NO: 34) FGFR3 G370C AGGAGCTGGTGGAGG CCGTAGCTGAGGATG CTGA CCTG (SEQ ID NO: 35) (SEQ ID NO: 36) FGFR3 Y373C GACGAGGCGGGCAGT GAAGAAGCCCACCCC G GTAG (SEQ ID NO: 37) (SEQ ID NO: 38)

[0048] Disclosed herein are primers having the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or any combination thereof.

[0049] Also disclosed herein are sets of primers having the sequences of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, or any combination thereof.

[0050] In some embodiments, the set of primers can have the sequence of SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, the set of primers can have the sequence of SEQ ID NO:7 and SEQ ID NO:8. In some embodiments, the set of primers can have the sequence of SEQ ID NO:9 and SEQ ID NO:10. In some embodiments, the set of primers can have the sequence of SEQ ID NO:11 and SEQ ID NO:12. In some embodiments, the set of primers can have the sequence of SEQ ID NO:13 and SEQ ID NO:14. In some embodiments, the set of primers can have the sequence of SEQ ID NO:15 and SEQ ID NO:16. In some embodiments, the set of primers can have the sequence of SEQ ID NO:17 and SEQ ID NO:18. In some embodiments, the set of primers can have the sequence of SEQ ID NO:19 and SEQ ID NO:20. In some embodiments, the set of primers can have the sequence of SEQ ID NO:21 and SEQ ID NO:22. In some embodiments, the set of primers can have the sequence of SEQ ID NO:23 and SEQ ID NO:24. In some embodiments, the set of primers can have the sequence of SEQ ID NO:25 and SEQ ID NO:26. In some embodiments, the set of primers can have the sequence of SEQ ID NO:27 and SEQ ID NO:28. In some embodiments, the set of primers can have the sequence of SEQ ID NO:29 and SEQ ID NO:30. In some embodiments, the set of primers can have the sequence of SEQ ID NO:31 and SEQ ID NO:32. In some embodiments, the set of primers can have the sequence of SEQ ID NO:33 and SEQ ID NO:34. In some embodiments, the set of primers can have the sequence of SEQ ID NO:35 and SEQ ID NO:36. In some embodiments, the set of primers can have the sequence of SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, the set of primers can have the sequences of any combination of the above sets of primers.

FGFR Inhibitors for Use in the Disclosed Methods

[0051] Suitable FGFR inhibitors for use in the disclosed methods are provided herein.

[0052] In some embodiments, if one or more FGFR mutants are present in the sample, the patient can be treated with a FGFR inhibitor disclosed in U.S. Publ. No. 2013/0072457 A1 (incorporated herein by reference), including any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof (suitable R groups are also disclosed in U.S. Publ. No. 2013/0072457 A1). In some aspects, for example, the patient can be treated with N-(3,5-dimethoxyphenyl)-N'-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl- )quinoxalin-6-yl]ethane-1,2-diamine (referred to herein as "JNJ-42756493" or "JNJ493"):

##STR00001##

including a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof. In some aspects, the pharmaceutically acceptable salt is a HCl salt. In some aspects, the patient can be treated with JNJ493 base.

[0053] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is N-[5-[2-(3,5-Dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3,5-diemthylpiper- azin-1-yl)benzamide (AZD4547), as described in Gavine, P. R., et al., AZD4547: An Orally Bioavailable, Potent, and Selective Inhibitor of the Fibroblast Growth Factor Receptor Tyrosine Kinase Family, Cancer Res. Apr. 15, 2012 72; 2045:

##STR00002##

including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0054] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is 3-(2,6-Dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-ph- enylamino]-pyrimid-4-yl}-1-methyl-urea (NVP-BGJ398) as described in Int'l Publ. No. WO2006/000420:

##STR00003##

including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0055] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is 4-amino-5-fluoro-3-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-q- uinolin-2-one (dovitinib) as described in Int't Publ. No. WO2006/127926:

##STR00004##

including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0056] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is 6-(7-((1-Aminocyclopropyl)-methoxy)-6-methoxyquinolin-4-yloxy)-N-methyl-1- -naphthamide (AL3810) (lucitanib; E-3810), as described in Bello, E. et al., E-3810 Is a Potent Dual Inhibitor of VEGFR and FGFR that Exerts Antitumor Activity in Multiple Preclinical Models, Cancer Res Feb. 15, 2011 71(A)1396-1405 and Int'l Publ. No. WO2008/112408:

##STR00005##

including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0057] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is an anti-FGFR2 antibody such as that described in WO2013/076186.

[0058] Additional suitable FGFR inhibitors include BAY1163877 (Bayer), BAY1179470 (Bayer), TAS-120 (Taiho), ARQ087 (ArQule), ASP5878 (Astellas), FF284 (Chugai), FP-1039 (GSK/FivePrime), Blueprint, LY-2874455 (Lilly), RG-7444 (Roche), or any combination thereof, including, when chemically possible, any tautomeric or stereochemically isomeric forms thereof, N-oxides thereof, pharmaceutically acceptable salts thereof, or solvates thereof.

[0059] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is BAY1163877 (Bayer), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.

[0060] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is BAY1179470 (Bayer), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.

[0061] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is TAS-120 (Taiho), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.

[0062] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is ARQ087 (ArQule), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.

[0063] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is ASP5878 (Astellas), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.

[0064] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is FF284 (Chugai), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.

[0065] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is FP-1039 (GSK/FivePrime), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.

[0066] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is Blueprint, including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.

[0067] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is LY-2874455 (Lilly), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.

[0068] In some embodiments, the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is RG-7444 (Roche), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.

[0069] Salts can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002, which is incorporated herein by reference. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used. The FGFR inhibitors for use in the disclosed methods may exist as mono- or di-salts depending upon the pKa of the acid from which the salt is formed.

[0070] Acid addition salts may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include salts formed with an acid including, but not limited to, acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulphonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)-(1S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulphonic, naphthalenesulphonic (e.g. naphthalene-2-sulphonic), naphthalene-1,5-disulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, pyruvic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, (+)-L-tartaric, thiocyanic, toluenesulphonic (e.g. p-toluenesulphonic), undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

[0071] One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulphuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulphonic, toluenesulphonic, methanesulphonic (mesylate), ethanesulphonic, naphthalenesulphonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. Another group of acid addition salts includes salts formed from acetic, adipic, ascorbic, aspartic, citric, DL-Lactic, fumaric, gluconic, glucuronic, hippuric, hydrochloric, glutamic, DL-malic, methanesulphonic, sebacic, stearic, succinic and tartaric acids.

[0072] If the compound is anionic, or has a functional group which may be anionic (e.g., --COOH may be --COO.sup.-), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na.sup.+ and K.sup.+, alkaline earth metal cations such as Ca²+ and Mg²+, and other cations such as Al³+. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH₄.sup.+) and substituted ammonium ions (e.g., NH₃R.sup.+, NH₂R₂.sup.+, NHR₃.sup.+, NR₄.sup.+).

[0073] Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄.sup.+.

[0074] Where the compounds contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the disclosed compounds. Compounds containing an amine function may also form N-oxides. A reference herein to a compound that contains an amine function also includes the N-oxide. Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle. N-Oxides can be formed by treatment of the corresponding amine with an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry, by Jerry March, 4^th Edition, Wiley Interscience, pages. More particularly, N-oxides can be made by the procedure of L. W. Deady (Syn. Comm. (1977), 7, 509-514) in which the amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent such as dichloromethane.

[0075] As used herein, the term "solvate" means a physical association of the compound with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. The term "solvate" is intended to encompass both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include the disclosed compounds in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, ethanolamine and the like. The compound may exert its biological effects while in solution.

[0076] Solvates are well known in pharmaceutical chemistry. They can be important to the processes for the preparation of a substance (e.g. in relation to their purification), the storage of the substance (e.g. its stability) and the ease of handling of the substance, and are often formed as part of the isolation or purification stages of a chemical synthesis. A person skilled in the art can determine by means of standard and long used techniques whether a hydrate or other solvate has formed by the isolation conditions or purification conditions used to prepare a given compound. Examples of such techniques include thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray crystallography (e.g. single crystal X-ray crystallography or X-ray powder diffraction) and Solid State NMR (SS-NMR, also known as Magic Angle Spinning NMR or MAS-NMR). Such techniques are as much a part of the standard analytical toolkit of the skilled chemist as NMR, IR, HPLC and MS. Alternatively the skilled person can deliberately form a solvate using crystallisation conditions that include an amount of the solvent required for the particular solvate. Thereafter the standard methods described above, can be used to establish whether solvates had formed. Also encompassed are any complexes (e.g. inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals) of the FGFR inhibitor.

[0077] Furthermore, the compound may have one or more polymorph (crystalline) or amorphous forms.

[0078] The compounds include compounds with one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element. For example, a reference to hydrogen includes within its scope ¹H, ²H (D), and ³H (T). Similarly, references to carbon and oxygen include within their scope respectively ¹²C, ¹3C and ¹⁴C and ¹6O and ¹⁸O. The isotopes may be radioactive or non-radioactive. In one embodiment, the compounds contain no radioactive isotopes. Such compounds are preferred for therapeutic use. In another embodiment, however, the compound may contain one or more radioisotopes. Compounds containing such radioisotopes may be useful in a diagnostic context.

[0079] In some embodiments, the patient is treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is N-(3,5-dimethoxyphenyl)-N'-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl- )quinoxalin-6-yl]ethane-1,2-diamine (referred to herein "JNJ-42756493"), or a pharmaceutically acceptable salt thereof or a solvate thereof.

Methods of Treating Cancer in a Patient

[0080] Disclosed herein are methods of treating cancer in a patient comprising: evaluating a biological sample from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel; and treating the patient with an FGFR inhibitor if one or more FGFR mutants are present in the sample.

[0081] The disclosed methods can be used to treat a variety of cancer types including, but not limited to, bladder cancer, metastatic bladder cancer, ovarian cancer, head and neck cancer, metastatic head and neck cancer, esophageal cancer, metastatic esophageal cancer, non-small-cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, prostate cancer, lung cancer, gastric cancer, urothelial carcinoma, small cell lung cancer, breast cancer, endometrial cancer, metastatic endometrial cancer, cholangiocarcinoma, hepatocellular carcinoma, glioblastoma, gliomas, colon carcinoma, sarcomas, solid tumors of squamous origin, and multiple myeloma.

[0082] The FGFR mutant panel that is used in the evaluating step is based, in part, on the patient's cancer type. For patients with bladder cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0083] For patients with metastatic bladder cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0084] For patients with ovarian cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0085] For patients with head and neck cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0086] For patients with metastatic head and neck cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastic head and neck cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0087] For patients with esophageal cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0088] For patients with metastatic esophageal cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0089] For patients with non-small cell lung (NSCL) adenocarcinoma, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0090] For patients with non-small cell lung (NSCL) squamous cell carcinoma, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0091] For patients with metastatic endometrial cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. in some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0092] For patients with breast cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0093] For patients with hepatocellular carcinoma, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0094] In some embodiments, the evaluating step comprises: isolating RNA from the biological sample; synthesizing cDNA from the isolated RNA; pre-amplifying the cDNA; and amplifying the pre-amplified cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel.

[0095] Isolating RNA from the biological sample can be performed by a number of procedures known to one skilled in the art. In one embodiment, RNA can be isolated from the biological sample using an AllPrep DNA/RNA FFPE Kit from Qiagen (product #80234)

[0096] Synthesizing cDNA from isolated RNA can be performed by a number of procedures known to one skilled in the art. In one embodiment, cDNA can be synthesized from isolated RNA using a High Capacity cDNA Reverse Transcriptase Kit with RNase Inhibitor from ABI (product #4374966).

[0097] Pre-amplification of cDNA can be performed by a number of procedures known to one skilled in the art. Amplification procedures are well known in the art. In one embodiment, cDNA can be pre-amplified using a TaqMan® PreAmp Master Mix (Life Technologies/Applied Biosystems® product #4391128).

[0098] In some embodiments, the amplifying step can comprise performing real-time PCR (qRT-PCR). Exemplary qRT-PCR procedures are discussed in the Example section herein. In some aspects, the qRT-PCR can be a Taqman® Real-Time PCR assay. qRT-PCR procedures can involve the use of probes to increase the specificity of the assay. Suitable probes for use in the qRT-PCR assay include any of the probes disclosed herein, for example, the probes disclosed in Table 15. In some embodiments, for example, the real-time PCR can be performed with one or more probes comprising SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55. In other embodiments, the real-time PCR can be performed with one or more probes consisting essentially of SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55. In other embodiments, the real-time PCR can be performed with one or more probes consisting of SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55. In other embodiments, the real-time PCR can be performed with one or more probes having SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55.

[0099] The qRT-PCR can be performed with one or more 3' blocking oligonucleotides. Exemplary qRT-PCR procedures using 3' blocking oligonucleotides are disclosed in the Example section herein. Suitable 3' blocking oligonucleotides include, for example, those disclosed in Table 8. In some embodiments, the qRT-PCR can be performed with one or more 3' blocking oligonucleotides comprising SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and/or SEQ ID NO: 42. In some embodiments, the qRT-PCR can be performed with one or more 3' blocking oligonucleotides consisting essentially of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and/or SEQ ID NO: 42. In some embodiments, the qRT-PCR can be performed with one or more 3' blocking oligonucleotides consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and/or SEQ ID NO: 42. In some embodiments, the qRT-PCR can be performed with one or more 3' blocking oligonucleotides having SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and/or SEQ ID NO: 42.

[0100] Suitable pairs of primers for use in the amplifying step include those disclosed in Table 3. For example, in some embodiments, the FGFR mutant and pair of primers can be FGFR3:TACC3 v1 and primers having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, the FGFR mutant and pair of primers can be FGFR3:TACC3 v3 and primers having the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8. In some embodiments, the FGFR mutant and pair of primers can be FGFR3:TACC3 Intron and primers having the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10. In some embodiments, the FGFR mutant and pair of primers can be FGFR3: BAIAP2L1 and primers having the amino acid sequences of SEQ ID NO:11 and SEQ ID NO:12. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:BICC1 and primers having the amino acid sequences of SEQ ID NO:13 and SEQ ID NO:14. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:AFF3 and primers having the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:16. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:CASP7 and primers having the amino acid sequences of SEQ ID NO:17 and SEQ ID NO:18. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:CCDC6 and primers having the amino acid sequences of SEQ ID NO:19 and SEQ ID NO:20. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:OFD1 and primers having the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22. In some embodiments, the FGFR mutant and pair of primers can be R248C and primers having the amino acid sequences of SEQ ID NO:23 and SEQ ID NO:24 or SEQ ID NO:31 and SEQ ID NO:32. In some embodiments, the FGFR mutant and pair of primers can be S249C and primers having the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:33 and SEQ ID NO:34. In some embodiments, the FGFR mutant and pair of primers can be G370C and primers having the amino acid sequences of SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:35 and SEQ ID NO:36. In some embodiments, the FGFR mutant and pair of primers can be Y373C and primers having the amino acid sequences of SEQ ID NO:29 and SEQ ID NO:30 or SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, the FGFR mutant and pair of primers can be any combination of the above disclosed FGFR mutants and corresponding pair of primers.

[0101] In some embodiments, the amplifying step can be performed with the following:

[0102] a. the pair of primers have the sequences SEQ ID NO:5 and SEQ ID NO:6 and the probe has the sequence of SEQ ID NO:43;

[0103] b. the pair of primers have the sequences SEQ ID NO:7 and SEQ ID NO:8 and the probe has the sequence of SEQ ID NO:44;

[0104] c. the pair of primers have the sequences SEQ ID NO:9 and SEQ ID NO:10 and the probe has the sequence of SEQ ID NO:46;

[0105] d. the pair of primers have the sequences SEQ ID NO:11 and SEQ ID NO:12 and the probe has the sequence of SEQ ID NO:47;

[0106] e. the pair of primers have the sequences SEQ ID NO:13 and SEQ ID NO:14 and the probe has the sequence of SEQ ID NO:45;

[0107] f. the pair of primers have the sequences SEQ ID NO:15 and SEQ ID NO:16 and the probe has the sequence of SEQ ID NO:48;

[0108] g. the pair of primers have the sequences SEQ ID NO:17 and SEQ ID NO:18 and the probe has the sequence of SEQ ID NO:49;

[0109] h. the pair of primers have the sequences SEQ ID NO:19 and SEQ ID NO:20 and the probe has the sequence of SEQ ID NO:50;

[0110] i. the pair of primers have the sequences SEQ ID NO:21 and SEQ ID NO:22 and the probe has the sequence of SEQ ID NO:51;

[0111] j. the pair of primers have the sequences SEQ ID NO:23 and SEQ ID NO:24 and the probe has the sequence of SEQ ID NO:52;

[0112] k. the pair of primers have the sequences SEQ ID NO:25 and SEQ ID NO:26 and the probe has the sequence of SEQ ID NO:53;

[0113] l. the pair of primers have the sequences SEQ ID NO:27 and SEQ ID NO:28 and the probe has the sequence of SEQ ID NO:54;

[0114] m. the pair of primers have the sequences SEQ ID NO:29 and SEQ ID NO:30 and the probe has the sequence of SEQ ID NO:55;

[0115] n. the pair of primers have the sequences SEQ ID NO:31 and SEQ ID NO:32, the probe has the sequence of SEQ ID NO:52, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:39;

[0116] o. the pair of primers have the sequences SEQ ID NO:33 and SEQ ID NO:34, the probe has the sequence of SEQ ID NO:53, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:40;

[0117] p. the pair of primers have the sequences SEQ ID NO:35 and SEQ ID NO:36, the probe has the sequence of SEQ ID NO:54, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:41;

[0118] q. the pair of primers have the sequences SEQ ID NO:37 and SEQ ID NO:38, the probe has the sequence of SEQ ID NO:55, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:42; or

[0119] r. any combination thereof.

[0120] The disclosed methods comprise treating a patient if one or more FGFR mutants are present in the sample. The presence of one or more FGFR mutants in the sample can be determined by, for example, sequencing the amplified cDNA.

[0121] Suitable FGFR inhibitors for use in the treatment methods include those previously described herein.

[0122] Also disclosed are FGFR inhibitors for use in the treatment of cancer in a patient wherein the patient is identified as being responsive to treatment with the FGFR inhibitor by evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel, wherein the presence of the one or more FGFR mutants in the sample is detected.

[0123] Further disclosed are FGFR inhibitors for use in the treatment of cancer in a patient wherein the patient is identified as being responsive to treatment with the FGFR inhibitor by evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel, wherein the one or more FGFR mutants are a FGFR fusion gene or a FGFR SNP, wherein the presence of the one or more FGFR mutants in the sample is detected, and wherein said evaluating comprises amplifying cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel.

[0124] Further disclosed are FGFR inhibitors for use in the treatment of cancer in a patient wherein the patient is identified as being responsive to treatment with the FGFR inhibitor by evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel, wherein the FGFR mutant is a FGFR fusion gene or a FGFR SNP, wherein the presence of one or more FGFR mutants in the sample is detected, and wherein said evaluating comprises amplifying a pre-amplified cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel.

Methods of Identifying a Cancer Patient that will be Responsive to Treatment with a Fibroblast Growth Factor Receptor (FGFR) Inhibitor

[0125] Disclosed herein are methods of identifying a cancer patient that will be responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor comprising: evaluating a biological sample from the patient for a FGFR mutant from a FGFR mutant gene panel, wherein the FGFR mutant is a FGFR fusion gene or a FGFR single nucleotide polymorphism, and wherein said evaluating comprises amplifying cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel and determining whether the one or more FGFR mutants from the gene panel are present in the sample, wherein the presence of the one or more FGFR mutants indicates that the patient will be responsive to treatment with the FGFR inhibitor.

[0126] Also provided are methods of identifying a cancer patient that is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor comprising: evaluating a biological sample from the patient for a FGFR mutant from a FGFR mutant gene panel, wherein the FGFR mutant is a FGFR fusion gene or a FGFR single nucleotide polymorphism, and wherein said evaluating comprises amplifying cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel and determining whether the one or more FGFR mutants from the gene panel are present in the sample, wherein the presence of the one or more FGFR mutants indicates that the patient is responsive to treatment with the FGFR inhibitor.

[0127] Further provided are methods of identifying a cancer patient that is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor comprising evaluating a biological sample from the patient for the presence of one or more FGFR mutant from a FGFR mutant gene panel, wherein the FGFR mutant is a FGFR fusion gene or a FGFR single nucleotide polymorphism, wherein the presence of the one or more FGFR mutants indicates that the patient is responsive to treatment with the FGFR inhibitor.

[0128] In some embodiments, the evaluating can comprise amplifying cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel. In some embodiments, the cDNA can be pre-amplified cDNA.

[0129] In some embodiments, the evaluating step comprises: isolating RNA from the biological sample and synthesizing cDNA from the isolated RNA. In some aspects, the evaluating step can be performed on preamplified cDNA. Thus, the evaluating step can further comprise pre-amplifying the cDNA prior to said amplifying step. Isolating RNA from a biological sample can be performed by a number of procedures known to one skilled in the art. In one embodiment, RNA can be isolated from the biological sample using an AllPrep DNA/RNA FFPE Kit from Qiagen (for example, product #80234). Synthesizing cDNA from isolated RNA can be performed by a number of procedures known to one skilled in the art. In one embodiment, cDNA can be synthesized from isolated RNA using a High Capacity cDNA Reverse Transcriptase Kit with RNase Inhibitor from ABI (for example, product #4374966). Pre-amplification of cDNA can be performed by a number of procedures known to one skilled in the art. Amplification procedures are well known in the art. In one embodiment, cDNA can be pre-amplified using a TaqMan® PreAmp Master Mix (Life Technologies/Applied Biosystems® product #4391128).

[0130] The disclosed methods can be used to identify patients with a number of different types of cancer that will be responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor including, but not limited to, bladder cancer, metastatic bladder cancer, ovarian cancer, head and neck cancer, esophageal cancer, non-small-cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, prostate cancer, lung cancer, gastric cancer, urothelial carcinoma, small cell lung cancer, breast cancer, endometrial cancer, cholangiocarcinoma, hepatocellular carcinoma, glioblastoma, gliomas, colon carcinoma, sarcomas, solid tumors of squamous origin, and multiple myeloma.

[0131] The FGFR mutant panel that is used in the evaluating step is based, in part, on the patient's cancer type. For patients with bladder cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants are present in a biological sample from a patient having bladder cancer.

[0132] For patients with metastatic bladder cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants are present in a biological sample from a patient having metastatic bladder cancer.

[0133] For patients with ovarian cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants is present in a biological sample from a patient having ovarian cancer.

[0134] For patients with head and neck cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having head and neck cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having head and neck cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having head and neck cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having head and neck cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having head and neck cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having head and neck cancer. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants is present in a biological sample from a patient having head and neck cancer.

[0135] For patients with metastatic head and neck cncer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastic head and neck cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0136] For patients with esophageal cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants is present in a biological sample from a patient having esophageal cancer.

[0137] For patients with metastatic esophageal cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0138] For patients with non-small-cell lung (NSCL) adenocarcinoma, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 Intron is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants is present in a biological sample from a patient having NSCL adenocarcinoma.

[0139] For patients with non-small cell lung (NSCL) squamous cell carcinoma, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR2:CCDC6 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants is present in a biological sample from a patient having NSCL squamous cell carcinoma.

[0140] For patients with metastatic endometrial cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0141] For patients with breast cancer, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0142] For patients with hepatocellular carcinoma, for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0143] Suitable pairs of primers for use in the amplifying step include those disclosed in Table 3. For example, in some embodiments, the FGFR mutant and pair of primers can be FGFR3:TACC3 v1 and primers having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, the FGFR mutant and pair of primers can be FGFR3:TACC3 v3 and primers having the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8. In some embodiments, the FGFR mutant and pair of primers can be FGFR3:TACC3 Intron and primers having the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10. In some embodiments, the FGFR mutant and pair of primers can be FGFR3:BAIAP2L1 and primers having the amino acid sequences of SEQ ID NO:11 and SEQ ID NO:12. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:BICC1 and primers having the amino acid sequences of SEQ ID NO:13 and SEQ ID NO:14. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:AFF3 and primers having the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:16. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:CASP7 and primers having the amino acid sequences of SEQ ID NO:17 and SEQ ID NO:18. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:CCDC6 and primers having the amino acid sequences of SEQ ID NO:19 and SEQ ID NO:20. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:OFD1 and primers having the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22. In some embodiments, the FGFR mutant and pair of primers can be R248C and primers having the amino acid sequences of SEQ ID NO:23 and SEQ ID NO:24 or SEQ ID NO:31 and SEQ ID NO:32. In some embodiments, the FGFR mutant and pair of primers can be S249C and primers having the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:33 and SEQ ID NO:34. In some embodiments, the FGFR mutant and pair of primers can be G370C and primers having the amino acid sequences of SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:35 and SEQ ID NO:36. In some embodiments, the FGFR mutant and pair of primers can be Y373C and primers having the amino acid sequences of SEQ ID NO:29 and SEQ ID NO:30 or SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, the FGFR mutant and pair of primers can be any combination of the above disclosed FGFR mutants and corresponding pair of primers.

[0144] The disclosed methods comprise determining whether the one or more FGFR mutants from the gene panel are present in the sample. In some embodiments, the determining step comprises sequencing the amplified cDNA.

[0145] In some embodiments, the method further comprises treating the patient with an FGFR inhibitor if the one or more FGFR mutants from the gene panel are present in the sample. Suitable FGFR inhibitors for use in the treatment methods include those previously described herein, in particular JNJ-42756493.

Kits for Identifying the Presence of FGFR Mutant Genes

[0146] Further disclosed are kits for identifying the presence of one or more FGFR mutant genes in a biological sample comprising: pairs of primers having the sequences of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or any combination thereof; and instructions for performing an assay to detect one or more FGFR mutant genes.

[0147] The kits can further comprise one or more probes, one or more 3' blocking oligonucleotides, or both. In some embodiments, the kits can further comprise one or more probes, for example, any one or more of the probes disclosed in Table 15. In some embodiments, the kits can further comprise one or more 3' blocking oligonucleotides, for example, any one or more of the 3' blocking oligonucleotides disclosed in Table 8. In some embodiments, the kits can further comprise one or more probes and one or more 3' blocking oligonucleotides. For example, in some embodiments, the kits can further comprise:

[0148] a. the pair of primers have the sequences SEQ ID NO:5 and SEQ ID NO:6 and the probe has the sequence of SEQ ID NO:43;

[0149] b. the pair of primers have the sequences SEQ ID NO:7 and SEQ ID NO:8 and the probe has the sequence of SEQ ID NO:44;

[0150] c. the pair of primers have the sequences SEQ ID NO:9 and SEQ ID NO:10 and the probe has the sequence of SEQ ID NO:46;

[0151] d. the pair of primers have the sequences SEQ ID NO:11 and SEQ ID NO:12 and the probe has the sequence of SEQ ID NO:47;

[0152] e. the pair of primers have the sequences SEQ ID NO:13 and SEQ ID NO:14 and the probe has the sequence of SEQ ID NO:45;

[0153] f. the pair of primers have the sequences SEQ ID NO:15 and SEQ ID NO:16 and the probe has the sequence of SEQ ID NO:48;

[0154] g. the pair of primers have the sequences SEQ ID NO:17 and SEQ ID NO:18 and the probe has the sequence of SEQ ID NO:49;

[0155] h. the pair of primers have the sequences SEQ ID NO:19 and SEQ ID NO:20 and the probe has the sequence of SEQ ID NO:50;

[0156] i. the pair of primers have the sequences SEQ ID NO:21 and SEQ ID NO:22 and the probe has the sequence of SEQ ID NO:51;

[0157] j. the pair of primers have the sequences SEQ ID NO:23 and SEQ ID NO:24 and the probe has the sequence of SEQ ID NO:52;

[0158] k. the pair of primers have the sequences SEQ ID NO:25 and SEQ ID NO:26 and the probe has the sequence of SEQ ID NO:53;

[0159] l. the pair of primers have the sequences SEQ ID NO:27 and SEQ ID NO:28 and the probe has the sequence of SEQ ID NO:54;

[0160] m. the pair of primers have the sequences SEQ ID NO:29 and SEQ ID NO:30 and the probe has the sequence of SEQ ID NO:55;

[0161] n. the pair of primers have the sequences SEQ ID NO:31 and SEQ ID NO:32, the probe has the sequence of SEQ ID NO:52, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:39;

[0162] o. the pair of primers have the sequences SEQ ID NO:33 and SEQ ID NO:34, the probe has the sequence of SEQ ID NO:53, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:40;

[0163] p. the pair of primers have the sequences SEQ ID NO:35 and SEQ ID NO:36, the probe has the sequence of SEQ ID NO:54, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:41;

[0164] q. the pair of primers have the sequences SEQ ID NO:37 and SEQ ID NO:38, the probe has the sequence of SEQ ID NO:55, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:42; or

[0165] r. any combination thereof.

Oligonucleotide Probes

[0166] Also disclosed are oligonucleotide probes having the sequence of any one of SEQ ID NOs:43-55. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:43. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:44. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:45. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:46. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:47. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:48. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:49. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:50. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:51. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:52. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:53. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:54. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:55.

3' Blocking Oligonucleotide

[0167] Also disclosed herein are oligonucleotides having the sequence of any one of SEQ ID NOs:39-42. In some embodiments, the 3' blocking oligonucleotide can have the sequence of SEQ ID NO:39. In some embodiments, the 3' blocking oligonucleotide can have the sequence of SEQ ID NO:40. In some embodiments, the 3' blocking oligonucleotide can have the sequence of SEQ ID NO:41. In some embodiments, the 3' blocking oligonucleotide can have the sequence of SEQ ID NO:42.

EXAMPLES

Example 1

Plasmid DNA Isolation and Purification

[0168] Below is an exemplary procedure for preparing FGFR fusion plasmid DNA.

[0169] Required equipment: centrifuge, capable of 1500×g; microcentrifuge; pipettors, positive-displacement or air-displacement; vortexer; nanodrop Spectrophotometer; 37° C. shaker/incubator; and an oven set to 37° C.

[0170] Required materials: frozen glycerol bacterial stock containing plasmid DNA; Kanamycin LB agar plates (Teknova #L1155); LB broth (Life Technologies #10855-021); Kanamycin (Sigma #K0254); plasmid purification kit (Qiagen #12123); absolute ethanol (Sigma Aldrich #E7023); isopropanol (Sigma Aldrich #W292907); Nuclease Free Water (Non-DEPC treated) (from IDT or Ambion #AM9932); RNase-free Barrier (Filter) Tips; RNase-free Microtube (1.5 to 2 mL VWR #10011-724); serological pipettes; and 14 ml Round bottom tubes (VWR #352057).

[0171] To recover bacteria from the glycerol stock, frozen bacteria were scraped off of the top of a glycerol stock tube using a sterile pipet tip, streaked onto a LB agar plate, and placed upside down in the oven at 37° C. overnight.

[0172] DNA plasmids were purified using Qiagen Plasmid DNA Purification protocol. Briefly, a single colony was picked from the streaked plate and incubated in a culture of 5 ml-LB medium containing 50 μg/ml Kanamycin overnight in a 37° C. shaker at approximately 300 rpm. The bacterial cells were harvested by centrifugation at 6000×g for 15 minutes at 4° C., and the pellet was resuspended in 300 μl of buffer P1. 300 μl of buffer P2 was added, mixed by inverting the tube 4-6 times, and incubated at RT (room temperature) for 5 minutes. 300 μl of chilled buffer P3 was added, mixed immediately by inverting 4-6 times, incubated on ice for 5 minutes, and centrifuged at maximum speed for 10 minutes. Supernatant containing plasmid DNA was removed promptly. A Qiagen-tip 20 was equilibrated by applying 1 ml of buffer QBT and allowed to empty by gravity flow. The supernatant was applied to the Qiagen-tip 20 and allowed to enter the resin by gravity flow. The Qiagen-tip 20 was washed with 2×2 ml of buffer QC and the DNA was eluted with 800 μl of buffer QF and the eluate was collected in a 1.5 ml Eppendorf tube. The DNA was precipitated by adding 0.7 volumes of isopropanol, mixed, and centrifuged immediately at 15000×g for 30 minutes in a microcentrifuge. The supernatant was decanted, and the DNA pellet was washed in 1 ml of 70% ethanol and centrifuged at 15000×g for 10 minutes. The supernatant was decanted. The pellet was air-dried for 5-10 minutes and the DNA was re-dissolved in 100 μl or suitable volume of nuclease-free water. Plasmid DNA was quantitated by Nanodrop and stored at -20° C. until further use.

Example 2

Generation of NRK Cell Lines

[0173] Expression vectors expressing each of the FGFR fusions were constructed. The expression vector was then transfected into normal rat kidney epithelial cells (NRK) cells. The stable cell lines were selected in media containing kanamycin following transfections. These cells were then grown and mRNA was isolated and subjected to FGFR fusion assays to confirm the presence of the specific FGFR fusions mRNA.

Example 3

FGFR-Fusion Cell Line Maintenance

[0174] The below protocol describes an exemplary procedure for culturing and maintaining the NRK FGFR-fusion over-expressing cell lines. Cell lines include, but are not limited to: NRK/FGFR3:TACC3v1, NRK/FGFR3:TACC3 v3, NRK/FGFR3:BAIAP2L1, NRK/FGFR2: BICC1, NRK/FGFR2:CASP7, NRK/FGFR2:CCDC6, NRK/FGFR2:AFF3, NRK/FGFR2:OFD1, and NRK/EMPTY VECTOR (plasmid control).

[0175] Required equipment: biosafety cabinet, fitted with vacuum aspiration system; CO₂ Incubator, set to 37° C. with 5% CO₂; -80° C. freezer; liquid nitrogen tank; water bath, set to 37° C.; and a microscope.

[0176] Required materials: serological pipettes; tissue culture flasks (T75 VWR #BD353136 and/or T150 VWR #15705-074); tissue culture 0.2 μm filtering units (Thermo Scientific #566-0020); DMEM (Dulbecco's Modified Eagle Medium) cell culture media (Life Technologies, #11965-084); Fetal Bovine Serum (FBS),certified, heat inactivated (Life Technologies, #10082147); PenStrep antibiotic solution (Life Technologies #15140-122); Trypsin-EDTA 0.25% solution (Life Technologies, #25200-056); DPBS (Dulbecco's Phosphate buffered solution, no calcium, no magnesium) (Life Technologies, #14190136); cell freezing container for cryopreservation; hand held pipetman; cell freezing media (Life Technologies, #12648-010); 15 ml conical tubes (VWR #62406-2); and cryovials (VWR #89094-800).

[0177] To prepare the cell culture media, DMEM medium was prepared by combining 445 ml of DMEM, 50 ml of FBS, and 5 ml of PenStrep. The prepared media was passed through a 0.2 μm filter unit and stored at 4° C.

[0178] To thaw frozen cells, prepared DMEM medium was warmed in the 37° C. water bath for at least 15 minutes and 15 ml of warmed medium was placed into a T75 flask. Cells were removed from liquid nitrogen tank and placed immediately in a 37° C. water bath until just thawed. Cryovials were sprayed generously with 70% alcohol and the excess was wiped with paper towels. The entire content was aliquoted into the T75 flask containing DMEM. Flask was swirled gently to mix and placed in incubator for 24 hours. If the cells were not ready for splitting, the media was changed to freshly prepared DMEM to remove residual freezing media. If cells were ready to split, each cell line was propagated once the flask achieved 80% confluency (splitting ratio for each cell line was dependent upon the experimental needs).

[0179] To freeze the cell lines, the cells were removed from the culture flask and spun down in a 15 ml conical tube for 5 minutes at 1500 RPM at RT. The medium was aspirated and 6 ml of cell freezing medium was added. The cells were mixed by pipetting up and down several times, and 1 ml of cell solution was aliquoted into each of 5 cryovials. Cryovials with cells were placed in a cryofreezing container, which was stored in a -80° C. freezer overnight, followed by long term storage in a liquid nitrogen tank.

Example 4

FFPET SNP Assay

[0180] An exemplary workflow and protocol for performing a FFPET SNP assay is described below. A similar procedure is performed for FFPET fusion assays, the results of which are shown in FIG. 2.

De-Parafinization of FFPET

[0181] Slides were subjected to increasing amounts of xylene followed by alcohol treatment in order to remove the paraffin.

FFPET RNA Extraction

[0182] The procedure for extracting RNA from breast cancer formalin fixed paraffin embedded tissue samples for downstream gene expression assay is described below.

[0183] Required equipment: centrifuge with plate adapter, capable of 1500×g; microcentrifuge; pipettors, positive-displacement or air-displacement; vortexer; NanoDrop 8000; heating block capable of incubation at 37° C., 56° C. and 80° C.; and pasteur pipette (Pipet Trans EX-FT 1.5 ml pk 500, VWR #14670-329).

[0184] Required Materials: AllPrep DNA/RNA FFPE Kit (Qiagen #80234); Absolute Ethanol (Sigma Aldrich #E7023); Isopropanol; Xylene; Nuclease Free Water (Non-DEPC treated) (from IDT or Ambion #AM9932); RNase-free Barrier (Filter) Tips; RNase-free; microtube (1.5 to 2 mL VWR #10011-724); and Qiagen AllPrep DNA/RNA FFPE Kit Handbook.

[0185] RNA was extracted using the AllPrep DNA/RNA FFPE Kit. Briefly, one 1-10 um section was placed in a 1.5 ml reaction tube and 800 μl of HemoDe or Xylene were added. The sample was vortexed for 4 seconds 3 times, incubated for 2 minutes, vortexed for 4 seconds for 3 times and incubated for 5 minutes.

[0186] The sample was centrifuged for 2 minutes at maximum speed (12,000-14,000×g) and the supernatant was discarded by aspiration. Tubes were capped immediately to avoid tissue from drying.

[0187] The above steps were repeated.

[0188] 800 μl ethanol abs. was added, the tube was flicked to dislodge the pellet, vortexed for 4 seconds 3 times, centrifuged for 2 minutes at maximum speed (12,000-14,000×g), and the supernatant was discarded by aspiration.

[0189] 800 μl 70% ethanol was added, the tube was flicked to dislodge the pellet, vortexed for 4 seconds 3 times, centrifuged for 2 minutes at maximum speed, and the supernatant was discarded by aspiration. After removal of 70% ethanol, the tube was re-spun for 10-20 seconds and the residual fluid was carefully removed with a fine bore pipet.

[0190] The open tubes were incubated in a heating block for 5-15 minutes at 37° C. to air dry the tissue pellet.

[0191] The pellet was resuspended by adding 150 μl Buffer PKD and the tube was flicked to loosen the pellet. 10 μl proteinase K was added and the tube was mixed by vortexing.

[0192] Tubes were incubated at 56° C. for 15 minutes, incubated on ice for 3 minutes, and centrifuged for 15 minutes at 20,000×g.

[0193] The supernatant was carefully transferred without disturbing the pellet to a new 1.5 ml microcentrifuge tube for RNA purification. The supernatant was incubated at 80° C. for 15 minutes. The tube was briefly centrifuged to remove drops from the inside of the lid. 320 μl Buffer RLT was added to adjust binding conditions, and the tube was mixed by vortexing or pipetting. 1120 μl ethanol (96-100%) was added and the tube was mixed well by vortexing or pipetting.

[0194] 700 μl of the sample, including any precipitate that may have formed, was transferred to an RNeasy MinElute spin column placed in a 2 ml collection tube, and centrifuged for 15 seconds at ≧8000×g (≧10,000 rpm). The flow-through was discarded. This step was repeated until the entire sample was passed through the RNeasy MinElute spin column.

[0195] 350 μl Buffer FRN was added to the RNeasy MinElute spin column and centrifuged for 15 seconds at ≧8000×g (≧10,000 rpm). Flow-through was discarded.

[0196] 10 μl DNase I stock solution was added to 70 μl Buffer RDD, mixed by gently inverting the tube, and centrifuged briefly to collect residual liquid from the sides of the tube.

[0197] The DNase I incubation mix (80 μl) was added directly to the RNeasy MinElute spin column membrane, and placed on the benchtop (20-30° C.) for 15 minutes.

[0198] 500 μl Buffer FRN was added to the RNeasy MinElute spin column and centrifuged for 15 seconds at ≧8000×g (≧10,000 rpm). The flow-through was saved for use in the next step, as it contains small RNAs.

[0199] The RNeasy MinElute spin column was placed in a new 2 ml collection tube (supplied). The flow-through from the previous step was applied to the spin column and centrifuged for 15 seconds at ≧8000×g (≧10,000 rpm). Flow-through was discarded.

[0200] 500 μl Buffer RPE was added to the RNeasy MinElute spin column and centrifuged for 15 second at ≧8000×g (≧10,000 rpm) to wash the spin column membrane. Flow-through was discarded.

[0201] 500 μl Buffer RPE was added to the RNeasy MinElute spin column and centrifuged for 15 seconds at ≧8000×g (≧10,000 rpm) to wash the spin column membrane. Collection tube with the flow-through was discarded.

[0202] The RNeasy MinElute spin column was placed in a new 2 ml collection tube and centrifuged at full speed for 5 minutes. The collection tube with the flow-through was discarded.

[0203] The RNeasy MinElute spin column was placed in a new 1.5 ml collection tube, 30 μl RNase-free water was added directly to the spin column membrane, incubated for 1 minute at room temperature, and centrifuged at full speed for 1 minute to elute the RNA.

[0204] The RNA samples were immediately stored in -80° C. freezer.

cDNA Synthesis

[0205] Disclosed below is a procedure of cDNA synthesis for the FFPET SNP Assays using Real time PCR (RT-PCR) analysis.

[0206] Required equipment: centrifuge with plate adapter, capable of 1500×g, microcentrifuge; pipettors (preferred single and multi-channel pipettor), positive-displacement or air-displacement; vortexer; and GeneAmp® PCR System 9700 (ABI #4314879) or equivalent.

[0207] Required materials: High Capacity cDNA Reverse Transcriptase Kit with RNase Inhibitor, 200 reactions (ABI #4374966); Nuclease Free Water (Non-DEPC treated) (from IDT) or equivalent; RNase-free Barrier (Filter) Tips; RNase-free Microtube (1.5 to 2 mL VWR #10011-724); MicroAmp® Optical 96-Well Reaction Plates (Life Technologies, #4306736); and sealing film (VWR #60941-072).

[0208] Following the RNA extraction (disclosed above) RNA sample tube(s) were kept on ice.

[0209] The kit components were used to prepare 2× Reverse Transcription (RT) Master Mix for all reactions, including 1 negative (water) control. Components were thawed on ice for approximately 15 minutes, gently inverted to mix and centrifuged briefly to bring down the solution. All reagents were returned to the ice. Tubes were not vortexed.

[0210] One Master Mix was prepared on ice in a 1.5 ml tube for the appropriate number of reactions (#reactions+10%, per 20-μL reaction) by combining the following amount of reagent per one reaction: 2 μl 10× RT Buffer Mix; 0.8 μl 25× dNTP Mix; 2 μl 10× RT Random Primers; 1 μl 50 U/μL MultiScribe Reverse Transcriptase; 1 μl RNase inhibitor; and 3.2 μl Nuclease/RNase free H₂O.

[0211] The Master Mix was vortexed several times (5 to 10) to mix and centrifuged briefly (1500×g, 5 to 10 seconds). 10 μl of the reaction mix was added to the appropriate wells of a 96-well plate.

[0212] The RNA samples were diluted to a concentration of 20 ng/μl. 10 μL of each RNA sample was added, including the water negative control, to the appropriate corresponding wells of the 96-well plate to a final reaction volume of 20 μL. The wells were mixed gently by pipetting up and down 3 times, sealed with a plate seal, and centrifuged briefly (1500×g for 60 seconds). Plates were kept on ice until ready to load in thermocycler.

[0213] The reaction plate was loaded into ABI 9700 Thermal Cycler in Clean Lab or Workstation and run using the following reverse-transcription program with a reaction volume of 20 82 l:

[0214] Step 1: 25° C. for 10 minutes

[0215] Step 2: 37° C. for 120 minutes

[0216] Step 3: 85° C. for 5 seconds

[0217] Step 4: 4° C. infinite hold

[0218] Synthesized cDNA was stored at -20° C. for next step of Pre-amplification.

Preamplification Assay Pool Mixture Preparation

[0219] The preamp assay pool mixture associated with the FFPET SNP Assay Pre-amplification Protocol was prepared as described below.

[0220] Required equipment: microcentrifuge; pipettors, positive-displacement or air-displacement; and vortexer.

[0221] Required materials: Nuclease Free Water (Non-DEPC treated) (from IDT) or equivalent; IDTE pH 8.0 (1× TE Solution) (IDT Technologies); RNase-free Barrier (Filter) Tips; and RNase-free Tubes (1.5 to 2 mL VWR #10011-724).

[0222] All TaqMan SNP Assays are ordered from Applied Biosystems, Life Technologies, Inc.

[0223] 100 μL of 20× SNP assays were prepared.

[0224] To prepare 0.2× Preamp Assay Pool, all assays were thawed on ice for approximately 15 minutes. The following volume of components was added to a 1.5 ml tube:

TABLE-US-00004 TABLE 4 Stock Volume Needed for 200 ul Target Concentration Preamp Stock (ul) Preamp Stock 1 FGFR3 S249C 20X 2 IDTE 198 Total Volume 200 Preamp Stock 2 FGFR3 R248C 20X 2 IDTE 198 Total Volume 200 Preamp Stock 3 FGFR3 Y373C 20X 2 IDTE 198 Total Volume 200 Note: The above volumes are for the preparation of 200 μl of 0.2X preamp assay pool. Volumes can be adjusted accordingly depending on the number of samples being tested.

[0225] The 0.2× PreAmp Assay Pool was vortexed briefly to mix (5 to 10 seconds) and centrifuged briefly (1500×g, 5-10 seconds). 100 μL of PreAmp Primer Pool was aliquoted into 1.5 ml tubes and stored at -20° C.

Pre-Amplification for the Breast Cancer Formalin-Fixed Paraffin Embedded Tissue SNP Assay Using Real Time PCR (RT-PCR) Analysis

[0226] Required equipment: centrifuge with plate adapter, capable of 1500×g; microcentrifuge; pipettors, positive-displacement or air-displacement; vortexer; GeneAmp® PCR System 9700 (ABI #4314879) or equivalent.

[0227] Required Materials: TaqMan® PreAmp Master Mix (2×) (Life Technologies #4391128); 0.2× Pooled Assay Mix (see Assay Preparation and Handling Protocol); 1× IDTE Buffer (10 mM Tris/0.1 mM EDTA, pH7.5, from IDT) or equivalent; Nuclease Free Water (Non-DEPC treated) (from IDT) or equivalent; RNase-free Barrier (Filter) Tips; RNase-free Microtube (1.5 to 2 mL VWR #10011-724); MicroAmp® Optical 96-Well Reaction Plates (Life Technologies, #4306736); MicroAmp® Optical Adhesive Film (Applied Biosystems PN 4311971); deep well plates (VWR #47734-788); foil seals (VWR #60941-126).

[0228] Samples were prepared by placing the cDNA and 0.2× assay mix pool on ice to thaw, approximately 5 minutes, and centrifuging the plate briefly (1500×g for 5 to 10 seconds).

[0229] The kit components were used to prepare 2× PreAmp Master Mix. The kit components were allowed to thaw on ice for approximately 5 minutes. After all reagents were thawed, the tubes were gently inverted to mix and briefly centrifuged to bring down the solution. All reagents were returned to the ice. The tubes were not vortexed.

[0230] In a Clean Lab or Biosafety hood, each Master Mix was prepared for the appropriate number of reactions on ice by combining the required volumes of reagents as indicated Table 5 below (#reactions+10%):

TABLE-US-00005 TABLE 5 Volume (μL) for One Component Reaction Master Mix 1 2X TaqMan PreAmp Master Mix 12.5 0.2X Assay Pool 1 6.25 Total volume 18.75 Master Mix 2 2X TaqMan PreAmp Master Mix 12.5 0.2X Assay Pool 2 6.25 Total volume 18.75 Master Mix 3 2X TaqMan PreAmp Master Mix 12.5 0.2X Assay Pool 3 6.25 Total volume 18.75 Assay pools contain primers and probes.

[0231] To prevent cross-priming of SNP assays, all 5 assays were split into 3 preamp reaction per sample.

[0232] Each Master Mix was vortexed several times (5 to 10) to mix, followed by a brief centrifuge (1500×g, 5 to 10 seconds). 18.75 μL of each Master Mix was aliquoted to the appropriate wells in a 96-well reaction plate. 6.25 μL of each cDNA samples, including water negative control well, was transferred into the appropriate wells in the Master Mix reaction plate for each preamp reaction. The sample was mixed gently by pipetting up and down 3 times and the cap was closed. The plate was briefly centrifuged (1500×g for 60 seconds) and kept on ice until ready to load in thermocycler.

[0233] The reaction plate ABI 9700 Thermal Cycler was loaded and run using the following program:

[0234] Step 1: 95° C. for 10 minutes

[0235] Step 2: 95° C. for 15 seconds

[0236] Step 3: 60° C. for 4 minutes

[0237] Step 4: Set Step 2-3 for 10 cycles

[0238] If a Gold or Silver Block was Used, Max Mode was Selected and Ramp Rate was set at 77%.

[0239] If an Aluminum Block was Used, Standard Mode (No Rate Change) was Selected.

[0240] Step 5: 4° C. infinite hold

[0241] Reaction volume set to 25 μL

[0242] The PreAmp reaction plate was centrifuged briefly (1500×g for 60 seconds) after PreAmp completion. 100 μl of IDTE was added to the appropriate wells of a new deep 96-well plate and 25 μL of each PreAmp product was transferred to the corresponding wells to have final dilution volume of 125 μL. The each well was mixed by pipetting up and down 3 times, the plate was sealed with foil adhesive, the plate was centrifuged briefly (1500×g for 5 to 10 seconds), and the PreAmp product was stored at -20° C. until further use.

FFPET SNP Assay--Real Time PCR

[0243] Disclosed below is the procedure for the Formalin-Fixed Paraffin Embedded Tissue SNP Assay using Real time PCR analysis.

[0244] Required equipment: centrifuge with plate adapter, capable of 1500×g; microcentrifuge; pipettors (preferred single and multi-channel pipettor), positive-displacement or air-displacement; vortexer; and ABI ViiA 7 real time PCR instrument (Life Technologies).

[0245] Required materials: TaqMan Genotyping Master Mix (Life Technologies #4371355); SNP Assays; Nuclease Free Water (Non-DEPC treated, from IDT) or equivalent; RNase-free Barrier (Filter) Tips; RNase-free Microtube (1.5 to 2 mL VWR #10011-724); MicroAmp® Optical Adhesive Film (Applied Biosystems PN 4311971); and MicroAmp® Optical 384-Well Reaction Plates.

[0246] Table 15 lists the sequences of the probes used during the Real Time PCR assays.

[0247] To prepare the samples, in a Clean Lab or Workstation, SNP assays were placed on ice to thaw for approximately 5 minutes. All reagents protected from light, to protect exposure of the fluorescent probes. Diluted PreAmp plates were placed on ice to thaw in a Dirty Lab or Workstation after preparing Genotyping Master Mix.

[0248] To prepare genotyping master mix, the Genotyping Master Mix was thawed on ice for approximately 5 minutes. The Master Mix (MM) was prepared in the required number of tubes on ice. The required volumes of reagents were combined in the appropriate labeled tubes as indicated in Table 6 below (# reactions+10%):

TABLE-US-00006 TABLE 6 Volume (μL) Component for One Reaction 2X Genotyping Master Mix 10 20X SNP Assay 1 RNase-free Water 4 Total volume 15 20X SNP assay mix contains primers, probes, and blocking oligos.

[0249] The Master Mix was vortexed several times (5 to 10) to mix and then centrifuged briefly (1500μg, 5 to 10 seconds). 15 μl of each Master Mix was added to the appropriate wells of a MicroAmp® Optical 384-Well Reaction Plates. The reaction plates were sealed with optical adhesive film.

[0250] The plate with 1:5 diluted PreAmp product was placed on ice for approximately 5-10 minutes to thaw. Using a multi-channel pipettor, 5 82 L of each diluted PreAmp product was transferred to the appropriate corresponding wells. The reaction plate was sealed with optical adhesive film and centrifuged briefly (1500×g for 60 seconds). Plates were kept on ice until ready to load in thermocycler.

[0251] The following conditions were run using the viiA 7 Software with the volume set at 20 μl:

TABLE-US-00007 TABLE 7 Stage Repetitions Process Temperature Time 1 1 Initial 60° C. 0.5 minutes 2 1 DNApol Activation 95° C. 10 minutes 3 40 Denature 95° C. 15 seconds Anneal/Extend 60° C. 1 minutes 4 1 Post-Read 60° C. 30 seconds

FGFR SNP-Specific qRT-PCR

[0252] The detection of rare somatic mutations in an excess of wild type alleles is increasingly important in cancer diagnosis. When the mutations of interest are close to each other, detection becomes challenging. To aid in the identification of FGFR SNPs from FFPET, a SNP-specific qRT-PCR assay was developed, in which SNP-specific amplification using Taqman MGB probes combined with the 3' dideoxy wild type (WT) allele blocker was used. The assay prevented non-specific binding, improved the number of on-target amplification, minimized the false positive signals from the WT alleles, and increased the sensitivity of the assay. This RNA based SNP detection assay, combined with the pre-amplification step in the assay, boosts the low or the rare mutant signals.

[0253] An exemplary strategy for SNP-specific qRT-PCR using a 3' dideoxy WT blocker oligonucleotide is shown in FIG. 3, and an exemplary FFPE sample validation strategy is illustrated in FIG. 4. Briefly, qRT-PCR was performed using the FGFR SNP primers in the presence of a 3' dideoxy WT blocker oligonucleotide, which was complementary to, and contained a short stretch of nucleotides flanking, the WT allele. Binding of the blocker oligonucleotide to the WT allele prevented applification of the WT allele, while the FGFR SNP primers bound to and specifically amplified the FGFR SNP. The 3' dideoxy WT blocker oligonucleotides used in the FGFR SNP-specific qRT-PCR are shown in Table 8. The FGFR SNP primers used in the FGFR SNP-specific qRT-PCR were: SEQ ID NO:31 and SEQ ID NO:32 (FGFR3 R248C); SEQ ID NO:33 and SEQ ID NO:34 (FGFR3 S249C); SEQ ID NO:35 and SEQ ID NO:36 (FGFR3 G370C); and SEQ ID NO:37 and SEQ ID NO:38 (FGFR3 Y373). Table 15 lists the sequences of the probes used during the real time PCR assays.

TABLE-US-00008 TABLE 8 3' dideoxy WT Target blocker oligonucleotide FGFR3 R248C TGGAGCGCTCCCCGCA-ddC (SEQ ID NO: 39) FGFR3 S249C GACGTGCTGGAGRGCTC-ddC (SEQ ID NO: 40)* FGFR3 G370C CTGACGAGGCGGGCAG-ddC (SEQ ID NO: 41) FGFR3 Y373 GTGTGTATGCAGGCATCCTCAG-ddC (SEQ ID NO: 42) *R can be A or G. 3' WT blocking oligo will have 50% A and 50% G at that particular position during the synthesis (purified by manufacturer to provide A or G at that particular position).

[0254] Samples for validatation studies were prepared as shown in Table 9. Exemplary validation data of the SNP-specific qRT-PCR using a 3' dideoxy WT blocker oligonucleotide for FGFR3 G370C, FGFR3 Y373, FGFR3 S249C, and FGFR3 R248C is illustrated in FIGS. 5A-5D, respectively. Raw Ct (cycle threshold) data for the FFPE samples with SNP-specific qRT-PCR with 3' dideoxy WT blocker oligonucleotides are shown in Table 10. The data derived from DNA and RNA using different platforms/techniques suggests that SNP-specific PCR with the 3' blocking nucleotide is a robust, reliable and a sensitive assay. The validation data suggests that one mutant allele/SNP can be detected in a large excess of WT-bearing genomic DNA, thus emphasizing the sensitivity and the specificity of each assay.

TABLE-US-00009 TABLE 9 Sample % Mutant 1 100 2 20 3 4 4 0.8 5 0 (100% WT) RNA from Stable cell lines expressing each FGFR3 SNPs (R248C, S249C, G370C, Y373C) and FGFR3 WT

TABLE-US-00010 TABLE 10 FGFR3 SNPs - SNP-Specific PCR with Janssen Dideoxy WT Blocker (Ct *) R&D Pt Id# R248C S249C G370C Y373C FMI/NGS ver1.0 7502 >35 28.03 >35 >35 S249C S249C 10000305 >35 >35 >35 >35 WT WT 33000127 >35 20.92 >35 >35 S249C S249C 33000118 >35 29.35 >35 >35 S249C S249C 10000306 >35 >35 >35 24.30 Y373C Y373C 34000226 >35 >35 >35 >35 WT WT 16446 >35 28.03 >35 >35 S249C S249C * Mean of two Cts FMI/NGS = Next generation Sequencing technique wherein DNA is used as an template to identify the mutations (without 3' blocking oligonucleotide); Janssen R&D = performed on RNA template (without the 3' blocking oligonucleotide); SNP-specific PCR performed on RNA template with the 3' blocking nucleotide.

Example 5

Validation of Custom FGFR Fusion Gene Detection Assay

Generation of Positive Controls for FGFR Fusion Assays

[0255] FGFR fusion "synthetic mini-genes," plasmids encoding FGFR fusions, and stable cell lines containing FGFR fusions were generated. Briefly, Synthetic mini genes were artificially constructed by linking a series of nucleotides, of about 100 base pairs, to each other corresponding to the target DNA sequence of the gene of interest. Plasmids encoding FGFR fusions were generated by cloning cDNA encoding the various FGFR fusion genes into an expression vector. Stable cell lines containing FGFR fusions were generated by transfecting plasmids encoding FGFR genes into normal rat kidney epithelial cells (NRK cells). The stable cell lines were selected under the G418 antibiotic. The FGFR fusion Taqman assay was performed on the total RNA isolated from these cell lines to confirm the successful generation of stable cell line(s) expressing the FGFR fusion(s). The stable cell lines expressing FGFR fusions are used a positive control. Table 15 lists the sequences of the probes used during the real time PCR assays.

Analysis of Lower Limit of Quantitation and Efficiency of FGFR Fusion Assays

[0256] To determine the lower limit of quantitation (LLOQ) and efficiency of the FGFR fusion gene assays, FGFR fusion products were generated by TaqMan PCR (as described in Example 4) and confirmed by Sanger Sequencing (FIG. 2). 100 pg of fusion positive DNA was mixed with normal human cDNA (confirmed fusion-negative), serially diluted 1:10, and analyzed using the Applied Biosystems ViiA7 Software v1.1. Efficiency standard curves are shown in FIG. 6. FGFR fusion LLOQ and efficiency are shown in Table 11.

TABLE-US-00011 TABLE 11 Assay LLOQ Efficiency FGFR3:TACC3 V1 1.0 fgm 104% FGFR3:TACC3 V3 10.0 fgm 104% FGFR3:TACC3 Intron 0.1 fgm 103% FGFR3:BAIAP2L1 1.0 fgm 101% FGFR2:AFF3 0.1 fgm 106% FGFR2:BICC1 10.0 fgm 105% FGFR2:CASP7 0.1 fgm 109% FGFR2:CCDC6 1.0 fgm 106% FGFR2:OFD1 0.1 fgm 96.6%

[0257] The FGFR fusion gene assay was next validated in fusion gene-positive cell lines. FGFR fusion gene expression, serial dilutions were prepared by spiking fusion protein-positive cells lines into a fusion protein-negative cell line. For example, a 1:2 serial dilution was prepared for both FGFR3:TACC3v1 and FGFR3:BAIAP2L1 and spiked into 1 million BAF cells. RNA was isolated (using Qiagen Rneasy kit), followed by RT-PCR, preamplification of cDNA, and TaqMan Real Time PCR for the targeted FGFR fusion gene. As shown in Table 12, both the FGFR3:TACC3v1 and FGFR3:BAIAP2L1 Fusion Gene TaqMan assays are able to detect the fusion target in 31 out of 1 million fusion-negative cells (sensitivity of 0.003%).

TABLE-US-00012 TABLE 12 Percent SW780 of Fusion-Positive RT112 FGFR3:BAIAP2L1 FGFR-fusion Cells vs FGFR3:TACC3v1 Average Ct Cell Count Background Average Ct (n = 2) (n = 2) Positive 1.00E+06 100% 17.56 20.35 Control 1000 0.1000% 27.95 28.61 500 0.0500% 29.11 28.91 250 0.0250% 29.62 30.14 125 0.0125% 30.26 31.43 62.5 0.0063% 31.19 31.69 LLOD 31.25 0.0031% 32.59 32.97 15.6 0.0016% 34.91 >40 0 0.0000% 0.00 >40 RT112 and SW780 = commercially available bladder cancer cell lines harboring the FGFR fusions (from American Type Culture Collection).

Example 6

Validation of Custom FGFR SNP Detection Assay

Evaluation of FGFR3 Mutations in Bladder Cancer

[0258] The R248C, S249C, and Y373C SNPs were observed in approximately 8%, approximately 61%, and approximately 19% of bladder cancer samples tested, respectively.

Example 7

Analysis of Cancer Samples

[0259] Samples were analyzed using the same procedure as described in example 4. The results are shown in Table 13 and FIG. 7. Table 13 shows the FGFR fusion prevalence in different cancers. FGFR fusions detected in FFPE samples from different cancers such as bladder (primary and metastatic), NSCLC (adenocarcinoma and squamous), ovarian, esophageal (primary and metastatic), head and neck (H&N; primary and metastatic), endometrial (metastatic), breast, and prostate cancer using the qRT-PCR method. All FGFR fusions tested were negative for prostate cancer samples. FGFR3:TACC3intron fusion was negative in bladder (primary), NSCLC (squamous), ovarian and esophageal (primary), H&N (primary and metastatic) and breast. FGFR2:OFD1 fusion was negative in bladder (primary and metastatic), NSCLC (adenocarcinoma), ovarian and esophageal (primary and metastatic). FGFR2:CCDC6 fusion was negative in bladder (primary and metastatic), NSCLC (adenocarcinoma), ovarian and esophageal (primary) and H&N (primary and metastatic)

[0260] FIG. 8 is an exemplary representation of FGFR fusion gene and mutation status in NSCLC adenocarcinoma and squamous cell carcinoma. In FGFR fusion positive NSCLC adenocarcinoma samples, 3/17 samples were positive for EGFR mutation, 3/17 samples were positive for KRAS mutation, and 1/17 samples were positive for cMET mutation. No EGFR, KRAS, or cMET mutations, however, were observed in FGFR fusion positive NSCLC squamous cell carcinoma samples.

TABLE-US-00013 TABLE 13 NSCLC Bladder Bladder NSCLC Squa- Ovar- Eso Eso H&N H&N Endo Pros- primary Mets Adeno mous ian Primary Mets Primary Mets Mets Breast tate (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) FGFR3: 1/22 5/48 3/89 2/125 4/94 2/41 2/42 1/37 0/40 5/46 3/112 0/72 TACC3v1 (4.55) (10.47) (3.37) (1.60) (4.26) (4.88) (4.76) (2.70) (0.00) (10.87) (2.69) (0.00) FGFR3: 1/22 2/48 9/89 5/129 5/94 1/41 10/42 0/37 0/40 2/46 6/112 0/72 TACC3v3 (4.55) (4.20) (13.90) (3.38) (5.32) (2.44) (23.81) (0.00) (0.00) (4.35) (5.36) (0.00) FGFR3: 0/22 0/48 3/89 0/125 0/94 0/41 1/42 0/37 0/40 2/46 0/112 0/72 TACC3In- (0.00) (0.00) (3.37) (0.00) (0.00) (0.00) (2.38) (0.00) (0.00) (4.35) (0.00) (0.00) tron FGFR3: 2/17 19/44 5/89 3/115 1/94 0/41 25/42 2/37 34/40 22/46 56/112 0/72 BAIAP2L1 (11.77) (43.18) (5.62) (2.61) (1.06) (0.00) (59.52) (5.41) (85.00) (47.83) (50.00) (0.00) FGFR2: 1/22 4/48 0/89 2/123 8/94 2/41 1/42 0/37 0/40 0/46 3/112 0/72 BICC1 (4.55) (8.33) (0.00) (1.63) (8.51) (4.88) (2.40) (0.00) (0.00) (0.00) (2.70) (0.00) FGFR2: 1/17 19/44 1/89 2/111 2/94 0/41 8/42 0/37 0/40 0/46 10/112 0/72 AFF3 (5.88) (43.18) (1.12) (1.80) (2.31) (0.00) (19.05) (0.00) (0.00) (0.00) (8.90) (0.00) FGFR2: 7/16 20/45 1/89 6/114 24/94 2/41 1/42 4/37 3/40 8/46 12/112 0/72 CASP7 (43.75) (44.44) (1.12) (5.26) (25.53) (4.88) (2.40) (10.81) (7.50) (17.40) (10.70) (0.00) FGFR2: 0/22 0/48 0/89 6/109 0/94 0/41 4/42 0/37 0/40 6/46 3/112 0/72 CCDC6 (0.00) (0.00) (0.00) (5.50) (0.00) (0.00) (9.52) (0.00) (0.00) (13.04) (2.70) (0.00) FGFR2: 0/17 0/44 0/89 1/121 0/94 0/41 1/42 0/37 3/40 3/46 10/112 0/72 OFD1 (0.00) (0.00) (0.00) (0.83) (0.00) (0.00) (2.40) (0.00) (7.50) (6.52) (8.90) (0.00) Eso = Esophageal; Endo = Endometerial

Example 8

Treatment of Patients with Advanced Solid Tumors

[0261] A clinical trial was conducted in which patients having various solid tumors expressing the FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:CCDC6 and FGFR2:BICC1 fusion genes were treated with JNJ-42756493. FIG. 9 illustrates exemplary results from phase I patient samples, in which FGFR fusions in Phase I JNJ-427493 (EDI10001) trial samples were detected using the qRT-PCR assay. All FGFR fusion assays were run simultaneously with positive controls (ST) and GAPDH for quality control assessment of the RNA. A) Graphical representation of the qRT-PCR data generated for pt #1000081: positive only for FGFR2:BICC1 fusion (inset shows details of the Ct values for FGFR2:BICC1 fusion, ST-positive control and GAPDH). B) Graphical representation of the qRT-PCR data generated for pt #33000158: positive only for FGFR3:TACC3v1 fusion (inset shows details of the Ct values for FGFR3:TACC3v1 fusion, ST-positive control and GAPDH). C) Graphical representation of the qRT-PCR data generated for pt #34000123: positive only for FGFR2:CCDC6 fusion (inset shows details of the Ct values for FGFR2:CCDC6 fusion, ST-positive control and GAPDH). D) Graphical representation of the qRT-PCR data generated for pt #340000115: positive for FGFR3:TACC3v1, FGFR3:TACC #v3 and FGFR2:CCDC6 fusions (inset shows details of the Ct values for FGFR fusions, ST-positive controls and GAPDH).

[0262] FIG. 10 represents an exemplary Phase I Study design for a First-In-Human Study of JNJ-42756493 in patients with advanced solid tumor. Shown is a graphical depiction of a traditional 3+3 design dose escalation method for the phase I clinical trial. The dose escalation phase aimed to establish the maximum tolerated dose (MTD) and recommended Phase II dose (RPD). The Part 1 arm was used to determine the intermittent dosing schedule, i.e., 7 days on and seven days off (10 mg/kg and 12 mg/kg). The Part 2 arm was used to determine the PD biomarkers (pharmacodynamics biomarkers; makers examined to link the effect of the drug to the target and biological tumor response) wherein the biopsy and blood sample were tested. The Part 3 arm was used the dose expansion cohort and included accrual of additional patients in specific indications (NSCLC,SCLC, breast and solid tumors) with different eligibility criteria (FGFR aberrations: translocation/mutation/amplifications) to further characterize the toxicity profiles of the JNJ493.

Evaluation of Clinical Activity

[0263] Significant clinical responses (RECIST) were observed at 9 mg dosing once a day (QD), 12 mg QD and 12 mg 7 d on/off in patients with the FGFR fusion genes. (FIG. 11; represents all dosing regimens).

Example 9

Generation of FGFR Fusion Stably Transfected RK3E Cells

FGFR Fusion Overexpressing Cell Lines

[0264] RK3E (rat kidney epithelial cells) cells were purchased from ATCC (Manassas, Va., USA) and cultured in DMEM supplemented with FBS and antibiotics (Invitrogen, Grand Island, N.Y., USA). FGFR fusion gene constructs were designed and cloned into the pReceiver expression vector (Genecopoeia, Rockville, Md., USA), which contains an HA-tag. Clones were transfected into RK3E cells using the Amaxa Cell Line Nucleofector (Lonza, Basel, Switzerland) following the manufacturer's protocol. The stably transfected cells were selected in complete medium with 800 ug/ml of G418 (Invitrogen). Overexpression of the fusions in the stably transfected cells was confirmed by real-time PCR and immunoblotting using an anti-pFGFR antibody (FIG. 12). As shown in FIG. 12, the stable cell lines showed expression of active FGFR fusion kinases, as exhibited by the expression of phosphorylation of FGFR.

Colony Formation Assay

[0265] Anchorage-independent growth of the FGFR fusion stably transfected RK3E cells was tested. 1 ml culture medium with 0.8% low melting point agarose was first plated into each of three wells of a six-well plate. After the agar solidified, each well received another 1 ml of 0.4% agar in culture medium containing 100 cells. After 14 days, colonies were fixed and stained with 0.1% cresyl crystal violet. The number of colonies was determined microscopically by manual counting from triplicate wells for each cell line. A representative view of each fusion-overexpressing cell line is shown in FIG. 13A. Anchorage-independent cell growth in soft-agar could be detected in the FGFR fusion stably transfected cells, but not in the empty vector control. FIG. 13B represents a quantitative analysis of colonies in soft agar for the FGFR fusion stably transfected RK3E cells and empty vector control. All experiments were carried out in duplicate and the results are expressed as colonies/100 cells plated. All of the FGFR fusions tested induced anchorage independent growth, highlighting their transforming ability

Downstream Target Expression

[0266] FGFR fusion stably transfected RK3E cells were plated in complete growth medium, serum starved overnight, then re-fed with 0.5% FBS growth media. Cells were treated with 1 μM of JNJ-42756493, AZD4547 or NVP-BGJ398 in the presence of ligands for 1 hour. For immunoblotting, whole cell lysates were collected in RIPA buffer (Thermo Scientific, Waltham, Mass., USA) and sample protein concentration was assayed using BCA Protein Assay (Thermo Scientific). Equal amounts of protein (30 pg per lane) were loaded onto on 4-12% Bis-Tris gels (Invitrogen) before an SDS-page was performed. Proteins were transferred to nitrocellulose membranes and probed with antibodies against p-FGFR, total-FGFR2, p-MAPK, total-MAPK, p-S6, total S6, B-actin (Cell Signaling Technology, Danvers, Mass., USA), and total-FGFR3 (Santa Cruz, Dallas, Tex., USA). The membranes were blocked with Odyssey blocking buffer for 1 h at room temperature and incubated overnight at 4° C. in a primary antibody solution diluted in Odyssey blocking buffer (1:1000). After three washes in 0.1% Tween tris buffered saline (TBST), the membranes were probed with goat anti-mouse or donkey anti-rabbit IR-Dye 670 or 800 cw labeled secondary antisera in Odyssey blocking buffer for 1 h at room temperature. Washes were repeated after secondary labeling and the membranes were imaged using a LiCor Odyssey scanner and the Odyssey 3.0 analytical software (LiCor, Lincoln, Nebr., USA). Effects of JNJ-42756493 was compared with AZD4547 and NVP-BGJ398. As shown in FIGS. 14A-14H, treatment with JNJ-42756493, AZD4547 and NVP-BGJ398 (lanes 2-4 in each blot) inhibited phosphorylation of FGFR and downstream targets i.e. MAPK and S6.

Drug Response Testing for FGFR fusion Overexpressing Cell Lines

[0267] FGFR fusion stably transfected RK3E cells were seeded into 96 well plates (1000 cells/well) in triplicates in complete growth medium plus and the ligands FGF-1 and FGF-2. After 24 hours, cells were serum starved overnight, then re-fed with 0.5% FBS growth media. 72 hours after plating, cells were treated with various concentrations of an 18 point 1:3 dilution series, starting at 10 μM, of JNJ493, AZD4547 (AZD), and NVP-BGJ398 (NVS). The Microtiter plates were then incubated for 72 hours and assayed for adenosine triphosphate (ATP; a marker of metabolically active cells) content using the Cell Titer-Glo® Luminescent Cell Viability assay (Promega Corp., Madison, Wis., USA) following the manufacturer's instructions, with modifications. Briefly, cells were allowed to equilibrate to room temperature, at which time a 1:1 mixture of Cell Titer-Glo® reagent was added. Cells were then placed on an orbital shaker for 2 minutes and incubated for 10 minutes at room temperature to stabilize the luminescent signal. The luminescence was quantified and measurements were conducted using an Envision Multilabel plate reader (Perkin Elmer; Waltham, Mass., USA). IC₅₀ values (shown in Table 14) were calculated using GraphPad Prism 5.0. As shown in Table 14, cells harboring the FGFR fusions showed sensitivity to the FGFR inhibitor JNJ-42756493, AZD4547 and NVP-BGJ398 in vitro, with JNJ-42756493 exhibiting enhanced sensitity (nanomolar concentration range) when compared to AZD4547 and NVP-BGJ398, whereas the empty vector control did not.

TABLE-US-00014 TABLE 14 Stimulated Proliferation (IC50) RK3E-Transgene JNJ493 (nM) AZD (nM) NVS (nM) Vector 7010 8011 >10 μM AFF3 0.1133 2.809 2.273 BAIA2PL1 0.3211 11.54 5.162 BICC1 0.3303 6.448 18.19 CASP7 0.4718 4.107 241.5 CCDC6 0.1894 13.36 10.72 OFD1 0.2303 7.259 15.99 TACC3-V1 0.2915 16.53 2.594 TACC3-V3 0.2706 8.664 4.092 FGFR2 >10 μM 6501 >10 μM FGFR3 >10 μM 5686 6344 KRAS 1621 1478 2136 AZD = AZD4547; NVS = NVP-BGJ398

TABLE-US-00015 TABLE 15 Target Probe Sequences FGFR3TACC3 V1 TCCACCGACGTAAAGG (SEQ ID NO: 43) FGFR3TACC3 V3 TCCACCGACGTGCCAG (SEQ ID NO: 44) FGFR2BICC1 CCAATGAGATCATGGAGG (SEQ ID NO: 45) FGFR3TACC3 Intron CCTTCTGGCCCAGGTG (SEQ ID NO: 46) FGFR3BAIAP2L1 CACCGACAATGTTATGG (SEQ ID NO: 47) FGFR2AFF3 TCACAACCAATGAGGAGAGT (SEQ ID NO: 48) FGFR2CASP7 CTGCCATCTCATTGGT (SEQ ID NO: 49) FGFR2CCDC6 AATGAGCAAGCCAGGGC (SEQ ID NO: 50) FGFR2OFD1 AAGTTGTGTCTCATTGGTT (SEQ ID NO: 51) FGFR3 R248C CTGGAGTGCTCCCC (SEQ ID NO: 52) FGFR3 S249C AGCGCTGCCCGCA (SEQ ID NO: 53) FGFR3 G370C GCGTGCAGTGTGTAT (SEQ ID NO: 54) FGFR3 Y373 CTGCACACACACTGC (SEQ ID NO: 55)

[0268] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

[0269] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in its entirety.

Nucleotide Sequence of FGFR Fusion Genes

[0270] The nucleotide sequences for the FGFR fusion cDNA that were engineered into expression vectors is provided in Table 16. The underlined sequences correspond to either FGFR3 or FGFR2, the sequences in normal font represent the fusion partners and the sequence in italic fonts represent the intron sequence of the FGFR3 gene.

TABLE-US-00016 TABLE 16 FGFR3:TACC3 v1 >ATGGGCGCCCCTGCCTGCGCCCTCGCGCT (3271 base pairs) CTGCGTGGCCGTGGCCATCGTGGCCGGCGC (SEQ ID NO: 56) CTCCTCGGAGTCCTTGGGGACGGAGCAGCG CGTCGTGGGGCGAGCGGCAGAAGTCCCGGG CCCAGAGCCCGGCCAGCAGGAGCAGTTGGT CTTCGGCAGCGGGGATGCTGTGGAGCTGAG CTGTCCCCCGCCCGGGGGTGGTCCCATGGG GCCCACTGTCTGGGTCAAGGATGGCACAGG GCTGGTGCCCTCGGAGCGTGTCCTGGTGGG GCCCCAGCGGCTGCAGGTGCTGAATGCCTC CCACGAGGACTCCGGGGCCTACAGCTGCCG GCAGCGGCTCACGCAGCGCGTACTGTGCCA CTTCAGTGTGCGGGTGACAGACGCTCCATC CTCGGGAGATGACGAAGACGGGGAGGACGA GGCTGAGGACACAGGTGTGGACACAGGGGC CCCTTACTGGACACGGCCCGAGCGGATGGA CAAGAAGCTGCTGGCCGTGCCGGCCGCCAA CACCGTCCGCTTCCGCTGCCCAGCCGCTGG CAACCCCACTCCCTCCATCTCCTGGCTGAA GAACGGCAGGGAGTTCCGCGGCGAGCACCG CATTGGAGGCATCAAGCTGCGGCATCAGCA GTGGAGCCTGGTCATGGAAAGCGTGGTGCC CTCGGACCGCGGCAACTACACCTGCGTCGT GGAGAACAAGTTTGGCAGCATCCGGCAGAC GTACACGCTGGACGTGCTGGAGCGCTCCCC GCACCGGCCCATCCTGCAGGCGGGGCTGCC GGCCAACCAGACGGCGGTGCTGGGCAGCGA CGTGGAGTTCCACTGCAAGGTGTACAGTGA CGCACAGCCCCACATCCAGTGGCTCAAGCA CGTGGAGGTGAATGGCAGCAAGGTGGGCCC GGACGGCACACCCTACGTTACCGTGCTCAA GACGGCGGGCGCTAACACCACCGACAAGGA GCTAGAGGTTCTCTCCTTGCACAACGTCAC CTTTGAGGACGCCGGGGAGTACACCTGCCT GGCGGGCAATTCTATTGGGTTTTCTCATCA CTCTGCGTGGCTGGTGGTGCTGCCAGCCGA GGAGGAGCTGGTGGAGGCTGACGAGGCGGG CAGTGTGTATGCAGGCATCCTCAGCTACGG GGTGGGCTTCTTCCTGTTCATCCTGGTGGT GGCGGCTGTGACGCTCTGCCGCCTGCGCAG CCCCCCCAAGAAAGGCCTGGGCTCCCCCAC CGTGCACAAGATCTCCCGCTTCCCGCTCAA GCGACAGGTGTCCCTGGAGTCCAACGCGTC CATGAGCTCCAACACACCACTGGTGCGCAT CGCAAGGCTGTCCTCAGGGGAGGGCCCCAC GCTGGCCAATGTCTCCGAGCTCGAGCTGCC TGCCGACCCCAAATGGGAGCTGTCTCGGGC CCGGCTGACCCTGGGCAAGCCCCTTGGGGA GGGCTGCTTCGGCCAGGTGGTCATGGCGGA GGCCATCGGCATTGACAAGGACCGGGCCGC CAAGCCTGTCACCGTAGCCGTGAAGATGCT GAAAGACGATGCCACTGACAAGGACCTGTC GGACCTGGTGTCTGAGATGGAGATGATGAA GATGATCGGGAAACACAAAAACATCATCAA CCTGCTGGGCGCCTGCACGCAGGGCGGGCC CCTGTACGTGCTGGTGGAGTACGCGGCCAA GGGTAACCTGCGGGAGTTTCTGCGGGCGCG GCGGCCCCCGGGCCTGGACTACTCCTTCGA CACCTGCAAGCCGCCCGAGGAGCAGCTCAC CTTCAAGGACCTGGTGTCCTGTGCCTACCA GGTGGCCCGGGGCATGGAGTACTTGGCCTC CCAGAAGTGCATCCACAGGGACCTGGCTGC CCGCAATGTGCTGGTGACCGAGGACAACGT GATGAAGATCGCAGACTTCGGGCTGGCCCG GGACGTGCACAACCTCGACTACTACAAGAA GACGACCAACGGCCGGCTGCCCGTGAAGTG GATGGCGCCTGAGGCCTTGTTTGACCGAGT CTACACTCACCAGAGTGACGTCTGGTCCTT TGGGGTCCTGCTCTGGGAGATCTTCACGCT GGGGGGCTCCCCGTACCCCGGCATCCCTGT GGAGGAGCTCTTCAAGCTGCTGAAGGAGGG CCACCGCATGGACAAGCCCGCCAACTGCAC ACACGACCTGTACATGATCATGCGGGAGTG CTGGCATGCCGCGCCCTCCCAGAGGCCCAC CTTCAAGCAGCTGGTGGAGGACCTGGACCG TGTCCTTACCGTGACGTCCACCGACGTAAA GGCGACACAGGAGGAGAACCGGGAGCTGAG GAGCAGGTGTGAGGAGCTCCACGGGAAGAA CCTGGAACTGGGGAAGATCATGGACAGGTT CGAAGAGGTTGTGTACCAGGCCATGGAGGA AGTTCAGAAGCAGAAGGAACTTTCCAAAGC TGAAATCCAGAAAGTTCTAAAAGAAAAAGA CCAACTTACCACAGATCTGAACTCCATGGA GAAGTCCTTCTCCGACCTCTTCAAGCGTTT TGAGAAACAGAAAGAGGTGATCGAGGGCTA CCGCAAGAACGAAGAGTCACTGAAGAAGTG CGTGGAGGATTACCTGGCAAGGATCACCCA GGAGGGCCAGAGGTACCAAGCCCTGAAGGC CCACGCGGAGGAGAAGCTGCAGCTGGCAAA CGAGGAGATCGCCCAGGTCCGGAGCAAGGC CCAGGCGGAAGCGTTGGCCCTCCAGGCCAG CCTGAGGAAGGAGCAGATGCGCATCCAGTC GCTGGAGAAGACAGTGGAGCAGAAGACTAA AGAGAACGAGGAGCTGACCAGGATCTGCGA CGACCTCATCTCCAAGATGGAGAAGATCTG A FGFR3:TACC3 v3 >ATGGGCGCCCCTGCCTGCGCCCTCGCGCT (3376 base pairs) CTGCGTGGCCGTGGCCATCGTGGCCGGCGC (SEQ ID NO: 57) CTCCTCGGAGTCCTTGGGGACGGAGCAGCG CGTCGTGGGGCGAGCGGCAGAAGTCCCGGG CCCAGAGCCCGGCCAGCAGGAGCAGTTGGT CTTCGGCAGCGGGGATGCTGTGGAGCTGAG CTGTCCCCCGCCCGGGGGTGGTCCCATGGG GCCCACTGTCTGGGTCAAGGATGGCACAGG GCTGGTGCCCTCGGAGCGTGTCCTGGTGGG GCCCCAGCGGCTGCAGGTGCTGAATGCCTC CCACGAGGACTCCGGGGCCTACAGCTGCCG GCAGCGGCTCACGCAGCGCGTACTGTGCCA CTTCAGTGTGCGGGTGACAGACGCTCCATC CTCGGGAGATGACGAAGACGGGGAGGACGA GGCTGAGGACACAGGTGTGGACACAGGGGC CCCTTACTGGACACGGCCCGAGCGGATGGA CAAGAAGCTGCTGGCCGTGCCGGCCGCCAA CACCGTCCGCTTCCGCTGCCCAGCCGCTGG CAACCCCACTCCCTCCATCTCCTGGCTGAA GAACGGCAGGGAGTTCCGCGGCGAGCACCG CATTGGAGGCATCAAGCTGCGGCATCAGCA GTGGAGCCTGGTCATGGAAAGCGTGGTGCC CTCGGACCGCGGCAACTACACCTGCGTCGT GGAGAACAAGTTTGGCAGCATCCGGCAGAC GTACACGCTGGACGTGCTGGAGCGCTCCCC GCACCGGCCCATCCTGCAGGCGGGGCTGCC GGCCAACCAGACGGCGGTGCTGGGCAGCGA CGTGGAGTTCCACTGCAAGGTGTACAGTGA CGCACAGCCCCACATCCAGTGGCTCAAGCA CGTGGAGGTGAATGGCAGCAAGGTGGGCCC GGACGGCACACCCTACGTTACCGTGCTCAA GACGGCGGGCGCTAACACCACCGACAAGGA GCTAGAGGTTCTCTCCTTGCACAACGTCAC CTTTGAGGACGCCGGGGAGTACACCTGCCT GGCGGGCAATTCTATTGGGTTTTCTCATCA CTCTGCGTGGCTGGTGGTGCTGCCAGCCGA GGAGGAGCTGGTGGAGGCTGACGAGGCGGG CAGTGTGTATGCAGGCATCCTCAGCTACGG GGTGGGCTTCTTCCTGTTCATCCTGGTGGT GGCGGCTGTGACGCTCTGCCGCCTGCGCAG CCCCCCCAAGAAAGGCCTGGGCTCCCCCAC CGTGCACAAGATCTCCCGCTTCCCGCTCAA GCGACAGGTGTCCCTGGAGTCCAACGCGTC CATGAGCTCCAACACACCACTGGTGCGCAT CGCAAGGCTGTCCTCAGGGGAGGGCCCCAC GCTGGCCAATGTCTCCGAGCTCGAGCTGCC TGCCGACCCCAAATGGGAGCTGTCTCGGGC CCGGCTGACCCTGGGCAAGCCCCTTGGGGA GGGCTGCTTCGGCCAGGTGGTCATGGCGGA GGCCATCGGCATTGACAAGGACCGGGCCGC CAAGCCTGTCACCGTAGCCGTGAAGATGCT GAAAGACGATGCCACTGACAAGGACCTGTC GGACCTGGTGTCTGAGATGGAGATGATGAA GATGATCGGGAAACACAAAAACATCATCAA CCTGCTGGGCGCCTGCACGCAGGGCGGGCC CCTGTACGTGCTGGTGGAGTACGCGGCCAA GGGTAACCTGCGGGAGTTTCTGCGGGCGCG GCGGCCCCCGGGCCTGGACTACTCCTTCGA CACCTGCAAGCCGCCCGAGGAGCAGCTCAC CTTCAAGGACCTGGTGTCCTGTGCCTACCA GGTGGCCCGGGGCATGGAGTACTTGGCCTC CCAGAAGTGCATCCACAGGGACCTGGCTGC CCGCAATGTGCTGGTGACCGAGGACAACGT GATGAAGATCGCAGACTTCGGGCTGGCCCG GGACGTGCACAACCTCGACTACTACAAGAA GACGACCAACGGCCGGCTGCCCGTGAAGTG GATGGCGCCTGAGGCCTTGTTTGACCGAGT CTACACTCACCAGAGTGACGTCTGGTCCTT TGGGGTCCTGCTCTGGGAGATCTTCACGCT GGGGGGCTCCCCGTACCCCGGCATCCCTGT GGAGGAGCTCTTCAAGCTGCTGAAGGAGGG CCACCGCATGGACAAGCCCGCCAACTGCAC ACACGACCTGTACATGATCATGCGGGAGTG CTGGCATGCCGCGCCCTCCCAGAGGCCCAC CTTCAAGCAGCTGGTGGAGGACCTGGACCG TGTCCTTACCGTGACGTCCACCGACGTGCC AGGCCCACCCCCAGGTGTTCCCGCGCCTGG GGGCCCACCCCTGTCCACCGGACCTATAGT GGACCTGCTCCAGTACAGCCAGAAGGACCT GGATGCAGTGGTAAAGGCGACACAGGAGGA GAACCGGGAGCTGAGGAGCAGGTGTGAGGA GCTCCACGGGAAGAACCTGGAACTGGGGAA GATCATGGACAGGTTCGAAGAGGTTGTGTA CCAGGCCATGGAGGAAGTTCAGAAGCAGAA GGAACTTTCCAAAGCTGAAATCCAGAAAGT TCTAAAAGAAAAAGACCAACTTACCACAGA TCTGAACTCCATGGAGAAGTCCTTCTCCGA CCTCTTCAAGCGTTTTGAGAAACAGAAAGA GGTGATCGAGGGCTACCGCAAGAACGAAGA GTCACTGAAGAAGTGCGTGGAGGATTACCT GGCAAGGATCACCCAGGAGGGCCAGAGGTA CCAAGCCCTGAAGGCCCACGCGGAGGAGAA GCTGCAGCTGGCAAACGAGGAGATCGCCCA GGTCCGGAGCAAGGCCCAGGCGGAAGCGTT GGCCCTCCAGGCCAGCCTGAGGAAGGAGCA GATGCGCATCCAGTCGCTGGAGAAGACAGT GGAGCAGAAGACTAAAGAGAACGAGGAGCT GACCAGGATCTGCGACGACCTCATCTCCAA GATGGAGAAGATCTGA FGFR3 Intron: >ATGGGCGCCCCTGCCTGCGCCCTCGCGCT TACC3 CTGCGTGGCCGTGGCCATCGTGGCCGGCGC (4463 base pairs) CTCCTCGGAGTCCTTGGGGACGGAGCAGCG (SEQ ID NO: 58) CGTCGTGGGGCGAGCGGCAGAAGTCCCGGG CCCAGAGCCCGGCCAGCAGGAGCAGTTGGT CTTCGGCAGCGGGGATGCTGTGGAGCTGAG CTGTCCCCCGCCCGGGGGTGGTCCCATGGG GCCCACTGTCTGGGTCAAGGATGGCACAGG GCTGGTGCCCTCGGAGCGTGTCCTGGTGGG GCCCCAGCGGCTGCAGGTGCTGAATGCCTC CCACGAGGACTCCGGGGCCTACAGCTGCCG GCAGCGGCTCACGCAGCGCGTACTGTGCCA CTTCAGTGTGCGGGTGACAGACGCTCCATC CTCGGGAGATGACGAAGACGGGGAGGACGA GGCTGAGGACACAGGTGTGGACACAGGGGC CCCTTACTGGACACGGCCCGAGCGGATGGA CAAGAAGCTGCTGGCCGTGCCGGCCGCCAA CACCGTCCGCTTCCGCTGCCCAGCCGCTGG CAACCCCACTCCCTCCATCTCCTGGCTGAA GAACGGCAGGGAGTTCCGCGGCGAGCACCG CATTGGAGGCATCAAGCTGCGGCATCAGCA GTGGAGCCTGGTCATGGAAAGCGTGGTGCC CTCGGACCGCGGCAACTACACCTGCGTCGT GGAGAACAAGTTTGGCAGCATCCGGCAGAC GTACACGCTGGACGTGCTGGAGCGCTCCCC GCACCGGCCCATCCTGCAGGCGGGGCTGCC GGCCAACCAGACGGCGGTGCTGGGCAGCGA CGTGGAGTTCCACTGCAAGGTGTACAGTGA CGCACAGCCCCACATCCAGTGGCTCAAGCA CGTGGAGGTGAATGGCAGCAAGGTGGGCCC GGACGGCACACCCTACGTTACCGTGCTCAA GACGGCGGGCGCTAACACCACCGACAAGGA GCTAGAGGTTCTCTCCTTGCACAACGTCAC CTTTGAGGACGCCGGGGAGTACACCTGCCT GGCGGGCAATTCTATTGGGTTTTCTCATCA CTCTGCGTGGCTGGTGGTGCTGCCAGCCGA GGAGGAGCTGGTGGAGGCTGACGAGGCGGG CAGTGTGTATGCAGGCATCCTCAGCTACGG GGTGGGCTTCTTCCTGTTCATCCTGGTGGT GGCGGCTGTGACGCTCTGCCGCCTGCGCAG CCCCCCCAAGAAAGGCCTGGGCTCCCCCAC CGTGCACAAGATCTCCCGCTTCCCGCTCAA GCGACAGGTGTCCCTGGAGTCCAACGCGTC CATGAGCTCCAACACACCACTGGTGCGCAT CGCAAGGCTGTCCTCAGGGGAGGGCCCCAC GCTGGCCAATGTCTCCGAGCTCGAGCTGCC TGCCGACCCCAAATGGGAGCTGTCTCGGGC CCGGCTGACCCTGGGCAAGCCCCTTGGGGA GGGCTGCTTCGGCCAGGTGGTCATGGCGGA GGCCATCGGCATTGACAAGGACCGGGCCGC CAAGCCTGTCACCGTAGCCGTGAAGATGCT

GAAAGACGATGCCACTGACAAGGACCTGTC GGACCTGGTGTCTGAGATGGAGATGATGAA GATGATCGGGAAACACAAAAACATCATCAA CCTGCTGGGCGCCTGCACGCAGGGCGGGCC CCTGTACGTGCTGGTGGAGTACGCGGCCAA GGGTAACCTGCGGGAGTTTCTGCGGGCGCG GCGGCCCCCGGGCCTGGACTACTCCTTCGA CACCTGCAAGCCGCCCGAGGAGCAGCTCAC CTTCAAGGACCTGGTGTCCTGTGCCTACCA GGTGGCCCGGGGCATGGAGTACTTGGCCTC CCAGAAGTGCATCCACAGGGACCTGGCTGC CCGCAATGTGCTGGTGACCGAGGACAACGT GATGAAGATCGCAGACTTCGGGCTGGCCCG GGACGTGCACAACCTCGACTACTACAAGAA GACGACCAACGGCCGGCTGCCCGTGAAGTG GATGGCGCCTGAGGCCTTGTTTGACCGAGT CTACACTCACCAGAGTGACGTCTGGTCCTT TGGGGTCCTGCTCTGGGAGATCTTCACGCT GGGGGGCTCCCCGTACCCCGGCATCCCTGT GGAGGAGCTCTTCAAGCTGCTGAAGGAGGG CCACCGCATGGACAAGCCCGCCAACTGCAC ACACGACCTGTACATGATCATGCGGGAGTG CTGGCATGCCGCGCCCTCCCAGAGGCCCAC CTTCAAGCAGCTGGTGGAGGACCTGGACCG TGTCCTTACCGTGACGTCCACCGACgtgag tgctggctctggcctggtgccacccgccta tgcccctccccctgccgtccccggccatcc tgccccccagagtgctgaggtgtggggcgg gccttTCTGGCCCAGGTGCCCTGGCTGACC TGGACTGCTCAAGCTCTTCCCAGAGCCCAG GAAGTTCTGAGAACCAAATGGTGTCTCCAG GAAAAGTGTCTGGCAGCCCTGAGCAAGCCG TGGAGGAAAACCTTAGTTCCTATTCCTTAG ACAGAAGAGTGACACCCGCCTCTGAGACCC TAGAAGACCCTTGCAGGACAGAGTCCCAGC ACAAAGCGGAGACTCCGCACGGAGCCGAGG AAGAATGCAAAGCGGAGACTCCGCACGGAG CCGAGGAGGAATGCCGGCACGGTGGGGTCT GTGCTCCCGCAGCAGTGGCCACTTCGCCTC CTGGTGCAATCCCTAAGGAAGCCTGCGGAG GAGCACCCCTGCAGGGTCTGCCTGGCGAAG CCCTGGGCTGCCCTGCGGGTGTGGGCACCC CCGTGCCAGCAGATGGCACTCAGACCCTTA CCTGTGCACACACCTCTGCTCCTGAGAGCA CAGCCCCAACCAACCACCTGGTGGCTGGCA GGGCCATGACCCTGAGTCCTCAGGAAGAAG TGGCTGCAGGCCAAATGGCCAGCTCCTCGA GGAGCGGACCTGTAAAACTAGAATTTGATG TATCTGATGGCGCCACCAGCAAAAGGGCAC CCCCACCAAGGAGACTGGGAGAGAGGTCCG GCCTCAAGCCTCCCTTGAGGAAAGCAGCAG TGAGGCAGCAAAAGGCCCCGCAGGAGGTGG AGGAGGACGACGGTAGGAGCGGAGCAGGAG AGGACCCCCCCATGCCAGCTTCTCGGGGCT CTTACCACCTCGACTGGGACAAAATGGATG ACCCAAACTTCATCCCGTTCGGAGGTGACA CCAAGTCTGGTTGCAGTGAGGCCCAGCCCC CAGAAAGCCCTGAGACCAGGCTGGGCCAGC CAGCGGCTGAACAGTTGCATGCTGGGCCTG CCACGGAGGAGCCAGGTCCCTGTCTGAGCC AGCAGCTGCATTCAGCCTCAGCGGAGGACA CGCCTGTGGTGCAGTTGGCAGCCGAGACCC CAACAGCAGAGAGCAAGGAGAGAGCCTTGA ACTCTGCCAGCACCTCGCTTCCCACAAGCT GTCCAGGCAGTGAGCCAGTGCCCACCCATC AGCAGGGGCAGCCTGCCTTGGAGCTGAAAG AGGAGAGCTTCAGAGACCCCGCTGAGGTTC TAGGCACGGGCGCGGAGGTGGATTACCTGG AGCAGTTTGGAACTTCCTCGTTTAAGGAGT CGGCCTTGAGGAAGCAGTCCTTATACCTCA AGTTCGACCCCCTCCTGAGGGACAGTCCTG GTAGACCAGTGCCCGTGGCCACCGAGACCA GCAGCATGCACGGTGCAAATGAGACTCCCT CAGGACGTCCGCGGGAAGCCAAGCTTGTGG AGTTCGATTTCTTGGGAGCACTGGACATTC CTGTGCCAGGCCCACCCCCAGGTGTTCCCG CGCCTGGGGGCCCACCCCTGTCCACCGGAC CTATAGTGGACCTGCTCCAGTACAGCCAGA AGGACCTGGATGCAGTGGTAAAGGCGACAC AGGAGGAGAACCGGGAGCTGAGGAGCAGGT GTGAGGAGCTCCACGGGAAGAACCTGGAAC TGGGGAAGATCATGGACAGGTTCGAAGAGG TTGTGTACCAGGCCATGGAGGAAGTTCAGA AGCAGAAGGAACTTTCCAAAGCTGAAATCC AGAAAGTTCTAAAAGAAAAAGACCAACTTA CCACAGATCTGAACTCCATGGAGAAGTCCT TCTCCGACCTCTTCAAGCGTTTTGAGAAAC AGAAAGAGGTGATCGAGGGCTACCGCAAGA ACGAAGAGTCACTGAAGAAGTGCGTGGAGG ATTACCTGGCAAGGATCACCCAGGAGGGCC AGAGGTACCAAGCCCTGAAGGCCCACGCGG AGGAGAAGCTGCAGCTGGCAAACGAGGAGA TCGCCCAGGTCCGGAGCAAGGCCCAGGCGG AAGCGTTGGCCCTCCAGGCCAGCCTGAGGA AGGAGCAGATGCGCATCCAGTCGCTGGAGA AGACAGTGGAGCAGAAGACTAAAGAGAACG AGGAGCTGACCAGGATCTGCGACGACCTCA TCTCCAAGATGGAGAAGATCTGA FGFR3:BAIAP2L1 >ATGGGCGCCCCTGCCTGCGCCCTCGCGCT (3765 base pairs) CTGCGTGGCCGTGGCCATCGTGGCCGGCGC (SEQ ID NO: 59) CTCCTCGGAGTCCTTGGGGACGGAGCAGCG CGTCGTGGGGCGAGCGGCAGAAGTCCCGGG CCCAGAGCCCGGCCAGCAGGAGCAGTTGGT CTTCGGCAGCGGGGATGCTGTGGAGCTGAG CTGTCCCCCGCCCGGGGGTGGTCCCATGGG GCCCACTGTCTGGGTCAAGGATGGCACAGG GCTGGTGCCCTCGGAGCGTGTCCTGGTGGG GCCCCAGCGGCTGCAGGTGCTGAATGCCTC CCACGAGGACTCCGGGGCCTACAGCTGCCG GCAGCGGCTCACGCAGCGCGTACTGTGCCA CTTCAGTGTGCGGGTGACAGACGCTCCATC CTCGGGAGATGACGAAGACGGGGAGGACGA GGCTGAGGACACAGGTGTGGACACAGGGGC CCCTTACTGGACACGGCCCGAGCGGATGGA CAAGAAGCTGCTGGCCGTGCCGGCCGCCAA CACCGTCCGCTTCCGCTGCCCAGCCGCTGG CAACCCCACTCCCTCCATCTCCTGGCTGAA GAACGGCAGGGAGTTCCGCGGCGAGCACCG CATTGGAGGCATCAAGCTGCGGCATCAGCA GTGGAGCCTGGTCATGGAAAGCGTGGTGCC CTCGGACCGCGGCAACTACACCTGCGTCGT GGAGAACAAGTTTGGCAGCATCCGGCAGAC GTACACGCTGGACGTGCTGGAGCGCTCCCC GCACCGGCCCATCCTGCAGGCGGGGCTGCC GGCCAACCAGACGGCGGTGCTGGGCAGCGA CGTGGAGTTCCACTGCAAGGTGTACAGTGA CGCACAGCCCCACATCCAGTGGCTCAAGCA CGTGGAGGTGAATGGCAGCAAGGTGGGCCC GGACGGCACACCCTACGTTACCGTGCTCAA GTCCTGGATCAGTGAGAGTGTGGAGGCCGA CGTGCGCCTCCGCCTGGCCAATGTGTCGGA GCGGGACGGGGGCGAGTACCTCTGTCGAGC CACCAATTTCATAGGCGTGGCCGAGAAGGC CTTTTGGCTGAGCGTTCACGGGCCCCGAGC AGCCGAGGAGGAGCTGGTGGAGGCTGACGA GGCGGGCAGTGTGTATGCAGGCATCCTCAG CTACGGGGTGGGCTTCTTCCTGTTCATCCT GGTGGTGGCGGCTGTGACGCTCTGCCGCCT GCGCAGCCCCCCCAAGAAAGGCCTGGGCTC CCCCACCGTGCACAAGATCTCCCGCTTCCC GCTCAAGCGACAGGTGTCCCTGGAGTCCAA CGCGTCCATGAGCTCCAACACACCACTGGT GCGCATCGCAAGGCTGTCCTCAGGGGAGGG CCCCACGCTGGCCAATGTCTCCGAGCTCGA GCTGCCTGCCGACCCCAAATGGGAGCTGTC TCGGGCCCGGCTGACCCTGGGCAAGCCCCT TGGGGAGGGCTGCTTCGGCCAGGTGGTCAT GGCGGAGGCCATCGGCATTGACAAGGACCG GGCCGCCAAGCCTGTCACCGTAGCCGTGAA GATGCTGAAAGACGATGCCACTGACAAGGA CCTGTCGGACCTGGTGTCTGAGATGGAGAT GATGAAGATGATCGGGAAACACAAAAACAT CATCAACCTGCTGGGCGCCTGCACGCAGGG CGGGCCCCTGTACGTGCTGGTGGAGTACGC GGCCAAGGGTAACCTGCGGGAGTTTCTGCG GGCGCGGCGGCCCCCGGGCCTGGACTACTC CTTCGACACCTGCAAGCCGCCCGAGGAGCA GCTCACCTTCAAGGACCTGGTGTCCTGTGC CTACCAGGTGGCCCGGGGCATGGAGTACTT GGCCTCCCAGAAGTGCATCCACAGGGACCT GGCTGCCCGCAATGTGCTGGTGACCGAGGA CAACGTGATGAAGATCGCAGACTTCGGGCT GGCCCGGGACGTGCACAACCTCGACTACTA CAAGAAGACGACCAACGGCCGGCTGCCCGT GAAGTGGATGGCGCCTGAGGCCTTGTTTGA CCGAGTCTACACTCACCAGAGTGACGTCTG GTCCTTTGGGGTCCTGCTCTGGGAGATCTT CACGCTGGGGGGCTCCCCGTACCCCGGCAT CCCTGTGGAGGAGCTCTTCAAGCTGCTGAA GGAGGGCCACCGCATGGACAAGCCCGCCAA CTGCACACACGACCTGTACATGATCATGCG GGAGTGCTGGCATGCCGCGCCCTCCCAGAG GCCCACCTTCAAGCAGCTGGTGGAGGACCT GGACCGTGTCCTTACCGTGACGTCCACCGA CAATGTTATGGAACAGTTCAATCCTGGGCT GCGAAATTTAATAAACCTGGGGAAAAATTA TGAGAAAGCTGTAAACGCTATGATCCTGGC AGGAAAAGCCTACTACGATGGAGTGGCCAA GATCGGTGAGATTGCCACTGGGTCCCCCGT GTCAACTGAACTGGGACATGTCCTCATAGA GATTTCAAGTACCCACAAGAAACTCAACGA GAGTCTTGATGAAAATTTTAAAAAATTCCA CAAAGAGATTATCCATGAGCTGGAGAAGAA GATAGAACTTGACGTGAAATATATGAACGC AACTCTAAAAAGATACCAAACAGAACACAA GAATAAATTAGAGTCTTTGGAGAAATCCCA AGCTGAGTTGAAGAAGATCAGAAGGAAAAG CCAAGGAAGCCGAAACGCACTCAAATATGA ACACAAAGAAATTGAGTATGTGGAGACCGT TACTTCTCGTCAGAGTGAAATCCAGAAATT CATTGCAGATGGTTGCAAAGAGGCTCTGCT TGAAGAGAAGAGGCGCTTCTGCTTTCTGGT TGATAAGCACTGTGGCTTTGCAAACCACAT ACATTATTATCACTTACAGTCTGCAGAACT ACTGAATTCCAAGCTGCCTCGGTGGCAGGA GACCTGTGTTGATGCCATCAAAGTGCCAGA GAAAATCATGAATATGATCGAAGAAATAAA GACCCCAGCCTCTACCCCCGTGTCTGGAAC TCCTCAGGCTTCACCCATGATCGAGAGAAG CAATGTGGTTAGGAAAGATTACGACACCCT TTCTAAATGCTCACCAAAGATGCCCCCCGC TCCTTCAGGCAGAGCATATACCAGTCCCTT GATCGATATGTTTAATAACCCAGCCACGGC TGCCCCGAATTCACAAAGGGTAAATAATTC AACAGGTACTTCCGAAGATCCCAGTTTACA GCGATCAGTTTCGGTTGCAACGGGACTGAA CATGATGAAGAAGCAGAAAGTGAAGACCAT CTTCCCGCACACTGCGGGCTCCAACAAGAC CTTACTCAGCTTTGCACAGGGAGATGTCAT CACGCTGCTCATCCCCGAGGAGAAGGATGG CTGGCTCTATGGAGAACACGACGTGTCCAA GGCGAGGGGTTGGTTCCCGTCGTCGTACAC GAAGTTGCTGGAAGAAAATGAGACAGAAGC AGTGACCGTGCCCACGCCAAGCCCCACACC AGTGAGAAGCATCAGCACCGTGAACTTGTC TGAGAATAGCAGTGTTGTCATCCCCCCACC CGACTACTTGGAATGCTTGTCCATGGGGGC AGCTGCCGACAGGAGAGCAGATTCGGCCAG GACGACATCCACCTTTAAGGCCCCAGCGTC CAAGCCCGAGACCGCGGCTCCTAACGATGC CAACGGGACTGCAAAGCCGCCTTTTCTCAG CGGAGAAAACCCCTTTGCCACTGTGAAACT CCGCCCGACTGTGACGAATGATCGCTCGGC ACCCATCATTCGATGA FGFR2:BICC1 >ATGGTCAGCTGGGGTCGTTTCATCTGCCT (5830 base pairs) GGTCGTGGTCACCATGGCAACCTTGTCCCT (SEQ ID NO: 60) GGCCCGGCCCTCCTTCAGTTTAGTTGAGGA TACCACATTAGAGCCAGAAGAGCCACCAAC CAAATACCAAATCTCTCAACCAGAAGTGTA CGTGGCTGCGCCAGGGGAGTCGCTAGAGGT GCGCTGCCTGTTGAAAGATGCCGCCGTGAT CAGTTGGACTAAGGATGGGGTGCACTTGGG GCCCAACAATAGGACAGTGCTTATTGGGGA GTACTTGCAGATAAAGGGCGCCACGCCTAG AGACTCCGGCCTCTATGCTTGTACTGCCAG TAGGACTGTAGACAGTGAAACTTGGTACTT CATGGTGAATGTCACAGATGCCATCTCATC CGGAGATGATGAGGATGACACCGATGGTGC GGAAGATTTTGTCAGTGAGAACAGTAACAA CAAGAGAGCACCATACTGGACCAACACAGA AAAGATGGAAAAGCGGCTCCATGCTGTGCC TGCGGCCAACACTGTCAAGTTTCGCTGCCC AGCCGGGGGGAACCCAATGCCAACCATGCG GTGGCTGAAAAACGGGAAGGAGTTTAAGCA GGAGCATCGCATTGGAGGCTACAAGGTACG AAACCAGCACTGGAGCCTCATTATGGAAAG TGTGGTCCCATCTGACAAGGGAAATTATAC CTGTGTAGTGGAGAATGAATACGGGTCCAT CAATCACACGTACCACCTGGATGTTGTGGA

GCGATCGCCTCACCGGCCCATCCTCCAAGC CGGACTGCCGGCAAATGCCTCCACAGTGGT CGGAGGAGACGTAGAGTTTGTCTGCAAGGT TTACAGTGATGCCCAGCCCCACATCCAGTG GATCAAGCACGTGGAAAAGAACGGCAGTAA ATACGGGCCCGACGGGCTGCCCTACCTCAA GGTTCTCAAGGCCGCCGGTGTTAACACCAC GGACAAAGAGATTGAGGTTCTCTATATTCG GAATGTAACTTTTGAGGACGCTGGGGAATA TACGTGCTTGGCGGGTAATTCTATTGGGAT ATCCTTTCACTCTGCATGGTTGACAGTTCT GCCAGCGCCTGGAAGAGAAAAGGAGATTAC AGCTTCCCCAGACTACCTGGAGATAGCCAT TTACTGCATAGGGGTCTTCTTAATCGCCTG TATGGTGGTAACAGTCATCCTGTGCCGAAT GAAGAACACGACCAAGAAGCCAGACTTCAG CAGCCAGCCGGCTGTGCACAAGCTGACCAA ACGTATCCCCCTGCGGAGACAGGTAACAGT TTCGGCTGAGTCCAGCTCCTCCATGAACTC CAACACCCCGCTGGTGAGGATAACAACACG CCTCTCTTCAACGGCAGACACCCCCATGCT GGCAGGGGTCTCCGAGTATGAACTTCCAGA GGACCCAAAATGGGAGTTTCCAAGAGATAA GCTGACACTGGGCAAGCCCCTGGGAGAAGG TTGCTTTGGGCAAGTGGTCATGGCGGAAGC AGTGGGAATTGACAAAGACAAGCCCAAGGA GGCGGTCACCGTGGCCGTGAAGATGTTGAA AGATGATGCCACAGAGAAAGACCTTTCTGA TCTGGTGTCAGAGATGGAGATGATGAAGAT GATTGGGAAACACAAGAATATCATAAATCT TCTTGGAGCCTGCACACAGGATGGGCCTCT CTATGTCATAGTTGAGTATGCCTCTAAAGG CAACCTCCGAGAATACCTCCGAGCCCGGAG GCCACCCGGGATGGAGTACTCCTATGACAT TAACCGTGTTCCTGAGGAGCAGATGACCTT CAAGGACTTGGTGTCATGCACCTACCAGCT GGCCAGAGGCATGGAGTACTTGGCTTCCCA AAAATGTATTCATCGAGATTTAGCAGCCAG AAATGTTTTGGTAACAGAAAACAATGTGAT GAAAATAGCAGACTTTGGACTCGCCAGAGA TATCAACAATATAGACTATTACAAAAAGAC CACCAATGGGCGGCTTCCAGTCAAGTGGAT GGCTCCAGAAGCCCTGTTTGATAGAGTATA CACTCATCAGAGTGATGTCTGGTCCTTCGG GGTGTTAATGTGGGAGATCTTCACTTTAGG GGGCTCGCCCTACCCAGGGATTCCCGTGGA GGAACTTTTTAAGCTGCTGAAGGAAGGACA CAGAATGGATAAGCCAGCCAACTGCACCAA CGAACTGTACATGATGATGAGGGACTGTTG GCATGCAGTGCCCTCCCAGAGACCAACGTT CAAGCAGTTGGTAGAAGACTTGGATCGAAT TCTCACTCTCACAACCAATGAGATCATGGA GGAAACAAATACGCAGATTGCTTGGCCATC AAAACTGAAGATCGGAGCCAAATCCAAGAA AGATCCCCATATTAAGGTTTCTGGAAAGAA AGAAGATGTTAAAGAAGCCAAGGAAATGAT CATGTCTGTCTTAGACACAAAAAGCAATCG AGTCACACTGAAGATGGATGTTTCACATAC AGAACATTCACATGTAATCGGCAAAGGTGG CAACAATATTAAAAAAGTGATGGAAGAAAC CGGATGCCATATCCACTTTCCAGATTCCAA CAGGAATAACCAAGCAGAAAAAAGCAACCA GGTATCTATAGCGGGACAACCAGCAGGAGT AGAATCTGCCCGAGTTAGAATTCGGGAGCT GCTTCCTTTGGTGCTGATGTTTGAGCTACC AATTGCTGGAATTCTTCAACCGGTTCCTGA TCCTAATTCCCCCTCTATTCAGCATATATC ACAAACGTACAATATTTCAGTATCATTTAA ACAGCGTTCCCGAATGTATGGTGCTACTGT CATAGTACGAGGGTCTCAGAATAACACTAG TGCTGTGAAGGAAGGAACTGCCATGCTGTT AGAACATCTTGCTGGGAGCTTAGCATCAGC TATTCCTGTGAGCACACAACTAGATATTGC AGCTCAACATCATCTCTTTATGATGGGTCG AAATGGGAGCAACATCAAACATATCATGCA GAGAACAGGTGCTCAGATCCACTTTCCTGA TCCCAGTAATCCACAAAAGAAATCTACCGT CTACCTCCAGGGCACCATTGAGTCTGTCTG TCTTGCAAGGCAATATCTCATGGGTTGTCT TCCTCTTGTGTTGATGTTTGATATGAAGGA AGAAATTGAAGTAGATCCACAATTCATTGC GCAGTTGATGGAACAGCTTGATGTCTTCAT CAGTATTAAACCAAAGCCCAAACAGCCAAG CAAGTCTGTGATTGTGAAAAGTGTTGAGCG AAATGCCTTAAATATGTATGAAGCAAGGAA ATGTCTCCTCGGACTTGAAAGCAGTGGGGT TACCATAGCAACCAGTCCATCCCCAGCATC CTGCCCTGCCGGCCTGGCATGTCCCAGCCT GGATATCTTAGCTTCAGCAGGCCTTGGACT CACTGGACTAGGTCTTTTGGGACCCACCAC CTTATCTCTGAACACTTCAACAACCCCAAA CTCACTCTTGAATGCTCTTAATAGCTCAGT CAGTCCTTTGCAAAGTCCAAGTTCTGGTAC ACCCAGCCCCACATTATGGGCACCCCCACT TGCTAATACTTCAAGTGCCACAGGTTTTTC TGCTATACCACACCTTATGATTCCATCTAC TGCCCAAGCCACATTAACTAATATTTTGTT GTCTGGAGTGCCCACCTATGGGCACACAGC TCCATCTCCCCCTCCTGGCTTGACTCCTGT TGATGTCCATATCAACAGTATGCAGACCGA AGGCAAAAAAATCTCTGCTGCTTTAAATGG ACATGCACAGTCTCCAGATATAAAATATGG TGCAATATCCACTTCATCACTTGGAGAAAA AGTGCTGAGTGCAAATCACGGGGATCCGTC CATCCAGACAAGTGGGTCTGAGCAGACATC TCCCAAATCAAGCCCCACTGAAGGTTGTAA TGATGCTTTTGTTGAAGTAGGCATGCCTCG AAGTCCTTCCCATTCTGGGAATGCTGGTGA CTTGAAACAGATGATGTGTCCCTCCAAGGT TTCCTGTGCCAAAAGGCAGACAGTGGAACT ATTGCAAGGCACGAAAAACTCACACTTACA CAGCACTGACAGGTTGCTCTCAGACCCTGA ACTGAGTGCTACCGAAAGCCCTTTGGCTGA CAAGAAGGCTCCAGGGAGTGAGCGCGCTGC AGAGAGGGCAGCAGCTGCCCAGCAAAACTC CGAAAGGGCCCACCTTGCTCCACGGTCATC ATATGTCAACATGCAGGCATTTGACTATGA ACAGAAGAAGCTATTAGCCACCAAAGCTAT GTTAAAGAAACCAGTGGTGACGGAGGTCAG AACGCCCACAAATACCTGGAGTGGCCTGGG TTTTTCTAAATCCATGCCAGCTGAAACTAT CAAGGAGTTGAGAAGGGCCAATCATGTGTC CTATAAGCCCACAATGACAACCACTTATGA GGGCTCATCCATGTCCCTTTCACGGTCCAA CAGTCGTGAGCACTTGGGAGGTGGAAGCGA ATCTGATAACTGGAGAGACCGAAATGGAAT TGGACCTGGAAGTCATAGTGAATTTGCAGC TTCTATTGGCAGCCCTAAGCGTAAACAAAA CAAATCAACGGAACACTATCTCAGCAGTAG CAATTACATGGACTGCATTTCCTCGCTGAC AGGAAGCAATGGCTGTAACTTAAATAGCTC TTTCAAAGGTTCTGACCTCCCTGAGCTCTT CAGCAAACTGGGCCTGGGCAAATACACAGA TGTTTTCCAGCAACAAGAGATCGATCTTCA GACATTCCTCACTCTCACAGATCAGGATCT GAAGGAGCTGGGAATAACTACTTTTGGTGC CAGGAGGAAAATGCTGCTTGCAATTTCAGA ACTAAATAAAAACCGAAGAAAGCTTTTTGA ATCGCCAAATGCACGCACCTCTTTCCTGGA AGGTGGAGCGAGTGGAAGGCTACCCCGTCA GTATCACTCAGACATTGCTAGTGTCAGTGG CCGCTGGTAG FGFR2:AFF3 >ATGGTCAGCTGGGGTCGTTTCATCTGCCT (5109 base pairs) GGTCGTGGTCACCATGGCAACCTTGTCCCT (SEQ ID NO: 61) GGCCCGGCCCTCCTTCAGTTTAGTTGAGGA TACCACATTAGAGCCAGAAGAGCCACCAAC CAAATACCAAATCTCTCAACCAGAAGTGTA CGTGGCTGCGCCAGGGGAGTCGCTAGAGGT GCGCTGCCTGTTGAAAGATGCCGCCGTGAT CAGTTGGACTAAGGATGGGGTGCACTTGGG GCCCAACAATAGGACAGTGCTTATTGGGGA GTACTTGCAGATAAAGGGCGCCACGCCTAG AGACTCCGGCCTCTATGCTTGTACTGCCAG TAGGACTGTAGACAGTGAAACTTGGTACTT CATGGTGAATGTCACAGATGCCATCTCATC CGGAGATGATGAGGATGACACCGATGGTGC GGAAGATTTTGTCAGTGAGAACAGTAACAA CAAGAGAGCACCATACTGGACCAACACAGA AAAGATGGAAAAGCGGCTCCATGCTGTGCC TGCGGCCAACACTGTCAAGTTTCGCTGCCC AGCCGGGGGGAACCCAATGCCAACCATGCG GTGGCTGAAAAACGGGAAGGAGTTTAAGCA GGAGCATCGCATTGGAGGCTACAAGGTACG AAACCAGCACTGGAGCCTCATTATGGAAAG TGTGGTCCCATCTGACAAGGGAAATTATAC CTGTGTAGTGGAGAATGAATACGGGTCCAT CAATCACACGTACCACCTGGATGTTGTGGA GCGATCGCCTCACCGGCCCATCCTCCAAGC CGGACTGCCGGCAAATGCCTCCACAGTGGT CGGAGGAGACGTAGAGTTTGTCTGCAAGGT TTACAGTGATGCCCAGCCCCACATCCAGTG GATCAAGCACGTGGAAAAGAACGGCAGTAA ATACGGGCCCGACGGGCTGCCCTACCTCAA GGTTCTCAAGGCCGCCGGTGTTAACACCAC GGACAAAGAGATTGAGGTTCTCTATATTCG GAATGTAACTTTTGAGGACGCTGGGGAATA TACGTGCTTGGCGGGTAATTCTATTGGGAT ATCCTTTCACTCTGCATGGTTGACAGTTCT GCCAGCGCCTGGAAGAGAAAAGGAGATTAC AGCTTCCCCAGACTACCTGGAGATAGCCAT TTACTGCATAGGGGTCTTCTTAATCGCCTG TATGGTGGTAACAGTCATCCTGTGCCGAAT GAAGAACACGACCAAGAAGCCAGACTTCAG CAGCCAGCCGGCTGTGCACAAGCTGACCAA ACGTATCCCCCTGCGGAGACAGGTAACAGT TTCGGCTGAGTCCAGCTCCTCCATGAACTC CAACACCCCGCTGGTGAGGATAACAACACG CCTCTCTTCAACGGCAGACACCCCCATGCT GGCAGGGGTCTCCGAGTATGAACTTCCAGA GGACCCAAAATGGGAGTTTCCAAGAGATAA GCTGACACTGGGCAAGCCCCTGGGAGAAGG TTGCTTTGGGCAAGTGGTCATGGCGGAAGC AGTGGGAATTGACAAAGACAAGCCCAAGGA GGCGGTCACCGTGGCCGTGAAGATGTTGAA AGATGATGCCACAGAGAAAGACCTTTCTGA TCTGGTGTCAGAGATGGAGATGATGAAGAT GATTGGGAAACACAAGAATATCATAAATCT TCTTGGAGCCTGCACACAGGATGGGCCTCT CTATGTCATAGTTGAGTATGCCTCTAAAGG CAACCTCCGAGAATACCTCCGAGCCCGGAG GCCACCCGGGATGGAGTACTCCTATGACAT TAACCGTGTTCCTGAGGAGCAGATGACCTT CAAGGACTTGGTGTCATGCACCTACCAGCT GGCCAGAGGCATGGAGTACTTGGCTTCCCA AAAATGTATTCATCGAGATTTAGCAGCCAG AAATGTTTTGGTAACAGAAAACAATGTGAT GAAAATAGCAGACTTTGGACTCGCCAGAGA TATCAACAATATAGACTATTACAAAAAGAC CACCAATGGGCGGCTTCCAGTCAAGTGGAT GGCTCCAGAAGCCCTGTTTGATAGAGTATA CACTCATCAGAGTGATGTCTGGTCCTTCGG GGTGTTAATGTGGGAGATCTTCACTTTAGG GGGCTCGCCCTACCCAGGGATTCCCGTGGA GGAACTTTTTAAGCTGCTGAAGGAAGGACA CAGAATGGATAAGCCAGCCAACTGCACCAA CGAACTGTACATGATGATGAGGGACTGTTG GCATGCAGTGCCCTCCCAGAGACCAACGTT CAAGCAGTTGGTAGAAGACTTGGATCGAAT TCTCACTCTCACAACCAATGAGGAGAGTAG ATCTGGAGAAACCAACAGCTGTGTTGAAGA AATAATCCGGGAGATGACCTGGCTTCCACC ACTTTCTGCTATTCAAGCACCTGGCAAAGT GGAACCAACCAAATTTCCATTTCCAAATAA GGACTCTCAGCTTGTATCCTCTGGACACAA TAATCCAAAGAAAGGTGATGCAGAGCCAGA GAGTCCAGACAGTGGCACATCGAATACATC AATGCTGGAAGATGACCTTAAGCTAAGCAG TGATGAAGAGGAGAATGAACAGCAGGCAGC TCAGAGAACGGCTCTCCGCGCTCTCTCTGA CAGCGCCGTGGTCCAGCAGCCCAACTGCAG AACCTCGGTGCCTTCCAGCAAGGGCAGCAG CAGCAGCAGCAGCAGCGGCAGCAGCAGCTC CTCCAGCGACTCAGAGAGCAGCTCCGGATC TGACTCGGAGACCGAGAGCAGCTCCAGCGA GAGTGAGGGCAGCAAGCCCCCCCACTTCTC CAGCCCCGAGGCTGAACCGGCATCCTCTAA CAAGTGGCAGCTGGATAAATGGCTAAACAA AGTTAATCCCCACAAGCCTCCTATTCTGAT CCAAAATGAAAGCCACGGGTCAGAGAGCAA TCAGTACTACAACCCGGTGAAAGAGGACGT CCAGGACTGTGGGAAAGTCCCCGACGTTTG CCAGCCCAGCCTGAGAGAGAAGGAGATCAA GAGCACTTGCAAGGAGGAGCAAAGGCCAAG GACAGCCAACAAGGCCCCTGGGAGTAAAGG CGTGAAGCAGAAGTCCCCGCCCGCGGCCGT GGCCGTGGCGGTGAGCGCAGCCGCCCCGCC ACCCGCAGTGCCCTGTGCGCCCGCGGAGAA CGCGCCCGCGCCTGCCCGGAGGTCCGCGGG CAAGAAGCCCACCAGGCGCACCGAGAGGAC CTCAGCCGGGGACGGCGCCAACTGCCACCG

GCCCGAGGAGCCCGCGGCCGCGGACGCGCT GGGGACGAGCGTGGTGGTCCCCCCGGAGCC CACCAAAACCAGGCCCTGTGGCAACAACAG AGCGAGCCACCGCAAGGAGCTGCGCTCCTC CGTGACCTGCGAGAAGCGCCGCACGCGGGG GCTAAGCAGGATCGTCCCCAAATCCAAGGA GTTCATTGAGACAGAGTCGTCATCTTCATC CTCCTCCTCGGACTCCGACCTGGAGTCCGA GCAGGAGGAGTACCCTCTGTCCAAAGCACA GACCGTGGCTGCCTCTGCCTCCTCCGGGAA TGATCAGAGGCTGAAGGAGGCCGCTGCCAA CGGGGGCAGTGGTCCTAGGGCCCCTGTAGG CTCCATCAACGCCAGGACCACCAGTGACAT CGCCAAGGAGCTGGAGGAGCAGTTCTACAC ACTGGTCCCCTTTGGCCGGAACGAACTTCT CTCCCCTCTAAAGGACAGTGATGAGATCAG GTCTCTCTGGGTCAAAATCGACCTGACCCT CCTGTCCAGGATCCCAGAACACCTGCCCCA GGAGCCAGGGGTATTGAGCGCCCCTGCCAC CAAGGACTCTGAGAGCGCACCGCCCAGCCA CACCTCGGACACACCTGCAGAAAAGGCTTT GCCAAAATCCAAGAGGAAACGCAAGTGTGA CAACGAAGACGACTACAGGGAGATCAAGAA GTCCCAGGGAGAGAAAGACAGCTCTTCAAG ACTGGCCACCTCCACCAGTAATACTTTGTC TGCAAACCACTGCAACATGAACATCAACAG TGTGGCAATACCAATAAATAAAAATGAAAA AATGCTTCGGTCGCCCATCTCACCCCTCTC TGATGCATCTAAACACAAATACACCAGCGA GGACTTAACTTCTTCCAGCCGACCTAATGG CAACAGTTTGTTTACTTCAGCCTCTTCCAG CAAAAAGCCTAAGGCCGACAGCCAGCTGCA GCCTCACGGCGGAGACCTCACGAAAGCAGC TCACAACAATTCTGAAAACATTCCCCTCCA CAAGTCACGGCCGCAGACGAAGCCGTGGTC TCCAGGCTCCAACGGCCACAGGGACTGCAA GAGGCAGAAACTTGTCTTCGATGATATGCC TCGCAGTGCCGATTATTTTATGCAAGAAGC TAAACGAATGAAGCATAAAGCAGATGCAAT GGTGGAAAAGTTTGGAAAGGCTTTGAACTA TGCTGAAGCAGCATTGTCGTTTATCGAGTG TGGAAATGCAATGGAACAAGGCCCCATGGA ATCCAAATCTCCTTATACGATGTATTCAGA AACAGTAGAGCTCATCAGGTATGCTATGAG ACTAAAAACCCACTCAGGCCCCAATGCCAC ACCAGAAGACAAACAACTGGCTGCATTATG TTACCGATGCCTGGCCCTCCTGTACTGGCG GATGTTTCGACTCAAAAGGGACCACGCTGT AAAGTATTCAAAAGCACTAATCGACTATTT CAAGAACTCATCTAAAGCCGCCCAAGCCCC ATCTCCGTGGGGGGCCAGTGGAAAGAGCAC TGGAACCCCATCCCCCATGTCTCCCAACCC CTCTCCCGCCAGCTCCGTGGGGTCTCAGGG CAGCCTCTCCAACGCCAGCGCCCTGTCCCC GTCGACCATCGTCAGCATCCCACAGCGCAT CCACCAGATGGCGGCCAACCACGTCAGCAT CACCAACAGCATCCTGCACAGCTACGACTA CTGGGAGATGGCCGACAACCTGGCCAAGGA AAACCGAGAATTCTTCAACGACCTGGATCT GCTCATGGGGCCGGTCACCCTGCACAGCAG CATGGAGCACCTGGTCCAGTACTCCCAACA GGGCCTGCACTGGCTGCGGAACAGCGCCCA CCTGTCATAG FGFR2:CASP7 >ATGGTCAGCTGGGGTCGTTTCATCTGCCT (3213 base pairs) GGTCGTGGTCACCATGGCAACCTTGTCCCT (SEQ ID NO: 62) GGCCCGGCCCTCCTTCAGTTTAGTTGAGGA TACCACATTAGAGCCAGAAGAGCCACCAAC CAAATACCAAATCTCTCAACCAGAAGTGTA CGTGGCTGCGCCAGGGGAGTCGCTAGAGGT GCGCTGCCTGTTGAAAGATGCCGCCGTGAT CAGTTGGACTAAGGATGGGGTGCACTTGGG GCCCAACAATAGGACAGTGCTTATTGGGGA GTACTTGCAGATAAAGGGCGCCACGCCTAG AGACTCCGGCCTCTATGCTTGTACTGCCAG TAGGACTGTAGACAGTGAAACTTGGTACTT CATGGTGAATGTCACAGATGCCATCTCATC CGGAGATGATGAGGATGACACCGATGGTGC GGAAGATTTTGTCAGTGAGAACAGTAACAA CAAGAGAGCACCATACTGGACCAACACAGA AAAGATGGAAAAGCGGCTCCATGCTGTGCC TGCGGCCAACACTGTCAAGTTTCGCTGCCC AGCCGGGGGGAACCCAATGCCAACCATGCG GTGGCTGAAAAACGGGAAGGAGTTTAAGCA GGAGCATCGCATTGGAGGCTACAAGGTACG AAACCAGCACTGGAGCCTCATTATGGAAAG TGTGGTCCCATCTGACAAGGGAAATTATAC CTGTGTAGTGGAGAATGAATACGGGTCCAT CAATCACACGTACCACCTGGATGTTGTGGA GCGATCGCCTCACCGGCCCATCCTCCAAGC CGGACTGCCGGCAAATGCCTCCACAGTGGT CGGAGGAGACGTAGAGTTTGTCTGCAAGGT TTACAGTGATGCCCAGCCCCACATCCAGTG GATCAAGCACGTGGAAAAGAACGGCAGTAA ATACGGGCCCGACGGGCTGCCCTACCTCAA GGTTCTCAAGGCCGCCGGTGTTAACACCAC GGACAAAGAGATTGAGGTTCTCTATATTCG GAATGTAACTTTTGAGGACGCTGGGGAATA TACGTGCTTGGCGGGTAATTCTATTGGGAT ATCCTTTCACTCTGCATGGTTGACAGTTCT GCCAGCGCCTGGAAGAGAAAAGGAGATTAC AGCTTCCCCAGACTACCTGGAGATAGCCAT TTACTGCATAGGGGTCTTCTTAATCGCCTG TATGGTGGTAACAGTCATCCTGTGCCGAAT GAAGAACACGACCAAGAAGCCAGACTTCAG CAGCCAGCCGGCTGTGCACAAGCTGACCAA ACGTATCCCCCTGCGGAGACAGGTAACAGT TTCGGCTGAGTCCAGCTCCTCCATGAACTC CAACACCCCGCTGGTGAGGATAACAACACG CCTCTCTTCAACGGCAGACACCCCCATGCT GGCAGGGGTCTCCGAGTATGAACTTCCAGA GGACCCAAAATGGGAGTTTCCAAGAGATAA GCTGACACTGGGCAAGCCCCTGGGAGAAGG TTGCTTTGGGCAAGTGGTCATGGCGGAAGC AGTGGGAATTGACAAAGACAAGCCCAAGGA GGCGGTCACCGTGGCCGTGAAGATGTTGAA AGATGATGCCACAGAGAAAGACCTTTCTGA TCTGGTGTCAGAGATGGAGATGATGAAGAT GATTGGGAAACACAAGAATATCATAAATCT TCTTGGAGCCTGCACACAGGATGGGCCTCT CTATGTCATAGTTGAGTATGCCTCTAAAGG CAACCTCCGAGAATACCTCCGAGCCCGGAG GCCACCCGGGATGGAGTACTCCTATGACAT TAACCGTGTTCCTGAGGAGCAGATGACCTT CAAGGACTTGGTGTCATGCACCTACCAGCT GGCCAGAGGCATGGAGTACTTGGCTTCCCA AAAATGTATTCATCGAGATTTAGCAGCCAG AAATGTTTTGGTAACAGAAAACAATGTGAT GAAAATAGCAGACTTTGGACTCGCCAGAGA TATCAACAATATAGACTATTACAAAAAGAC CACCAATGGGCGGCTTCCAGTCAAGTGGAT GGCTCCAGAAGCCCTGTTTGATAGAGTATA CACTCATCAGAGTGATGTCTGGTCCTTCGG GGTGTTAATGTGGGAGATCTTCACTTTAGG GGGCTCGCCCTACCCAGGGATTCCCGTGGA GGAACTTTTTAAGCTGCTGAAGGAAGGACA CAGAATGGATAAGCCAGCCAACTGCACCAA CGAACTGTACATGATGATGAGGGACTGTTG GCATGCAGTGCCCTCCCAGAGACCAACGTT CAAGCAGTTGGTAGAAGACTTGGATCGAAT TCTCACTCTCACAACCAATGAGATGGCAGA TGATCAGGGCTGTATTGAAGAGCAGGGGGT TGAGGATTCAGCAAATGAAGATTCAGTGGA TGCTAAGCCAGACCGGTCCTCGTTTGTACC GTCCCTCTTCAGTAAGAAGAAGAAAAATGT CACCATGCGATCCATCAAGACCACCCGGGA CCGAGTGCCTACATATCAGTACAACATGAA TTTTGAAAAGCTGGGCAAATGCATCATAAT AAACAACAAGAACTTTGATAAAGTGACAGG TATGGGCGTTCGAAACGGAACAGACAAAGA TGCCGAGGCGCTCTTCAAGTGCTTCCGAAG CCTGGGTTTTGACGTGATTGTCTATAATGA CTGCTCTTGTGCCAAGATGCAAGATCTGCT TAAAAAAGCTTCTGAAGAGGACCATACAAA TGCCGCCTGCTTCGCCTGCATCCTCTTAAG CCATGGAGAAGAAAATGTAATTTATGGGAA AGATGGTGTCACACCAATAAAGGATTTGAC AGCCCACTTTAGGGGGGATAGATGCAAAAC CCTTTTAGAGAAACCCAAACTCTTCTTCAT TCAGGCTTGCCGAGGGACCGAGCTTGATGA TGGCATCCAGGCCGACTCGGGGCCCATCAA TGACACAGATGCTAATCCTCGATACAAGAT CCCAGTGGAAGCTGACTTCCTCTTCGCCTA TTCCACGGTTCCAGGCTATTACTCGTGGAG GAGCCCAGGAAGAGGCTCCTGGTTTGTGCA AGCCCTCTGCTCCATCCTGGAGGAGCACGG AAAAGACCTGGAAATCATGCAGATCCTCAC CAGGGTGAATGACAGAGTTGCCAGGCACTT TGAGTCTCAGTCTGATGACCCACACTTCCA TGAGAAGAAGCAGATCCCCTGTGTGGTCTC CATGCTCACCAAGGAACTCTACTTCAGTCA ATAG FGFR2:CCDC6 >ATGGTCAGCTGGGGTCGTTTCATCTGCCT (3423 base pairs) GGTCGTGGTCACCATGGCAACCTTGTCCCT (SEQ ID NO: 63) GGCCCGGCCCTCCTTCAGTTTAGTTGAGGA TACCACATTAGAGCCAGAAGAGCCACCAAC CAAATACCAAATCTCTCAACCAGAAGTGTA CGTGGCTGCGCCAGGGGAGTCGCTAGAGGT GCGCTGCCTGTTGAAAGATGCCGCCGTGAT CAGTTGGACTAAGGATGGGGTGCACTTGGG GCCCAACAATAGGACAGTGCTTATTGGGGA GTACTTGCAGATAAAGGGCGCCACGCCTAG AGACTCCGGCCTCTATGCTTGTACTGCCAG TAGGACTGTAGACAGTGAAACTTGGTACTT CATGGTGAATGTCACAGATGCCATCTCATC CGGAGATGATGAGGATGACACCGATGGTGC GGAAGATTTTGTCAGTGAGAACAGTAACAA CAAGAGAGCACCATACTGGACCAACACAGA AAAGATGGAAAAGCGGCTCCATGCTGTGCC TGCGGCCAACACTGTCAAGTTTCGCTGCCC AGCCGGGGGGAACCCAATGCCAACCATGCG GTGGCTGAAAAACGGGAAGGAGTTTAAGCA GGAGCATCGCATTGGAGGCTACAAGGTACG AAACCAGCACTGGAGCCTCATTATGGAAAG TGTGGTCCCATCTGACAAGGGAAATTATAC CTGTGTAGTGGAGAATGAATACGGGTCCAT CAATCACACGTACCACCTGGATGTTGTGGA GCGATCGCCTCACCGGCCCATCCTCCAAGC CGGACTGCCGGCAAATGCCTCCACAGTGGT CGGAGGAGACGTAGAGTTTGTCTGCAAGGT TTACAGTGATGCCCAGCCCCACATCCAGTG GATCAAGCACGTGGAAAAGAACGGCAGTAA ATACGGGCCCGACGGGCTGCCCTACCTCAA GGTTCTCAAGGCCGCCGGTGTTAACACCAC GGACAAAGAGATTGAGGTTCTCTATATTCG GAATGTAACTTTTGAGGACGCTGGGGAATA TACGTGCTTGGCGGGTAATTCTATTGGGAT ATCCTTTCACTCTGCATGGTTGACAGTTCT GCCAGCGCCTGGAAGAGAAAAGGAGATTAC AGCTTCCCCAGACTACCTGGAGATAGCCAT TTACTGCATAGGGGTCTTCTTAATCGCCTG TATGGTGGTAACAGTCATCCTGTGCCGAAT GAAGAACACGACCAAGAAGCCAGACTTCAG CAGCCAGCCGGCTGTGCACAAGCTGACCAA ACGTATCCCCCTGCGGAGACAGGTAACAGT TTCGGCTGAGTCCAGCTCCTCCATGAACTC CAACACCCCGCTGGTGAGGATAACAACACG CCTCTCTTCAACGGCAGACACCCCCATGCT GGCAGGGGTCTCCGAGTATGAACTTCCAGA GGACCCAAAATGGGAGTTTCCAAGAGATAA GCTGACACTGGGCAAGCCCCTGGGAGAAGG TTGCTTTGGGCAAGTGGTCATGGCGGAAGC AGTGGGAATTGACAAAGACAAGCCCAAGGA GGCGGTCACCGTGGCCGTGAAGATGTTGAA AGATGATGCCACAGAGAAAGACCTTTCTGA TCTGGTGTCAGAGATGGAGATGATGAAGAT GATTGGGAAACACAAGAATATCATAAATCT TCTTGGAGCCTGCACACAGGATGGGCCTCT CTATGTCATAGTTGAGTATGCCTCTAAAGG CAACCTCCGAGAATACCTCCGAGCCCGGAG GCCACCCGGGATGGAGTACTCCTATGACAT TAACCGTGTTCCTGAGGAGCAGATGACCTT CAAGGACTTGGTGTCATGCACCTACCAGCT GGCCAGAGGCATGGAGTACTTGGCTTCCCA AAAATGTATTCATCGAGATTTAGCAGCCAG AAATGTTTTGGTAACAGAAAACAATGTGAT GAAAATAGCAGACTTTGGACTCGCCAGAGA TATCAACAATATAGACTATTACAAAAAGAC CACCAATGGGCGGCTTCCAGTCAAGTGGAT GGCTCCAGAAGCCCTGTTTGATAGAGTATA CACTCATCAGAGTGATGTCTGGTCCTTCGG GGTGTTAATGTGGGAGATCTTCACTTTAGG GGGCTCGCCCTACCCAGGGATTCCCGTGGA GGAACTTTTTAAGCTGCTGAAGGAAGGACA CAGAATGGATAAGCCAGCCAACTGCACCAA CGAACTGTACATGATGATGAGGGACTGTTG GCATGCAGTGCCCTCCCAGAGACCAACGTT CAAGCAGTTGGTAGAAGACTTGGATCGAAT TCTCACTCTCACAACCAATGAGCAAGCCAG GGCTGAGCAGGAAGAAGAATTCATTAGTAA

CACTTTATTCAAGAAAATTCAGGCTTTGCA GAAGGAGAAAGAAACCCTTGCTGTAAATTA TGAGAAAGAAGAAGAATTCCTCACTAATGA GCTCTCCAGAAAATTGATGCAGTTGCAGCA TGAGAAAGCCGAACTAGAACAGCATCTTGA ACAAGAGCAGGAATTTCAGGTCAACAAACT GATGAAGAAAATTAAAAAACTGGAGAATGA CACCATTTCTAAGCAACTTACATTAGAACA GTTGAGACGGGAGAAGATTGACCTTGAAAA TACATTGGAACAAGAACAAGAAGCACTAGT TAATCGCCTCTGGAAAAGGATGGATAAGCT TGAAGCTGAAAAGCGAATCCTGCAGGAAAA ATTAGACCAGCCCGTCTCTGCTCCACCATC GCCTAGAGATATCTCCATGGAGATTGATTC TCCAGAAAATATGATGCGTCACATCAGGTT TTTAAAGAATGAAGTGGAACGGCTGAAGAA GCAACTGAGAGCTGCTCAGTTACAGCATTC AGAGAAAATGGCACAGTATCTGGAGGAGGA ACGTCACATGAGAGAAGAGAACTTGAGGCT CCAGAGGAAGCTGCAGAGGGAGATGGAGAG AAGAGAAGCCCTCTGTCGACAGCTCTCCGA GAGTGAGTCCAGCTTAGAAATGGACGACGA AAGGTATTTTAATGAGATGTCTGCACAAGG ATTAAGACCTCGCACTGTGTCCAGCCCGAT CCCTTACACACCTTCTCCGAGTTCAAGCAG GCCTATATCACCTGGTCTATCATATGCAAG TCACACGGTTGGTTTCACGCCACCAACTTC ACTGACTAGAGCTGGAATGTCTTATTACAA TTCCCCGGGTCTTCACGTGCAGCACATGGG AACATCCCATGGTATCACAAGGCCTTCACC ACGGAGAAGCAACAGTCCTGACAAATTCAA ACGGCCCACGCCGCCTCCATCTCCCAACAC ACAGACCCCAGTCCAGCCACCTCCGCCTCC ACCTCCGCCACCCATGCAGCCCACGGTCCC CTCAGCAGCCACCTCGCAGCCTACTCCTTC GCAACATTCGGCGCACCCCTCCTCCCAGCC TTAA FGFR2:OFD1 >ATGGTCAGCTGGGGTCGTTTCATCTGCCT (5229 base pairs) GGTCGTGGTCACCATGGCAACCTTGTCCCT (SEQ ID NO: 64) GGCCCGGCCCTCCTTCAGTTTAGTTGAGGA TACCACATTAGAGCCAGAAGAGCCACCAAC CAAATACCAAATCTCTCAACCAGAAGTGTA CGTGGCTGCGCCAGGGGAGTCGCTAGAGGT GCGCTGCCTGTTGAAAGATGCCGCCGTGAT CAGTTGGACTAAGGATGGGGTGCACTTGGG GCCCAACAATAGGACAGTGCTTATTGGGGA GTACTTGCAGATAAAGGGCGCCACGCCTAG AGACTCCGGCCTCTATGCTTGTACTGCCAG TAGGACTGTAGACAGTGAAACTTGGTACTT CATGGTGAATGTCACAGATGCCATCTCATC CGGAGATGATGAGGATGACACCGATGGTGC GGAAGATTTTGTCAGTGAGAACAGTAACAA CAAGAGAGCACCATACTGGACCAACACAGA AAAGATGGAAAAGCGGCTCCATGCTGTGCC TGCGGCCAACACTGTCAAGTTTCGCTGCCC AGCCGGGGGGAACCCAATGCCAACCATGCG GTGGCTGAAAAACGGGAAGGAGTTTAAGCA GGAGCATCGCATTGGAGGCTACAAGGTACG AAACCAGCACTGGAGCCTCATTATGGAAAG TGTGGTCCCATCTGACAAGGGAAATTATAC CTGTGTAGTGGAGAATGAATACGGGTCCAT CAATCACACGTACCACCTGGATGTTGTGGA GCGATCGCCTCACCGGCCCATCCTCCAAGC CGGACTGCCGGCAAATGCCTCCACAGTGGT CGGAGGAGACGTAGAGTTTGTCTGCAAGGT TTACAGTGATGCCCAGCCCCACATCCAGTG GATCAAGCACGTGGAAAAGAACGGCAGTAA ATACGGGCCCGACGGGCTGCCCTACCTCAA GGTTCTCAAGGCCGCCGGTGTTAACACCAC GGACAAAGAGATTGAGGTTCTCTATATTCG GAATGTAACTTTTGAGGACGCTGGGGAATA TACGTGCTTGGCGGGTAATTCTATTGGGAT ATCCTTTCACTCTGCATGGTTGACAGTTCT GCCAGCGCCTGGAAGAGAAAAGGAGATTAC AGCTTCCCCAGACTACCTGGAGATAGCCAT TTACTGCATAGGGGTCTTCTTAATCGCCTG TATGGTGGTAACAGTCATCCTGTGCCGAAT GAAGAACACGACCAAGAAGCCAGACTTCAG CAGCCAGCCGGCTGTGCACAAGCTGACCAA ACGTATCCCCCTGCGGAGACAGGTAACAGT TTCGGCTGAGTCCAGCTCCTCCATGAACTC CAACACCCCGCTGGTGAGGATAACAACACG CCTCTCTTCAACGGCAGACACCCCCATGCT GGCAGGGGTCTCCGAGTATGAACTTCCAGA GGACCCAAAATGGGAGTTTCCAAGAGATAA GCTGACACTGGGCAAGCCCCTGGGAGAAGG TTGCTTTGGGCAAGTGGTCATGGCGGAAGC AGTGGGAATTGACAAAGACAAGCCCAAGGA GGCGGTCACCGTGGCCGTGAAGATGTTGAA AGATGATGCCACAGAGAAAGACCTTTCTGA TCTGGTGTCAGAGATGGAGATGATGAAGAT GATTGGGAAACACAAGAATATCATAAATCT TCTTGGAGCCTGCACACAGGATGGGCCTCT CTATGTCATAGTTGAGTATGCCTCTAAAGG CAACCTCCGAGAATACCTCCGAGCCCGGAG GCCACCCGGGATGGAGTACTCCTATGACAT TAACCGTGTTCCTGAGGAGCAGATGACCTT CAAGGACTTGGTGTCATGCACCTACCAGCT GGCCAGAGGCATGGAGTACTTGGCTTCCCA AAAATGTATTCATCGAGATTTAGCAGCCAG AAATGTTTTGGTAACAGAAAACAATGTGAT GAAAATAGCAGACTTTGGACTCGCCAGAGA TATCAACAATATAGACTATTACAAAAAGAC CACCAATGGGCGGCTTCCAGTCAAGTGGAT GGCTCCAGAAGCCCTGTTTGATAGAGTATA CACTCATCAGAGTGATGTCTGGTCCTTCGG GGTGTTAATGTGGGAGATCTTCACTTTAGG GGGCTCGCCCTACCCAGGGATTCCCGTGGA GGAACTTTTTAAGCTGCTGAAGGAAGGACA CAGAATGGATAAGCCAGCCAACTGCACCAA CGAACTGTACATGATGATGAGGGACTGTTG GCATGCAGTGCCCTCCCAGAGACCAACGTT CAAGCAGTTGGTAGAAGACTTGGATCGAAT TCTCACTCTCACAACCAATGAGACACAACT TCGAAACCAGCTAATTCATGAGTTGATGCA CCCTGTATTGAGTGGAGAACTGCAGCCTCG GTCCATTTCAGTAGAAGGGAGCTCCCTCTT AATAGGCGCCTCTAACTCTTTAGTGGCAGA TCACTTACAAAGATGTGGCTATGAATATTC ACTTTCTGTTTTCTTTCCAGAAAGTGGTTT GGCAAAAGAAAAGGTATTTACTATGCAGGA TCTATTACAACTCATTAAAATCAACCCTAC TTCCAGTCTCTACAAATCACTGGTTTCAGG ATCTGATAAAGAAAATCAAAAAGGTTTTCT TATGCATTTTTTAAAAGAATTGGCAGAATA TCATCAAGCTAAAGAGAGTTGTAATATGGA AACTCAGACAAGTTCGACATTTAACAGAGA TTCTCTGGCTGAGAAGCTTCAGCTTATTGA TGATCAGTTTGCAGATGCTTACCCTCAGCG TATCAAGTTCGAATCTTTAGAAATAAAGCT AAATGAGTATAAGAGAGAAATAGAAGAGCA ACTTCGGGCAGAAATGTGTCAAAAGTTGAA GTTTTTTAAAGATACCGAGATAGCAAAAAT TAAAATGGAAGCAAAAAAAAAGTATGAAAA GGAGTTAACCATGTTCCAGAATGATTTTGA AAAAGCTTGTCAAGCAAAATCTGAAGCTCT CGTTCTTCGGGAAAAGAGTACCCTTGAAAG AATTCACAAGCACCAAGAGATTGAAACAAA AGAAATTTATGCTCAAAGGCAACTTTTACT AAAAGATATGGATTTGCTAAGAGGAAGAGA AGCAGAGCTGAAGCAAAGAGTTGAAGCTTT TGAATTGAACCAGAAGCTCCAGGAAGAAAA ACATAAAAGCATAACTGAGGCACTTAGGAG ACAGGAGCAGAATATAAAGAGTTTTGAGGA GACCTATGACCGAAAGCTCAAGAATGAACT TCTAAAGTATCAACTTGAACTGAAGGATGA CTACATCATTAGAACTAATCGACTGATTGA AGATGAAAGGAAGAATAAAGAAAAAGCTGT TCATTTGCAAGAGGAGCTCATAGCTATTAA TTCAAAAAAGGAGGAACTCAATCAATCTGT AAATCGTGTGAAAGAACTTGAGCTTGAATT AGAGTCTGTCAAAGCCCAGTCTTTGGCAAT AACAAAACAAAACCATATGCTGAATGAAAA GGTTAAAGAGATGAGTGATTATTCACTACT AAAAGAAGAGAAACTGGAGCTTCTGGCACA AAATAAATTACTTAAACAACAACTGGAAGA GAGTAGAAATGAAAACCTGCGTCTCCTAAA CCGCCTAGCTCAGCCGGCTCCTGAACTTGC AGTCTTTCAGAAAGAACTACGGAAAGCCGA AAAGGCTATAGTGGTTGAGCATGAGGAGTT CGAAAGCTGCAGGCAAGCTCTGCACAAACA ACTGCAAGACGAAATTGAGCATTCTGCACA GCTGAAGGCCCAGATTCTAGGTTACAAAGC TTCTGTAAAGAGTTTAACTACTCAGGTTGC CGATTTAAAATTGCAACTGAAGCAAACTCA GACAGCCCTAGAGAATGAAGTGTACTGCAA TCCAAAGCAGTCTGTGATCGATCGTTCTGT CAATGGATTAATAAATGGCAATGTGGTGCC TTGCAATGGTGAGATAAGTGGGGATTTCTT GAACAATCCTTTTAAACAGGAAAACGTTCT AGCACGTATGGTTGCATCAAGGATCACAAA TTATCCAACTGCATGGGTGGAGGGTAGTTC CCCTGATTCTGACCTTGAGTTTGTAGCCAA TACTAAGGCAAGGGTCAAAGAGCTTCAGCA AGAGGCCGAACGCTTGGAAAAGGCTTTCAG AAGTTACCATCGGAGAGTCATTAAAAACTC TGCCAAAAGCCCACTAGCAGCAAAGAGCCC ACCATCTCTGCACTTGCTGGAAGCCTTCAA AAACATTACTTCCAGTTCCCCGGAAAGACA TATTTTTGGAGAGGACAGAGTTGTCTCTGA GCAGCCTCAAGTGGGCACACTTGAAGAAAG GAATGACGTCGTGGAAGCACTGACAGGCAG TGCAGCCTCGAGGCTCCGCGGGGGCACTTC CTCCAGACGCCTCTCTTCCACACCCCTTCC AAAAGCAAAAAGAAGCCTCGAAAGTGAAAT GTATCTGGAAGGTCTGGGCAGATCACACAT TGCTTCCCCCAGTCCTTGTCCTGACAGAAT GCCCCTACCATCACCCACTGAGTCTAGGCA CAGCCTCTCCATCCCTCCTGTCTCCAGCCC TCCGGAGCAGAAAGTGGGTCTTTATCGAAG ACAAACTGAACTTCAAGACAAAAGTGAATT TTCAGATGTGGACAAGCTAGCTTTTAAGGA TAATGAGGAGTTTGAATCATCTTTTGAATC TGCAGGGAACATGCCAAGGCAGTTGGAAAT GGGCGGGCTTTCTCCTGCCGGGGATATGTC TCATGTGGACGCTGCTGCAGCTGCTGTGCC CCTCTCATATCAGCACCCAAGTGTAGATCA GAAACAAATTGAAGAACAAAAGGAAGAAGA AAAAATACGGGAACAGCAAGTGAAAGAACG AAGGCAGAGAGAAGAAAGAAGGCAGAGTAA CCTACAAGAAGTTTTAGAAAGGGAACGAAG AGAACTAGAAAAACTGTATCAGGAAAGGAA GATGATTGAAGAATCACTGAAGATTAAAAT AAAAAAGGAATTAGAAATGGAAAATGAATT AGAAATGAGTAATCAAGAAATAAAAGACAA ATCTGCTCACAGTGAAAATCCTTTAGAGAA ATACATGAAAATCATCCAGCAGGAGCAAGA CCAGGAGTCGGCAGATAAGAGCTCAAAAAA GATGGTCCAAGAAGGCTCCCTAGTGGACAC GCTGCAATCTAGTGACAAAGTCGAAAGTTT AACAGGCTTTTCTCATGAAGAACTAGACGA CTCTTGGTAA

Sequence CWU 1

1

731268DNAHomo sapiens 1tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 60tacacgctgg acgtgctgga gtgctccccg caccggccca tcctgcaggc ggggctgccg 120gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 180gcacagcccc acatccagtg gctcaagcac gtggaggtga atggcagcaa ggtgggcccg 240gacggcacac cctacgttac cgtgctca 2682378DNAHomo sapiens 2gaccgcggca actacacctg cgtcgtggag aacaagtttg gcagcatccg gcagacgtac 60acgctggacg tgctgggtga gggccctggg gcggcgcggg ggtgggggcg gcagtggcgg 120tggtggtgag ggagggggtg gcccctgagc gtcatctgcc cccacagagc gctgcccgca 180ccggcccatc ctgcaggcgg ggctgccggc caaccagacg gcggtgctgg gcagcgacgt 240ggagttccac tgcaaggtgt acagtgacgc acagccccac atccagtggc tcaagcacgt 300ggaggtgaat ggcagcaagg tgggcccgga cggcacaccc tacgttaccg tgctcaaggt 360gggccaccgt gtgcacgt 3783234DNAHomo sapiens 3gcgggcaatt ctattgggtt ttctcatcac tctgcgtggc tggtggtgct gccagccgag 60gaggagctgg tggaggctga cgaggcgtgc agtgtgtatg caggcatcct cagctacggg 120gtgggcttct tcctgttcat cctggtggtg gcggctgtga cgctctgccg cctgcgcagc 180ccccccaaga aaggcctggg ctcccccacc gtgcacaaga tctcccgctt cccg 2344301DNAHomo sapiens 4ctagaggttc tctccttgca caacgtcacc tttgaggacg ccggggagta cacctgcctg 60gcgggcaatt ctattgggtt ttctcatcac tctgcgtggc tggtggtgct gccagccgag 120gaggagctgg tggaggctga cgaggcgggc agtgtgtgtg caggcatcct cagctacggg 180gtgggcttct tcctgttcat cctggtggtg gcggctgtga cgctctgccg cctgcgcagc 240ccccccaaga aaggcctggg ctcccccacc gtgcacaaga tctcccgctt cccgctcaag 300c 301521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 5gacctggacc gtgtccttac c 21621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 6cttccccagt tccaggttct t 21720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 7aggacctgga ccgtgtcctt 20820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 8tataggtccg gtggacaggg 20915DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 9ggccatcctg ccccc 151020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 10gagcagtcca ggtcagccag 201120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 11ctggaccgtg tccttaccgt 201220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 12gcagcccagg attgaactgt 201323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 13tggatcgaat tctcactctc aca 231421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 14gccaagcaat ctgcgtattt g 211525DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 15tggtagaaga cttggatcga attct 251623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 16tctcccggat tatttcttca aca 231723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 17gctcttcaat acagccctga tca 231824DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 18acttggatcg aattctcact ctca 241923DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 19tggatcgaat tctcactctc aca 232025DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 20gcaaagcctg aattttcttg aataa 252124DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 21agggtgcatc aactcatgaa ttag 242224DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 22acttggatcg aattctcact ctca 242318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 23gcatccggca gacgtaca 182415DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 24ccccgcctgc aggat 152518DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 25gcatccggca gacgtaca 182615DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 26ccccgcctgc aggat 152719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 27aggagctggt ggaggctga 192819DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 28ccgtagctga ggatgcctg 192918DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 29ctggtggagg ctgacgag 183016DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 30agcccacccc gtagct 163121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 31gtcgtggaga acaagtttgg c 213217DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 32gtctggttgg ccggcag 173321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 33gtcgtggaga acaagtttgg c 213417DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 34gtctggttgg ccggcag 173519DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 35aggagctggt ggaggctga 193619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 36ccgtagctga ggatgcctg 193716DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 37gacgaggcgg gcagtg 163819DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 38gaagaagccc accccgtag 193917DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 39tggagcgctc cccgcac 174018DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 40gacgtgctgg agrgctcc 184117DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 41ctgacgaggc gggcagc 174223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 42gtgtgtatgc aggcatcctc agc 234316DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 43tccaccgacg taaagg 164416DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 44tccaccgacg tgccag 164518DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 45ccaatgagat catggagg 184616DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 46ccttctggcc caggtg 164717DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 47caccgacaat gttatgg 174820DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 48tcacaaccaa tgaggagagt 204916DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 49ctgccatctc attggt 165017DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 50aatgagcaag ccagggc 175119DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 51aagttgtgtc tcattggtt 195214DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 52ctggagtgct cccc 145313DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 53agcgctgccc gca 135415DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 54gcgtgcagtg tgtat 155515DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 55ctgcacacac actgc 15562850DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 56atgggcgccc ctgcctgcgc cctcgcgctc tgcgtggccg tggccatcgt ggccggcgcc 60tcctcggagt ccttggggac ggagcagcgc gtcgtggggc gagcggcaga agtcccgggc 120ccagagcccg gccagcagga gcagttggtc ttcggcagcg gggatgctgt ggagctgagc 180tgtcccccgc ccgggggtgg tcccatgggg cccactgtct gggtcaagga tggcacaggg 240ctggtgccct cggagcgtgt cctggtgggg ccccagcggc tgcaggtgct gaatgcctcc 300cacgaggact ccggggccta cagctgccgg cagcggctca cgcagcgcgt actgtgccac 360ttcagtgtgc gggtgacaga cgctccatcc tcgggagatg acgaagacgg ggaggacgag 420gctgaggaca caggtgtgga cacaggggcc ccttactgga cacggcccga gcggatggac 480aagaagctgc tggccgtgcc ggccgccaac accgtccgct tccgctgccc agccgctggc 540aaccccactc cctccatctc ctggctgaag aacggcaggg agttccgcgg cgagcaccgc 600attggaggca tcaagctgcg gcatcagcag tggagcctgg tcatggaaag cgtggtgccc 660tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 720tacacgctgg acgtgctgga gcgctccccg caccggccca tcctgcaggc ggggctgccg 780gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 840gcacagcccc acatccagtg gctcaagcac gtggaggtga atggcagcaa ggtgggcccg 900gacggcacac cctacgttac cgtgctcaag acggcgggcg ctaacaccac cgacaaggag 960ctagaggttc tctccttgca caacgtcacc tttgaggacg ccggggagta cacctgcctg 1020gcgggcaatt ctattgggtt ttctcatcac tctgcgtggc tggtggtgct gccagccgag 1080gaggagctgg tggaggctga cgaggcgggc agtgtgtatg caggcatcct cagctacggg 1140gtgggcttct tcctgttcat cctggtggtg gcggctgtga cgctctgccg cctgcgcagc 1200ccccccaaga aaggcctggg ctcccccacc gtgcacaaga tctcccgctt cccgctcaag 1260cgacaggtgt ccctggagtc caacgcgtcc atgagctcca acacaccact ggtgcgcatc 1320gcaaggctgt cctcagggga gggccccacg ctggccaatg tctccgagct cgagctgcct 1380gccgacccca aatgggagct gtctcgggcc cggctgaccc tgggcaagcc ccttggggag 1440ggctgcttcg gccaggtggt catggcggag gccatcggca ttgacaagga ccgggccgcc 1500aagcctgtca ccgtagccgt gaagatgctg aaagacgatg ccactgacaa ggacctgtcg 1560gacctggtgt ctgagatgga gatgatgaag atgatcggga aacacaaaaa catcatcaac 1620ctgctgggcg cctgcacgca gggcgggccc ctgtacgtgc tggtggagta cgcggccaag 1680ggtaacctgc gggagtttct gcgggcgcgg cggcccccgg gcctggacta ctccttcgac 1740acctgcaagc cgcccgagga gcagctcacc ttcaaggacc tggtgtcctg tgcctaccag 1800gtggcccggg gcatggagta cttggcctcc cagaagtgca tccacaggga cctggctgcc 1860cgcaatgtgc tggtgaccga ggacaacgtg atgaagatcg cagacttcgg gctggcccgg 1920gacgtgcaca acctcgacta ctacaagaag acgaccaacg gccggctgcc cgtgaagtgg 1980atggcgcctg aggccttgtt tgaccgagtc tacactcacc agagtgacgt ctggtccttt 2040ggggtcctgc tctgggagat cttcacgctg gggggctccc cgtaccccgg catccctgtg 2100gaggagctct tcaagctgct gaaggagggc caccgcatgg acaagcccgc caactgcaca 2160cacgacctgt acatgatcat gcgggagtgc tggcatgccg cgccctccca gaggcccacc 2220ttcaagcagc tggtggagga cctggaccgt gtccttaccg tgacgtccac cgacgtaaag 2280gcgacacagg aggagaaccg ggagctgagg agcaggtgtg aggagctcca cgggaagaac 2340ctggaactgg ggaagatcat ggacaggttc gaagaggttg tgtaccaggc catggaggaa 2400gttcagaagc agaaggaact ttccaaagct gaaatccaga aagttctaaa agaaaaagac 2460caacttacca cagatctgaa ctccatggag aagtccttct ccgacctctt caagcgtttt 2520gagaaacaga aagaggtgat cgagggctac cgcaagaacg aagagtcact gaagaagtgc 2580gtggaggatt acctggcaag gatcacccag gagggccaga ggtaccaagc cctgaaggcc 2640cacgcggagg agaagctgca gctggcaaac gaggagatcg cccaggtccg gagcaaggcc 2700caggcggaag cgttggccct ccaggccagc ctgaggaagg agcagatgcg catccagtcg 2760ctggagaaga cagtggagca gaagactaaa gagaacgagg agctgaccag gatctgcgac 2820gacctcatct ccaagatgga gaagatctga 2850572955DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 57atgggcgccc ctgcctgcgc cctcgcgctc tgcgtggccg tggccatcgt ggccggcgcc 60tcctcggagt ccttggggac ggagcagcgc gtcgtggggc gagcggcaga agtcccgggc 120ccagagcccg gccagcagga gcagttggtc ttcggcagcg gggatgctgt ggagctgagc 180tgtcccccgc ccgggggtgg tcccatgggg cccactgtct gggtcaagga tggcacaggg 240ctggtgccct cggagcgtgt cctggtgggg ccccagcggc tgcaggtgct gaatgcctcc 300cacgaggact ccggggccta cagctgccgg cagcggctca cgcagcgcgt actgtgccac 360ttcagtgtgc gggtgacaga cgctccatcc tcgggagatg acgaagacgg ggaggacgag 420gctgaggaca caggtgtgga cacaggggcc ccttactgga cacggcccga gcggatggac 480aagaagctgc tggccgtgcc ggccgccaac accgtccgct tccgctgccc agccgctggc 540aaccccactc cctccatctc ctggctgaag aacggcaggg agttccgcgg cgagcaccgc 600attggaggca tcaagctgcg gcatcagcag tggagcctgg tcatggaaag cgtggtgccc 660tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 720tacacgctgg acgtgctgga gcgctccccg caccggccca tcctgcaggc ggggctgccg 780gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 840gcacagcccc acatccagtg gctcaagcac gtggaggtga atggcagcaa ggtgggcccg 900gacggcacac cctacgttac cgtgctcaag acggcgggcg ctaacaccac cgacaaggag 960ctagaggttc tctccttgca caacgtcacc tttgaggacg ccggggagta cacctgcctg 1020gcgggcaatt ctattgggtt ttctcatcac tctgcgtggc tggtggtgct gccagccgag 1080gaggagctgg tggaggctga cgaggcgggc agtgtgtatg caggcatcct cagctacggg 1140gtgggcttct tcctgttcat cctggtggtg gcggctgtga cgctctgccg cctgcgcagc 1200ccccccaaga aaggcctggg ctcccccacc gtgcacaaga tctcccgctt cccgctcaag 1260cgacaggtgt ccctggagtc caacgcgtcc atgagctcca acacaccact ggtgcgcatc 1320gcaaggctgt cctcagggga gggccccacg ctggccaatg tctccgagct cgagctgcct 1380gccgacccca aatgggagct gtctcgggcc cggctgaccc tgggcaagcc ccttggggag 1440ggctgcttcg gccaggtggt catggcggag gccatcggca ttgacaagga ccgggccgcc 1500aagcctgtca ccgtagccgt gaagatgctg aaagacgatg ccactgacaa ggacctgtcg 1560gacctggtgt ctgagatgga gatgatgaag atgatcggga aacacaaaaa catcatcaac 1620ctgctgggcg cctgcacgca gggcgggccc ctgtacgtgc tggtggagta cgcggccaag 1680ggtaacctgc gggagtttct gcgggcgcgg cggcccccgg gcctggacta ctccttcgac 1740acctgcaagc cgcccgagga gcagctcacc ttcaaggacc tggtgtcctg tgcctaccag 1800gtggcccggg gcatggagta cttggcctcc cagaagtgca tccacaggga cctggctgcc 1860cgcaatgtgc tggtgaccga ggacaacgtg atgaagatcg cagacttcgg gctggcccgg 1920gacgtgcaca acctcgacta ctacaagaag acgaccaacg gccggctgcc cgtgaagtgg 1980atggcgcctg aggccttgtt tgaccgagtc tacactcacc agagtgacgt ctggtccttt 2040ggggtcctgc tctgggagat cttcacgctg gggggctccc cgtaccccgg catccctgtg 2100gaggagctct tcaagctgct gaaggagggc caccgcatgg acaagcccgc caactgcaca 2160cacgacctgt acatgatcat gcgggagtgc tggcatgccg cgccctccca gaggcccacc 2220ttcaagcagc tggtggagga cctggaccgt gtccttaccg tgacgtccac cgacgtgcca 2280ggcccacccc caggtgttcc cgcgcctggg ggcccacccc tgtccaccgg acctatagtg 2340gacctgctcc agtacagcca gaaggacctg gatgcagtgg taaaggcgac acaggaggag 2400aaccgggagc tgaggagcag gtgtgaggag ctccacggga agaacctgga actggggaag 2460atcatggaca ggttcgaaga ggttgtgtac caggccatgg aggaagttca gaagcagaag 2520gaactttcca aagctgaaat ccagaaagtt ctaaaagaaa aagaccaact taccacagat 2580ctgaactcca tggagaagtc cttctccgac ctcttcaagc gttttgagaa acagaaagag 2640gtgatcgagg gctaccgcaa gaacgaagag tcactgaaga agtgcgtgga ggattacctg 2700gcaaggatca cccaggaggg ccagaggtac caagccctga aggcccacgc ggaggagaag 2760ctgcagctgg caaacgagga gatcgcccag gtccggagca aggcccaggc ggaagcgttg 2820gccctccagg ccagcctgag gaaggagcag atgcgcatcc agtcgctgga gaagacagtg 2880gagcagaaga ctaaagagaa cgaggagctg accaggatct gcgacgacct catctccaag 2940atggagaaga tctga 2955584462DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 58atgggcgccc ctgcctgcgc cctcgcgctc tgcgtggccg tggccatcgt ggccggcgcc 60tcctcggagt ccttggggac ggagcagcgc gtcgtggggc gagcggcaga agtcccgggc 120ccagagcccg gccagcagga gcagttggtc ttcggcagcg gggatgctgt ggagctgagc 180tgtcccccgc ccgggggtgg tcccatgggg cccactgtct gggtcaagga tggcacaggg 240ctggtgccct cggagcgtgt cctggtgggg ccccagcggc tgcaggtgct gaatgcctcc 300cacgaggact ccggggccta cagctgccgg cagcggctca cgcagcgcgt actgtgccac 360ttcagtgtgc gggtgacaga cgctccatcc tcgggagatg acgaagacgg ggaggacgag 420gctgaggaca caggtgtgga cacaggggcc ccttactgga cacggcccga gcggatggac 480aagaagctgc tggccgtgcc ggccgccaac accgtccgct tccgctgccc agccgctggc 540aaccccactc cctccatctc ctggctgaag aacggcaggg agttccgcgg cgagcaccgc 600attggaggca tcaagctgcg gcatcagcag tggagcctgg tcatggaaag cgtggtgccc 660tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 720tacacgctgg acgtgctgga gcgctccccg

caccggccca tcctgcaggc ggggctgccg 780gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 840gcacagcccc acatccagtg gctcaagcac gtggaggtga atggcagcaa ggtgggcccg 900gacggcacac cctacgttac cgtgctcaag acggcgggcg ctaacaccac cgacaaggag 960ctagaggttc tctccttgca caacgtcacc tttgaggacg ccggggagta cacctgcctg 1020gcgggcaatt ctattgggtt ttctcatcac tctgcgtggc tggtggtgct gccagccgag 1080gaggagctgg tggaggctga cgaggcgggc agtgtgtatg caggcatcct cagctacggg 1140gtgggcttct tcctgttcat cctggtggtg gcggctgtga cgctctgccg cctgcgcagc 1200ccccccaaga aaggcctggg ctcccccacc gtgcacaaga tctcccgctt cccgctcaag 1260cgacaggtgt ccctggagtc caacgcgtcc atgagctcca acacaccact ggtgcgcatc 1320gcaaggctgt cctcagggga gggccccacg ctggccaatg tctccgagct cgagctgcct 1380gccgacccca aatgggagct gtctcgggcc cggctgaccc tgggcaagcc ccttggggag 1440ggctgcttcg gccaggtggt catggcggag gccatcggca ttgacaagga ccgggccgcc 1500aagcctgtca ccgtagccgt gaagatgctg aaagacgatg ccactgacaa ggacctgtcg 1560gacctggtgt ctgagatgga gatgatgaag atgatcggga aacacaaaaa catcatcaac 1620ctgctgggcg cctgcacgca gggcgggccc ctgtacgtgc tggtggagta cgcggccaag 1680ggtaacctgc gggagtttct gcgggcgcgg cggcccccgg gcctggacta ctccttcgac 1740acctgcaagc cgcccgagga gcagctcacc ttcaaggacc tggtgtcctg tgcctaccag 1800gtggcccggg gcatggagta cttggcctcc cagaagtgca tccacaggga cctggctgcc 1860cgcaatgtgc tggtgaccga ggacaacgtg atgaagatcg cagacttcgg gctggcccgg 1920gacgtgcaca acctcgacta ctacaagaag acgaccaacg gccggctgcc cgtgaagtgg 1980atggcgcctg aggccttgtt tgaccgagtc tacactcacc agagtgacgt ctggtccttt 2040ggggtcctgc tctgggagat cttcacgctg gggggctccc cgtaccccgg catccctgtg 2100gaggagctct tcaagctgct gaaggagggc caccgcatgg acaagcccgc caactgcaca 2160cacgacctgt acatgatcat gcgggagtgc tggcatgccg cgccctccca gaggcccacc 2220ttcaagcagc tggtggagga cctggaccgt gtccttaccg tgacgtccac cgacgtgagt 2280gctggctctg gcctggtgcc acccgcctat gcccctcccc ctgccgtccc cggccatcct 2340gccccccaga gtgctgaggt gtggggcggg cctttctggc ccaggtgccc tggctgacct 2400ggactgctca agctcttccc agagcccagg aagttctgag aaccaaatgg tgtctccagg 2460aaaagtgtct ggcagccctg agcaagccgt ggaggaaaac cttagttcct attccttaga 2520cagaagagtg acacccgcct ctgagaccct agaagaccct tgcaggacag agtcccagca 2580caaagcggag actccgcacg gagccgagga agaatgcaaa gcggagactc cgcacggagc 2640cgaggaggaa tgccggcacg gtggggtctg tgctcccgca gcagtggcca cttcgcctcc 2700tggtgcaatc cctaaggaag cctgcggagg agcacccctg cagggtctgc ctggcgaagc 2760cctgggctgc cctgcgggtg tgggcacccc cgtgccagca gatggcactc agacccttac 2820ctgtgcacac acctctgctc ctgagagcac agccccaacc aaccacctgg tggctggcag 2880ggccatgacc ctgagtcctc aggaagaagt ggctgcaggc caaatggcca gctcctcgag 2940gagcggacct gtaaaactag aatttgatgt atctgatggc gccaccagca aaagggcacc 3000cccaccaagg agactgggag agaggtccgg cctcaagcct cccttgagga aagcagcagt 3060gaggcagcaa aaggccccgc aggaggtgga ggaggacgac ggtaggagcg gagcaggaga 3120ggaccccccc atgccagctt ctcggggctc ttaccacctc gactgggaca aaatggatga 3180cccaaacttc atcccgttcg gaggtgacac caagtctggt tgcagtgagg cccagccccc 3240agaaagccct gagaccaggc tgggccagcc agcggctgaa cagttgcatg ctgggcctgc 3300cacggaggag ccaggtccct gtctgagcca gcagctgcat tcagcctcag cggaggacac 3360gcctgtggtg cagttggcag ccgagacccc aacagcagag agcaaggaga gagccttgaa 3420ctctgccagc acctcgcttc ccacaagctg tccaggcagt gagccagtgc ccacccatca 3480gcaggggcag cctgccttgg agctgaaaga ggagagcttc agagaccccg ctgaggttct 3540aggcacgggc gcggaggtgg attacctgga gcagtttgga acttcctcgt ttaaggagtc 3600ggccttgagg aagcagtcct tatacctcaa gttcgacccc ctcctgaggg acagtcctgg 3660tagaccagtg cccgtggcca ccgagaccag cagcatgcac ggtgcaaatg agactccctc 3720aggacgtccg cgggaagcca agcttgtgga gttcgatttc ttgggagcac tggacattcc 3780tgtgccaggc ccacccccag gtgttcccgc gcctgggggc ccacccctgt ccaccggacc 3840tatagtggac ctgctccagt acagccagaa ggacctggat gcagtggtaa aggcgacaca 3900ggaggagaac cgggagctga ggagcaggtg tgaggagctc cacgggaaga acctggaact 3960ggggaagatc atggacaggt tcgaagaggt tgtgtaccag gccatggagg aagttcagaa 4020gcagaaggaa ctttccaaag ctgaaatcca gaaagttcta aaagaaaaag accaacttac 4080cacagatctg aactccatgg agaagtcctt ctccgacctc ttcaagcgtt ttgagaaaca 4140gaaagaggtg atcgagggct accgcaagaa cgaagagtca ctgaagaagt gcgtggagga 4200ttacctggca aggatcaccc aggagggcca gaggtaccaa gccctgaagg cccacgcgga 4260ggagaagctg cagctggcaa acgaggagat cgcccaggtc cggagcaagg cccaggcgga 4320agcgttggcc ctccaggcca gcctgaggaa ggagcagatg cgcatccagt cgctggagaa 4380gacagtggag cagaagacta aagagaacga ggagctgacc aggatctgcg acgacctcat 4440ctccaagatg gagaagatct ga 4462593765DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 59atgggcgccc ctgcctgcgc cctcgcgctc tgcgtggccg tggccatcgt ggccggcgcc 60tcctcggagt ccttggggac ggagcagcgc gtcgtggggc gagcggcaga agtcccgggc 120ccagagcccg gccagcagga gcagttggtc ttcggcagcg gggatgctgt ggagctgagc 180tgtcccccgc ccgggggtgg tcccatgggg cccactgtct gggtcaagga tggcacaggg 240ctggtgccct cggagcgtgt cctggtgggg ccccagcggc tgcaggtgct gaatgcctcc 300cacgaggact ccggggccta cagctgccgg cagcggctca cgcagcgcgt actgtgccac 360ttcagtgtgc gggtgacaga cgctccatcc tcgggagatg acgaagacgg ggaggacgag 420gctgaggaca caggtgtgga cacaggggcc ccttactgga cacggcccga gcggatggac 480aagaagctgc tggccgtgcc ggccgccaac accgtccgct tccgctgccc agccgctggc 540aaccccactc cctccatctc ctggctgaag aacggcaggg agttccgcgg cgagcaccgc 600attggaggca tcaagctgcg gcatcagcag tggagcctgg tcatggaaag cgtggtgccc 660tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 720tacacgctgg acgtgctgga gcgctccccg caccggccca tcctgcaggc ggggctgccg 780gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 840gcacagcccc acatccagtg gctcaagcac gtggaggtga atggcagcaa ggtgggcccg 900gacggcacac cctacgttac cgtgctcaag tcctggatca gtgagagtgt ggaggccgac 960gtgcgcctcc gcctggccaa tgtgtcggag cgggacgggg gcgagtacct ctgtcgagcc 1020accaatttca taggcgtggc cgagaaggcc ttttggctga gcgttcacgg gccccgagca 1080gccgaggagg agctggtgga ggctgacgag gcgggcagtg tgtatgcagg catcctcagc 1140tacggggtgg gcttcttcct gttcatcctg gtggtggcgg ctgtgacgct ctgccgcctg 1200cgcagccccc ccaagaaagg cctgggctcc cccaccgtgc acaagatctc ccgcttcccg 1260ctcaagcgac aggtgtccct ggagtccaac gcgtccatga gctccaacac accactggtg 1320cgcatcgcaa ggctgtcctc aggggagggc cccacgctgg ccaatgtctc cgagctcgag 1380ctgcctgccg accccaaatg ggagctgtct cgggcccggc tgaccctggg caagcccctt 1440ggggagggct gcttcggcca ggtggtcatg gcggaggcca tcggcattga caaggaccgg 1500gccgccaagc ctgtcaccgt agccgtgaag atgctgaaag acgatgccac tgacaaggac 1560ctgtcggacc tggtgtctga gatggagatg atgaagatga tcgggaaaca caaaaacatc 1620atcaacctgc tgggcgcctg cacgcagggc gggcccctgt acgtgctggt ggagtacgcg 1680gccaagggta acctgcggga gtttctgcgg gcgcggcggc ccccgggcct ggactactcc 1740ttcgacacct gcaagccgcc cgaggagcag ctcaccttca aggacctggt gtcctgtgcc 1800taccaggtgg cccggggcat ggagtacttg gcctcccaga agtgcatcca cagggacctg 1860gctgcccgca atgtgctggt gaccgaggac aacgtgatga agatcgcaga cttcgggctg 1920gcccgggacg tgcacaacct cgactactac aagaagacga ccaacggccg gctgcccgtg 1980aagtggatgg cgcctgaggc cttgtttgac cgagtctaca ctcaccagag tgacgtctgg 2040tcctttgggg tcctgctctg ggagatcttc acgctggggg gctccccgta ccccggcatc 2100cctgtggagg agctcttcaa gctgctgaag gagggccacc gcatggacaa gcccgccaac 2160tgcacacacg acctgtacat gatcatgcgg gagtgctggc atgccgcgcc ctcccagagg 2220cccaccttca agcagctggt ggaggacctg gaccgtgtcc ttaccgtgac gtccaccgac 2280aatgttatgg aacagttcaa tcctgggctg cgaaatttaa taaacctggg gaaaaattat 2340gagaaagctg taaacgctat gatcctggca ggaaaagcct actacgatgg agtggccaag 2400atcggtgaga ttgccactgg gtcccccgtg tcaactgaac tgggacatgt cctcatagag 2460atttcaagta cccacaagaa actcaacgag agtcttgatg aaaattttaa aaaattccac 2520aaagagatta tccatgagct ggagaagaag atagaacttg acgtgaaata tatgaacgca 2580actctaaaaa gataccaaac agaacacaag aataaattag agtctttgga gaaatcccaa 2640gctgagttga agaagatcag aaggaaaagc caaggaagcc gaaacgcact caaatatgaa 2700cacaaagaaa ttgagtatgt ggagaccgtt acttctcgtc agagtgaaat ccagaaattc 2760attgcagatg gttgcaaaga ggctctgctt gaagagaaga ggcgcttctg ctttctggtt 2820gataagcact gtggctttgc aaaccacata cattattatc acttacagtc tgcagaacta 2880ctgaattcca agctgcctcg gtggcaggag acctgtgttg atgccatcaa agtgccagag 2940aaaatcatga atatgatcga agaaataaag accccagcct ctacccccgt gtctggaact 3000cctcaggctt cacccatgat cgagagaagc aatgtggtta ggaaagatta cgacaccctt 3060tctaaatgct caccaaagat gccccccgct ccttcaggca gagcatatac cagtcccttg 3120atcgatatgt ttaataaccc agccacggct gccccgaatt cacaaagggt aaataattca 3180acaggtactt ccgaagatcc cagtttacag cgatcagttt cggttgcaac gggactgaac 3240atgatgaaga agcagaaagt gaagaccatc ttcccgcaca ctgcgggctc caacaagacc 3300ttactcagct ttgcacaggg agatgtcatc acgctgctca tccccgagga gaaggatggc 3360tggctctatg gagaacacga cgtgtccaag gcgaggggtt ggttcccgtc gtcgtacacg 3420aagttgctgg aagaaaatga gacagaagca gtgaccgtgc ccacgccaag ccccacacca 3480gtgagaagca tcagcaccgt gaacttgtct gagaatagca gtgttgtcat ccccccaccc 3540gactacttgg aatgcttgtc catgggggca gctgccgaca ggagagcaga ttcggccagg 3600acgacatcca cctttaaggc cccagcgtcc aagcccgaga ccgcggctcc taacgatgcc 3660aacgggactg caaagccgcc ttttctcagc ggagaaaacc cctttgccac tgtgaaactc 3720cgcccgactg tgacgaatga tcgctcggca cccatcattc gatga 3765604989DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 60atggtcagct ggggtcgttt catctgcctg gtcgtggtca ccatggcaac cttgtccctg 60gcccggccct ccttcagttt agttgaggat accacattag agccagaaga gccaccaacc 120aaataccaaa tctctcaacc agaagtgtac gtggctgcgc caggggagtc gctagaggtg 180cgctgcctgt tgaaagatgc cgccgtgatc agttggacta aggatggggt gcacttgggg 240cccaacaata ggacagtgct tattggggag tacttgcaga taaagggcgc cacgcctaga 300gactccggcc tctatgcttg tactgccagt aggactgtag acagtgaaac ttggtacttc 360atggtgaatg tcacagatgc catctcatcc ggagatgatg aggatgacac cgatggtgcg 420gaagattttg tcagtgagaa cagtaacaac aagagagcac catactggac caacacagaa 480aagatggaaa agcggctcca tgctgtgcct gcggccaaca ctgtcaagtt tcgctgccca 540gccgggggga acccaatgcc aaccatgcgg tggctgaaaa acgggaagga gtttaagcag 600gagcatcgca ttggaggcta caaggtacga aaccagcact ggagcctcat tatggaaagt 660gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg agaatgaata cgggtccatc 720aatcacacgt accacctgga tgttgtggag cgatcgcctc accggcccat cctccaagcc 780ggactgccgg caaatgcctc cacagtggtc ggaggagacg tagagtttgt ctgcaaggtt 840tacagtgatg cccagcccca catccagtgg atcaagcacg tggaaaagaa cggcagtaaa 900tacgggcccg acgggctgcc ctacctcaag gttctcaagg ccgccggtgt taacaccacg 960gacaaagaga ttgaggttct ctatattcgg aatgtaactt ttgaggacgc tggggaatat 1020acgtgcttgg cgggtaattc tattgggata tcctttcact ctgcatggtt gacagttctg 1080ccagcgcctg gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt 1140tactgcatag gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg 1200aagaacacga ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa 1260cgtatccccc tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc 1320aacaccccgc tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg 1380gcaggggtct ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag 1440ctgacactgg gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca 1500gtgggaattg acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa 1560gatgatgcca cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg 1620attgggaaac acaagaatat cataaatctt cttggagcct gcacacagga tgggcctctc 1680tatgtcatag ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg 1740ccacccggga tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc 1800aaggacttgg tgtcatgcac ctaccagctg gccagaggca tggagtactt ggcttcccaa 1860aaatgtattc atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg 1920aaaatagcag actttggact cgccagagat atcaacaata tagactatta caaaaagacc 1980accaatgggc ggcttccagt caagtggatg gctccagaag ccctgtttga tagagtatac 2040actcatcaga gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 2100ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 2160agaatggata agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg 2220catgcagtgc cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt 2280ctcactctca caaccaatga gatcatggag gaaacaaata cgcagattgc ttggccatca 2340aaactgaaga tcggagccaa atccaagaaa gatccccata ttaaggtttc tggaaagaaa 2400gaagatgtta aagaagccaa ggaaatgatc atgtctgtct tagacacaaa aagcaatcga 2460gtcacactga agatggatgt ttcacataca gaacattcac atgtaatcgg caaaggtggc 2520aacaatatta aaaaagtgat ggaagaaacc ggatgccata tccactttcc agattccaac 2580aggaataacc aagcagaaaa aagcaaccag gtatctatag cgggacaacc agcaggagta 2640gaatctgccc gagttagaat tcgggagctg cttcctttgg tgctgatgtt tgagctacca 2700attgctggaa ttcttcaacc ggttcctgat cctaattccc cctctattca gcatatatca 2760caaacgtaca atatttcagt atcatttaaa cagcgttccc gaatgtatgg tgctactgtc 2820atagtacgag ggtctcagaa taacactagt gctgtgaagg aaggaactgc catgctgtta 2880gaacatcttg ctgggagctt agcatcagct attcctgtga gcacacaact agatattgca 2940gctcaacatc atctctttat gatgggtcga aatgggagca acatcaaaca tatcatgcag 3000agaacaggtg ctcagatcca ctttcctgat cccagtaatc cacaaaagaa atctaccgtc 3060tacctccagg gcaccattga gtctgtctgt cttgcaaggc aatatctcat gggttgtctt 3120cctcttgtgt tgatgtttga tatgaaggaa gaaattgaag tagatccaca attcattgcg 3180cagttgatgg aacagcttga tgtcttcatc agtattaaac caaagcccaa acagccaagc 3240aagtctgtga ttgtgaaaag tgttgagcga aatgccttaa atatgtatga agcaaggaaa 3300tgtctcctcg gacttgaaag cagtggggtt accatagcaa ccagtccatc cccagcatcc 3360tgccctgccg gcctggcatg tcccagcctg gatatcttag cttcagcagg ccttggactc 3420actggactag gtcttttggg acccaccacc ttatctctga acacttcaac aaccccaaac 3480tcactcttga atgctcttaa tagctcagtc agtcctttgc aaagtccaag ttctggtaca 3540cccagcccca cattatgggc acccccactt gctaatactt caagtgccac aggtttttct 3600gctataccac accttatgat tccatctact gcccaagcca cattaactaa tattttgttg 3660tctggagtgc ccacctatgg gcacacagct ccatctcccc ctcctggctt gactcctgtt 3720gatgtccata tcaacagtat gcagaccgaa ggcaaaaaaa tctctgctgc tttaaatgga 3780catgcacagt ctccagatat aaaatatggt gcaatatcca cttcatcact tggagaaaaa 3840gtgctgagtg caaatcacgg ggatccgtcc atccagacaa gtgggtctga gcagacatct 3900cccaaatcaa gccccactga aggttgtaat gatgcttttg ttgaagtagg catgcctcga 3960agtccttccc attctgggaa tgctggtgac ttgaaacaga tgatgtgtcc ctccaaggtt 4020tcctgtgcca aaaggcagac agtggaacta ttgcaaggca cgaaaaactc acacttacac 4080agcactgaca ggttgctctc agaccctgaa ctgagtgcta ccgaaagccc tttggctgac 4140aagaaggctc cagggagtga gcgcgctgca gagagggcag cagctgccca gcaaaactcc 4200gaaagggccc accttgctcc acggtcatca tatgtcaaca tgcaggcatt tgactatgaa 4260cagaagaagc tattagccac caaagctatg ttaaagaaac cagtggtgac ggaggtcaga 4320acgcccacaa atacctggag tggcctgggt ttttctaaat ccatgccagc tgaaactatc 4380aaggagttga gaagggccaa tcatgtgtcc tataagccca caatgacaac cacttatgag 4440ggctcatcca tgtccctttc acggtccaac agtcgtgagc acttgggagg tggaagcgaa 4500tctgataact ggagagaccg aaatggaatt ggacctggaa gtcatagtga atttgcagct 4560tctattggca gccctaagcg taaacaaaac aaatcaacgg aacactatct cagcagtagc 4620aattacatgg actgcatttc ctcgctgaca ggaagcaatg gctgtaactt aaatagctct 4680ttcaaaggtt ctgacctccc tgagctcttc agcaaactgg gcctgggcaa atacacagat 4740gttttccagc aacaagagat cgatcttcag acattcctca ctctcacaga tcaggatctg 4800aaggagctgg gaataactac ttttggtgcc aggaggaaaa tgctgcttgc aatttcagaa 4860ctaaataaaa accgaagaaa gctttttgaa tcgccaaatg cacgcacctc tttcctggaa 4920ggtggagcga gtggaaggct accccgtcag tatcactcag acattgctag tgtcagtggc 4980cgctggtag 4989615109DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 61atggtcagct ggggtcgttt catctgcctg gtcgtggtca ccatggcaac cttgtccctg 60gcccggccct ccttcagttt agttgaggat accacattag agccagaaga gccaccaacc 120aaataccaaa tctctcaacc agaagtgtac gtggctgcgc caggggagtc gctagaggtg 180cgctgcctgt tgaaagatgc cgccgtgatc agttggacta aggatggggt gcacttgggg 240cccaacaata ggacagtgct tattggggag tacttgcaga taaagggcgc cacgcctaga 300gactccggcc tctatgcttg tactgccagt aggactgtag acagtgaaac ttggtacttc 360atggtgaatg tcacagatgc catctcatcc ggagatgatg aggatgacac cgatggtgcg 420gaagattttg tcagtgagaa cagtaacaac aagagagcac catactggac caacacagaa 480aagatggaaa agcggctcca tgctgtgcct gcggccaaca ctgtcaagtt tcgctgccca 540gccgggggga acccaatgcc aaccatgcgg tggctgaaaa acgggaagga gtttaagcag 600gagcatcgca ttggaggcta caaggtacga aaccagcact ggagcctcat tatggaaagt 660gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg agaatgaata cgggtccatc 720aatcacacgt accacctgga tgttgtggag cgatcgcctc accggcccat cctccaagcc 780ggactgccgg caaatgcctc cacagtggtc ggaggagacg tagagtttgt ctgcaaggtt 840tacagtgatg cccagcccca catccagtgg atcaagcacg tggaaaagaa cggcagtaaa 900tacgggcccg acgggctgcc ctacctcaag gttctcaagg ccgccggtgt taacaccacg 960gacaaagaga ttgaggttct ctatattcgg aatgtaactt ttgaggacgc tggggaatat 1020acgtgcttgg cgggtaattc tattgggata tcctttcact ctgcatggtt gacagttctg 1080ccagcgcctg gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt 1140tactgcatag gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg 1200aagaacacga ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa 1260cgtatccccc tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc 1320aacaccccgc tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg 1380gcaggggtct ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag 1440ctgacactgg gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca 1500gtgggaattg acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa 1560gatgatgcca cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg 1620attgggaaac acaagaatat cataaatctt cttggagcct gcacacagga tgggcctctc 1680tatgtcatag ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg 1740ccacccggga tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc 1800aaggacttgg tgtcatgcac ctaccagctg gccagaggca tggagtactt ggcttcccaa 1860aaatgtattc atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg 1920aaaatagcag actttggact cgccagagat atcaacaata tagactatta caaaaagacc 1980accaatgggc ggcttccagt caagtggatg gctccagaag ccctgtttga tagagtatac 2040actcatcaga gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 2100ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 2160agaatggata agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg

2220catgcagtgc cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt 2280ctcactctca caaccaatga ggagagtaga tctggagaaa ccaacagctg tgttgaagaa 2340ataatccggg agatgacctg gcttccacca ctttctgcta ttcaagcacc tggcaaagtg 2400gaaccaacca aatttccatt tccaaataag gactctcagc ttgtatcctc tggacacaat 2460aatccaaaga aaggtgatgc agagccagag agtccagaca gtggcacatc gaatacatca 2520atgctggaag atgaccttaa gctaagcagt gatgaagagg agaatgaaca gcaggcagct 2580cagagaacgg ctctccgcgc tctctctgac agcgccgtgg tccagcagcc caactgcaga 2640acctcggtgc cttccagcaa gggcagcagc agcagcagca gcagcggcag cagcagctcc 2700tccagcgact cagagagcag ctccggatct gactcggaga ccgagagcag ctccagcgag 2760agtgagggca gcaagccccc ccacttctcc agccccgagg ctgaaccggc atcctctaac 2820aagtggcagc tggataaatg gctaaacaaa gttaatcccc acaagcctcc tattctgatc 2880caaaatgaaa gccacgggtc agagagcaat cagtactaca acccggtgaa agaggacgtc 2940caggactgtg ggaaagtccc cgacgtttgc cagcccagcc tgagagagaa ggagatcaag 3000agcacttgca aggaggagca aaggccaagg acagccaaca aggcccctgg gagtaaaggc 3060gtgaagcaga agtccccgcc cgcggccgtg gccgtggcgg tgagcgcagc cgccccgcca 3120cccgcagtgc cctgtgcgcc cgcggagaac gcgcccgcgc ctgcccggag gtccgcgggc 3180aagaagccca ccaggcgcac cgagaggacc tcagccgggg acggcgccaa ctgccaccgg 3240cccgaggagc ccgcggccgc ggacgcgctg gggacgagcg tggtggtccc cccggagccc 3300accaaaacca ggccctgtgg caacaacaga gcgagccacc gcaaggagct gcgctcctcc 3360gtgacctgcg agaagcgccg cacgcggggg ctaagcagga tcgtccccaa atccaaggag 3420ttcattgaga cagagtcgtc atcttcatcc tcctcctcgg actccgacct ggagtccgag 3480caggaggagt accctctgtc caaagcacag accgtggctg cctctgcctc ctccgggaat 3540gatcagaggc tgaaggaggc cgctgccaac gggggcagtg gtcctagggc ccctgtaggc 3600tccatcaacg ccaggaccac cagtgacatc gccaaggagc tggaggagca gttctacaca 3660ctggtcccct ttggccggaa cgaacttctc tcccctctaa aggacagtga tgagatcagg 3720tctctctggg tcaaaatcga cctgaccctc ctgtccagga tcccagaaca cctgccccag 3780gagccagggg tattgagcgc ccctgccacc aaggactctg agagcgcacc gcccagccac 3840acctcggaca cacctgcaga aaaggctttg ccaaaatcca agaggaaacg caagtgtgac 3900aacgaagacg actacaggga gatcaagaag tcccagggag agaaagacag ctcttcaaga 3960ctggccacct ccaccagtaa tactttgtct gcaaaccact gcaacatgaa catcaacagt 4020gtggcaatac caataaataa aaatgaaaaa atgcttcggt cgcccatctc acccctctct 4080gatgcatcta aacacaaata caccagcgag gacttaactt cttccagccg acctaatggc 4140aacagtttgt ttacttcagc ctcttccagc aaaaagccta aggccgacag ccagctgcag 4200cctcacggcg gagacctcac gaaagcagct cacaacaatt ctgaaaacat tcccctccac 4260aagtcacggc cgcagacgaa gccgtggtct ccaggctcca acggccacag ggactgcaag 4320aggcagaaac ttgtcttcga tgatatgcct cgcagtgccg attattttat gcaagaagct 4380aaacgaatga agcataaagc agatgcaatg gtggaaaagt ttggaaaggc tttgaactat 4440gctgaagcag cattgtcgtt tatcgagtgt ggaaatgcaa tggaacaagg ccccatggaa 4500tccaaatctc cttatacgat gtattcagaa acagtagagc tcatcaggta tgctatgaga 4560ctaaaaaccc actcaggccc caatgccaca ccagaagaca aacaactggc tgcattatgt 4620taccgatgcc tggccctcct gtactggcgg atgtttcgac tcaaaaggga ccacgctgta 4680aagtattcaa aagcactaat cgactatttc aagaactcat ctaaagccgc ccaagcccca 4740tctccgtggg gggccagtgg aaagagcact ggaaccccat cccccatgtc tcccaacccc 4800tctcccgcca gctccgtggg gtctcagggc agcctctcca acgccagcgc cctgtccccg 4860tcgaccatcg tcagcatccc acagcgcatc caccagatgg cggccaacca cgtcagcatc 4920accaacagca tcctgcacag ctacgactac tgggagatgg ccgacaacct ggccaaggaa 4980aaccgagaat tcttcaacga cctggatctg ctcatggggc cggtcaccct gcacagcagc 5040atggagcacc tggtccagta ctcccaacag ggcctgcact ggctgcggaa cagcgcccac 5100ctgtcatag 5109623213DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 62atggtcagct ggggtcgttt catctgcctg gtcgtggtca ccatggcaac cttgtccctg 60gcccggccct ccttcagttt agttgaggat accacattag agccagaaga gccaccaacc 120aaataccaaa tctctcaacc agaagtgtac gtggctgcgc caggggagtc gctagaggtg 180cgctgcctgt tgaaagatgc cgccgtgatc agttggacta aggatggggt gcacttgggg 240cccaacaata ggacagtgct tattggggag tacttgcaga taaagggcgc cacgcctaga 300gactccggcc tctatgcttg tactgccagt aggactgtag acagtgaaac ttggtacttc 360atggtgaatg tcacagatgc catctcatcc ggagatgatg aggatgacac cgatggtgcg 420gaagattttg tcagtgagaa cagtaacaac aagagagcac catactggac caacacagaa 480aagatggaaa agcggctcca tgctgtgcct gcggccaaca ctgtcaagtt tcgctgccca 540gccgggggga acccaatgcc aaccatgcgg tggctgaaaa acgggaagga gtttaagcag 600gagcatcgca ttggaggcta caaggtacga aaccagcact ggagcctcat tatggaaagt 660gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg agaatgaata cgggtccatc 720aatcacacgt accacctgga tgttgtggag cgatcgcctc accggcccat cctccaagcc 780ggactgccgg caaatgcctc cacagtggtc ggaggagacg tagagtttgt ctgcaaggtt 840tacagtgatg cccagcccca catccagtgg atcaagcacg tggaaaagaa cggcagtaaa 900tacgggcccg acgggctgcc ctacctcaag gttctcaagg ccgccggtgt taacaccacg 960gacaaagaga ttgaggttct ctatattcgg aatgtaactt ttgaggacgc tggggaatat 1020acgtgcttgg cgggtaattc tattgggata tcctttcact ctgcatggtt gacagttctg 1080ccagcgcctg gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt 1140tactgcatag gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg 1200aagaacacga ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa 1260cgtatccccc tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc 1320aacaccccgc tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg 1380gcaggggtct ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag 1440ctgacactgg gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca 1500gtgggaattg acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa 1560gatgatgcca cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg 1620attgggaaac acaagaatat cataaatctt cttggagcct gcacacagga tgggcctctc 1680tatgtcatag ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg 1740ccacccggga tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc 1800aaggacttgg tgtcatgcac ctaccagctg gccagaggca tggagtactt ggcttcccaa 1860aaatgtattc atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg 1920aaaatagcag actttggact cgccagagat atcaacaata tagactatta caaaaagacc 1980accaatgggc ggcttccagt caagtggatg gctccagaag ccctgtttga tagagtatac 2040actcatcaga gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 2100ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 2160agaatggata agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg 2220catgcagtgc cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt 2280ctcactctca caaccaatga gatggcagat gatcagggct gtattgaaga gcagggggtt 2340gaggattcag caaatgaaga ttcagtggat gctaagccag accggtcctc gtttgtaccg 2400tccctcttca gtaagaagaa gaaaaatgtc accatgcgat ccatcaagac cacccgggac 2460cgagtgccta catatcagta caacatgaat tttgaaaagc tgggcaaatg catcataata 2520aacaacaaga actttgataa agtgacaggt atgggcgttc gaaacggaac agacaaagat 2580gccgaggcgc tcttcaagtg cttccgaagc ctgggttttg acgtgattgt ctataatgac 2640tgctcttgtg ccaagatgca agatctgctt aaaaaagctt ctgaagagga ccatacaaat 2700gccgcctgct tcgcctgcat cctcttaagc catggagaag aaaatgtaat ttatgggaaa 2760gatggtgtca caccaataaa ggatttgaca gcccacttta ggggggatag atgcaaaacc 2820cttttagaga aacccaaact cttcttcatt caggcttgcc gagggaccga gcttgatgat 2880ggcatccagg ccgactcggg gcccatcaat gacacagatg ctaatcctcg atacaagatc 2940ccagtggaag ctgacttcct cttcgcctat tccacggttc caggctatta ctcgtggagg 3000agcccaggaa gaggctcctg gtttgtgcaa gccctctgct ccatcctgga ggagcacgga 3060aaagacctgg aaatcatgca gatcctcacc agggtgaatg acagagttgc caggcacttt 3120gagtctcagt ctgatgaccc acacttccat gagaagaagc agatcccctg tgtggtctcc 3180atgctcacca aggaactcta cttcagtcaa tag 3213633423DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 63atggtcagct ggggtcgttt catctgcctg gtcgtggtca ccatggcaac cttgtccctg 60gcccggccct ccttcagttt agttgaggat accacattag agccagaaga gccaccaacc 120aaataccaaa tctctcaacc agaagtgtac gtggctgcgc caggggagtc gctagaggtg 180cgctgcctgt tgaaagatgc cgccgtgatc agttggacta aggatggggt gcacttgggg 240cccaacaata ggacagtgct tattggggag tacttgcaga taaagggcgc cacgcctaga 300gactccggcc tctatgcttg tactgccagt aggactgtag acagtgaaac ttggtacttc 360atggtgaatg tcacagatgc catctcatcc ggagatgatg aggatgacac cgatggtgcg 420gaagattttg tcagtgagaa cagtaacaac aagagagcac catactggac caacacagaa 480aagatggaaa agcggctcca tgctgtgcct gcggccaaca ctgtcaagtt tcgctgccca 540gccgggggga acccaatgcc aaccatgcgg tggctgaaaa acgggaagga gtttaagcag 600gagcatcgca ttggaggcta caaggtacga aaccagcact ggagcctcat tatggaaagt 660gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg agaatgaata cgggtccatc 720aatcacacgt accacctgga tgttgtggag cgatcgcctc accggcccat cctccaagcc 780ggactgccgg caaatgcctc cacagtggtc ggaggagacg tagagtttgt ctgcaaggtt 840tacagtgatg cccagcccca catccagtgg atcaagcacg tggaaaagaa cggcagtaaa 900tacgggcccg acgggctgcc ctacctcaag gttctcaagg ccgccggtgt taacaccacg 960gacaaagaga ttgaggttct ctatattcgg aatgtaactt ttgaggacgc tggggaatat 1020acgtgcttgg cgggtaattc tattgggata tcctttcact ctgcatggtt gacagttctg 1080ccagcgcctg gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt 1140tactgcatag gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg 1200aagaacacga ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa 1260cgtatccccc tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc 1320aacaccccgc tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg 1380gcaggggtct ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag 1440ctgacactgg gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca 1500gtgggaattg acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa 1560gatgatgcca cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg 1620attgggaaac acaagaatat cataaatctt cttggagcct gcacacagga tgggcctctc 1680tatgtcatag ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg 1740ccacccggga tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc 1800aaggacttgg tgtcatgcac ctaccagctg gccagaggca tggagtactt ggcttcccaa 1860aaatgtattc atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg 1920aaaatagcag actttggact cgccagagat atcaacaata tagactatta caaaaagacc 1980accaatgggc ggcttccagt caagtggatg gctccagaag ccctgtttga tagagtatac 2040actcatcaga gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 2100ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 2160agaatggata agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg 2220catgcagtgc cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt 2280ctcactctca caaccaatga gcaagccagg gctgagcagg aagaagaatt cattagtaac 2340actttattca agaaaattca ggctttgcag aaggagaaag aaacccttgc tgtaaattat 2400gagaaagaag aagaattcct cactaatgag ctctccagaa aattgatgca gttgcagcat 2460gagaaagccg aactagaaca gcatcttgaa caagagcagg aatttcaggt caacaaactg 2520atgaagaaaa ttaaaaaact ggagaatgac accatttcta agcaacttac attagaacag 2580ttgagacggg agaagattga ccttgaaaat acattggaac aagaacaaga agcactagtt 2640aatcgcctct ggaaaaggat ggataagctt gaagctgaaa agcgaatcct gcaggaaaaa 2700ttagaccagc ccgtctctgc tccaccatcg cctagagata tctccatgga gattgattct 2760ccagaaaata tgatgcgtca catcaggttt ttaaagaatg aagtggaacg gctgaagaag 2820caactgagag ctgctcagtt acagcattca gagaaaatgg cacagtatct ggaggaggaa 2880cgtcacatga gagaagagaa cttgaggctc cagaggaagc tgcagaggga gatggagaga 2940agagaagccc tctgtcgaca gctctccgag agtgagtcca gcttagaaat ggacgacgaa 3000aggtatttta atgagatgtc tgcacaagga ttaagacctc gcactgtgtc cagcccgatc 3060ccttacacac cttctccgag ttcaagcagg cctatatcac ctggtctatc atatgcaagt 3120cacacggttg gtttcacgcc accaacttca ctgactagag ctggaatgtc ttattacaat 3180tccccgggtc ttcacgtgca gcacatggga acatcccatg gtatcacaag gccttcacca 3240cggagaagca acagtcctga caaattcaaa cggcccacgc cgcctccatc tcccaacaca 3300cagaccccag tccagccacc tccgcctcca cctccgccac ccatgcagcc cacggtcccc 3360tcagcagcca cctcgcagcc tactccttcg caacattcgg cgcacccctc ctcccagcct 3420taa 3423645229DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 64atggtcagct ggggtcgttt catctgcctg gtcgtggtca ccatggcaac cttgtccctg 60gcccggccct ccttcagttt agttgaggat accacattag agccagaaga gccaccaacc 120aaataccaaa tctctcaacc agaagtgtac gtggctgcgc caggggagtc gctagaggtg 180cgctgcctgt tgaaagatgc cgccgtgatc agttggacta aggatggggt gcacttgggg 240cccaacaata ggacagtgct tattggggag tacttgcaga taaagggcgc cacgcctaga 300gactccggcc tctatgcttg tactgccagt aggactgtag acagtgaaac ttggtacttc 360atggtgaatg tcacagatgc catctcatcc ggagatgatg aggatgacac cgatggtgcg 420gaagattttg tcagtgagaa cagtaacaac aagagagcac catactggac caacacagaa 480aagatggaaa agcggctcca tgctgtgcct gcggccaaca ctgtcaagtt tcgctgccca 540gccgggggga acccaatgcc aaccatgcgg tggctgaaaa acgggaagga gtttaagcag 600gagcatcgca ttggaggcta caaggtacga aaccagcact ggagcctcat tatggaaagt 660gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg agaatgaata cgggtccatc 720aatcacacgt accacctgga tgttgtggag cgatcgcctc accggcccat cctccaagcc 780ggactgccgg caaatgcctc cacagtggtc ggaggagacg tagagtttgt ctgcaaggtt 840tacagtgatg cccagcccca catccagtgg atcaagcacg tggaaaagaa cggcagtaaa 900tacgggcccg acgggctgcc ctacctcaag gttctcaagg ccgccggtgt taacaccacg 960gacaaagaga ttgaggttct ctatattcgg aatgtaactt ttgaggacgc tggggaatat 1020acgtgcttgg cgggtaattc tattgggata tcctttcact ctgcatggtt gacagttctg 1080ccagcgcctg gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt 1140tactgcatag gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg 1200aagaacacga ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa 1260cgtatccccc tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc 1320aacaccccgc tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg 1380gcaggggtct ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag 1440ctgacactgg gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca 1500gtgggaattg acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa 1560gatgatgcca cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg 1620attgggaaac acaagaatat cataaatctt cttggagcct gcacacagga tgggcctctc 1680tatgtcatag ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg 1740ccacccggga tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc 1800aaggacttgg tgtcatgcac ctaccagctg gccagaggca tggagtactt ggcttcccaa 1860aaatgtattc atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg 1920aaaatagcag actttggact cgccagagat atcaacaata tagactatta caaaaagacc 1980accaatgggc ggcttccagt caagtggatg gctccagaag ccctgtttga tagagtatac 2040actcatcaga gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 2100ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 2160agaatggata agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg 2220catgcagtgc cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt 2280ctcactctca caaccaatga gacacaactt cgaaaccagc taattcatga gttgatgcac 2340cctgtattga gtggagaact gcagcctcgg tccatttcag tagaagggag ctccctctta 2400ataggcgcct ctaactcttt agtggcagat cacttacaaa gatgtggcta tgaatattca 2460ctttctgttt tctttccaga aagtggtttg gcaaaagaaa aggtatttac tatgcaggat 2520ctattacaac tcattaaaat caaccctact tccagtctct acaaatcact ggtttcagga 2580tctgataaag aaaatcaaaa aggttttctt atgcattttt taaaagaatt ggcagaatat 2640catcaagcta aagagagttg taatatggaa actcagacaa gttcgacatt taacagagat 2700tctctggctg agaagcttca gcttattgat gatcagtttg cagatgctta ccctcagcgt 2760atcaagttcg aatctttaga aataaagcta aatgagtata agagagaaat agaagagcaa 2820cttcgggcag aaatgtgtca aaagttgaag ttttttaaag ataccgagat agcaaaaatt 2880aaaatggaag caaaaaaaaa gtatgaaaag gagttaacca tgttccagaa tgattttgaa 2940aaagcttgtc aagcaaaatc tgaagctctc gttcttcggg aaaagagtac ccttgaaaga 3000attcacaagc accaagagat tgaaacaaaa gaaatttatg ctcaaaggca acttttacta 3060aaagatatgg atttgctaag aggaagagaa gcagagctga agcaaagagt tgaagctttt 3120gaattgaacc agaagctcca ggaagaaaaa cataaaagca taactgaggc acttaggaga 3180caggagcaga atataaagag ttttgaggag acctatgacc gaaagctcaa gaatgaactt 3240ctaaagtatc aacttgaact gaaggatgac tacatcatta gaactaatcg actgattgaa 3300gatgaaagga agaataaaga aaaagctgtt catttgcaag aggagctcat agctattaat 3360tcaaaaaagg aggaactcaa tcaatctgta aatcgtgtga aagaacttga gcttgaatta 3420gagtctgtca aagcccagtc tttggcaata acaaaacaaa accatatgct gaatgaaaag 3480gttaaagaga tgagtgatta ttcactacta aaagaagaga aactggagct tctggcacaa 3540aataaattac ttaaacaaca actggaagag agtagaaatg aaaacctgcg tctcctaaac 3600cgcctagctc agccggctcc tgaacttgca gtctttcaga aagaactacg gaaagccgaa 3660aaggctatag tggttgagca tgaggagttc gaaagctgca ggcaagctct gcacaaacaa 3720ctgcaagacg aaattgagca ttctgcacag ctgaaggccc agattctagg ttacaaagct 3780tctgtaaaga gtttaactac tcaggttgcc gatttaaaat tgcaactgaa gcaaactcag 3840acagccctag agaatgaagt gtactgcaat ccaaagcagt ctgtgatcga tcgttctgtc 3900aatggattaa taaatggcaa tgtggtgcct tgcaatggtg agataagtgg ggatttcttg 3960aacaatcctt ttaaacagga aaacgttcta gcacgtatgg ttgcatcaag gatcacaaat 4020tatccaactg catgggtgga gggtagttcc cctgattctg accttgagtt tgtagccaat 4080actaaggcaa gggtcaaaga gcttcagcaa gaggccgaac gcttggaaaa ggctttcaga 4140agttaccatc ggagagtcat taaaaactct gccaaaagcc cactagcagc aaagagccca 4200ccatctctgc acttgctgga agccttcaaa aacattactt ccagttcccc ggaaagacat 4260atttttggag aggacagagt tgtctctgag cagcctcaag tgggcacact tgaagaaagg 4320aatgacgtcg tggaagcact gacaggcagt gcagcctcga ggctccgcgg gggcacttcc 4380tccagacgcc tctcttccac accccttcca aaagcaaaaa gaagcctcga aagtgaaatg 4440tatctggaag gtctgggcag atcacacatt gcttccccca gtccttgtcc tgacagaatg 4500cccctaccat cacccactga gtctaggcac agcctctcca tccctcctgt ctccagccct 4560ccggagcaga aagtgggtct ttatcgaaga caaactgaac ttcaagacaa aagtgaattt 4620tcagatgtgg acaagctagc ttttaaggat aatgaggagt ttgaatcatc ttttgaatct 4680gcagggaaca tgccaaggca gttggaaatg ggcgggcttt ctcctgccgg ggatatgtct 4740catgtggacg ctgctgcagc tgctgtgccc ctctcatatc agcacccaag tgtagatcag 4800aaacaaattg aagaacaaaa ggaagaagaa aaaatacggg aacagcaagt gaaagaacga 4860aggcagagag aagaaagaag gcagagtaac ctacaagaag ttttagaaag ggaacgaaga 4920gaactagaaa aactgtatca ggaaaggaag atgattgaag aatcactgaa gattaaaata 4980aaaaaggaat tagaaatgga aaatgaatta gaaatgagta atcaagaaat aaaagacaaa 5040tctgctcaca gtgaaaatcc tttagagaaa tacatgaaaa tcatccagca ggagcaagac 5100caggagtcgg cagataagag ctcaaaaaag

atggtccaag aaggctccct agtggacacg 5160ctgcaatcta gtgacaaagt cgaaagttta acaggctttt ctcatgaaga actagacgac 5220tcttggtaa 52296518DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 65tccaccgacg taaaggcg 186618DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 66taccgtgacg tccaccga 186718DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 67gccttctggc ccaggtgc 186818DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 68tccaccgaca atgttatg 186918DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 69accaatgagg agagtaga 187018DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 70accaatgaga tcatggag 187116DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 71accaatgaga tggcag 167218DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 72accaatgagc aagccagg 187318DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 73accaatgaga cacaactt 18

Claims

1. A method of identifying a cancer patient that is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor comprising: evaluating a biological sample from the patient for a FGFR mutant from a FGFR mutant gene panel, wherein the FGFR mutant is a FGFR fusion gene or a FGFR single nucleotide polymorphism, and wherein said evaluating comprises amplifying cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel; and determining whether the one or more FGFR mutants from the FGFR mutant gene panel are present in the sample, wherein the presence of the one or more FGFR mutants indicates that the patient is responsive to treatment with the FGFR inhibitor.

2. The method of claim 1, wherein the FGFR fusion gene comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof.

3. The method of claim 1, wherein the FGFR single nucleotide polymorphism comprises R248C, S249C, G370C, or Y373C, or any combination thereof.

4. The method of claim 1, wherein the cancer is bladder cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

5. The method of claim 1, wherein the cancer is metastatic bladder cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

6. The method of claim 1, wherein the cancer is ovarian cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

7. The method of claim 1, wherein the cancer is head and neck cancer and the FGFR mutant gene panel comprises FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

8. The method of claim 1, wherein the cancer is metastatic head and neck cancer and the FGFR mutant gene panel comprises FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof.

9. The method of claim 1, wherein the cancer is esophageal cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

10. The method of claim 1, wherein the cancer is metastatic esophageal cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.

11. The method of claim 1, wherein the cancer is non-small-cell lung carcinoma adenocarcinoma and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

12. The method of claim 1, wherein the cancer is non-small cell lung carcinoma squamous cell carcinoma and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

13. The method of claim 1, wherein the cancer is metastatic endometrial cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof.

14. The method of claim 1, wherein the cancer is breast cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.

15. The method of claim 1, wherein the cancer is hepatocellular carcinoma and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

16. The method of claim 1, wherein the FGFR mutant and pair of primers are: FGFR3:TACC3 v1 and primers having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6; FGFR3:TACC3 v3 and primers having the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8; FGFR3:TACC3 Intron and primers having the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10; FGFR3:BAIAP2L1 and primers having the amino acid sequences of SEQ ID NO:11 and SEQ ID NO:12; FGFR2:BICC1 and primers having the amino acid sequences of SEQ ID NO:13 and SEQ ID NO:14; FGFR2:AFF3 and primers having the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:16; FGFR2:CASP7 and primers having the amino acid sequences of SEQ ID NO:17 and SEQ ID NO:18; FGFR2:CCDC6 and primers having the amino acid sequences of SEQ ID NO:19 and SEQ ID NO:20; FGFR2:OFD1 and primers having the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22; R248C and primers having the amino acid sequences of SEQ ID NO:23 and SEQ ID NO:24 or SEQ ID NO:31 and SEQ ID NO:32; S249C and primers having the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:33 and SEQ ID NO:34; G370C and primers having the amino acid sequences of SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:35 and SEQ ID NO:36; Y373C and primers having the amino acid sequences of SEQ ID NO:29 and SEQ ID NO:30 or SEQ ID NO:37 and SEQ ID NO:38; or any combination thereof.

17. The method of claim 1, wherein the evaluating comprises: isolating RNA from the biological sample and synthesizing cDNA from the isolated RNA.

18. The method of claim 17, further comprising pre-amplifying the cDNA prior to the amplifying step.

19. The method of claim 1, wherein the cDNA is preamplified.

20. The method of claim 1, wherein the amplifying step comprises performing real-time PCR.

21. The method of claim 20, wherein the real-time PCR is performed with one or more probes comprising SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, and/or SEQ ID NO:55.

22. The method of claim 21, wherein the real-time PCR is further performed with one or more 3' blocking oligonucleotides comprising SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and/or SEQ ID NO:42.

23. The method of claim 1, wherein said determining step comprises sequencing the amplified cDNA.

24. A kit for identifying the presence of one or more FGFR mutant genes in a biological sample comprising: pairs of primers having the sequences of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, or any combination thereof; and instructions for performing an assay to detect one or more FGFR mutant genes.

25. The kit of claim 24, further comprising one or more probes, one or more 3' blocking oligonucleotides, or both.

26. The kit of claim 25, wherein a. the pair of primers have the sequences SEQ ID NO:5 and SEQ ID NO:6 and the probe has the sequence of SEQ ID NO:43; b. the pair of primers have the sequences SEQ ID NO:7 and SEQ ID NO:8 and the probe has the sequence of SEQ ID NO:44; c. the pair of primers have the sequences SEQ ID NO:9 and SEQ ID NO:10 and the probe has the sequence of SEQ ID NO:46; d. the pair of primers have the sequences SEQ ID NO:11 and SEQ ID NO:12 and the probe has the sequence of SEQ ID NO:47; e. the pair of primers have the sequences SEQ ID NO:13 and SEQ ID NO:14 and the probe has the sequence of SEQ ID NO:45; f. the pair of primers have the sequences SEQ ID NO:15 and SEQ ID NO:16 and the probe has the sequence of SEQ ID NO:48; g. the pair of primers have the sequences SEQ ID NO:17 and SEQ ID NO:18 and the probe has the sequence of SEQ ID NO:49; h. the pair of primers have the sequences SEQ ID NO:19 and SEQ ID NO:20 and the probe has the sequence of SEQ ID NO:50; i. the pair of primers have the sequences SEQ ID NO:21 and SEQ ID NO:22 and the probe has the sequence of SEQ ID NO:51; j. the pair of primers have the sequences SEQ ID NO:23 and SEQ ID NO:24 and the probe has the sequence of SEQ ID NO:52; k. the pair of primers have the sequences SEQ ID NO:25 and SEQ ID NO:26 and the probe has the sequence of SEQ ID NO:53; l. the pair of primers have the sequences SEQ ID NO:27 and SEQ ID NO:28 and the probe has the sequence of SEQ ID NO:54; m. the pair of primers have the sequences SEQ ID NO:29 and SEQ ID NO:30 and the probe has the sequence of SEQ ID NO:55; n. the pair of primers have the sequences SEQ ID NO:31 and SEQ ID NO:32, the probe has the sequence of SEQ ID NO:52, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:39; o. the pair of primers have the sequences SEQ ID NO:33 and SEQ ID NO:34, the probe has the sequence of SEQ ID NO:53, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:40; p. the pair of primers have the sequences SEQ ID NO:35 and SEQ ID NO:36, the probe has the sequence of SEQ ID NO:54, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:41; q. the pair of primers have the sequences SEQ ID NO:37 and SEQ ID NO:38, the probe has the sequence of SEQ ID NO:55, and the 3' blocking oligonucleotide has the sequence of SEQ ID NO:42; or r. any combination thereof.

27. A primer having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or any combination thereof.

28. A set of primers having the sequences of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, or any combination thereof.

29. An oligonucleotide probe having the sequence of any one of SEQ ID NOs:43-55, or any combination thereof.

30. An oligonucleotide having the sequence of any one of SEQ ID NOs:39-42, or any combination thereof.

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