Using FGFR mutation genomes to identify cancer patients who will respond to treatment with FGFR inhibitors

Abstract

This article discloses methods for identifying cancer patients who will respond to treatment with fibroblast growth factor receptor (FGFR) inhibitors, as well as methods for treating cancer patients. The methods include assessing the presence of one or more FGFR mutants from an FGFR-mutant genome in a biological sample from the patient. This article also discloses kits and primers for identifying the presence of one or more FGFR-mutant genes in a biological sample.

Description

[0001] This application is a divisional application of the Chinese national application filed on September 18, 2015, with application number 201580064601.1, entitled "Identification of Cancer Patients Responding to Treatment with FGFR Inhibitors Using FGFR Mutant Genomes".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 056,159, filed September 26, 2014, the disclosure of which is incorporated herein by reference in its entirety.

[0004] sequence list

[0005] This application includes a sequence list that has been electronically submitted in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on August 6, 2015, named 103693.000782_SL.txt, and is 66,185 bytes in size. Technical Field

[0006] This article provides methods for identifying cancer patients who will respond to treatment with fibroblast growth factor receptor inhibitors, as well as methods for treating said cancer patients. Background Technology

[0007] Identifying genetic abnormalities can help select one or more appropriate treatment options for cancer patients. This is also useful for cancer patients whose cancer type cannot be treated with a primary therapy (first-line therapy), especially in situations where there is no recognized standard of care for second-line and subsequent treatments. Fibroblast growth factor receptor (FGFR) belongs to the receptor tyrosine kinase family and is involved in regulating cell survival, proliferation, migration, and differentiation. Alterations in FGFR have been observed in some cancers. Currently, there are no approved therapies that can effectively treat patients with altered FGFR. Summary of the Invention

[0008] This article discloses a method for identifying cancer patients who will respond to treatment with fibroblast growth factor receptor (FGFR) inhibitors. The method includes: assessing an FGFR mutant from an FGFR-mutant genome in a biological sample from the patient, wherein the FGFR mutant is an FGFR fusion gene or an FGFR single nucleotide polymorphism, and wherein the assessment includes amplifying cDNA using primer pairs that bind to and amplify one or more FGFR mutants from the FGFR-mutant genome; and determining the presence of one or more FGFR mutants from the genome in the sample, wherein the presence of one or more FGFR mutants indicates that the patient will respond to treatment with an FGFR inhibitor.

[0009] This article also discloses a method for treating cancer in patients, which includes: assessing the presence of one or more FGFR mutants from an FGFR-mutant genome in a biological sample from the patient; and if one or more FGFR mutants are present in the sample, treating the patient with an FGFR inhibitor.

[0010] This article also provides kits and primers for identifying the presence of one or more FGFR mutant genes in biological samples. Attached Figure Description

[0011] The invention and the detailed embodiments described below can be further understood when read in conjunction with the accompanying drawings. Exemplary embodiments of the disclosed methods, kits, and primers are shown in the drawings to illustrate the disclosed methods, kits, and primers; however, the methods, kits, and primers are not limited to the specific embodiments disclosed. In the drawings:

[0012] Figure 1 This is an illustration of an exemplary FGFR fusion gene; the presence of at least one of these genes indicates that the patient will respond to treatment with an FGFR inhibitor. The illustration also shows (thin arrows) exemplary primer positions used to amplify the fusion gene.

[0013] Figure 2 (including) Figures 2A to 2I () indicates a positive result from Sanger sequencing of the following FFPET samples: 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.

[0014] Figure 3 An exemplary strategy for SNP-specific qRT-PCR using 3' dideoxy wild-type (WT) blocking oligonucleotides is shown.

[0015] Figure 4 An exemplary analytical validation strategy for detecting FGFR SNPs is shown. Experiments were performed in an engineered RK3E cell line expressing the FGFR fusion and diluted to a wild-type cell line that did not contain the FGFR3 / FGFR2 fusion.

[0016] Figure 5 (including) Figures 5A to 5D The following shows SNP-specific PCR results for dideoxyWT inhibitors (a) G370C, (B) Y373C, (C) S249C and (D) R248C.

[0017] Figure 6 (including) Figures 6A to 6I The following groups are represented by the efficiency standard curves for FGFR fusion gene assays: A) FGFR3:TACC3 v1; B) FGFR3:TACC3 v3; C) FGFR3:TACC3 introns; D) FGFR3:BAIAP2L1; E) FGFR2:AFF3; F) FGFR2:BICC1; G) FGFR2:CASP7; H) FGFR2:CCDC6 and I) FGFR2:OFD1.

[0018] Figure 7 This is an exemplary representation of the FGFR fusion gene status in bladder cancer (primary and metastatic), NSCLC (adenocarcinoma and squamous cell carcinoma), ovarian cancer, esophageal cancer (primary and metastatic), head and neck cancer (H&N, primary and metastatic), endometrial cancer (metastatic), breast cancer, and prostate cancer.

[0019] Figure 8 This is an exemplary representation of FGFR fusion genes and mutation states in NSCLC adenocarcinoma and squamous cell carcinoma.

[0020] Figure 9 (including) Figures 9A to 9D The numbers () represent exemplary results from samples from stage I patients. Determination was performed using synthetic template assay control (ST), GAPDH (quality control sample) primers, or specific primers from the following groups: A) FGFR2:BICC1 fusion; B) FGFR3:TACC3 (exon 18:exon 1) fusion; C) FGFR2:CCDC6 fusion; or D) FGFR3:TACC3 v1, FGFR3:TACC3 v3, or FGFR2:CCDC6 fusion. Patient samples were from the following groups: A—urothelial carcinoma; B—bladder cancer; C—cholangiocarcinoma; and D—adrenal carcinoma.

[0021] Figure 10 This describes an exemplary Phase I study design for a first-in-human study of JNJ-42756493 in patients with advanced solid tumors.

[0022] Figure 11 This represents the maximum percentage of inhibition of the total target lesion diameter reduction from baseline at a dose level greater than or equal to 6 mg. Patients with solid tumors were treated with different doses of the FGFR inhibitor JNJ-42756493 according to daily or intermittent dosing regimens (7 days dosing / 7 days off). The figure shows the dose and tumor type. Tumor reduction was measured according to RECIST criteria. Patients with FGFR gene translocations and mutations in their tumors appeared to be more sensitive to the FGFR inhibitor JNJ-42756493.

[0023] Figure 12 The expression of various FGFR fusions in RK3E cells stably transfected with the indicated FGFR fusions is shown.

[0024] Figure 13 (including) Figures 13A to 13B The results of colony formation assays in RK3E cells stably transfected with the FGFR fusion strain shown are presented. (A) Staining with 0.1% toluene crystal violet in a 6-well plate and (B) Bar graph showing the number of colonies per 100 inoculated cells. The results represent two independent experiments.

[0025] Figure 14 (including) Figures 14A to 14H The image shows the expression of an exemplary downstream target in RK3E cells stably transfected with the FGFR fusion variant shown. Detailed Implementation

[0026] The disclosed methods, kits, and primers can be more readily understood by referring to the accompanying drawings, which form a part of this disclosure, and the detailed embodiments described below. It should be understood that the disclosed methods, kits, and primers are not limited to the specific methods, kits, and primers described and / or illustrated herein, and the terminology used herein is for illustrative purposes only and is not intended to limit the methods, kits, and primers protected by the claims.

[0027] Mentioning a specific numerical value includes at least that specific value, unless the context explicitly specifies otherwise. When referring to a range of values, another embodiment includes from one specific value and / or to other specific values. Furthermore, when referring to values ​​expressed as a range, each value within that range is included. All ranges refer to closed intervals and can be combined.

[0028] It should be understood that, for clarity, certain features of the disclosed methods, kits, and primers are described in the context of individual embodiments, but may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed methods, kits, and primers are described in the context of individual embodiments, but may also be provided individually or in any sub-combination.

[0029] As used in this article, the singular forms “a,” “a,” and “the” include the plural forms.

[0030] The following abbreviations are used throughout the instruction manual: FGFR (fibroblast growth factor receptor); LLOQ (lower limit of quantitation); FGFR3:TACC3 (fusion of the gene encoding FGFR3 with protein 3 containing a transformed acid coil-coil); FGFR3:BAIAP2L1 (fusion of the gene encoding FGFR3 with protein 1, a brain-specific angiogenesis inhibitor 1-associated protein 2); FGFR2:AFF3 (fusion of the gene encoding FGFR2 with member 3 of the AF4 / FMR2 family); FGFR2:BICC1 (fusion of the gene encoding FGFR3 with protein 1, a brain-specific angiogenesis inhibitor 1-associated protein 2); FGFR2:AFF3 (fusion of the gene encoding FGFR2 with member 3 of the AF4 / FMR2 family); FGFR2:BICC1 (fusion of the gene encoding FGFR3 with protein 3 containing a transformed acid coil-coil). The following are considered fusions of FGFR2 gene and bi-tailed C homologue: FGFR2:CASP7 (FGFR2 gene and caspase 7), FGFR2:CCDC6 (FGFR2 gene and protein 6 containing a coiled-coil domain), FGFR2:OFD1 (FGFR2 gene and orofacial-digital syndrome 1), FFPET (formalin-fixed paraffin-embedded tissue), SNP (single nucleotide polymorphism), NSCLC (non-small cell lung cancer), and ct (circulatory threshold).

[0031] As used herein, “treatment” and similar terms mean reducing the severity and / or frequency of cancer symptoms, eliminating cancer symptoms and / or the root cause of said symptoms, reducing the frequency or likelihood of cancer symptoms and / or their root cause, and improving or remedying damage caused directly or indirectly by cancer.

[0032] "Biosample" refers to any sample from a patient from which cancer cells can be obtained and RNA isolated. Suitable biosamples include, but are not limited to, blood, lymph, bone marrow, solid tumor samples, or any combination thereof. In some embodiments, the biosample may be an FFPET.

[0033] As used herein, "pre-amplification" refers to a PCR procedure performed prior to the amplification step, the purpose of which is to increase the amount of template cDNA used in the amplification step. The pre-amplification step may, for example, use... Pre-amplified master mixture (LifeTechnologies / Applied) Product No. 4391128) was used.

[0034] As used herein, "amplification" and similar terms refer to the production of many identical copies of a nucleic acid sample. Suitable techniques for amplifying nucleic acid samples include, but are not limited to, polymerase chain reaction (PCR) and real-time polymerase chain reaction (RT-PCR). In some embodiments, the amplification step includes RT-PCR.

[0035] FGFR mutant

[0036] As used in this article, the phrase “FGFR mutant” refers to an FGFR fusion gene, an FGFR single nucleotide polymorphism, or both.

[0037] "FGFR fusion" or "FGFR fusion gene" refers to either of these genes formed by a translocation between a gene encoding an FGFR (e.g., FGRF2 or FGFR3) or a portion thereof and one or a portion thereof of a fusion partner disclosed herein. The disclosed methods can be used to determine the presence of one or more of the following FGFR fusion genes in a biological sample from a patient: FGFR3:TACC3v1, FGFR3:TACC3v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, or any combination thereof. Table 1 provides FGFR fusion genes and the exons of fused FGFRs and fusion partners. Figure 1 Illustrations of various FGFR fusion genes are provided. The sequences of each FGFR fusion gene are disclosed in Table 16.

[0038] Table 1

[0039]

[0040]

[0041] An FGFR single nucleotide polymorphism (SNP) is a difference in a single nucleotide in the FGFR2 or FGFR3 gene between individuals. Specifically, an FGFR single nucleotide polymorphism (SNP) is a difference in a single nucleotide in the FGFR3 gene between individuals. The disclosed methods can be used to determine the presence of one or more of the following FGFR SNPs in a biological sample from a patient: FGFR3R248C, FGFR3 S249C, FGFR3 G370C, FGFR3 Y373C, or any combination thereof. The sequences of the FGFR SNPs are provided in Table 2.

[0042] Table 2

[0043]

[0044]

[0045] The sequence corresponds to nucleotides 920-1510 of FGFR3 (Genebank ID#NM_000142.4).

[0046] Nucleotides marked in bold and underlined represent SNPs.

[0047] *Sometimes it is mistakenly identified as Y375C in the literature.

[0048] As used herein, “FGFR mutant genome” includes one or more of the listed FGFR mutants. In some implementations, the FGFR mutant genome depends on the patient’s cancer type.

[0049] The FGFR mutant set used in the evaluation steps of the disclosed method is based to some extent on the patient's cancer type. For bladder cancer patients, suitable FGFR mutant genomes may include 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.

[0050] For patients with metastatic bladder cancer, suitable FGFR mutation genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0051] For ovarian cancer patients, suitable FGFR mutation genomes may include FGFR3:TACC3 v1, FGFR3:TACC3v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0052] For patients with head and neck cancer, suitable FGFR mutation genomes may include FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0053] For patients with metastatic head and neck cancer, suitable FGFR mutation genomes may include FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof.

[0054] For esophageal cancer patients, suitable FGFR mutation genomes may include FGFR3:TACC3 v1, FGFR3:TACC3v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0055] For patients with metastatic esophageal cancer, suitable FGFR mutation genomes may include 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.

[0056] For patients with non-small cell lung adenocarcinoma, suitable FGFR mutated genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0057] For patients with non-small cell lung squamous cell carcinoma, suitable FGFR mutation genomes may include FGFR3:TACC3v1, FGFR3:TACC3v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0058] For patients with metastatic endometrial cancer, suitable FGFR mutated genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof.

[0059] For breast cancer patients, suitable FGFR mutated genomes may include FGFR3:TACC3v1, FGFR3:TACC3v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.

[0060] Primers for amplifying the FGFR mutant

[0061] Those skilled in the art will understand that nucleic acid amplification requires primers that are complementary to and bind to the 5' and 3' regions of the nucleic acid strands flanking the region to be amplified. As used herein, a "primer pair" refers to the forward and reverse primers used in the amplification step. Primer pairs suitable for performing the disclosed methods are listed in Table 3.

[0062] Table 3

[0063]

[0064] Disclosed herein are primers having the following nucleic acid sequences: 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.

[0065] This paper also discloses primer sets having the following sequences: 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. NO:38, or any combination thereof.

[0066] In some embodiments, the primer set may have the sequences of SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, the primer set may have the sequences of SEQ ID NO:7 and SEQ ID NO:8. In some embodiments, the primer set may have the sequences of SEQ ID NO:9 and SEQ ID NO:10. In some embodiments, the primer set may have the sequences of SEQ ID NO:11 and SEQ ID NO:12. In some embodiments, the primer set may have the sequences of SEQ ID NO:13 and SEQ ID NO:14. In some embodiments, the primer set may have the sequences of SEQ ID NO:15 and SEQ ID NO:16. In some embodiments, the primer set may have the sequences of SEQ ID NO:17 and SEQ ID NO:18. In some embodiments, the primer set may have the sequences of SEQ ID NO:19 and SEQ ID NO:20. In some embodiments, the primer set may have the sequences of SEQ ID NO:21 and SEQ ID NO:22. In some embodiments, the primer set may have the sequences of SEQ ID NO:23 and SEQ ID NO:24. In some embodiments, the primer set may have the sequences of SEQ ID NO:25 and SEQ ID NO:26. In some embodiments, the primer set may have the sequences of SEQ ID NO:27 and SEQ ID NO:28. In some embodiments, the primer set may have the sequences of SEQ ID NO:29 and SEQ ID NO:30. In some embodiments, the primer set may have the sequences of SEQ ID NO:31 and SEQ ID NO:32. In some embodiments, the primer set may have the sequences of SEQ ID NO:33 and SEQ ID NO:34. In some embodiments, the primer set may have the sequences of SEQ ID NO:35 and SEQ ID NO:36. In some embodiments, the primer set may have the sequences of SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, the primer set may have any combination of the sequences described above.

[0067] FGFR inhibitors used in the disclosed method

[0068] This article provides suitable FGFR inhibitors for the disclosed methods.

[0069] In some embodiments, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor disclosed in U.S. Publication No. 2013 / 0072457A1 (incorporated herein by reference), said FGFR inhibitor comprising any tautomer or stereochemical isomer thereof and its N-oxide, pharmaceutically acceptable salt thereof, or solvate thereof (the suitable R group is disclosed in U.S. Publication No. 2013 / 0072457A1). In some aspects, for example, the patient may 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”):

[0070]

[0071] This includes its N-oxide, its pharmaceutically acceptable salt, or its solvates. In some respects, the pharmaceutically acceptable salt is the HCl salt. In some respects, the JNJ493 base can be used to treat patients.

[0072] In some implementations, if a patient sample contains one or more FGFR mutants, the patient can be treated with an FGFR inhibitor, wherein the FGFR inhibitor is N-[5-[2-(3,5-dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3,5-dimethylpiperazin-1-yl)benzamide (AZD4547), as described by Gavine, PR et al. in “AZD4547: An Orally Bioavailable, Potent, and Selective Inhibitor of the Fibroblast Growth Factor Receptor Tyrosine Kinase Family, Cancer Res, April 15, 2012, Vol. 72, p. 2045”:

[0073]

[0074] Where chemically possible, this includes any of its tautomers or stereochemical isomers and its N-oxides, pharmaceutically acceptable salts or solvates thereof.

[0075] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is 3-(2,6-dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]pyrimidin-4-yl}-1-methyl-urea (NVP-BGJ398), as described in International Publication No. WO2006 / 000420:

[0076]

[0077] Where chemically possible, this includes any of its tautomers or stereochemical isomers and its N-oxides, pharmaceutically acceptable salts or solvates thereof.

[0078] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is 4-amino-5-fluoro-3-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-quinolin-2-one (dovirtinib), as described in International Publication No. WO2006 / 127926:

[0079]

[0080] Where chemically possible, this includes any of its tautomers or stereochemical isomers and its N-oxides, pharmaceutically acceptable salts or solvates thereof.

[0081] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient can be treated with an FGFR inhibitor, wherein the FGFR inhibitor is 6-(7-((1-aminocyclopropyl)-methoxy)-6-methoxyquinoline-4-yloxy)-N-methyl-1-naphthylcarboxamide (AL3810) (deritinib, E-3810), as described by Bello, E. et al. in “E-3810 Is a Potent Dual Inhibitor of VEGFR and FGFR that Exerts Antitumor Activity in Multiple Preclinical Models, Cancer Res, February 15, 2011, Vol. 71(A), pp. 1396-1405” and International Publication No. WO2008 / 112408:

[0082]

[0083] Where chemically possible, this includes any of its tautomers or stereochemical isomers and its N-oxides, pharmaceutically acceptable salts or solvates thereof.

[0084] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is an anti-FGFR2 antibody, as described in WO2013 / 076186.

[0085] Other 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, where chemically possible, any of their tautomers or stereochemical isomers, their N-oxides, their pharmaceutically acceptable salts, or their solvates.

[0086] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is BAY1163877 (Bayer), including, where chemically possible, any of its tautomers or stereochemical isomers, its N-oxide, its pharmaceutically acceptable salts, or solvates thereof.

[0087] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is BAY1179470 (Bayer), including, where chemically possible, any of its tautomers or stereochemical isomers, its N-oxides, its pharmaceutically acceptable salts, or solvates thereof.

[0088] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is TAS-120 (Taiho), including, where chemically possible, any of its tautomers or stereochemical isomers, its N-oxide, its pharmaceutically acceptable salt, or its solvates.

[0089] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is ARQ087 (ArQule), including, where chemically possible, any of its tautomers or stereochemical isomers, its N-oxide, its pharmaceutically acceptable salts, or solvates thereof.

[0090] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is ASP5878 (Astellas), including, where chemically possible, any of its tautomers or stereochemical isomers, its N-oxides, its pharmaceutically acceptable salts, or solvates thereof.

[0091] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is FF284 (Chugai), including, where chemically possible, any tautomer or stereochemical isomer thereof, and, N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0092] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is FP-1039 (GSK / FivePrime), including, where chemically possible, any of its tautomers or stereochemical isomers, its N-oxide, its pharmaceutically acceptable salts, or solvates thereof.

[0093] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is a Blueprint, including, where chemically possible, any tautomer or stereochemical isomer of it, its N-oxide, its pharmaceutically acceptable salt or solvation thereof.

[0094] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is LY-2874455 (Lilly), including, where chemically possible, any of its tautomers or stereochemical isomers, its N-oxide, its pharmaceutically acceptable salts, or solvates thereof.

[0095] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient may be treated with an FGFR inhibitor, wherein the FGFR inhibitor is RG-7444 (Roche), including, where chemically possible, any of its tautomers or stereochemical isomers, its N-oxide, its pharmaceutically acceptable salts, or solvates thereof.

[0096] Salts can be synthesized from parent compounds containing basic or acidic moieties using conventional chemical methods, such as those described in "Pharmaceutical Salts: Properties, Selection, and Use, edited by P. Heinrich Stahl and Camille G. Wermuth, ISBN: 3-90639-026-8, hardcover, page 388, August 2002," which is incorporated herein by reference. Typically, these salts are prepared by reacting the free acidic or basic form of these compounds with a suitable base or acid in water, an organic solvent, or a mixture of both, generally using a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. Depending on the pKa of the acid forming the salt, the FGFR inhibitors used in the disclosed methods can exist in single or disalt form.

[0097] Acid addition salts can be formed from a variety of inorganic and organic acids. Examples of acid addition salts include salts formed from acids, including but not limited to: acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+)camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactobionic acid, gentian acid, glucoheponic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutarate, glycolic acid, hippuric acid, hydrogen... Bromic acid, hydrochloric acid, hydroiodic acid, hydroxyethanesulfonic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalenesulfonic acid (e.g., naphthalene-2-sulfonic acid), naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanate, toluenesulfonic acid (e.g., p-toluenesulfonic acid), undecenoic acid and valeric acid, as well as acetylated amino acids and cation exchange resins.

[0098] One special group of salts consists of acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, ethanesulfonic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid (methanesulfonates), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid, and lactobionic acid. Another group of acid addition salts includes those formed from acetic acid, adipic acid, ascorbic acid, aspartic acid, citric acid, DL-lactic acid, fumaric acid, gluconic acid, glucuronic acid, hippuric acid, hydrochloric acid, glutamic acid, DL-malic acid, methanesulfonic acid, sebacic acid, stearic acid, succinic acid, and tartaric acid.

[0099] If the compound is anionic or has a functional group that can be anionic (e.g., -COOH can be -COO), - If a suitable inorganic cation is present, then the salt can be formed using a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+. + and K + Alkaline earth metal cations such as Ca 2+ and Mg 2+ and other cations such as Al 3+ Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4+). + ) and substituted ammonium ions (e.g., NH3R) + NH2R2 + NHR3 + NR4 + ).

[0100] Examples of suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, aminobutanetriol, and amino acids such as lysine and arginine. A common example of a quaternary ammonium ion is N(CH3)4. + .

[0101] When compounds contain amine functional groups, they can form quaternary ammonium salts, for example, by reacting with an alkylating agent according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of compounds disclosed in this invention. Compounds containing amine functional groups can also form N-oxides. Compounds containing amine functional groups mentioned herein also include N-oxides. In the case of compounds containing several amine functional groups, one or more nitrogen atoms can be oxidized to form N-oxides. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen atoms in nitrogen-containing heterocycles. N-oxides can be formed by treating the corresponding amine with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid), see, for example, Advanced Organic Chemistry, Jerry, 4th edition, Wiley Interscience, several pages. More specifically, N-oxides can be prepared by the LWDeady procedure (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.

[0102] As used herein, the term "solvent" refers to the physical association of a compound with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate will be separable, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. The term "solvent" is intended to encompass both the solution phase and the separable solvate. Non-limiting examples of suitable solvates include combinations of the compounds disclosed in this invention with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, ethanolamine, etc. The compound can exert its biological activity in solution.

[0103] Solvates are well known in pharmaceutical chemistry. They can be important for the preparation of substances (e.g., in relation to their purification), storage (e.g., their stability), and ease of handling, and are often formed as part of an isolated phase or purification stage of chemical synthesis. Those skilled in the art can determine whether hydrates or other solvates are formed by the isolation or purification conditions used to prepare a given compound using standard and long-established techniques. Examples of these 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 nuclear magnetic resonance (SS-NMR, also known as magic-angle rotating NMR or MAS-NMR). These techniques, such as NMR, IR, HPLC, and MS, are also part of the standard analytical toolkit for skilled chemists. Alternatively, those skilled in the art can intentionally form solvates using crystallization conditions that include the amount of solvent required for a particular solvate. The standard methods described above can then be used to determine whether the solvate has formed. This also includes any complexes of FGFR inhibitors (e.g., inclusion complexes or inclusion compounds with compounds such as cyclodextrins, or complexes with metals).

[0104] In addition, the compound may have one or more polymorphs (crystalline) or amorphous forms.

[0105] The term "compound" includes compounds having one or more isotopic substitutions, and when referring to a specific element, it includes all isotopes of that element within its scope. For example, when referring to hydrogen, it includes... 1 H, 2 H(D) and 3 H(T). Similarly, when referring to carbon and oxygen, their respective ranges include: 12 C 13 C and 14 C and 16 O and 18 O. The isotope may be radioactive or non-radioactive. In one embodiment, the compound does not contain a radioactive isotope. These compounds are preferably intended for therapeutic use. However, in another embodiment, the compound may contain one or more radioactive isotopes. Compounds containing such radioactive isotopes may be useful in diagnostic situations.

[0106] In some implementations, if one or more FGFR mutants are present in a patient sample, the patient is treated with an FGFR inhibitor, 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 as "JNJ-42756493"), or a pharmaceutically acceptable salt thereof or a solvation thereof.

[0107] Methods of treating cancer patients

[0108] This article discloses a method for treating cancer patients, which includes: assessing the presence of one or more FGFR mutants from an FGFR-mutant genome in a biological sample from the patient; and if one or more FGFR mutants are present in the sample, treating the patient with an FGFR inhibitor.

[0109] The disclosed methods can be used to treat various types of cancer, 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, bile duct cancer, hepatocellular carcinoma, glioblastoma, glioma, colon cancer, sarcoma, primary squamous solid tumor, and multiple myeloma.

[0110] The FGFR mutant set used in the evaluation steps is based to some extent on the patient's cancer type. For example, for a bladder cancer patient, suitable FGFR mutant genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3G370C, or FGFR3 Y373C, or any combination thereof. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3R248C is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3S249C is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3G370C is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3Y373C is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the bladder cancer patient is treated with an FGFR inhibitor.

[0111] For example, for patients with metastatic bladder cancer, suitable FGFR mutant genomes may include 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. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3 R248C is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3 S249C is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3 G370C is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3 Y373C is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the patient with metastatic bladder cancer is treated with an FGFR inhibitor.

[0112] For example, for ovarian cancer patients, suitable FGFR-mutated genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3R248C is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3S249C is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3G370C is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3Y373C is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the ovarian cancer patient is treated with an FGFR inhibitor.

[0113] For example, for head and neck cancer patients, suitable FGFR mutated genomes may include FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Therefore, in some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the head and neck cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the head and neck cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3 R248C is present in the sample, the head and neck cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3 S249C is present in the sample, the head and neck cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3 G370C is present in the sample, the head and neck cancer patient is treated with an FGFR inhibitor. In some implementations, if FGFR3 Y373C is present in the sample, the patient with head and neck cancer is treated with an FGFR inhibitor. In some implementations, if any combination of the above-mentioned FGFR mutants is present in the sample, the patient with head and neck cancer is treated with an FGFR inhibitor.

[0114] For example, for patients with metastatic head and neck cancer, suitable FGFR mutant genomes may include FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof. Therefore, in some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the patient with metastatic head and neck cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the patient with metastatic head and neck cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:OFD1 is present in the sample, the patient with metastatic head and neck cancer is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the patient with metastatic head and neck cancer is treated with an FGFR inhibitor.

[0115] For example, for esophageal cancer patients, suitable FGFR mutant genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3Y373C, or any combination thereof. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the esophageal cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the esophageal cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the esophageal cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the esophageal cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3 R248C is present in the sample, the esophageal cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3 S249C is present in the sample, the esophageal cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3 G370C is present in the sample, the esophageal cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3 Y373C is present in the sample, the esophageal cancer patient is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-described FGFR mutants is present in the sample, the esophageal cancer patient is treated with an FGFR inhibitor.

[0116] For example, for patients with metastatic esophageal cancer, suitable FGFR-mutated genomes may include 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. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 intron is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CCD6 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:OFD1 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor.

[0117] For example, for patients with non-small cell lung (NSCL) adenocarcinoma, suitable FGFR-mutated genomes may include 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. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 intron is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3R248C is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3S249C is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3G370C is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3Y373C is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor. In some implementations, if any combination of the above-mentioned FGFR mutants is present in the sample, the NSCL adenocarcinoma patient is treated with an FGFR inhibitor.

[0118] For example, for patients with non-small cell lung (NSCL) squamous cell carcinoma, suitable FGFR-mutated genomes may include 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. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the NSCL squamous cell carcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the NSCL squamous cell carcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the NSCL squamous cell carcinoma patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the patient with NSCL squamous cell carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the patient with NSCL squamous cell carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the patient with NSCL squamous cell carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CCDC6 is present in the sample, the patient with NSCL squamous cell carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 R248C is present in the sample, the patient with NSCL squamous cell carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 S249C is present in the sample, the patient with NSCL squamous cell carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 G370C is present in the sample, the patient with NSCL squamous cell carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 Y373C is present in the sample, the patient with NSCL squamous cell carcinoma is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the patient with NSCL squamous cell carcinoma is treated with an FGFR inhibitor.

[0119] For example, for patients with metastatic endometrial cancer, suitable FGFR-mutated genomes may include FGFR3:TACC3v1, FGFR3:TACC3v3, FGFR3:TACC3 introns, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof. Therefore, in some embodiments, if FGFR3:TACC3v1 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3v3 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 introns are present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CCDC6 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:OFD1 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor.

[0120] For example, for breast cancer patients, suitable FGFR-mutated genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 introns, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 introns are present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CCD6 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:OFD1 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the breast cancer patient is treated with an FGFR inhibitor.

[0121] For example, for patients with hepatocellular carcinoma, suitable FGFR mutant genomes may include 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, FGFR3G370C, or FGFR3 Y373C, or any combination thereof. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 intron is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CCDC6 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:OFD1 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 R248C is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 S249C is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 G370C is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 Y373C is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-described FGFR mutants is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor.

[0122] In some implementations, the evaluation steps include: isolating RNA from a biological sample; synthesizing cDNA from the isolated RNA; pre-amplifying the cDNA; and amplifying the pre-amplified cDNA with primer pairs that bind to and amplify one or more FGFR mutants from an FGFR mutant genome.

[0123] RNA can be isolated from biological samples using a number of procedures known to those skilled in the art. In one embodiment, RNA can be isolated from biological samples using the AllPrep DNA / RNA FFPE kit (product number 80234) from Qiagen.

[0124] The synthesis of cDNA from isolated RNA can be performed through several procedures known to those skilled in the art. In one embodiment, cDNA can be synthesized from isolated RNA using a high-capacity cDNA reverse transcriptase kit (product number 4374966) with an RNase inhibitor from ABI.

[0125] Pre-amplification of cDNA can be performed using several procedures known to those skilled in the art. Amplification procedures are well known in the art. In one embodiment, a method can be used... Pre-amplified master mixture (Life Technologies / Applied) Pre-amplified cDNA (Product No. 4391128).

[0126] In some implementations, the amplification step may include performing real-time PCR (qRT-PCR). Exemplary qRT-PCR procedures are discussed in the Examples section of this document. In some aspects, qRT-PCR may be... Real-Time PCR Assay. qRT-PCR procedures may involve the use of probes to increase the specificity of the assay. Suitable probes for qRT-PCR assays include any probes disclosed herein, such as those disclosed in Table 15. In some embodiments, real-time PCR may be performed using one or more probes, including: 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, real-time PCR can be performed using one or more probes, said probes being substantially composed of the following: 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, real-time PCR can be performed using one or more probes, said probes being composed of the following: 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, real-time PCR can be performed using one or more probes having the following characteristics: 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.

[0127] qRT-PCR can be performed using one or more 3' blocking oligonucleotides. Exemplary qRT-PCR procedures using 3' blocking oligonucleotides are disclosed in the Examples section of this document. Suitable 3' blocking oligonucleotides include, for example, those disclosed in Table 8. In some embodiments, qRT-PCR can be performed using one or more 3' blocking oligonucleotides, said 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, qRT-PCR can be performed using one or more 3' blocking oligonucleotides, said 3' blocking oligonucleotides consisting substantially of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and / or SEQ ID NO:42. In some embodiments, qRT-PCR can be performed using one or more 3' blocking oligonucleotides, said 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 implementations, qRT-PCR can be performed using 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.

[0128] Suitable primer pairs for the amplification step include those disclosed in Table 3. For example, in some embodiments, the FGFR mutant and primer pair may 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 primer pair may 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 primer pair may be FGFR3:TACC3 introns and primers having the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10. In some embodiments, the FGFR mutant and primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair 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 primer pair can be any combination of the FGFR mutant and corresponding primer pair disclosed above.

[0129] In some implementations, the amplification step can be performed in the following ways:

[0130] a. The primer pair has the sequences of SEQ ID NO:5 and SEQ ID NO:6, and the probe has the sequence of SEQ ID NO:43;

[0131] b. The primer pair has the sequences of SEQ ID NO:7 and SEQ ID NO:8, and the probe has the sequence of SEQ ID NO:44;

[0132] c. The primer pair has the sequences of SEQ ID NO:9 and SEQ ID NO:10, and the probe has the sequence of SEQ ID NO:46;

[0133] d. The primer pair has the sequences of SEQ ID NO:11 and SEQ ID NO:12, and the probe has the sequence of SEQ ID NO:47;

[0134] e. The primer pair has the sequences of SEQ ID NO:13 and SEQ ID NO:14, and the probe has the sequence of SEQ ID NO:45;

[0135] f. The primer pair has the sequences of SEQ ID NO:15 and SEQ ID NO:16, and the probe has the sequence of SEQ ID NO:48;

[0136] g. The primer pair has the sequences of SEQ ID NO:17 and SEQ ID NO:18, and the probe has the sequence of SEQ ID NO:49;

[0137] h. The primer pair has the sequences of SEQ ID NO:19 and SEQ ID NO:20, and the probe has the sequence of SEQ ID NO:50;

[0138] i. The primer pair has the sequences of SEQ ID NO:21 and SEQ ID NO:22, and the probe has the sequence of SEQ ID NO:51;

[0139] j. The primer pair has the sequences of SEQ ID NO:23 and SEQ ID NO:24, and the probe has the sequence of SEQ ID NO:52;

[0140] k. The primer pair has the sequences of SEQ ID NO:25 and SEQ ID NO:26, and the probe has the sequence of SEQ ID NO:53;

[0141] l. The primer pair has the sequences of SEQ ID NO:27 and SEQ ID NO:28, and the probe has the sequence of SEQ ID NO:54;

[0142] m. The primer pair has the sequences of SEQ ID NO:29 and SEQ ID NO:30, and the probe has the sequence of SEQ ID NO:55;

[0143] n. The primer pair has the sequences of 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.

[0144] sequence;

[0145] o. The primer pair has the sequences of 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;

[0146] p. The primer pair has the sequences of 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;

[0147] q. The primer pair has the sequences of 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

[0148] r. Any combination of them.

[0149] The disclosed method includes treating a patient in the presence of one or more FGFR mutants in a sample. The presence of one or more FGFR mutants in a sample can be determined, for example, by sequencing the amplified cDNA.

[0150] Suitable FGFR inhibitors for treatment include those previously described in this article.

[0151] This article also discloses an FGFR inhibitor for treating cancer in patients, wherein a patient’s response to treatment with an FGFR inhibitor is identified by assessing the presence of one or more FGFR mutants from an FGFR-mutant genome in a biological sample obtained from the patient, wherein one or more FGFR mutants are detected in the sample.

[0152] This article also discloses an FGFR inhibitor for treating cancer in patients, wherein a patient’s responsiveness to treatment with an FGFR inhibitor is determined by assessing the presence of one or more FGFR mutants from an FGFR-mutant genome in a biological sample obtained from the patient, wherein the one or more FGFR mutants are FGFR fusion genes or FGFR SNPs, wherein the presence of one or more FGFR mutants in the sample is detected, and wherein the assessment includes amplifying cDNA with primer pairs that bind to and amplify one or more FGFR mutants from an FGFR-mutant genome.

[0153] This article also discloses an FGFR inhibitor for treating cancer in patients, wherein a patient’s responsiveness to treatment with an FGFR inhibitor is determined by assessing the presence of one or more FGFR mutants from an FGFR-mutant genome in a biological sample obtained from the patient, wherein the FGFR mutant is an FGFR fusion gene or an FGFR SNP, wherein the presence of one or more FGFR mutants in the sample is detected, and wherein the assessment includes amplifying pre-amplified cDNA with primer pairs that bind to and amplify one or more FGFR mutants from an FGFR-mutant genome.

[0154] Identify cancer patients who will respond to treatment with fibroblast growth factor receptor (FGFR) inhibitors. Method

[0155] This article discloses a method for identifying cancer patients who will respond to treatment with fibroblast growth factor receptor (FGFR) inhibitors, the method comprising: evaluating FGFR mutants from an FGFR-mutant genome in a biological sample from the patient, wherein the FGFR mutant is an FGFR fusion gene or an FGFR single nucleotide polymorphism, and wherein the evaluation comprises amplifying cDNA with primer pairs that bind to and amplify one or more FGFR mutants from the FGFR-mutant genome, and determining the presence of one or more FGFR mutants from the genome in the sample, wherein the presence of one or more FGFR mutants indicates that the patient will respond to treatment with an FGFR inhibitor.

[0156] This article also provides a method for identifying cancer patients who respond to treatment with fibroblast growth factor receptor (FGFR) inhibitors, the method comprising: evaluating FGFR mutants from an FGFR-mutant genome in a biological sample from the patient, wherein the FGFR mutant is an FGFR fusion gene or an FGFR single nucleotide polymorphism, and wherein the evaluation comprises amplifying cDNA with primer pairs that bind to and amplify one or more FGFR mutants from the FGFR-mutant genome, and determining the presence of one or more FGFR mutants from the genome in the sample, wherein the presence of one or more FGFR mutants indicates that the patient has responded to treatment with an FGFR inhibitor.

[0157] The method also provides for identifying cancer patients who respond to treatment with fibroblast growth factor receptor (FGFR) inhibitors, the method comprising: assessing the presence of one or more FGFR mutants from an FGFR-mutant genome in a biological sample from the patient, wherein the FGFR mutant is an FGFR fusion gene or an FGFR single nucleotide polymorphism, wherein the presence of one or more FGFR mutants indicates that the patient has responded to treatment with an FGFR inhibitor.

[0158] In some embodiments, the evaluation may include amplifying cDNA using primer pairs that bind to and amplify one or more FGFR mutants from an FGFR mutant genome. In some embodiments, the cDNA may be pre-amplified cDNA.

[0159] In some embodiments, the evaluation step includes isolating RNA from a biological sample and synthesizing cDNA from the isolated RNA. In some aspects, an evaluation step may be performed on pre-amplified cDNA. Therefore, the evaluation step may also include pre-amplifying the cDNA prior to the amplification step. RNA can be isolated from a biological sample using a number of procedures known to those skilled in the art. In one embodiment, RNA can be isolated from a biological sample using the AllPrep DNA / RNA FFPE kit from Qiagen (e.g., product number 80234). Synthesizing cDNA from the isolated RNA can be performed using a number of procedures known to those skilled in the art. In one embodiment, cDNA can be synthesized from the isolated RNA using a high-capacity cDNA reverse transcriptase kit with an RNase inhibitor from ABI (e.g., product number 4374966). Pre-amplification of the cDNA can be performed using a number of procedures known to those skilled in the art. Amplification procedures are well known in the art. In one embodiment, a pre-amplification step may be performed using a high-capacity cDNA reverse transcriptase kit with an RNase inhibitor from ABI (e.g., product number 4374966). Pre-amplified master mixture (Life Technologies / Applied) Pre-amplified cDNA (Product No. 4391128).

[0160] The disclosed method can be used to identify patients with a variety of different types of cancer who will respond to treatment with fibroblast growth factor receptor (FGFR) inhibitors, 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, bile duct cancer, hepatocellular carcinoma, glioblastoma, glioma, colon cancer, sarcoma, primary squamous solid tumor, and multiple myeloma.

[0161] The FGFR mutant set used in the evaluation step is based to some extent on the patient's cancer type. For example, for a bladder cancer patient, suitable FGFR mutant genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3G370C, or FGFR3 Y373C, or any combination thereof. Therefore, in some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v1 in a biological sample from a bladder cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v3 in a biological sample from a bladder cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3:BAIAP2L1 in a biological sample from a bladder cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:BICC1 in a biological sample from a bladder cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:AFF3 in a biological sample from a bladder cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:CASP7 in a biological sample from a bladder cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 R248C in a biological sample from a bladder cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 S249C in a biological sample from a bladder cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 G370C in a biological sample from a bladder cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 Y373C in a biological sample from a bladder cancer patient. In some embodiments, the evaluation step includes determining the presence of any combination of the above-described FGFR mutants in a biological sample from a bladder cancer patient.

[0162] For example, for patients with metastatic bladder cancer, suitable FGFR mutant genomes may include 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. Therefore, in some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v1 in a biological sample from a patient with metastatic bladder cancer. In some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v3 in a biological sample from a patient with metastatic bladder cancer. In some embodiments, the evaluation step includes determining the presence of FGFR3:BAIAP2L1 in a biological sample from a patient with metastatic bladder cancer. In some embodiments, the evaluation step includes determining the presence of FGFR2:BICC1 in a biological sample from a patient with metastatic bladder cancer. In some embodiments, the evaluation step includes determining the presence of FGFR2:AFF3 in a biological sample from a patient with metastatic bladder cancer. In some embodiments, the evaluation step includes determining the presence of FGFR2:CASP7 in a biological sample from a patient with metastatic bladder cancer. In some embodiments, the evaluation step includes determining the presence of FGFR3 R248C in a biological sample from a patient with metastatic bladder cancer. In some embodiments, the evaluation step includes determining the presence of FGFR3 S249C in a biological sample from a patient with metastatic bladder cancer. In some embodiments, the evaluation step includes determining the presence of FGFR3 G370C in a biological sample from a patient with metastatic bladder cancer. In some embodiments, the evaluation step includes determining the presence of any combination of the above-described FGFR mutants in a biological sample from a patient with metastatic bladder cancer.

[0163] For example, for ovarian cancer patients, suitable FGFR mutant genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Therefore, in some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v1 in a biological sample from an ovarian cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v3 in a biological sample from an ovarian cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3:BAIAP2L1 in a biological sample from an ovarian cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:BICC1 in a biological sample from an ovarian cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:AFF3 in a biological sample from an ovarian cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:CASP7 in a biological sample from an ovarian cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3R248C in a biological sample from an ovarian cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 S249C in a biological sample from an ovarian cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 G370C in a biological sample from an ovarian cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 Y373C in a biological sample from an ovarian cancer patient. In some embodiments, the evaluation step includes determining the presence of any combination of the above-described FGFR mutants in a biological sample from an ovarian cancer patient.

[0164] For example, for head and neck cancer patients, suitable FGFR mutated genomes may include FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Therefore, in some embodiments, the evaluation step includes determining the presence of FGFR3:BAIAP2L1 in a biological sample from a head and neck cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:CASP7 in a biological sample from a head and neck cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 R248C in a biological sample from a head and neck cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 S249C in a biological sample from a head and neck cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 G370C in a biological sample from a head and neck cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 Y373C in a biological sample from a head and neck cancer patient. In some implementations, the evaluation step includes determining whether any combination of the aforementioned FGFR mutants is present in biological samples from head and neck cancer patients.

[0165] For example, for patients with metastatic head and neck cancer, suitable FGFR mutant genomes may include FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof. Therefore, in some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the patient with metastatic head and neck cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the patient with metastatic head and neck cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:OFD1 is present in the sample, the patient with metastatic head and neck cancer is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the patient with metastatic head and neck cancer is treated with an FGFR inhibitor.

[0166] For example, for esophageal cancer patients, suitable FGFR mutant genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3Y373C, or any combination thereof. Therefore, in some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v1 in a biological sample from an esophageal cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v3 in a biological sample from an esophageal cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:BICC1 in a biological sample from an esophageal cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:CASP7 in a biological sample from an esophageal cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 R248C in a biological sample from an esophageal cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 S249C in a biological sample from an esophageal cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 G370C in a biological sample from an esophageal cancer patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 Y373C in a biological sample from an esophageal cancer patient. In some embodiments, the evaluation step includes determining the presence of any combination of the above-described FGFR mutants in a biological sample from an esophageal cancer patient.

[0167] For example, for patients with metastatic esophageal cancer, suitable FGFR-mutated genomes may include 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. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 intron is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CCD6 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:OFD1 is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the patient with metastatic esophageal cancer is treated with an FGFR inhibitor.

[0168] For patients with non-small cell lung (NSCL) adenocarcinoma, suitable FGFR-mutated genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 introns, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Therefore, in some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v1 in a biological sample from an NSCL adenocarcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v3 in a biological sample from an NSCL adenocarcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 introns in a biological sample from an NSCL adenocarcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR3:BAIAP2L1 in a biosample from an NSCL adenocarcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:AFF3 in a biosample from an NSCL adenocarcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:CASP7 in a biosample from an NSCL adenocarcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 R248C in a biosample from an NSCL adenocarcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 S249C in a biosample from an NSCL adenocarcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 G370C in a biosample from an NSCL adenocarcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR3 Y373C in a biosample from an NSCL adenocarcinoma patient. In some embodiments, the evaluation step includes determining the presence of any combination of the above-described FGFR mutants in a biosample from an NSCL adenocarcinoma patient.

[0169] For example, for patients with non-small cell lung (NSCL) squamous cell carcinoma, suitable FGFR mutant genomes may include 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. Therefore, in some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v1 in a biological sample from an NSCL squamous cell carcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR3:TACC3 v3 in a biological sample from an NSCL squamous cell carcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR3:BAIAP2L1 in a biological sample from an NSCL squamous cell carcinoma patient. In some embodiments, the evaluation step includes determining the presence of FGFR2:BICC1 in a biological sample from a patient with NSCL squamous cell carcinoma. In some embodiments, the evaluation step includes determining the presence of FGFR2:AFF3 in a biological sample from a patient with NSCL squamous cell carcinoma. In some embodiments, the evaluation step includes determining the presence of FGFR2:CASP7 in a biological sample from a patient with NSCL squamous cell carcinoma. In some embodiments, the evaluation step includes determining the presence of FGFR2:CCDC6 in a biological sample from a patient with NSCL squamous cell carcinoma. In some embodiments, the evaluation step includes determining the presence of FGFR3 R248C in a biological sample from a patient with NSCL squamous cell carcinoma. In some embodiments, the evaluation step includes determining the presence of FGFR3 S249C in a biological sample from a patient with NSCL squamous cell carcinoma. In some embodiments, the evaluation step includes determining the presence of FGFR3 G370C in a biological sample from a patient with NSCL squamous cell carcinoma. In some embodiments, the evaluation step includes determining the presence of FGFR3 Y373C in a biological sample from a patient with NSCL squamous cell carcinoma. In some implementations, the evaluation step includes determining whether any combination of the aforementioned FGFR mutants is present in a biological sample from a patient with NSCL squamous cell carcinoma.

[0170] For example, for patients with metastatic endometrial cancer, suitable FGFR-mutated genomes may include FGFR3:TACC3v1, FGFR3:TACC3v3, FGFR3:TACC3 introns, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof. Therefore, in some embodiments, if FGFR3:TACC3v1 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3v3 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 introns are present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CCDC6 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if FGFR2:OFD1 is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the patient with metastatic endometrial cancer is treated with an FGFR inhibitor.

[0171] For example, for breast cancer patients, suitable FGFR-mutated genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 introns, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 introns are present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CCD6 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if FGFR2:OFD1 is present in the sample, the breast cancer patient is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-mentioned FGFR mutants is present in the sample, the breast cancer patient is treated with an FGFR inhibitor.

[0172] For example, for patients with hepatocellular carcinoma, suitable FGFR-mutated genomes may include FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 introns, 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. Therefore, in some embodiments, if FGFR3:TACC3 v1 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 v3 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3:TACC3 introns are present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3:BAIAP2L1 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:BICC1 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:AFF3 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CASP7 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:CCDC6 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR2:OFD1 is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 R248C is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 S249C is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 G370C is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if FGFR3 Y373C is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor. In some embodiments, if any combination of the above-described FGFR mutants is present in the sample, the patient with hepatocellular carcinoma is treated with an FGFR inhibitor.

[0173] Suitable primer pairs for the amplification step include those disclosed in Table 3. For example, in some embodiments, the FGFR mutant and primer pair may 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 primer pair may 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 primer pair may be FGFR3:TACC3 introns and primers having the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10. In some embodiments, the FGFR mutant and primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair may 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 primer pair 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 primer pair can be any combination of the FGFR mutant and corresponding primer pair disclosed above.

[0174] The disclosed methods include determining the presence of one or more FGFR mutants from the genome in a sample. In some embodiments, the determination step includes sequencing amplified cDNA.

[0175] In some implementations, if one or more FGFR mutants from the genome are present in the sample, the method also includes treating the patient with an FGFR inhibitor. Suitable FGFR inhibitors for this treatment include those previously described herein, particularly JNJ-42756493.

[0176] Kit for identifying the presence of FGFR mutant genes

[0177] This article also discloses a kit for identifying the presence of one or more FGFR mutant genes in biological samples, the kit comprising primer pairs having the following sequences: 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:3 ... NO:36, SEQ ID NO:37, SEQ ID NO:38 or any combination thereof; and instructions for use in performing assays to detect one or more FGFR mutant genes.

[0178] The kit may also include one or more probes, one or more 3' blocking oligonucleotides, or both. In some embodiments, the kit may also include one or more probes, such as any one or more probes disclosed in Table 15. In some embodiments, the kit may also include one or more 3' blocking oligonucleotides, such as any one or more 3' blocking oligonucleotides disclosed in Table 8. In some embodiments, the kit may also include one or more probes and one or more 3' blocking oligonucleotides. For example, in some embodiments, the kit may also include:

[0179] a. The primer pair has the sequences of SEQ ID NO:5 and SEQ ID NO:6, and the probe has the sequence of SEQ ID NO:43;

[0180] b. The primer pair has the sequences of SEQ ID NO:7 and SEQ ID NO:8, and the probe has the sequence of SEQ ID NO:44;

[0181] c. The primer pair has the sequences of SEQ ID NO:9 and SEQ ID NO:10, and the probe has the sequence of SEQ ID NO:46;

[0182] d. The primer pair has the sequences of SEQ ID NO:11 and SEQ ID NO:12, and the probe has the sequence of SEQ ID NO:47;

[0183] e. The primer pair has the sequences of SEQ ID NO:13 and SEQ ID NO:14, and the probe has the sequence of SEQ ID NO:45;

[0184] f. The primer pair has the sequences of SEQ ID NO:15 and SEQ ID NO:16, and the probe has the sequence of SEQ ID NO:48;

[0185] g. The primer pair has the sequences of SEQ ID NO:17 and SEQ ID NO:18, and the probe has the sequence of SEQ ID NO:49;

[0186] h. The primer pair has the sequences of SEQ ID NO:19 and SEQ ID NO:20, and the probe has the sequence of SEQ ID NO:50;

[0187] i. The primer pair has the sequences of SEQ ID NO:21 and SEQ ID NO:22, and the probe has the sequence of SEQ ID NO:51;

[0188] j. The primer pair has the sequences of SEQ ID NO:23 and SEQ ID NO:24, and the probe has the sequence of SEQ ID NO:52;

[0189] k. The primer pair has the sequences of SEQ ID NO:25 and SEQ ID NO:26, and the probe has the sequence of SEQ ID NO:53;

[0190] l. The primer pair has the sequences of SEQ ID NO:27 and SEQ ID NO:28, and the probe has the sequence of SEQ ID NO:54;

[0191] m. The primer pair has the sequences of SEQ ID NO:29 and SEQ ID NO:30, and the probe has the sequence of SEQ ID NO:55;

[0192] n. The primer pair has the sequences of 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;

[0193] o. The primer pair has the sequences of 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;

[0194] p. The primer pair has the sequences of 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;

[0195] q. The primer pair has the sequences of 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

[0196] r. Any combination of them.

[0197] Oligonucleotide probes

[0198] This document also discloses oligonucleotide probes having sequences of any one of SEQ ID NO:43-55. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:43. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:44. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:45. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:46. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:47. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:48. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:49. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:50. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:51. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:52. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:53. In some embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO:54. In some implementations, the oligonucleotide probe may have the sequence SEQ ID NO:55.

[0199] 3' Blocking Oligonucleotides

[0200] This document also discloses oligonucleotides having sequences of any one of SEQ ID NO:39-42. In some embodiments, the 3' blocking oligonucleotide may have the sequence of SEQ ID NO:39. In some embodiments, the 3' blocking oligonucleotide may have the sequence of SEQ ID NO:40. In some embodiments, the 3' blocking oligonucleotide may have the sequence of SEQ ID NO:41. In some embodiments, the 3' blocking oligonucleotide may have the sequence of SEQ ID NO:42.

[0201] Example

[0202] Example 1—Plasmid DNA Isolation and Purification

[0203] The following is an exemplary procedure for preparing FGFR fusion plasmid DNA.

[0204] Required equipment: centrifuge capable of 1500×g; microcentrifuge; pipette, volumetric or degassing type; vortex mixer; nanodrop spectrophotometer; 37°C shaker / incubator; and oven set to 37°C.

[0205] Materials required: Frozen glycerol bacterial stock containing plasmid DNA; kanamycin LB agar plates (Teknova#L1155); LB liquid medium (Life Technologies#10855-021); kanamycin (Sigma#K0254); plasmid purification kit (Qiagen#12123); pure ethanol (Sigma Aldrich#E7023); isopropanol (Sigma Aldrich#W292907); nuclease-free purified water (untreated with DEPC) (from IDT or Ambion#AM9932); RNase-free barrier (filter) pipette tips; RNase-free microtubes (1.5 to 2 mL VWR#10011-724); serum pipettes; and 14 mL round-bottom tubes (VWR#352057).

[0206] To recover bacteria from the glycerol stock, frozen bacteria were scraped from the top of the glycerol stock tube using the tip of a sterile pipette, streaked onto an LB agar plate, and then inverted in a 37°C oven overnight.

[0207] DNA plasmids were purified using the Qiagen plasmid DNA purification protocol. In brief, single colonies were picked from streak plates and incubated overnight at approximately 300 rpm in 5 ml-LB medium containing 50 μg / ml kanamycin on a 37°C shaker. Bacterial cells were collected by centrifugation at 6000 × g for 15 minutes at 4°C, and the precipitate was resuspended in 300 μl of buffer P1. 300 μl of buffer P2 was added, mixed 4–6 times by inverting the tube, and incubated at RT (room temperature) for 5 minutes. 300 μl of cooling buffer P3 was added, mixed immediately by inverting the tube 4–6 times, incubated on ice for 5 minutes, and centrifuged at maximum speed for 10 minutes. The supernatant containing plasmid DNA was quickly removed. Qiagen-tip 20 was equilibrated by adding 1 ml of buffer QBT and then drained by gravity. The supernatant was added to Qiagen-tip 20 and then added to the resin by gravity. Qiagen-tip 20 was washed with 2 × 2 ml QC buffer, and the DNA was eluted with 800 μl QF buffer, collecting the eluent in a 1.5 ml Eppendorf tube. DNA was precipitated by adding 0.7 volume of isopropanol, mixing, and immediately centrifuging at 15000 × g for 30 min in a microcentrifuge. The supernatant was decanted, and the DNA precipitate was washed with 1 ml of 70% ethanol and centrifuged at 15000 × g for 10 min. The supernatant was decanted again. The precipitate was air-dried for 5–10 min, and the DNA was redissolved in 100 μl or an appropriate volume of nuclease-free pure water. The plasmid DNA was quantified using Nanodrop and stored at -20°C until further use.

[0208] Example 2—Generation of NRK cell lines

[0209] Expression vectors expressing each FGFR fusion variant were constructed. These expression vectors were then transfected into normal rat kidney epithelial (NRK) cells. After transfection, stable cell lines were selected in kanamycin-containing medium. These cells were then grown, and mRNA was isolated and subjected to FGFR fusion assays to confirm the presence of specific FGFR fusion mRNAs.

[0210] Example 3—Maintenance of FGFR fusion cell line

[0211] The following protocol describes an exemplary method for culturing and maintaining NRK FGFR fusion-overexpressing cell lines. Cell lines include, but are not limited to: NRK / FGFR3:TACC3v1, NRK / FGFR3:TACC3v3, NRK / FGFR3:BAIAP2L1, NRK / FGFR2:BICC1, NRK / FGFR2:CASP7, NRK / FGFR2:CCDC6, NRK / FGFR2:AFF3, NRK / FGFR2:OFD1, and NRK / EMPTY VECTOR (plasmid control).

[0212] Required equipment: Biosafety cabinet with vacuum aspiration system; CO2 incubator set to 5% CO2 and 37°C; -80°C freezer; liquid nitrogen tank; water bath set to 37°C; and microscope.

[0213] Required materials: Serum pipettes; tissue culture flasks (T75 VWR#BD353136 and / or T150 VWR#15705-074); 0.2μm tissue culture filter unit (Thermo Scientific#566-0020); DMEM (Dalbeco Modified Eagle Medium) cell culture medium (Life Technologies,#11965-084); Fetal bovine serum (FBS), certified and heat-inactivated (Life Technologies,#10082147); PenStrep antibiotic solution (Life Technologies#15140-122); Trypsin-EDTA 0.25% solution (Life Technologies,#25200-056); DPBS (Duborough Phosphate Buffered Solution, calcium-free, magnesium-free) (Life Technologies,#14190136); cell freezing containers for cryopreservation; handheld pipettes; cell freezing medium (Life Technologies). Technologies, #12648-010); 15ml conical tubes (VWR#62406-2); and cryogenic flasks (VWR#89094-800).

[0214] To prepare cell culture medium, DMEM medium was prepared by combining 445 ml DMEM, 50 ml FBS, and 5 ml PenStrep. The prepared medium was passed through a 0.2 μm filter unit and stored at 4 °C.

[0215] To thaw frozen cells, heat the prepared DMEM medium in a 37°C water bath for at least 15 minutes, and place 15 ml of the warmed medium in a T75 flask. Remove the cells from the liquid nitrogen container and immediately place them in a 37°C water bath until thawed. Spray the frozen flask with a large amount of 70% ethanol and wipe off any excess ethanol with a paper towel. Aliquot all contents into T75 flasks containing DMEM. Gently swirl the flasks to mix and incubate for 24 hours. If the cells are not ready to divide, replace the medium with freshly prepared DMEM to remove any residual frozen medium. If the cells are ready to divide, each cell line will proliferate once the flasks reach 80% confluence (the division rate for each cell line depends on experimental needs).

[0216] To freeze the cell line, remove the cells from the culture flask and centrifuge at 1500 RPM for 5 minutes in a 15 ml conical tube at room temperature. Aspirate the culture medium and add 6 ml of cell freezing medium. Mix the cells several times by pipetting up and down, and aliquot 1 ml of the cell solution into each of five freezing flasks. Place the freezing flasks containing the cells in a cryogenic container and store them overnight in a -80°C freezer, then store them long-term in liquid nitrogen.

[0217] Example 4—FFPET SNP Determination

[0218] The following describes an exemplary workflow and protocol for performing FFPET SNP determination. A similar procedure is performed for FFPET fusion determination, and the results are shown in Figure 2.

[0219] FFPET debounding

[0220] The tablets were treated with an increased amount of xylene, and then treated with ethanol to remove the paraffin.

[0221] FFPETRNA extraction

[0222] The procedure for extracting RNA from formalin-fixed paraffin-embedded tissue samples from breast cancer for downstream gene expression assays is described below.

[0223] Required equipment: Centrifuge with plate adapter, capable of 1500×g; microcentrifuge; pipettes, volumetric or degassing type; vortex mixer; NanoDrop 8000; heating blocks capable of incubation at 37°C, 56°C and 80°C; and Pasteur pipettes (Pipet Trans EX-FT 1.5ml pk 500, VWR#14670-329).

[0224] Required materials: AllPrep DNA / RNA FFPE Kit (Qiagen #80234); pure ethanol (SigmaAldrich #E7023); isopropanol; xylene; nuclease-free purified water (untreated with DEPC) (from IDT or Ambion #AM9932); RNase-free barrier (filter) pipette tips; RNase-free microtubes (1.5 to 2 mL VWR #10011-724); and the Qiagen AllPrep DNA / RNA FFPE Kit manual.

[0225] RNA was extracted using the AllPrep DNA / RNA FFPE kit. Briefly, a 1–10 μm fraction was placed in a 1.5 ml reaction tube, and 800 μl of HemoDe or xylene was added. The sample was vortexed 3 times for 4 seconds, incubated for 2 minutes, then vortexed 3 times for 4 seconds and incubated for 5 minutes.

[0226] Centrifuge the sample at maximum speed (12,000-14,000 × g) for 2 minutes and discard the supernatant by aspiration. Immediately cap the tube to prevent the tissue from drying out.

[0227] Repeat the steps above.

[0228] Add 800 μl of pure ethanol to the test tube, gently tap the test tube to remove the precipitate, vortex for 4 seconds 3 times, centrifuge at the maximum speed (12,000-14,000×g) for 2 minutes, and discard the supernatant by suction.

[0229] Add 800 μl of 70% ethanol to a test tube, gently tap the tube to remove the precipitate, vortex 3 times for 4 seconds, centrifuge at maximum speed for 2 minutes, and discard the supernatant by suction. After removing the 70% ethanol, rotate the tube for another 10-20 seconds and carefully remove any remaining fluid using a fine pipette.

[0230] The opened tube was incubated in a heating block at 37°C for 5-15 minutes to air-dry the tissue precipitate.

[0231] Resuspend the precipitate by adding 150 μl of buffer PKD and gently tap the tube to loosen the precipitate. Add 10 μl of proteinase K and vortex the tube.

[0232] Incubate the tube at 56°C for 15 minutes, then on ice for 3 minutes, and centrifuge at 20,000×g for 15 minutes.

[0233] Without disturbing the precipitate, carefully transfer the supernatant to a new 1.5 ml microcentrifuge tube for RNA purification. Incubate the supernatant at 80°C for 15 minutes. Briefly centrifuge the tube to remove droplets from the inside of the cap. Add 320 μl of RLT buffer to adjust binding conditions and mix the tube by vortexing or pipetting. Add 1120 μl of ethanol (96–100%) and mix thoroughly by vortexing or pipetting.

[0234] Transfer 700 μl of sample (including any precipitate that may have formed) to an RNeasy MinElute rotary column placed in a 2 ml collection tube and centrifuge at ≥8000×g (≥10,000 rpm) for 15 seconds. Discard the influent portion. Repeat this step until the entire sample has passed through the RNeasy MinElute rotary column.

[0235] Add 350 μl of buffer FRN to an RNeasy MinElute rotating column and centrifuge at ≥8000×g (≥10,000 rpm) for 15 seconds. Discard the influent portion.

[0236] Add 10 μl of DNase I stock solution to 70 μl of buffer RDD, mix by gently inverting the tube, and briefly centrifuge to collect residual liquid from the side of the tube.

[0237] Add the DNA enzyme I incubation mixture (80 μl) directly to the RNeasy MinElute rotating column membrane and place it on the table for 15 minutes (20-30°C).

[0238] Add 500 μl of buffer FRN to an RNeasy MinElute rotating column and centrifuge at ≥8000×g (≥10,000 rpm) for 15 seconds. Reserve the influent portion for the next step, as it contains small RNA.

[0239] Place the RNeasy MinElute spinning column into a new 2ml collection tube (provided). Apply the inflow portion from the previous step to the spinning column and centrifuge at ≥8000×g (≥10,000rpm) for 15 seconds. Discard the inflow portion.

[0240] Add 500 μl of RPE buffer to an RNeasy MinElute rotating column and centrifuge at ≥8000×g (≥10,000 rpm) for 15 seconds to wash the rotating column membrane. Discard the influent portion.

[0241] Add 500 μl of buffer RPE to an RNeasy MinElute rotating column and centrifuge at ≥8000×g (≥10,000 rpm) for 15 seconds to wash the rotating column membrane. Discard the collection tube containing the inflow portion.

[0242] Place the RNeasy MinElute rotating column into a new 2ml collection tube and centrifuge at full speed for 5 minutes. Discard the collection tube containing the inflow portion.

[0243] Place the RNeasy MinElute spinning column into a new 1.5ml collection tube, add 30μl of RNase-free water directly to the spinning column membrane, incubate at room temperature for 1 minute, and centrifuge at full speed for 1 minute to elute RNA.

[0244] Store RNA samples immediately in a -80°C freezer.

[0245] cDNA synthesis

[0246] The following describes the cDNA synthesis process for FFPET SNP assay using real-time PCR (RT-PCR) analysis.

[0247] Required equipment: a centrifuge with a plate adapter capable of 1500×g, a microcentrifuge; pipettes (preferably single-channel and multi-channel pipettes), volumetric or degassing type; a vortex mixer; and more. PCR system 9700 (ABI#4314879) or equivalent.

[0248] Required materials: High-capacity cDNA reverse transcriptase kit with RNase inhibitor, 200 reactions (ABI#4374966); nuclease-free pure water (untreated with DEPC) (from IDT) or equivalent; RNase-free barrier (filter) pipette tips; RNase-free microtubes (1.5 to 2 mL VWR#10011-724); MicroAmp TM Optical 96-well reaction plate (LifeTechnologies, #4306736); and sealing film (VWR#60941-072).

[0249] After RNA extraction (disclosed above), keep the RNA sample tube on ice.

[0250] Use the kit components to prepare a 2× reverse transcription (RT) master mixture for all reactions, including one negative (water) control. Thaw the components on ice for approximately 15 minutes, gently invert to mix, and briefly centrifuge to degrade the solution. Return all reagents to ice. Tubes were not vortexed.

[0251] A master mixture was prepared on ice in a 1.5 ml tube for an appropriate number of reactions (reaction number + 10% / 20 μL reaction) by mixing the following amounts of reagents in each reaction: 2 μl 10× room temperature buffer mixture; 0.8 μl 25× dNTP mixture; 2 μl 10× room temperature random primers; 1 μl 50 U / μL MultiScribe reverse transcriptase; 1 μl RNase inhibitor; and 3.2 μl H2O-free nuclease / RNase.

[0252] Vortex the main mixture several times (5 to 10 times) to mix and then briefly centrifuge (1500×g, 5 to 10 seconds). Add 10 μl of the reaction mixture to the appropriate well of a 96-well plate.

[0253] Dilute the RNA sample to a concentration of 20 ng / μl. Add 10 μL of each RNA sample, including the water negative control, to the appropriate well of a 96-well plate, for a final reaction volume of 20 μL. Gently mix the wells three times by pipetting up and down, seal with the plate sealer, and briefly centrifuge (1500×g, 60 sec). Keep the plate on ice until ready to be loaded into a thermal cycler.

[0254] Load the reaction plate into an ABI 9700 thermal cycler in a clean laboratory or workstation and run the following reverse transcription program (reaction volume 20 μl):

[0255] Step 1: 25℃ for 10 minutes

[0256] Step 2: 37℃ for 120 minutes

[0257] Step 3: 85℃ for 5 seconds

[0258] Step 4: 4℃ and maintain

[0259] The synthesized cDNA is stored at -20°C for the next step of pre-amplification.

[0260] Preparation of pre-amplification assay mixture

[0261] The pre-amplification assay cell mixture associated with the FFPET SNP assay pre-amplification protocol was prepared as follows.

[0262] Required equipment: microcentrifuge; pipettes, volumetric or vented; and vortex mixer.

[0263] Required materials: nuclease-free pure water (untreated with DEPC) (from IDT) or equivalent; IDTE pH 8.0 (1×TE solution) (IDT Technologies); RNase-free barrier (filter) pipette tips; and RNase-free tubes (1.5 to 2 mL VWR#10011-724).

[0264] All TaqMan SNP assays were ordered from Applied Biosystems, Life Technologies, Inc.

[0265] Prepare 100 μL of 20×SNP assay sample.

[0266] To prepare the 0.2× pre-amplification assay cell, all analytes were thawed on ice for approximately 15 minutes. The following volumes of the components were added to a 1.5 ml tube:

[0267] Table 4

[0268]

[0269] Note: The above volume is for preparing a 200 μl 0.2× pre-amplification assay cell. The volume can be adjusted according to the number of samples being tested.

[0270] Briefly vortex the 0.2× preamplification assay cell (5 to 10 seconds) and briefly centrifuge (1500×g, 5–10 seconds). Aliquot 100 μL of the preamplification primer cell into 1.5 ml tubes and store at -20°C.

[0271] Preliminary analysis of SNPs in formalin-fixed paraffin-embedded breast cancer tissue using real-time PCR (RT-PCR) analysis. Amplification

[0272] Required equipment: Centrifuge with plate adapter, capable of 1500×g; microcentrifuge; pipettes, volumetric or degassing type; vortex mixer; PCR system 9700 (ABI#4314879) or equivalent.

[0273] Required materials: Preamplification master mixture (2×) (Life Technologies #4391128); 0.2× cell assay mixture (see Assay Preparation and Handling Protocol); 1× IDTE buffer (10mM Tris / 0.1mM EDTA, pH 7.5, from IDT) or equivalent; nuclease-free purified water (untreated with DEPC) (from IDT) or equivalent; RNase-free barrier (filter) pipette tips; RNase-free microtubes (1.5 to 2 mL VWR #10011-724); MicroAmp TM Optical 96-well reaction plate (Life Technologies, #4306736); Optical adhesive film (Applied Biosystems PN 4311971); deep hole plate (VWR#47734-788); foil sealant (VWR#60941-126).

[0274] Samples were prepared by thawing the cDNA and 0.2× assay mixture in a cell on ice for about 5 minutes and then briefly centrifuging the plate (1500×g for 5 to 10 seconds).

[0275] The kit components are used to prepare a 2× preamplification master mixture. Thaw the kit components on ice for approximately 5 minutes. After thawing all reagents, gently invert the tube to mix and briefly centrifuge to degrade the solution. Return all reagents to ice. Do not vortex the tubes.

[0276] In a clean laboratory or biosafety enclosure, prepare appropriate quantities of each master mixture of reactions on ice by combining the required volumes of reagents (as shown in Table 5 below, reaction number + 10%):

[0277] Table 5

[0278]

[0279] The assay cell contains primers and probes.

[0280] To prevent cross-activation of SNP assays, all five assays were divided into three pre-amplification reactions for each sample.

[0281] Vortex each master mixture several times (5 to 10 times) to mix, then briefly centrifuge (1500×g, 5 to 10 seconds). Aliquot 18.75 μL of each master mixture into the appropriate wells of a 96-well plate. For each pre-amplification reaction, transfer 6.25 μL of each cDNA sample (including the water negative control well) to the appropriate well of the master mixture plate. Gently mix the sample by pipetting up and down 3 times, then seal the plate. Briefly centrifuge the plate (1500×g, 60 seconds) and keep it on ice until ready to be loaded into a thermal cycler.

[0282] Use the following procedure to load and run the ABI 9700 thermal cycler:

[0283] Step 1: 95℃ for 10 minutes

[0284] Step 2: 95℃ for 15 seconds

[0285] Step 3: 60℃ for 4 minutes

[0286] Step 4: Set steps 2-3 to continue for 10 cycles.

[0287] If using gold or silver ingots, select the maximum mode and set the heating rate to 77%. If using aluminum ingots, select the standard mode (no rate change).

[0288] Step 5: 4℃ and maintain

[0289] The reaction volume was set to 25 μL.

[0290] After pre-amplification, briefly centrifuge the pre-amplification reaction plate (1500×g, 60 seconds). Add 100 μl of IDTE to the appropriate wells of a new deep 96-well plate, and transfer 25 μl of each pre-amplification product to the corresponding well to bring the final dilution volume to 125 μL. Mix each well three times by up-and-down pipetting, seal the plate with foil adhesive, briefly centrifuge the plate (1500×g, 5–10 seconds), and store the pre-amplification product at -20°C until further use.

[0291] FFPETSNP assay—real-time PCR

[0292] The procedure for determining SNPs in formalin-fixed paraffin-embedded tissues using real-time PCR analysis is disclosed below.

[0293] Required equipment: a centrifuge with a plate adapter capable of 1500×g; a microcentrifuge; pipettes (preferably single-channel and multi-channel pipettes), volumetric or degassing type; a vortex mixer; and an ABI ViiA 7 real-time PCR instrument (LifeTechnologies).

[0294] Required materials: TaqMan genotyping master mixture (Life Technologies #4371355); SNP assay; nuclease-free pure water (untreated by DEPC, from IDT) or equivalent; RNase-free barrier (filter) pipette tips; RNase-free microtubes (1.5 to 2 mL VWR #10011-724); Optical adhesive film (Applied Biosystems PN4311971); and MicroAmp TM Optical 384-well reaction plate.

[0295] Table 15 lists the sequences of the probes used in real-time PCR assays.

[0296] To prepare samples, thaw the SNP assay on ice for approximately 5 minutes in a clean lab or workstation. Prevent all reagents from being exposed to light to protect the fluorescent probes from exposure. Place the diluted pre-amplified plate on ice to thaw in a dirty lab or workstation after preparing the genotyping master mixture.

[0297] To prepare the genotyping master mixture, thaw it on ice for approximately 5 minutes. Prepare the master mixture (MM) into the required number of tubes on ice. Combine the required volumes of reagents into appropriate labeled tubes as shown in Table 6 below (reaction number + 10%):

[0298] Table 6

[0299] Components Volume of a single reaction (μL) 2× master mixture of genotyping 10 20×SNP determination 1 RNase-free pure water 4 Total volume 15

[0300] The 20×SNP assay mixture contains primers, probes, and blocking oligonucleotides.

[0301] Vortex the master mixtures several times (5 to 10 times) to combine, then briefly centrifuge (1500×g, 5 to 10 seconds). Add 15 μl of each master mixture to MicroAmp. TM The appropriate holes in the optical 384-well reaction plate are used. The reaction plate is sealed with an optical adhesive film.

[0302] Place the plate with the 1:5 dilution of pre-amplified product on ice for approximately 5–10 minutes to thaw. Using a multichannel pipette, transfer 5 μL of each dilution of pre-amplified product into the appropriate well. Seal the reaction plate with an optical adhesive membrane and briefly centrifuge (1500 × g, 60 sec). Keep the plate on ice until ready to be loaded into a thermal cycler.

[0303] Run the following conditions using viiA 7 software with a volume set to 20 μl:

[0304] Table 7

[0305]

[0306] FGFR SNP-specific qRT-PCR

[0307] Detection of rare somatic mutations in excess wild-type alleles is increasingly important in cancer diagnosis. Detection becomes challenging when mutations of interest are close together. To aid in the identification of FGFR SNPs from FFPET, a SNP-specific qRT-PCR assay was developed, which utilizes SNP-specific amplification using a combination of Taqman MGB probes and a 3' dideoxy wild-type (WT) allele inhibitor. This assay prevents nonspecific binding, improves the number of target amplifications, minimizes false-positive signals from WT alleles, and enhances the sensitivity of the assay. This RNA-based SNP detection assay, combined with a pre-amplification step in the assay, enhances the signal of low- or rare mutations.

[0308] An exemplary strategy for SNP-specific qRT-PCR using 3' dideoxyWT blocking oligonucleotides is shown in Figure 3 In the middle, and Figure 4 An exemplary FFPE sample validation strategy is illustrated. In short, qRT-PCR is performed using FGFR SNP primers in the presence of a 3' dideoxyWT blocking oligonucleotide that is complementary to the WT allele and contains a nucleotide segment flanked by the WT allele. The binding of the blocking oligonucleotide to the WT allele prevents the application of the WT allele, while the FGFR SNP primers bind to and specifically amplify the FGFR SNP. The 3' dideoxyWT blocking oligonucleotides used in FGFR SNP-specific qRT-PCR are shown in Table 8. The FGFR SNP primers used for FGFR SNP-specific qRT-PCR are: 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 in the real-time PCR assay.

[0309] Table 8

[0310]

[0311] *R can be either A or G. During synthesis, the 3'WT blocking oligonucleotide will have 50% A and 50% G at that specific position (purified by the manufacturer to provide either A or G at that specific position).

[0312] Samples used for validation studies were prepared as shown in Table 9. Exemplary validation data using SNP-specific qRT-PCR of 3' dideoxyWT inhibitor oligonucleotides targeting FGFR3 G370C, FGFR3 Y373, FGFR3 S249C, and FGFR3 R248C are shown in Table 9. Figures 5A to 5D Raw Ct (cycle threshold) data for SNP-specific qRT-PCR of FFPE samples containing 3' dideoxyWT blocking oligonucleotides are shown in Table 10. Data obtained from DNA and RNA using different platforms / technologies demonstrate that SNP-specific PCR with 3' blocking nucleotides is a robust, reliable, and sensitive assay. Validation data show that a single mutant allele / SNP can be detected in large amounts of WT-carrying genomic DNA, highlighting the sensitivity and specificity of each assay.

[0313] Table 9

[0314] sample mutation% 1 100 2 20 3 4 4 0.8 5 0 (100% WT)

[0315] RNA expressing each FGFR3 SNP (R248C, S249C, G370C, Y373C) and FGFR3 WT in stable cell lines

[0316] Table 10

[0317]

[0318] *Average of two Ct values

[0319] FMI / NGS = Next-Generation Sequencing, in which DNA is used as a template to identify mutations (without 3' blocking oligonucleotides); Janssen R&D = RNA templates (without 3' blocking oligonucleotides); SNP-Specific PCR = RNA templates containing 3' blocking nucleotides.

[0320] Example 5—Validation of Customized FGFR Fusion Gene Detection

[0321] Generation of positive controls for FGFR fusion analysis

[0322] The process involved generating a synthetic small gene for FGFR fusion, a plasmid encoding the FGFR fusion, and stable cell lines containing the FGFR fusion. In short, a synthetic small gene was artificially constructed by linking a series of nucleotides with approximately 100 base pairs (corresponding to the target DNA sequence of the gene of interest) together. A plasmid encoding the FGFR fusion was generated by cloning the cDNA encoding each FGFR fusion gene into an expression vector. Stable cell lines containing the FGFR fusion were generated by transfecting the plasmid encoding the FGFR gene into normal rat kidney epithelial cells (NRK cells). Stable cell lines were selected using the G418 antibiotic. Total RNA isolated from these cell lines was subjected to a TaqMan assay for the FGFR fusion to confirm the successful generation of stable cell lines expressing the FGFR fusion. Stable cell lines expressing the FGFR fusion were used as positive controls. Table 15 lists the probe sequences used in the real-time PCR assay.

[0323] Analysis of the limit of quantitation and efficiency of FGFR fusion assay

[0324] To determine the limit of quantitation (LLOQ) and efficiency of FGFR fusion gene assays, FGFR fusion products were generated by TaqMan PCR (as described in Example 4) and confirmed by Sanger sequencing (Figure 2). 100 pg of fusion-positive DNA was mixed with normal human cDNA (confirmed fusion-negative), serially diluted to 1:10, and analyzed using Applied Biosystems ViiA7 Software v1.1. The efficiency standard curve is shown in Figure 6. The LLOQ and efficiency of the FGFR fusion are shown in Table 11.

[0325] Table 11

[0326] Measurement LLOQ efficiency FGFR3:TACC3V1 1.0fgm 104％ FGFR3:TACC3V3 10.0fgm 104％ FGFR3:TACC3 intron 0.1fgm 103％ FGFR3:BAIAP2L1 1.0fgm 101％ FGFR2:AFF3 0.1fgm 106％ FGFR2:BICC1 10.0fgm 105％ FGFR2:CASP7 0.1fgm 109％ FGFR2:CCDC6 1.0fgm 106％ FGFR2:OFD1 0.1fgm 96.6％

[0327] Next, the FGFR fusion gene assay was validated in fusion gene-positive cell lines. FGFR fusion gene expression serial dilutions were prepared by incorporating fusion protein-positive cell lines into fusion protein-negative cell lines. For example, 1:2 serial dilutions of FGFR3:TACC3v1 and FGFR3:BAIAP2L1 were prepared and incorporated into 1 million BAF cells. RNA was isolated (using the Qiagen Rneasy kit), followed by RT-PCR, pre-amplification of cDNA, and TaqMan real-time PCR targeting the FGFR fusion gene. As shown in Table 12, both the FGFR3:TACC3v1 and FGFR3:BAIAP2L1 fusion gene TaqMan assays detected 31 fusion targets in 1 million fusion-negative cells (sensitivity 0.003%).

[0328] Table 12

[0329]

[0330] RT112 and SW780 are commercially available bladder cancer cell lines containing FGFR fusions (from the U.S. Type Culture Collection).

[0331] Example 6—Validation of Customized FGFR SNP Detection

[0332] Assessment of FGFR3 mutations in bladder cancer

[0333] In the bladder cancer samples tested, R248C, S249C, and Y373C SNPs were observed in approximately 8%, 61%, and 19%, respectively.

[0334] Example 7—Analysis of Cancer Samples

[0335] The samples were analyzed using the same procedure as described in Example 4. The results are shown in Table 13 and... Figure 7 The prevalence of FGFR fusions in different cancers is shown in Table 13. FGFR fusions in FFPE samples from different cancers (such as bladder cancer (primary and metastatic), NSCLC (adenocarcinoma and squamous cell carcinoma), ovarian cancer, esophageal cancer (primary and metastatic), head and neck cancer (H&N, primary and metastatic), endometrial cancer (metastatic), breast cancer, and prostate cancer) were detected using qRT-PCR. All tested FGFR fusions were negative for prostate cancer samples. The FGFR3:TACC3 intron fusion was negative in bladder cancer (primary), NSCLC (squamous cell carcinoma), ovarian cancer, esophageal cancer (primary), H&N cancer (primary and metastatic), and breast cancer. The FGFR2:OFD1 fusion was negative in bladder cancer (primary and metastatic), NSCLC (adenocarcinoma), ovarian cancer, and esophageal cancer (primary and metastatic). The FGFR2:CCDC6 fusion was negative in bladder cancer (primary and metastatic), NSCLC (adenocarcinoma), ovarian cancer and esophageal cancer (primary), and H&N cancer (primary and metastatic).

[0336] Figure 8 This 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 EGFR mutation-positive, 3 / 17 samples were KRAS mutation-positive, and 1 / 17 samples were cMET mutation-positive. However, EGFR, KRAS, or cMET mutations were not observed in FGFR fusion-positive NSCLC squamous cell carcinoma samples.

[0337]

[0338] Example 8—Treatment of patients with advanced solid tumors

[0339] Clinical trials were conducted in which patients with various solid tumors expressing the FGFR3:TACC3 v1, FGFR3:TACC3v3, FGFR2:CCDC6, and FGFR2:BICC1 fusion genes were treated with JNJ-42756493. Figure 9 shows exemplary results from Phase I patient samples, where FGFR fusions were detected in Phase I JNJ-427493 (EDI10001) test samples using qRT-PCR assays. All FGFR fusion assays were performed concurrently with a positive control (ST) and GAPDH for RNA quality control assessment. A) Graphical representation of qRT-PCR data generated for pt#1000081: Only the FGFR2:BICC1 fusion was positive (the inset shows details of the Ct values ​​for the FGFR2:BICC1 fusion, ST positive control, and GAPDH). B) Graphical representation of qRT-PCR data generated for pt#33000158: Only the FGFR3:TACC3v1 fusion was positive (inset showing details of Ct values ​​for the FGFR3:TACC3v1 fusion, ST positive control, and GAPDH). C) Graphical representation of qRT-PCR data generated for pt#34000123: Only the FGFR2:CCDC6 fusion was positive (inset showing details of Ct values ​​for the FGFR2:CCDC6 fusion, ST positive control, and GAPDH). D) Graphical representation of qRT-PCR data generated for pt#340000115: The FGFR3:TACC3v1, FGFR3:TACC#v3, and FGFR2:CCDC6 fusions were positive (inset showing details of Ct values ​​for the FGFR fusion, ST positive control, and GAPDH).

[0340] Figure 10 This diagram illustrates an exemplary Phase I study design for a first-in-human study of JNJ-42756493 in patients with advanced solid tumors. The figure shows a graphical representation of the conventional 3+3 dose-escalation design in a Phase I clinical trial. The purpose of the dose-escalation phase is to determine the maximum tolerated dose (MTD) and the recommended Phase II dose (RPD). Part 1 is used to determine the intermittent dosing regimen, i.e., 7 days of dosing and 7 days of withdrawal (10 mg / kg and 12 mg / kg). Part 2 is used to determine PD biomarkers (pharmacodynamic biomarkers; markers examined to correlate drug efficacy with target and biological tumor response), which were tested on biopsy and blood samples. Part 3 is used to expand the dose and include additional patients with specific indications (NSCLC, SCLC, breast cancer, and solid tumors) according to different eligibility criteria (FGFR aberrations: translocation / mutation / amplification) to further characterize the toxicity profile of JNJ493.

[0341] Assessment of clinical activities

[0342] In patients with the FGFR fusion gene, significant clinical responses (RECIST) were observed with once-daily dosing of 9 mg (QD), 12 mg QD, and 12 mg dosing / discontinuation over 7 days. Figure 11 (This indicates all dosing regimens).

[0343] Example 9—Production of RK3E cells stably transfected with FGFR fusion variant

[0344] FGFR fusion overexpressing cell lines

[0345] RK3E (rat kidney epithelial cells) were purchased from ATCC (Manassas, VA, USA) and cultured in DMEM supplemented with FBS and antibiotics (Invitrogen, Grand Island, NY, USA). An FGFR fusion gene construct was designed and cloned into a pReceiver expression vector containing an HA tag (Genecopoeia, Rockville, MD, USA). The clone was transfected into RK3E cells using Amaxa cell line nuclear transfection (Lonza, Basel, Switzerland) according to the manufacturer's protocol. Stably transfected cells were selected in complete medium containing 800 μg / ml G418 (Invitrogen). Overexpression of the fusion gene in stably transfected cells was confirmed by real-time PCR and Western blotting using an anti-pFGFR antibody. Figure 12 ).like Figure 12 As shown, stable cell lines demonstrate the expression of active FGFR fusion kinase, as indicated by the phosphorylation of FGFR.

[0346] Colony formation assay

[0347] Anchorage-independent growth of RK3E cells stably transfected with the FGFR fusion variant was tested. First, 1 ml of medium containing 0.8% low-melting-point agarose was seeded into three wells of a six-well plate. After the agar solidified, an additional 1 ml of medium containing 0.4% agarose and 100 cells per well was added to each well. After 14 days, colonies were fixed and stained with 0.1% toluene crystal violet. Colony counts were determined by manual counting in three wells for each cell line under a microscope. Representative views of each fusion-overexpressing cell line are shown in [image / image / image]. Figure 13A As shown in the figure, anchorage-independent growth in soft agar can be detected in cells stably transfected with the FGFR fusion, but not in the empty vector control. Figure 13BThis represents a quantitative analysis of colonies in soft agar cells stably transfected with FGFR fusions and those in empty vector control. All experiments were performed in duplicate, and results are expressed as colony counts per 100 inoculated cells. All FGFR fusions tested induced anchorage-independent growth, demonstrating their transforming capability.

[0348] Downstream target expression

[0349] RK3E cells stably transfected with the FGFR fusion were seeded into complete growth medium, serum starved overnight, and then refeeded with 0.5% FBS growth medium. In the presence of ligands, cells were treated for 1 hour with 1 μM JNJ-42756493, AZD4547, or NVP-BGJ398. For Western blotting, whole-cell lysates were collected in RIPA buffer (Thermo Scientific, Waltham, MA, USA), and protein concentration was determined using the BCA protein assay (Thermo Scientific). Equal volumes of protein (30 μg / lane) were first loaded onto 4–12% Bis-Tris gels (Invitrogen), followed by SDS-page. Proteins were transferred to nitrocellulose membranes and hybridized with antibodies against p-FGFR, total FGFR2, p-MAPK, total MAPK, p-S6, total S6, and β-actin (Cell Signaling Technology, Danvers, MA, USA) and against total FGFR3 (Santa Cruz, Dallas, TX, USA). The membranes were blocked with Odyssey blocking buffer for 1 hour at room temperature and incubated overnight at 4°C in a 1:1000 solution of primary antibody diluted with Odyssey blocking buffer. After washing three times in 0.1% Tween Tris buffer (TBST), the membranes were hybridized with secondary antibody serum labeled with goat anti-mouse or donkey anti-rabbit IR-Dye 670 or 800cw in Odyssey blocking buffer for 1 hour at room temperature. Washing was repeated after the second labeling, and the membranes were imaged using a LiCor Odyssey scanner and Odyssey 3.0 analysis software (LiCor, Lincoln, NE, USA). The effects of JNJ-42756493 were compared with those of AZD4547 and NVP-BGJ398. For example... Figures 14A to 14H As shown, treatment with JNJ-42756493, AZD4547, and NVP-BGJ398 (with 2 to 4 lanes per blot) inhibited phosphorylation of FGFR and downstream targets (i.e., MAPK and S6).

[0350] Drug response testing of FGFR fusion overexpression cell lines

[0351] RK3E cells stably transfected with the FGFR fusion were seeded in triplicate into 96-well plates (1000 cells / well), each well containing complete growth medium and ligands FGF-1 and FGF-2. After 24 hours, the cells were starved of serum overnight and then refeeded with 0.5% FBS growth medium. 72 hours after seeding, the cells were treated with 18 serially diluted 1:3 concentrations of JNJ493, AZD4547 (AZD), and NVP-BGJ398 (NVS) at an initial concentration of 10 μM. The cells were then incubated in microtiter plates for 72 hours and treated with Cell... The Luminescent Cell Viability assay (PromegaCorp., Madison, WI, USA) measures adenosine triphosphate (ATP, a marker of metabolically active cells) levels according to the manufacturer's instructions, with slight modifications. In short, cells are equilibrated to room temperature, at which point Cell... The reagent mixture was prepared. Cells were then placed on a fixed-track shaker for 2 minutes and incubated at room temperature for 10 minutes to stabilize the luminescence signal. Quantitative analysis of the luminescence was performed using an Envision Multilabel microplate reader (Perkin Elmer; Waltham, MA, USA). IC50 was calculated using a GraphPad Prism 5.0. 50 The values ​​are shown in Table 14. As shown in Table 14, cells containing the FGFR fusion variant showed sensitivity to the FGFR inhibitors JNJ-42756493, AZD4547, and NVP-BGJ398 in vitro. Among them, JNJ-42756493 showed enhanced sensitivity (nanomolar concentration range) compared to AZD4547 and NVP-BGJ398, while the empty vector control showed no sensitivity.

[0352] Table 14

[0353]

[0354] AZD=AZD4547; NVS=NVP-BGJ398

[0355] Table 15

[0356]

[0357]

[0358] Those skilled in the art will understand that many changes and modifications can be made to the preferred embodiments of the invention, and such changes and modifications can be made without departing from the spirit of the invention. Therefore, the appended claims are intended to cover all such equivalent changes that fall within the true spirit and scope of the invention.

[0359] Every patent, patent application, and patent disclosure cited or described in this document is incorporated herein by reference in its entirety.

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372] Implementation Plan

[0373] The implementation schemes listed below are intended to complement, rather than replace or substitute for, the preceding description.

[0374] Implementation Scheme 1. A method for identifying cancer patients who have responded to treatment with fibroblast growth factor receptor (FGFR) inhibitors, the method comprising:

[0375] Evaluate FGFR mutants from FGFR-mutant genomes in biological samples from patients, wherein the FGFR mutant is an FGFR fusion gene or an FGFR single nucleotide polymorphism, and wherein the evaluation includes:

[0376] amplifying cDNA using primer pairs that bind to and amplify one or more FGFR mutants from an FGFR mutant genome; and

[0377] To determine whether the sample contains one or more FGFR mutants from an FGFR-mutant genome, the presence of one or more FGFR mutants indicates that the patient has responded to treatment with an FGFR inhibitor.

[0378] Implementation Scheme 2. A method for identifying cancer patients who have responded to treatment with fibroblast growth factor receptor (FGFR) inhibitors, the method comprising:

[0379] Assess the presence of one or more FGFR mutants from an FGFR-mutant genome in a biological sample from a patient, wherein the FGFR mutant is an FGFR fusion gene or an FGFR single nucleotide polymorphism, and the presence of one or more FGFR mutants indicates that the patient has responded to treatment with an FGFR inhibitor.

[0380] Implementation Scheme 3. The method according to Implementation Scheme 1 or 2, wherein the FGFR fusion gene includes FGFR3:TACC3v1, FGFR3:TACC3v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6 or FGFR2:OFD1, or any combination thereof.

[0381] Implementation Scheme 4. The method according to Implementation Scheme 1 or 2, wherein the FGFR single nucleotide polymorphism includes R248C, S249C, G370C or Y373C, or any combination thereof.

[0382] Implementation Scheme 5. The method according to Implementation Scheme 1 or 2, wherein the cancer is bladder cancer, and the FGFR mutated genome includes 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.

[0383] Implementation Scheme 6. The method according to Implementation Scheme 1 or 2, wherein the cancer is metastatic bladder cancer, and the FGFR mutated genome includes 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.

[0384] Implementation Scheme 7. The method according to Implementation Scheme 1 or 2, wherein the cancer is ovarian cancer, and the FGFR mutated genome includes 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.

[0385] Implementation Scheme 8. The method according to Implementation Scheme 1 or 2, wherein the cancer is head and neck cancer, and the FGFR mutated genome includes FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[0386] Implementation Scheme 9. The method according to Implementation Scheme 1 or 2, wherein the cancer is metastatic head and neck cancer, and the FGFR mutated genome includes FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof.

[0387] Implementation Scheme 10. The method according to Implementation Scheme 1 or 2, wherein the cancer is esophageal cancer, and the FGFR mutated genome includes FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[0388] Implementation Scheme 11. The method according to Implementation Scheme 1 or 2, wherein the cancer is metastatic esophageal cancer, and the FGFR mutated genome includes 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.

[0389] Implementation Scheme 12. The method according to Implementation Scheme 1 or 2, wherein the cancer is non-small cell lung cancer adenocarcinoma, and the FGFR mutated genome includes FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3Y373C, or any combination thereof.

[0390] Implementation Scheme 13. The method according to Implementation Scheme 1 or 2, wherein the cancer is non-small cell lung cancer or squamous cell carcinoma, and the FGFR mutated genome includes FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3G370C, or FGFR3 Y373C, or any combination thereof.

[0391] Implementation Scheme 14. The method according to Implementation Scheme 1 or 2, wherein the cancer is metastatic endometrial cancer, and the FGFR mutated genome includes FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof.

[0392] Implementation Scheme 15. The method according to Implementation Scheme 1 or 2, wherein the cancer is breast cancer, and the FGFR mutated genome includes 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.

[0393] Implementation Scheme 16. The method according to Implementation Scheme 1 or 2, wherein the cancer is hepatocellular carcinoma, and the FGFR mutated genome includes FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0394] Implementation Scheme 17. The method according to any one of Implementation Schemes 2 to 16, wherein the evaluation includes amplifying cDNA with primer pairs that bind to and amplify one or more FGFR mutants from the FGFR mutant genome.

[0395] Implementation Scheme 18. The method according to Implementation Scheme 17, wherein the cDNA is pre-amplified cDNA.

[0396] Implementation Scheme 19. The method according to any one of the foregoing implementation schemes, wherein the FGFR mutant and primer pair are:

[0397] FGFR3:TACC3 v1 and primers having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6;

[0398] FGFR3:TACC3 v3 and primers having the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8;

[0399] FGFR3:TACC3 intron and primers having the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10;

[0400] FGFR3:BAIAP2L1 and primers having the amino acid sequences of SEQ ID NO:11 and SEQ ID NO:12;

[0401] FGFR2:BICC1 and primers having the amino acid sequences of SEQ ID NO:13 and SEQ ID NO:14;

[0402] Primers for FGFR2:AFF3 and the amino acid sequences having SEQ ID NO:15 and SEQ ID NO:16;

[0403] Primers for FGFR2:CASP7 and amino acid sequences having SEQ ID NO:17 and SEQ ID NO:18;

[0404] Primers for FGFR2:CCDC6 and the amino acid sequences having SEQ ID NO:19 and SEQ ID NO:20;

[0405] FGFR2:OFD1 and primers having the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22;

[0406] R248C and primers having amino acid sequences of SEQ ID NO:23 and SEQ ID NO:24 or SEQ ID NO:31 and SEQ ID NO:32;

[0407] S249C and primers having amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:33 and SEQ ID NO:34;

[0408] G370C and primers having amino acid sequences of SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:35 and SEQ ID NO:36;

[0409] Y373C and primers having amino acid sequences of SEQ ID NO:29 and SEQ ID NO:30 or SEQ ID NO:37 and SEQ ID NO:38;

[0410] Or any combination thereof.

[0411] Implementation Scheme 20. The method according to any one of the foregoing implementation schemes, wherein the evaluation includes:

[0412] RNA is isolated from biological samples and cDNA is synthesized from the isolated RNA.

[0413] Implementation Scheme 21. The method according to Implementation Scheme 20 further includes pre-amplifying the cDNA prior to the amplification step.

[0414] Implementation Scheme 22. The method according to any one of Implementation Schemes 1 or 3 to 21, wherein the cDNA is pre-amplified.

[0415] Implementation Scheme 23. The method according to any one of Implementation Schemes 1 or 3 to 22, wherein the amplification step includes performing real-time PCR.

[0416] Implementation Scheme 24. The method according to Implementation Scheme 23, wherein real-time PCR is performed using one or more probes, said probes including 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.

[0417] Implementation Scheme 25. The method according to Implementation Scheme 23 or 24, wherein real-time PCR is performed using one or more 3' blocking oligonucleotides, said 3' blocking oligonucleotides including SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 and / or SEQ ID NO:42.

[0418] Implementation Scheme 26. The method according to any one of Implementation Schemes 1 or 3 to 25, wherein the assay step includes sequencing the amplified cDNA.

[0419] Implementation Scheme 27. A kit for identifying the presence of one or more FGFR mutant genes in a biological sample, the kit comprising:

[0420] Primer pairs having the following sequences: 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. NO:38, or any combination thereof; and

[0421] Instructions for use in assays to detect one or more FGFR mutant genes.

[0422] Implementation Scheme 28. The kit according to Implementation Scheme 27 further includes one or more probes, one or more 3' blocking oligonucleotides, or both.

[0423] Implementation Scheme 29. The reagent kit according to Implementation Scheme 28, wherein

[0424] a. The primer pair has the sequences of SEQ ID NO:5 and SEQ ID NO:6, and the probe has the sequence of SEQ ID NO:43;

[0425] b. The primer pair has the sequences of SEQ ID NO:7 and SEQ ID NO:8, and the probe has the sequence of SEQ ID NO:44;

[0426] c. The primer pair has the sequences of SEQ ID NO:9 and SEQ ID NO:10, and the probe has the sequence of SEQ ID NO:46;

[0427] d. The primer pair has the sequences of SEQ ID NO:11 and SEQ ID NO:12, and the probe has the sequence of SEQ ID NO:47;

[0428] e. The primer pair has the sequences of SEQ ID NO:13 and SEQ ID NO:14, and the probe has the sequence of SEQ ID NO:45;

[0429] f. The primer pair has the sequences of SEQ ID NO:15 and SEQ ID NO:16, and the probe has the sequence of SEQ ID NO:48;

[0430] g. The primer pair has the sequences of SEQ ID NO:17 and SEQ ID NO:18, and the probe has the sequence of SEQ ID NO:49;

[0431] h. The primer pair has the sequences of SEQ ID NO:19 and SEQ ID NO:20, and the probe has the sequence of SEQ ID NO:50;

[0432] i. The primer pair has the sequences of SEQ ID NO:21 and SEQ ID NO:22, and the probe has the sequence of SEQ ID NO:51;

[0433] j. The primer pair has the sequences of SEQ ID NO:23 and SEQ ID NO:24, and the probe has the sequence of SEQ ID NO:52;

[0434] k. The primer pair has the sequences of SEQ ID NO:25 and SEQ ID NO:26, and the probe has the sequence of SEQ ID NO:53;

[0435] l. The primer pair has the sequences of SEQ ID NO:27 and SEQ ID NO:28, and the probe has the sequence of SEQ ID NO:54;

[0436] m. The primer pair has the sequences of SEQ ID NO:29 and SEQ ID NO:30, and the probe has the sequence of SEQ ID NO:55;

[0437] n. The primer pair has the sequences of 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;

[0438] o. The primer pair has the sequences of 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;

[0439] p. The primer pair has the sequences of 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;

[0440] q. The primer pair has the sequences of 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

[0441] r. Any combination of them.

[0442] Embodiment 30. A primer having the following amino acid sequence: 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.

[0443] Implementation Scheme 31. A primer set having the following sequences: 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. NO:38, or any combination thereof.

[0444] Implementation Scheme 32. An oligonucleotide probe having a sequence having any one of SEQ ID NO:43 to 55 or any combination thereof.

[0445] Implementation Scheme 33. An oligonucleotide having a sequence having any one of SEQ ID NO:39 to 42 or any combination thereof. <110> Jensen Pharmaceuticals Co., Ltd. <120> Using FGFR mutation genomes to identify cancer patients who will respond to treatment with FGFR inhibitors <130> 103693.000782 <140> <141> <150> 62 / 056,159 <151> 2014-09-26 <160> 73 <170> Patent version 3.5 <210> 1 <211> 268 <212> DNA <213> Homo sapiens <400> 1 tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 60 tacacgctgg acgtgctgga gtgctccccg caccggccca tcctgcaggc ggggctgccg 120 gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 180 gcacagcccc acatccagtg gctcaagcac gtggaggtga atggcagcaa ggtgggcccg 240 gacggcacac cctacgttac cgtgctca 268 <210> 2 <211> 378 <212> DNA <213> Homo sapiens <400> 2 gaccgcggca actacacctg cgtcgtggag aacaagtttg gcagcatccg gcagacgtac 60 acgctggacg tgctgggtga gggccctggg gcggcgcggg ggtgggggcg gcagtggcgg 120 tggtggtgag ggagggggtg gcccctgagc gtcatctgcc cccacagagc gctgcccgca 180 ccggcccatc ctgcaggcgg ggctgccggc caaccagacg gcggtgctgg gcagcgacgt 240 ggagttccac tgcaaggtgt acagtgacgc acagccccac atccagtggc tcaagcacgt 300 ggaggtgaat ggcagcaagg tgggcccgga cggcacaccc tacgttaccg tgctcaaggt 360 gggccaccgt gtgcacgt 378 <210> 3 <211> 234 <212> DNA <213> Homo sapiens <400> 3 gcgggcaatt ctattgggtt ttctcatcac tctgcgtggc tggtggtgct gccagccgag 60 gaggagctgg tggaggctga cgaggcgtgc agtgtgtatg caggcatcct cagctacggg 120 gtgggcttct tcctgttcat cctggtggtg gcggctgtga cgctctgccg cctgcgcagc 180 ccccccaaga aaggcctggg ctcccccacc gtgcacaaga tctcccgctt cccg 234 <210> 4 <211> 301 <212> DNA <213> Homo sapiens <400> 4 ctagaggttc tctccttgca caacgtcacc tttgaggacg ccggggagta cacctgcctg 60 gcgggcaatt ctattgggtt ttctcatcac tctgcgtggc tggtggtgct gccagccgag 120 gaggagctgg tggaggctga cgaggcgggc agtgtgtgtg caggcatcct cagctacggg 180 gtgggcttct tcctgttcat cctggtggtg gcggctgtga cgctctgccg cctgcgcagc 240 ccccccaaga aaggcctggg ctcccccacc gtgcacaaga tctcccgctt cccgctcaag 300 c 301 <210> 5 <211> twenty one <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 5 gacctggacc gtgtccttac c 21 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 6 cttccccagt tccaggttct t 21 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 7 aggacctgga ccgtgtcctt 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 8 tataggtccg gtggacaggg 20 <210> 9 <211> 15 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 9 ggccatcctg ccccc 15 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 10 gagcagtcca ggtcagccag 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 11 ctggaccgtg tccttaccgt 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 12 gcagcccagg attgaactgt 20 <210> 13 <211> twenty three <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 13 tggatcgaat tctcactctc aca 23 <210> 14 <211> twenty one <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 14 gccaagcaat ctgcgtattt g 21 <210> 15 <211> 25 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 15 tggtagaaga cttggatcga attct 25 <210> 16 <211> twenty three <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 16 tctcccggat tatttcttca aca 23 <210> 17 <211> twenty three <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 17 gctcttcaat acagccctga tca 23 <210> 18 <211> twenty four <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 18 acttggatcg aattctcact ctca 24 <210> 19 <211> twenty three <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 19 tggatcgaat tctcactctc aca 23 <210> 20 <211> 25 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 20 gcaaagcctg aattttcttg aataa 25 <210> twenty one <211> twenty four <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> twenty one agggtgcatc aactcatgaa ttag 24 <210> twenty two <211> twenty four <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> twenty two acttggatcg aattctcact ctca 24 <210> twenty three <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> twenty three gcatccggca gacgtaca 18 <210> twenty four <211> 15 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> twenty four ccccgcctgc aggat 15 <210> 25 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 25 gcatccggca gacgtaca 18 <210> 26 <211> 15 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 26 ccccgcctgc aggat 15 <210> 27 <211> 19 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 27 aggagctggt ggaggctga 19 <210> 28 <211> 19 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 28 ccgtagctga ggatgcctg 19 <210> 29 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 29 ctggtggagg ctgacgag 18 <210> 30 <211> 16 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 30 agcccacccc gtagct 16 <210> 31 <211> twenty one <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 31 gtcgtggaga acaagtttgg c 21 <210> 32 <211> 17 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 32 gtctggttgg ccggcag 17 <210> 33 <211> twenty one <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 33 gtcgtggaga acaagtttgg c 21 <210> 34 <211> 17 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 34 gtctggttgg ccggcag 17 <210> 35 <211> 19 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 35 aggagctggt ggaggctga 19 <210> 36 <211> 19 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 36 ccgtagctga ggatgcctg 19 <210> 37 <211> 16 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 37 gacgaggcgg gcagtg 16 <210> 38 <211> 19 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic primers" <400> 38 gaagaagccc accccgtag 19 <210> 39 <211> 17 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 39 tggagcgctc cccgcac 17 <210> 40 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 40 gacgtgctgg agrgctcc 18 <210> 41 <211> 17 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 41 ctgacgaggc gggcagc 17 <210> 42 <211> twenty three <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 42 gtgtgtatgc aggcatcctc agc 23 <210> 43 <211> 16 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 43 tccaccgacg taaagg 16 <210> 44 <211> 16 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 44 tccaccgacg tgccag 16 <210> 45 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 45 ccaatgagat catggagg 18 <210> 46 <211> 16 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 46 ccttctggcc caggtg 16 <210> 47 <211> 17 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 47 caccgacaat gttatgg 17 <210> 48 <211> 20 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 48 tcacaaccaa tgaggagagt 20 <210> 49 <211> 16 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 49 ctgccatctc attggt 16 <210> 50 <211> 17 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 50 aatgagcaag ccagggc 17 <210> 51 <211> 19 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 51 aagttgtgtc tcattggtt 19 <210> 52 <211> 14 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 52 ctggagtgct cccc 14 <210> 53 <211> 13 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 53 agcgctgccc gca 13 <210> 54 <211> 15 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 54 gcgtgcagtg tgtat 15 <210> 55 <211> 15 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic probe" <400> 55 ctgcacacac actgc 15 <210> 56 <211> 2850 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 56 atgggcgccc ctgcctgcgc cctcgcgctc tgcgtggccg tggccatcgt ggcggcgcc 60 tccggagt ccttggggac ggagcagcgc gtcgtggggc gagcggcaga agtcccgggc 120 ccagagcccg gccagcagga gcagttggtc ttcggcagcg gggatgctgt ggagctgagc 180 tgtccccccgc ccgggggtgg tcccatgggg cccactgtct gggtcaagga tggcacaggg 240 ctggtgccct cggagcgtgt cctggtgggg ccccagcggc tgcaggtgct gaatgcctcc 300 cacgaggact ccggggccta cagctgccgg cagcggctca cgcagcgcgt actgtgccac 360 ttcagtgtgc gggtgacaga cgctccatcc tcgggagatg acgaagacgg ggaggacgag 420 gctgaggaca caggtgtgga cacaggggcc ccttactgga cacggcccga gcggatggac 480 aagaagctgc tggccgtgcc ggccgccaac accgtccgct tccgctgcc agccgctggc 540 aaccccactc cctccatctc ctggctgaag aacggcaggg agttccgcgg cgagcaccgc 600 attggaggca tcaagctgcg gcatcagcag tggagcctgg tcatggaaag cgtggtgccc 660 tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 720 tacacgctgg acgtgctgga gcgctccccg caccggccca tcctgcaggc ggggctgccg 780 gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 840 gcacagcccc acatccagtg gctcaagcac gtggaggtga atggcagcaa ggtgggcccg 900 gacggcacac cctacgttac cgtgctcaag acggcgggcg ctaacaccac cgacaaggag 960 ctagaggttc tctccttgca caacgtcacc tttgaggacg ccggggagta cacctgcctg 1020 gcgggcaatt ctattgggtt ttctcatcac tctgcgtggc tggtggtgct gccagccgag 1080 gaggagctgg tggaggctga cgaggcgggc agtgtgtatg caggcatcct cagctacggg 1140 gtgggcttct tcctgttcat cctggtggtg gcggctgtga cgctctgccg cctgcgcagc 1200 ccccccaaga aaggcctggg ctcccccacc gtgcacaaga tctcccgctt cccgctcaag 1260 cgacaggtgt ccctggagtc caacgcgtcc atgagctcca acacaccact ggtgcgcatc 1320 gcaaggctgt cctcagggga gggccccacg ctggccaatg tctccgagct cgagctgcct 1380 gccgacccca aatgggagct gtctcgggcc cggctgaccc tgggcaagcc ccttggggag 1440 ggctgcttcg gccaggtggt catggcggag gccatcggca ttgacaagga ccgggccgcc 1500 aagcctgtca ccgtagccgt gaagatgctg aaagacgatg ccactgacaa ggacctgtcg 1560 gacctggtgt ctgagatgga gatgatgaag atgatcggga aacacaaaaa catcatcaac 1620 ctgctgggcg cctgcacgca gggcgggccc ctgtacgtgc tggtggagta cgcggccaag 1680 ggtaacctgc gggagtttct gcgggcgcgg cggcccccgg gcctggacta ctccttcgac 1740 acctgcaagc cgcccgagga gcagctcacc ttcaaggacc tggtgtcctg tgcctaccag 1800 gtggcccggg gcatggagta cttggcctcc cagaagtgca tccacaggga cctggctgcc 1860 cgcaatgtgc tggtgaccga ggacaacgtg atgaagatcg cagacttcgg gctggcccgg 1920 gacgtgcaca acctcgacta ctacaagaag acgaccaacg gccggctgcc cgtgaagtgg 1980 atggcgcctg aggccttgtt tgaccgagtc tacactcacc agagtgacgt ctggtccttt 2040 ggggtcctgc tctgggagat cttcacgctg gggggctccc cgtaccccgg catccctgtg 2100 gaggagctct tcaagctgct gaaggagggc caccgcatgg acaagcccgc caactgcaca 2160 cacgacctgt acatgatcat gcgggagtgc tggcatgccg cgccctccca gaggcccacc 2220 ttcaagcagc tggtggagga cctggaccgt gtccttaccg tgacgtccac cgacgtaaag 2280 gcgacacagg aggagaaccg ggagctgagg agcaggtgtg aggagctcca cgggaagaac 2340 ctggaactgg ggaagatcat ggacaggttc gaagaggttg tgtaccaggc catggagagaa 2400 gttcagaagc agaaggaact ttccaaagct gaaatccaga aagttctaaa agaaaaagac 2460 caacttacca cagatctgaa ctccatggag aagtccttct ccgacctctt caagcgtttt 2520 gagaacaga aagaggtgat cgagggctac cgcaagaacg aagagtcact gaagaagtgc 2580 gtggaggatt acctggcaag gatcacccag gagggccaga ggtaccaagc cctgaaggcc 2640 cacgcggagg agaagctgca gctggcaaac gaggagatcg cccaggtccg gagcaaggcc 2700 caggggaag cgttggccct ccaggccagc ctgaggaagg agcagatgcg catccagtcg 2760 2820 gacctcatct ccaagatgga gaagatctga 2850 <210> 57 <211> 2955 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Note="Description of artificial sequence: synthetic polynucleotide" <400> 57 atgggcgccc ctgcctgcgc cctcgcgctc tgcgtggccg tggccatcgt ggccggcgcc 60 tcctcggagt ccttggggac ggagcagcgc gtcgtggggc gagcggcaga agtcccgggc 120 ccagagcccg gccagcagga gcagttggtc ttcggcagcg gggatgctgt ggagctgagc 180 tgtcccccgc ccgggggtgg tcccatgggg cccactgtct gggtcaagga tggcacaggg 240 ctggtgccct cggagcgtgt cctggtgggg ccccagcggc tgcaggtgct gaatgcctcc 300 cacgaggact ccggggccta cagctgccgg cagcggctca cgcagcgcgt actgtgccac 360 ttcagtgtgc gggtgacaga cgctccatcc tcgggagatg acgaagacgg ggaggacgag 420 gctgaggaca caggtgtgga cacaggggcc ccttactgga cacggcccga gcggatggac 480 aagaagctgc tggccgtgcc ggccgccaac accgtccgct tccgctgccc agccgctggc 540 aaccccactc cctccatctc ctggctgaag aacggcaggg agttccgcgg cgagcaccgc 600 attggaggca tcaagctgcg gcatcagcag tggagcctgg tcatggaaag cgtggtgccc 660 tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 720 tacacgctgg acgtgctgga gcgctccccg caccggccca tcctgcaggc ggggctgccg 780 gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 840 gcacagcccc acatccagtg gctcaagcac gtggaggtga atggcagcaa ggtgggcccg 900 gacggcacac cctacgttac cgtgctcaag acggcgggcg ctaacaccac cgacaaggag 960 ctagaggttc tctccttgca caacgtcacc tttgaggacg ccggggagta cacctgcctg 1020 gcgggcaatt ctattgggtt ttctcatcac tctgcgtggc tggtggtgct gccagccgag 1080 gaggagctgg tggaggctga cgaggcgggc agtgtgtatg caggcatcct cagctacggg 1140 gtgggcttct tcctgttcat cctggtggtg gcggctgtga cgctctgccg cctgcgcagc 1200 ccccccaaga aaggcctggg ctcccccacc gtgcacaaga tctcccgctt cccgctcaag 1260 cgacaggtgt ccctggagtc caacgcgtcc atgagctcca acacaccact ggtgcgcatc 1320 gcaaggctgt cctcagggga gggccccacg ctggccaatg tctccgagct cgagctgcct gccgacccca aatgggagct gtctcgggcc cggctgaccc tgggcaagcc ccttggggag 1440 ggctgcttcg gccaggtggt catggcggag gccatcggca ttgacaagga ccggggccgcc 1500. aagcctgtca ccgtagccgt gaagatgctg aaagacgatg ccactgacaa ggacctgtcg gacctggtgt ctgagatgga gatgatgaag atgatcggga aacacaaaaa catcatcaac ctgctgggcg cctgcacgca gggcgggccc ctgtacgtgc tggtggagta cgcggccaag 1680. ggtaacctgc gggagtttct gcggggcgcgg cggccccgg gcctggacta ctccttcgac 1740 acctgcaagc cgcccgagga gcagctcacc ttcaaggacc tggtgtcctg tgcctaccag gtggcccggg gcatggagta cttggcctcc cagaagtgca tccacaggga cctggctgcc 1860. cgcaatgtgc tggtgaccga ggacaacgtg atgaagatcg cagacttcgg gctggcccgg gacgtgcaca acctcgacta ctacaagaag acgaccaacg gccggctgcc cgtgaagtgg atggcgcctg aggccttgtt tgaccgagtc tacactcacc aggtgacgt ctggtccttt ggggtcctgc tctgggagat cttcacgctg gggggctccc cgtaccccgg catccctgtg 2100 gaggagctct tcaagctgct gaaggagggc caccgcatgg acaagcccgc caactgcaca 2160 cacgacctgt acatgatcat gcgggagtgc tggcatgccg cgccctccca gaggcccacc 2220 ttcaagcagc tggtggagga cctggaccgt gtccttaccg tgacgtccac cgacgtgcca 2280 ggcccacccc caggtgttcc cgcgcctggg ggcccacccc tgtccaccgg acctatagtg 2340 gacctgctcc agtacagcca gaaggacctg gatgcagtgg taaaggcgac acaggaggag 2400 aaccgggagc tgaggagcag gtgtgaggag ctccacggga agaacctgga actggggaag 2460 atcatggaca ggttcgaaga ggttgtgtac caggccatgg aggaagttca gaagcagaag 2520 gaactttcca aagctgaaat ccagaaagtt ctaaaagaaa aagaccaact taccacagat 2580 ctgaactcca tggagaagtc cttctccgac ctcttcaagc gttttgagaa acagaaagag 2640 gtgatcgagg gctaccgcaa gaacgaagag tcactgaaga agtgcgtgga ggattacctg 2700 gcaaggatca cccaggaggg ccagaggtac caagccctga aggcccacgc ggaggagaag 2760 ctgcagctgg caaacgagga gatcgcccag gtccggagca aggcccaggc ggaagcgttg 2820 gccctccagg ccagcctgag gaaggagcag atgcgcatcc agtcgctgga gaagacagtg 2880 gagcagaaga ctaaagagaa cgaggagctg accaggatct gcgacgacct catctccaag 2940 atggagaaga tctga 2955 <210> 58 <211> 4462 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic polynucleotide” <400> 58 atgggcgccc ctgcctgcgc cctcgcgctc tgcgtggccg tggccatcgt ggccggcgcc 60 tcctcggagt ccttggggac ggagcagcgc gtcgtggggc gagcggcaga agtcccgggc 120 ccagagcccg gccagcagga gcagttggtc ttcggcagcg gggatgctgt ggagctgagc 180 tgtcccccgc ccgggggtgg tcccatgggg cccactgtct gggtcaagga tggcacaggg 240 ctggtgccct cggagcgtgt cctggtgggg ccccagcggc tgcaggtgct gaatgcctcc 300 cacgaggact ccggggccta cagctgccgg cagcggctca cgcagcgcgt actgtgccac 360 ttcagtgtgc gggtgacaga cgctccatcc tcgggagatg acgaagacgg ggaggacgag 420 gctgaggaca caggtgtgga cacaggggcc ccttactgga cacggcccga gcggatggac 480 aagaagctgc tggccgtgcc ggccgccaac accgtccgct tccgctgcc agccgctggc 540 aaccccactc cctccatctc ctggctgaag aacggcaggg agttccgcgg cgagcaccgc 600 attggaggca tcaagctgcg gcatcagcag tggagcctgg tcatggaaag cgtggtgccc 660 tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 720 tacacgctgg acgtgctgga gcgctccccg caccggccca tctgcaggc ggggctgccg 780 gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 840 gcacagcccc acatccagtg gctcaagcac gtggaggtga atggcagcaa ggtgggcccg 900 gacggcacac cctacgttac cgtgctcaag acggcgggcg ctaacaccac cgacaaggag 960 ctagaggttc tctccttgca caacgtcacc tttgaggacg ccggggagta cacctgcctg 1020 gcgggcaatt ctattgggtt ttctcatcac tctgcgtggc tggtggtgct gccagccgag 1080 gaggagctgg tggaggctga cgaggcgggc agtgtgtatg caggcatcct cagctacggg 1140 gtgggcttct tcctgttcat cctggtggtg gcggctgtga cgctctgccg cctgcgcagc 1200 ccccccaaga aaggcctggg ctccccccacc gtgcacaaga tctcccgctt cccgctcaag 1260 cgacaggtgt ccctggagtc caacgcgtcc atgagctcca acacaccact ggtgcgcatc gcaaggctgt cctcagggga gggccccacg ctggccaatg tctccgagct cgagctgcct gccgacccca aatgggagct gtctcgggcc cggctgaccc tgggcaagcc ccttggggag 1440 ggctgcttcg gccaggtggt catggcggag gccatcggca ttgacaagga ccggggccgcc 1500. aagcctgtca ccgtagccgt gaagatgctg aaagacgatg ccactgacaa ggacctgtcg gacctggtgt ctgagatgga gatgatgaag atgatcggga aacacaaaaa catcatcaac ctgctgggcg cctgcacgca gggcgggccc ctgtacgtgc tggtggagta cgcggccaag 1680. ggtaacctgc gggagtttct gcggggcgcgg cggccccgg gcctggacta ctccttcgac 1740 acctgcaagc cgcccgagga gcagctcacc ttcaaggacc tggtgtcctg tgcctaccag gtggcccggg gcatggagta cttggcctcc cagaagtgca tccacaggga cctggctgcc 1860 cgcaatgtgc tggtgaccga ggacaacgtg atgaagatcg cagacttcgg gctggcccgg 1920 gacgtgcaca acctcgacta ctacaagaag acgaccaacg gccggctgcc cgtgaagtgg 1980 atggcgcctg aggccttgtt tgaccgagtc tacactcacc agagtgacgt ctggtccttt 2040 ggggtcctgc tctgggagat cttcacgctg gggggctccc cgtaccccgg catccctgtg 2100 gaggagctct tcaagctgct gaaggagggc caccgcatgg acaagcccgc caactgcaca 2160 cacgacctgt acatgatcat gcgggagtgc tggcatgccg cgccctccca gaggcccacc 2220 ttcaagcagc tggtggagga cctggaccgt gtccttaccg tgacgtccac cgacgtgagt 2280 gctggctctg gcctggtgcc acccgcctat gcccctcccc ctgccgtccc cggccatcct 2340 gccccccaga gtgctgaggt gtggggcggg cctttctggc ccaggtgccc tggctgacct 2400 ggactgctca agctcttccc agagcccagg aagttctgag aaccaaatgg tgtctccagg 2460 aaaagtgtct ggcagccctg agcaagccgt ggaggaaaac cttagttcct attccttaga 2520 cagaagagtg acacccgcct ctgagaccct agagaccct tgcaggacag agtcccagca 2580 haaagcggag actccgcacg gagccgagga agaatgcaaa gcggagactc cgcacggagc 2640 cgaggaggaa tgccggcacg gtggggtctg tgctcccgca gcagtggcca cttcgcctcc 2700 tggtgcaatc cctaggaag cctgcggagg agcaccctg cagggtctgc ctggcgaagc 2760 cctgggctgc cctgcgggtg tgggcacccc cgtgccagca gatggcactc agacccttac 2820 ctgtgcacac acctctgctc ctgagagcac agccccaacc aaccacctgg tggctggcag 2880 ggccatgacc ctgagtccctc aggaagt ggctgcaggc caaatggcca gctcctcgag 2940 gagcggacct gtaaaactag aatttgatgt atctgatggc gccaccagca aaagggcacc 3000 cccaccaagg agactgggag agaggtccgg cctcaagcct cccttgagga aagcagcagt 3060 gaggcagcaa aaggccccgc aggaggtgga ggaggacgac ggtaggagcg gagcaggaga 3120 ggaccccccc atgccagctt ctcggggctc ttaccacctc gactgggaca aaatggatga 3180 cccaaacttc atcccgttcg gaggtgacac caagtctggt tgcagtgagg cccagccccc 3240 agaaagccct gagaccaggc tgggccagcc agcggctgaa cagttgcatg ctgggcctgc 3300 cacggaggag ccaggtccct gtctgagcca gcagctgcat tcagcctcag cggaggacac 3360 gcctgtggtg cagttggcag ccgagacccc aacagcagag agcaaggaga gagccttgaa 3420 ctctgccagc acctcgcttc ccacaagctg tccaggcagt gagccagtgc ccacccatca 3480 gcaggggcag cctgccttgg agctgaaaga ggagagcttc agagaccccg ctgaggttct 3540 aggcacgggc gcggaggtgg attacctgga gcagtttgga acttcctcgt ttaaggagtc 3600 ggccttgagg aagcagtcct tatacctcaa gttcgacccc ctcctgaggg acagtcctgg 3660 tagaccagtg cccgtggcca ccgagaccag cagcatgcac ggtgcaaatg agactccctc 3720 aggacgtccg cgggaagcca agcttgtgga gttcgatttc ttgggagcac tggacattcc 3780 tgtgccaggc ccacccccag gtgttcccgc gcctgggggc ccacccctgt ccaccggacc 3840 tatagtggac ctgctccagt acagccagaa ggacctggat gcagtggtaa aggcgacaca 3900 ggaggagaac cgggagctga ggagcaggtg tgaggagctc cacgggaaga acctggaact 3960 ggggaagatc atggacaggt tcgaagaggt tgtgtaccag gccatggagg aagttcagaa 4020 gcagaaggaa ctttccaaag ctgaaatcca gaaagttcta aaagaaaaag accaacttac 4080<000!796>cacagatctg aactccatgg agaagtcctt ctccgacctc ttcaagcgtt ttgagaaaca 4140 gaaagaggtg atcgagggct accgcaagaa cgaagagtca ctgaagaagt gcgtggagga 4200 ttacctggca aggatcaccc aggagggcca gaggtaccaa gccctgaagg cccacgcgga 4260 ggagaagctg cagctggcaa acgaggagat cgcccaggtc cggagcaagg cccaggcgga 4320 agcgttggcc ctccaggcca gcctgaggaa ggagcagatg cgcatccagt cgctggagaa 4380[[ID=!3]] gacagtggag cagaagacta aagagaacga ggagctgacc aggatctgcg acgacctcat 4440 ctccaagatg gagaagatct ga 4462 <210> 59 <211> 3765<00018(5><212> DNA <213> Artificial Sequence <220> <221! Source <223> / Note= "Description of artificial sequence: Synthetic polynucleotide" <400> 59 atgggcgccc ctgcctgcgc cctcgcgctc tgcgtggccg tggccatcgt ggccggcgcc 60 It should be noted that there seems to be an error in tag "000!796" in the original text, which is translated as "<000!796>" here. It should be corrected to the correct tag number for accurate translation.tccggagt ccttggggac ggagcagcgc gtcgtggggc gagcggcaga agtcccgggc 120 ccagagcccg gccagcagga gcagttggtc ttcggcagcg gggatgctgt ggagctgagc 180 tgtccccccgc ccgggggtgg tcccatgggg cccactgtct gggtcaagga tggcacaggg 240 ctggtgccct cggagcgtgt cctggtgggg ccccagcggc tgcaggtgct gaatgcctcc 300 cacgaggact ccggggccta cagctgccgg cagcggctca cgcagcgcgt actgtgccac 360 ttcagtgtgc gggtgacaga cgctccatcc tcgggagatg acgaagacgg ggaggacgag 420 gctgaggaca caggtgtgga cacaggggcc ccttactgga cacggcccga gcggatggac 480 aagaagctgc tggccgtgcc ggccgccaac accgtccgct tccgctgcc agccgctggc 540 aaccccactc cctccatctc ctggctgaag aacggcaggg agttccgcgg cgagcaccgc 600 attggaggca tcaagctgcg gcatcagcag tggagcctgg tcatggaaag cgtggtgccc 660 tcggaccgcg gcaactacac ctgcgtcgtg gagaacaagt ttggcagcat ccggcagacg 720 tacacgctgg acgtgctgga gcgctccccg caccggccca tctgcaggc ggggctgccg 780 gccaaccaga cggcggtgct gggcagcgac gtggagttcc actgcaaggt gtacagtgac 840 gcacagcccc acatccagtg gctcaagcac gtggagagtga atggcagcaa ggtgggcccg 900 gacggcacac cctacgttac cgtgctcaag tcctggatca gtgagagtgt ggaggccgac 960 gtgcgcctcc gcctggccaa tgtgtcggag cggacgggg gcgagtacct ctgtcgagcc 1020 accaatttca taggcgtggc cgagaaggcc ttttggctga gcgttcacgg gccccgagca 1080 gccgaggagg agctggtgga ggctgacgag gcgggcagtg tgtatgcagg catcctcagc 1140 tacggggtgg gcttcttcct gttcatcctg gtggtggcgg ctgtgacgct ctgccgcctg 1200 cgcagcccccc ccaagaaagg cctgggctcc cccaccgtgc acaagatctc ccgcttcccg 1260 ctcaagcgac aggtgtccct ggagtccaac gcgtccatga gctccaacac accactggtg 1320 cgcatcgcaa ggctctcctc aggggagggc cccacgctgg ccaatgtctc cgagctcgag 1380 ctgcctgccg accccaaatg ggagctgtct cgggcccggc tgaccctggg caagcccctt 1440 ggggagggct gcttcggcca ggtggtcatg gcggaggcca tcggcattga caaggaccgg 1500 gccgccaagc ctgtcaccgt agccgtgaag atgctgaaag acgatgccac tgacaaggac 1560 ctgtcggacc tggtgtctga gatggagatg atgaagatga tcgggaaaca caaaaacatc 1620 atcaacctgc tgggcgcctg cacgcagggc gggcccctgt acgtgctggt ggagtacgcg 1680 gccaagggta acctgcggga gtttctgcgg gcgcggcggc ccccgggcct ggactactcc 1740 ttcgacacct gcaagccgcc cgaggagcag ctcaccttca aggacctggt gtcctgtgcc 1800 taccaggtgg cccggggcat ggagtacttg gcctcccaga agtgcatcca cagggacctg 1860 gctgcccgca atgtgctggt gaccgaggac aacgtgatga agatcgcaga cttcgggctg 1920 gcccgggacg tgcacaacct cgactactac aagaagacga ccaacggccg gctgcccgtg 1980 aagtggatgg cgcctgaggc cttgtttgac cgagtctaca ctcaccagag tgacgtctgg 2040 tcctttgggg tcctgctctg ggagatcttc acgctggggg gctccccgta ccccggcatc 2100 cctgtggagg agctcttcaa gctgctgaag gagggccacc gcatggacaa gcccgccaac 2160 tgcacacacg acctgtacat gatcatgcgg gagtgctggc atgccgcgcc ctcccagagg 2220 cccaccttca agcagctggt ggaggacctg gaccgtgtcc ttaccgtgac gtccaccgac 2280 aatgttatgg aacagttca tcctgggctg cgaatttaa taaacctggg gaaaaattat 2340 gagaaagctg taaacgctat gatcctggca ggaaaagcct actacgatgg agtggccaag 2400 atcggtgaga ttgccactgg gtcccccgtg tcactgaac tgggacatgt cctcatagag 2460 atttcaagta cccacaagaa actcaacgag agtcttgatg aaaattta aaaattccac 2520 aaagagatta tccatgagct gggagaag atagaacttg acgtgaaata tatgaacgca 2580 actctaaaa gatcaaac agacacaag aaaattag agtctttgga gaatcccaa 2640 gctgagttga agagatcag aaggaaagc aaggaagcc gaacgcact caatatgaa 2700 cacaagaaaa ttgagtatgt ggagaccgtt acttctcgtc agagtgaat ccagaaatttc 2760 attgcagatg gttgcaaga ggctctgctt gagagaga ggcgctctg ctttctggtt 2820 gataagcact gtggctttgc aaaccacata cattattatc acttacagtc tgcagaacta 2880 ctgaattcca agctgcctcg gtggcaggag acctgtgttg atgccatcaa agtgccagag 2940 aaaatcatga atatgatcga agaataag acccagcct ctacccccgt gtctggaact 3000 cctcaggctt cacccatgat cgagagaagc atgtggtta ggaaagatta cgacaccctt 3060 tctaaatgct caccaagat gccccccgct ccttcaggca gagcatatac cagtccctg 3120 atcgatatgt ttaataaccc agccacggct gccccgaatt cacaagggt aaataattca 3180 acaggtactt ccgagatcc cagtttacag cgatcagttt cggttgcac gggactgaac 3240 multiplying agcagaagt gagaccacatc ttcccgcaca ctgcggggctc siacaagacc 3300 ttactcagct ttgcacagggg agatgtcatc acgctgctca tccccgagga gaaggatggc 3360 tggctctatg gagaacacga cgtgtccaag gcgaggggtt gtcgtacacg 3420 aagttgctgg aagaaaatga gagagagca gtgaccgtgc ccacgccaag ccccacacca 3480 gtgagaagca tcagcaccgt gaacttgtct gagaatagca gtgttgtcat cccccaccc 3540 gactacttgg aatgcttgtc catgggggca gctgccgaca ggagagcaga ttcggccagg 3600 acgacatcca cctttaaggc cccagcgtcc aagcccgaga ccgcggctcc taacgatgcc 3660 aacgggactg caaagccgcc ttttctcagc ggagaaaacc cctttgccac tgtgaaactc 3720 cgcccgactg tgacgaatga tcgctcggca cccatcattc gatga 3765 <210> 60 <211> 4989 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 60 atggtcagct ggggtcgttt catctgcctg gtcgtggtca ccatggcaac cttgtccctg 60 gcccggccct ccttcagttt agttgaggat accacattag agccagaaga gccaccaacc 120 aaataccaaa tctctcaacc agaagtgtac gtggctgcgc caggggagtc gctagaggtg 180 cgctgcctgt tgaaagatgc cgccgtgatc agttggacta aggatggggt gcacttgggg 240 cccaacaata ggacagtgct tattggggag tacttgcaga taaagggcgc cacgcctaga 300 gactccggcc tctatgcttg tactgccagt aggactgtag acagtgaaac ttggtacttc 360 atggtgaatg tcacagatgc catctcatcc ggagatgatg aggatgacac cgatggtgcg 420 gaagattttg tcagtgagaa cagtaacaac aagagagcac catactggac caacacagaa 480 aagatggaaa agcggctcca tgctgtgcct gcggccaaca ctgtcaagtt tcgctgccca 540 gccgggggga acccaatgcc aaccatgcgg tggctgaaaa acgggaagga gtttaagcag 600 gagcatcgca ttggaggcta caaggtacga aaccagcact ggagcctcat tatggaaagt 660 gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg agaatgaata cgggtccatc 720 aatcacacgt accacctgga tgttgtggag cgatcgcctc accggcccat cctccaagcc 780 ggactgccgg caaatgcctc cacagtggtc ggaggagacg tagagtttgt ctgcaaggtt 840 tacagtgatg cccagcccca catccagtgg atcaagcacg tggaaaagaa cggcagtaaa 900 tacgggcccg acgggctgcc ctacctcaag gttctcaagg ccgccggtgt taacaccacg 960 gacaaagaga ttgaggttct ctatattcgg aatgtaactt ttgaggacgc tggggaatat 1020 acgtgcttgg cgggtaattc tattgggata tcctttcact ctgcatggtt gacagttctg 1080 ccagcgcctg gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt 1140 tactgcatag gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg 1200 aagaacacga ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa cgtatccccc tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc aacaccccgc tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg gcaggggtct ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag ctgacactgg gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca gtgggaattg acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa gatgatgcca cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg attgggaac acaagaat cataaatctt cttggagcct gcacacagga tgggcctctc tatgtcatag ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg 1740 ccacccggga tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc aaggacttgg tgtcatgcac ctaccagctg gccagaggca tggagtactt ggcttcccaa aaatgtattc atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg aaatagcag actttggact cgccagagat atcaacaata tagactatta caaaaagacc 1980 accaatgggc ggcttccagt caagtggatg gctccagaag ccctgtttga tagagtatac 2040 actcatcaga gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 2100 ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 2160 agaatggata agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg 2220 catgcagtgc cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt 2280 ctcactctca caaccaatga gatcatggag gaaacaaata cgcagattgc ttggccatca 2340 aaactgaaga tcggagccaa atccaagaaa gatccccata ttaaggtttc tggaaagaaa 2400 gaagatgtta aagaagccaa ggaaatgatc atgtctgtct tagacacaaa aagcaatcga 2460 gtcacactga agatggatgt ttcacataca gaacattcac atgtaatcgg caaaggtggc 2520 aacaatatta aaaaagtgat ggaagaaacc ggatgccata tccactttcc agattccaac 2580 aggaataacc aagcagaaaa aagcaaccag gtatctatag cgggacaacc agcaggagta 2640 gaatctgccc gagttagaat tcgggagctg cttcctttgg tgctgatgtt tgagctacca 2700 attgctggaa ttcttcaacc ggttcctgat cctaattccc cctctattca gcatatatca 2760 caaacgtaca atatttcagt atcatttaaa cagcgttccc gaatgtatgg tgctactgtc 2820 atagtacgag ggtctcagaa taacactagt gctgtgaagg aaggaactgc catgctgtta 2880 gaacatcttg ctgggagctt agcatcagct attcctgtga gcacacaact agatattgca 2940 gctcaacatc atctctttat gatgggtcga aatgggagca acatcaaaca tatcatgcag 3000 agaacaggtg ctcagatcca ctttcctgat cccagtaatc cacaaaagaa atctaccgtc 3060 tacctccagg gcaccattga gtctgtctgt cttgcaaggc aatatctcat gggttgtctt 3120 cctcttgtgt tgatgtttga tatgaaggaa gaaattgaag tagatccaca attcattgcg 3180 cagttgatgg aacagcttga tgtcttcatc agtattaaac caaagcccaa acagccaagc 3240 aagtctgtga ttgtgaaaag tgttgagcga aatgccttaa atatgtatga agcaaggaaa 3300 tgtctcctcg gacttgaaag cagtggggtt accatagcaa ccagtccatc cccagcatcc 3360 tgccctgccg gcctggcatg tcccagcctg gatatcttag cttcagcagg ccttggactc 3420 actggactag gtcttttggg acccaccacc ttatctctga acacttcaac aaccccaaac 3480 tcactcttga atgctcttaa tagctcagtc agtcctttgc aaagtccaag ttctggtaca 3540 cccagcccca cattatgggc acccccactt gctaatactt caagtgccac aggtttttct 3600 gctataccac accttatgat tccatctact gcccaagcca cattaactaa tattttgttg 3660 tctggagtgc ccacctatgg gcacacagct ccatctcccc ctcctggctt gactcctgtt 3720 gatgtccata tcaacagtat gcagaccgaa ggcaaaaaaa tctctgctgc tttaaatgga 3780 catgcacagt ctccagatat aaaatatggt gcaatatcca cttcatcact tggagaaaaa 3840 gtgctgagtg caaatcacgg ggatccgtcc atccagacaa gtgggtctga gcagacatct 3900 cccaaatcaa gccccactga aggttgtaat gatgcttttg ttgaagtagg catgcctcga 3960 agtccttccc attctgggaa tgctggtgac ttgaaacaga tgatgtgtcc ctccaaggtt 4020 tcctgtgcca aaaggcagac agtggaacta ttgcaaggca cgaaaaactc acacttacac 4080 agcactgaca ggttgctctc agaccctgaa ctgagtgcta ccgaaagccc tttggctgac 4140 aagaaggctc cagggagtga gcgcgctgca gagagggcag cagctgccca gcaaaactcc 4200 gaaagggccc accttgctcc acggtcatca tatgtcaaca tgcaggcatt tgactatgaa 4260 cagaagaagc tattagccac caaagctatg ttaaagaaac cagtggtgac ggaggtcaga 4320 acgcccacaa atacctggag tggcctgggt ttttctaaat ccatgccagc tgaaactatc 4380 aaggagttga gaagggccaa tcatgtgtcc tataagccca caatgacaac cacttatgag 4440 ggctcatcca tgtccctttc acggtccaac agtcgtgagc acttgggagg tggaagcgaa 4500 tctgataact ggagagaccg aaatggaatt ggacctggaa gtcatagtga atttgcagct 4560 tctattggca gccctaagcg taaacaaaac aaatcaacgg aacactatct cagcagtagc 4620 aattacatgg actgcatttc ctcgctgaca ggaagcaatg gctgtaactt aatagctct 4680 ttcaaaggtt ctgacctccc tgagctcttc agcaaactgg gcctgggcaa atacacagat 4740 gttttccagc aacaagagat cgatcttcag acattcctca ctctcacaga tcaggatctg 4800 aaggagctgg gaataactac ttttggtgcc aggaggaaaa tgctgcttgc aatttcagaa 4860 ctaaataaaa accgaagaaa gctttttgaa tcgccaaatg cacgcacctc tttcctggaa 4920 ggtggagcga gtggaaggct accccgtcag tatcactcag acattgctag tgtcagtggc 4980 cgctggtag 4989 <210> 61 <211> 5109 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic polynucleotide” <400> 61 atggtcagct ggggtcgttt catctgcctg gtcgtggtca ccatggcaac cttgtccctg 60 gcccggccct ccttcagttt agttgaggat accacattag agccagaaga gccaccaacc 120 aaataccaaa tctctcaacc agaagtgtac gtggctgcgc caggggagtc gctagaggtg 180 cgctgcctgt tgaaagatgc cgccgtgatc agttggacta aggatggggt gcacttgggg 240 cccaacaata ggacagtgct tattggggag tacttgcaga taaagggcgc cacgcctaga 300 gactccggcc tctatgcttg tactgccagt aggactgtag acagtgaaac ttggtacttc 360 atggtgaatg tcacagatgc catctcatcc ggagatgat aggatgacac cgatggtgcg 420 gaagattttg tcagtgagaa cagtacaac agagagcac catactggac cacaca 480 aagatggaaa agcggctcca tgctgtgcct gcggccaaca ctgtcaagtt tcgctgccca 540 gccgggggga acccaatgcc aaccatgcgg tggctgaaaa acgggaagga gtttaagcag 600 gagcatcgca tggaggta caagtacga aaccagcact ggaggctcat tatggaagt 660 gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg agaatgaata cgggtccatc 720 aatcacacgt accacctgga tgttgtggag cgatcgccctc accggccat cctccaagcc 780 ggactgccgg caatgccctc cacagtggtc ggaggacg tagagttgt ctgcaaggtt 840 tacagtgatg cccagcccca catccagtgg atcagcacg tggaaaagaa cggcagtaaa 900 tacggccccg acgggctgcc ctacctcaag gttctcagg ccgccggtgt taacaccacg 960 gawaagaga tgaggttct ctatattcgg atgtactt ttgaggacgc tgggaat 1020 acgtgcttgg cgggtaattc tattgggata tcctttcact ctgcatggttzgagttctg 1080 ccagcgcctg gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt tactgcatag gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg aagaacacga ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa cgtatccccc tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc aacaccccgc tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg gcaggggtct ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag ctgacactgg gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca gtgggaattg acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa gatgatgcca cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg attgggaac acaagaat cataaatctt cttggagcct gcacacagga tgggcctctc tatgtcatag ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg 1740 ccacccggga tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc aggacttgg tgtcatgcac ctaccagctg gccagaggca tggagtactt ggcttcccaa 1860 aaatgtattc atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg 1920 aaaatagcag actttggact cgccagagat atcaacaata tagactatta caaaaagacc 1980 accaatgggc ggcttccagt caagtggatg gctccagaag ccctgtttga tagtatac 2040 actcatcaga gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 2100 ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 2160 agaatggata agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg 2220 catgcagtgc cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt 2280 ctcactctca caaccaatga ggagagtaga tctggagaaa ccaacagctg tgttgaagaa 2340 ataatccggg agatgacctg gcttccacca ctttctgcta ttcaagcacc tggcaaagtg 2400 gaaccaacca aatttccatt tccaaataag gactctcagc ttgtatcctc tggacacaat 2460 aatccaaaga aaggtgatgc agagccagag agtccagaca gtggcacatc gaatacatca 2520 atgctggaag atgaccttaa gctaagcagt gatgaaggg agaatgaca gcaggcagct 2580 cagagaacgg ctctccgcgc tctctgac agcgccgtgg tccagcagcc caacgcaga 2640 acctcggtgc cttccagcag gggcagcagc agcagcagca gcagcggcag cagcagctcc 2700 tccagcgact cagagagcag ctcggatct gactcggaga ccgagagcag ctccagcgag 2760 agtgagggca gcaagcccccc ccactctcc agccccgagg ctgaccggc atcctctaac 2820 aagtggcagc tggataatg gctaaacaa gttaatcccc acagcctcc tattctgatc 2880 caaaatgaaa gccacggggtc agagagcaat cagtactaca acccggtgaa aggaggacgtc 2940 caggactgtg ggaaagtccc cgacgttttgc cagcccagcc tgagagagaa ggagatcaag 3000 agcacttgca aggaggagca aaggccaagg acagccaca aggccctgg gagtaaggc 3060 gtgaagcaga agtccccgcc cgcggccgtg gccgtggcgg tgagcgcagc cgccccgcca 3120 cccgcagtgc cctgtgcgcc cgcggagac gcgcccgcgc ctgcccggag gtccgcgggc 3180 aagaagccca cccaggcgcac cgagaggacc tcagccgggg acggcgccaa ctgccaccgg 3240 cccgaggagc ccgcggccgc ggacgcgctg gggacgagcg tggtggtccc cccggagccc 3300 accaaaacca ggccctgtgg caacaacaga gcgagccacc gcaaggagct gcgctcctcc 3360 gtgacctgcg agaagcgccg cacgcggggg ctaagcagga tcgtccccaa atccaaggag 3420 ttcattgaga cagagtcgtc atcttcatcc tcctcctcgg actccgacct ggagtccgag 3480 caggaggagt accctctgtc caaagcacag accgtggctg cctctgcctc ctccgggaat 3540 gatcagaggc tgaaggaggc cgctgccaac gggggcagtg gtcctagggc ccctgtaggc 3600 tccatcaacg ccaggaccac cagtgacatc gccaaggagc tggaggagca gttctacaca 3660 ctggtcccct ttggccggaa cgaacttctc tcccctctaa aggacagtga tgagatcagg 3720 tctctctggg tcaaaatcga cctgaccctc ctgtccagga tcccagaaca cctgccccag 3780 gagccagggg tattgagcgc ccctgccacc aaggactctg agagcgcacc gcccagccac 3840 acctcggaca cacctgcaga aaaggctttg ccaaaatcca agaggaaacg caagtgtgac 3900 aacgaagacg actacaggga gatcaagaag tcccagggag agaaagacag ctcttcaaga 3960 ctggccacct ccaccagtaa tactttgtct gcaaaccact gcaacatgaa catcaacagt 4020 gtggcaatac caataaataa aaatgaaaaa atgcttcggt cgcccatctc acccctctct 4080 gatgcatcta aacacaaata caccagcgag gacttaactt cttccagccg acctaatggc 4140 aacagtttgt ttacttcagc ctcttccagc aaaaagccta aggccgacag ccagctgcag 4200 cctcacggcg gagacctcac gaaagcagct cacaacaatt ctgaaaacat tcccctccac 4260 aagtcacggc cgcagacgaa gccgtggtct ccaggctcca acggccacag ggactgcaag 4320 aggcagaaac ttgtcttcga tgatatgcct cgcagtgccg attattttat gcaagaagct 4380 aaacgaatga agcataaagc agatgcaatg gtggaaaagt ttggaaaggc tttgaactat 4440 gctgaagcag cattgtcgtt tatcgagtgt ggaaatgcaa tggaacaagg ccccatggaa 4500 tccaaatctc cttatacgat gtattcagaa acagtagagc tcatcaggta tgctatgaga 4560 ctaaaaaccc actcaggccc caatgccaca ccagaagaca aacaactggc tgcattatgt 4620 taccgatgcc tggccctcct gtactggcgg atgtttcgac tcaaaaggga ccacgctgta 4680 aagtattcaa aagcactaat cgactatttc aagaactcat ctaaagccgc ccaagcccca 4740 tctccgtggg gggccagtgg aaagagcact ggaaccccat cccccatgtc tcccaacccc 4800 tctcccgcca gctccgtggg gtctcagggc agcctctcca acgccagcgc cctgtccccg 4860 tcgaccatcg tcagcatccc acagcgcatc caccagatgg cggccaacca cgtcagcatc 4920 accaacagca tcctgcacag ctacgactac tgggagatgg ccgacaacct ggccaaggaa 4980 aaccgagaat tcttcaacga cctggatctg ctcatggggc cggtcaccct gcacagcagc 5040 atggagcacc tggtccagta ctcccaacag ggcctgcact ggctgcggaa cagcgcccac 5100 ctgtcatag 5109 <210> 62 <211> 3213 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Note="Description of artificial sequence: synthetic polynucleotide" <400> 62 atggtcagct ggggtcgttt catctgcctg gtcgtggtca ccatggcaac cttgtccctg 60 gcccggccct ccttcagttt agttgaggat accacattag agccagaaga gccaccaacc 120 aaataccaaa tctctcacc agaagtgtac gtggctgcgc caggggagtc gctagaggtg 180 cgctgcctgt tgaagatgc cgccgtgatc agttggacta aggatggggt gcacttgggg 240 cccaacaata ggacagtgct tattgggg tacttgcaga taaagggcgc cacgcctaga 300 gactccggcc tcttagcttg tactgccagt aggactgtag acagtgaac tggtacttc 360 atggtgaatg tcacagatgc catctcatcc ggagatgat aggatgacac cgatggtgcg 420 gaagattttg tcagtgagaa cagtacaac agagagcac catactggac cacaca 480 aagatggaaa agcggctcca tgctgtgcct gcggccaaca ctgtcaagtt tcgctgccca 540 gccgggggga acccaatgcc aaccatgcgg tggctgaaaa acgggaagga gtttaagcag 600 gagcatcgca tggaggta caagtacga aaccagcact ggaggctcat tatggaagt 660 gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg agaatgaata cgggtccatc 720 aatcacacgt accacctgga tgttgtggag cgatcgccctc accggccat cctccaagcc 780 ggactgccgg caatgccctc cacagtggtc ggaggacg tagagttgt ctgcaaggtt 840 tacagtgatg cccagcccca catccagtgg atcaagcacg tggaaaagaa cggcagtaaa 900 tacgggcccg acgggctgcc ctacctcaag gttctcaagg ccgccggtgt taacaccacg 960 gaaaagaga ttgaggttct ctatattcgg aatgtaactt ttgaggacgc tggggaatat 1020 acgtgcttgg cgggtaattc tattgggata tcctttcact ctgcatggtt gacagttctg 1080 ccagcgcctg gaagagaaa ggagattaca gcttccccag actacctgga gatagccatt 1140 tactgcatag gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg 1200 aagaacacga ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa 1260 cgtatcccc tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc 1320 aacaccccgc tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg 1380 gcaggggtct ccgagtatga acttccagag gaccaaaat gggagtttcc aagagataag 1440 ctgacactgg gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca 1500 gtgggaattg acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa 1560 gatgatgcca cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg 1620 attgggaaac acaagaatat cataaatctt cttggagcct gcacacagga tgggcctctc 1680 tatgtcatag ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg 1740 ccacccggga tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc 1800 aggacttgg tgtcatgcac ctaccagctg gccagaggca tggagtactt ggcttcccaa 1860 aaatgtattc atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg 1920 aaaatagcag actttggact cgccagagat atcaacaata tagactatta caaaaagacc 1980 accaatgggc ggcttccagt caagtggatg gctccagaag ccctgtttga tagtatac 2040 actcatcaga gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 2100 ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 2160 agaatggata agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg 2220 catgcagtgc cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt 2280 ctcactctca caaccaatga gatggcagat gatcagggct gtattgaaga gcaggggggtt 2340 gaggattcag caaatgaaga ttcagtggat gctaagccag accggtcctc gtttgtaccg 2400 tccctcttca gtaagaagaa gaaaaatgtc accatgcgat ccatcaagac cacccgggac 2460 cgagtgccta catatcagta caacatgaat tttgaaaagc tgggcaaatg catcataata 2520 aacaacaaga actttgataa agtgacaggt atgggcgttc gaaacggaac agacaaagat 2580 gccgaggcgc tcttcaagtg cttccgaagc ctgggttttg acgtgattgt ctataatgac 2640 tgctcttgtg ccaagatgca agatctgctt aaaaaagctt ctgaagagga ccatacaaat 2700 gccgcctgct tcgcctgcat cctcttaagc catggagaag aaaatgtaat ttatgggaaa 2760 gatggtgtca caccaataaa ggatttgaca gccacttta ggggggatag atgcaaaacc 2820 cttttagaga aacccaaact cttcttcatt caggcttgcc gagggaccga gcttgatgat 2880 ggcatccagg ccgactcggg gcccatcaat gacacagatg ctaatcctcg atacaagatc 2940 ccagtggaag ctgacttcct cttcgcctat tccacggttc caggctatta ctcgtggagg 3000 agcccaggaa gaggctcctg gtttgtgcaa gccctctgct ccatcctgga ggagcacgga 3060 aaagacctgg aaatcatgca gatcctcacc agggtgaatg acagagttgc caggcacttt 3120 gagtctcagt ctgatgaccc acacttccat gagaagaagc agatcccctg tgtggtctcc 3180 atgctcacca aggaactcta cttcagtcaa tag 3213 <210> 63 <211> 3423 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic polynucleotide” <400> 63 atggtcagct ggggtcgttt catctgcctg gtcgtggtca ccatggcaac cttgtccctg 60 gcccggccct ccttcagttt agttgaggat accacattag agccagaaga gccaccaacc 120 aaataccaaa tctctcaacc agaagtgtac gtggctgcgc caggggagtc gctagaggtg 180 cgctgcctgt tgaaagatgc cgccgtgatc agttggacta aggatggggt gcacttgggg 240 cccaacaata ggacagtgct tattggggag tacttgcaga taaagggcgc cacgcctaga 300 gactccggcc tctatgcttg tactgccagt aggactgtag acagtgaaac ttggtacttc 360 atggtgaatg tcacagatgc catctcatcc ggagatgat aggatgacac cgatggtgcg 420 gaagattttg tcagtgagaa cagtacaac agagagcac catactggac cacaca 480 aagatggaaa agcggctcca tgctgtgcct gcggccaaca ctgtcaagtt tcgctgccca 540 gccgggggga acccaatgcc aaccatgcgg tggctgaaaa acgggaagga gtttaagcag 600 gagcatcgca tggaggta caagtacga aaccagcact ggaggctcat tatggaagt 660 gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg agaatgaata cgggtccatc 720 aatcacacgt accacctgga tgttgtggag cgatcgccctc accggccat cctccaagcc 780 ggactgccgg caatgccctc cacagtggtc ggaggacg tagagttgt ctgcaaggtt 840 tacagtgatg cccagcccca catccagtgg atcagcacg tggaaaagaa cggcagtaaa 900 tacggccccg acgggctgcc ctacctcaag gttctcagg ccgccggtgt taacaccacg 960 gawaagaga tgaggttct ctatattcgg atgtactt ttgaggacgc tgggaat 1020 acgtgcttgg cgggtaattc tattgggata tcctttcact ctgcatggttzgagttctg 1080 ccagcgcctg gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt tactgcatag gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg aagaacacga ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa cgtatccccc tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc aacaccccgc tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg gcaggggtct ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag ctgacactgg gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca gtgggaattg acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa gatgatgcca cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg attgggaac acaagaat cataaatctt cttggagcct gcacacagga tgggcctctc tatgtcatag ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg 1740 ccacccggga tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc aaggacttgg tgtcatgcac ctaccagctg gccagaggca tggagtactt ggcttcccaa 1860 aaatgtattc atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg 1920 aaatagcag actttggact cgccagagat atcaacaata tagactatta caaaaagacc 1980 accaatgggc ggcttccagt caagtggatg gctccagaag ccctgtttga tagagtatac 2040 actcatcaga gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 2100 ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 2160 agaatggata agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg 2220 catgcagtgc cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt 2280 ctcactctca caaccaatga gcaagccagg gctgagcagg aagaagaatt cattagtaac 2340 actttattca agaaaattca ggctttgcag aaggagaaag aaacccttgc tgtaaattat 2400 gagaaagaag aagaattcct cactaatgag ctctccagaa aattgatgca gttgcagcat 2460 gagaaagccg aactagaaca gcatcttgaa caagagcagg aatttcaggt caacaaactg 2520 atgaagaaaa ttaaaaaact ggagaatgac accattcta agcaacttac attagacag 2580 ttgagacgggg agagattga ccttgaaaat acatggaac aagaacaaga accactagtt 2640 aatcgccctct ggaataagctt gaagctgaaa agcgaatccct gcaggaaaaa 2700 ttagaccagc ccgtctctgc tccaccatcg cctagagata tctccatgga gattgattct 2760 ccagaaaata tgatgcgtca catcaggttt ttaagaatg aagtggaacg gctgaagaag 2820 cacagtact ctgctcagtt acaccattca gagaaatgg cacagtatct ggaggaggaa 2880 cgtcacatga gagagagaa cttgaggctc cagaggaagc tgcagaggga gagagaga 2940 agagaagccc tctgtcgaca gctctccgag agtgagtcca gcttagaat ggacgacgaa 3000 aggtatttta atgagatgtc tgcacaagga ttaagaccctc gcactgtgtc cagcccgatc 3060 ccttacacac cttctccgag ttcaagcagg cctatatcac ctggtctatc atatgcaagt 3120 cacacggttg gttcacgcc accaacttca ctgactgag ctggaatgtc ttattacaat 3180 tcccggggtc ttcacgtgca gcacatggga acatcccatg gtatcacaag gccttcacca 3240 cggagaagca acagtcctga caaattcaaa cggcccacgc cgcctccatc tcccaacaca 3300 cagaccccag tccagccacc tccgcctcca cctccgccac ccatgcagcc cacggtcccc 3360 tcagcagcca cctcgcagcc tactccttcg caacattcgg cgcacccctc ctcccagcct 3420 taa 3423 <210> 64 <211> 5229 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 64 atggtcagct ggggtcgttt catctgcctg gtcgtggtca ccatggcaac cttgtccctg 60 gcccggccct ccttcagttt agttgaggat accacattag agccagaaga gccaccaacc 120 aaataccaaa tctctcaacc agaagtgtac gtggctgcgc caggggagtc gctagaggtg 180 cgctgcctgt tgaaagatgc cgccgtgatc agttggacta aggatggggt gcacttgggg 240 cccaacaata ggacagtgct tattggggag tacttgcaga taaagggcgc cacgcctaga 300 gactccggcc tctatgcttg tactgccagt aggactgtag acagtgaaac ttggtacttc 360 atggtgaatg tcacagatgc catctcatcc ggagatgat aggatgacac cgatggtgcg 420 gaagattttg tcagtgagaa cagtacaac agagagcac catactggac cacaca 480 aagatggaaa agcggctcca tgctgtgcct gcggccaaca ctgtcaagtt tcgctgccca 540 gccgggggga acccaatgcc aaccatgcgg tggctgaaaa acgggaagga gtttaagcag 600 gagcatcgca tggaggta caagtacga aaccagcact ggaggctcat tatggaagt 660 gtggtcccat ctgacaaggg aaattatacc tgtgtagtgg agaatgaata cgggtccatc 720 aatcacacgt accacctgga tgttgtggag cgatcgccctc accggccat cctccaagcc 780 ggactgccgg caatgccctc cacagtggtc ggaggacg tagagttgt ctgcaaggtt 840 tacagtgatg cccagcccca catccagtgg atcagcacg tggaaaagaa cggcagtaaa 900 tacggccccg acgggctgcc ctacctcaag gttctcagg ccgccggtgt taacaccacg 960 gawaagaga tgaggttct ctatattcgg atgtactt ttgaggacgc tgggaat 1020 acgtgcttgg cgggtaattc tattgggata tcctttcact ctgcatggttzgagttctg 1080 ccagcgcctg gaagagaaaa ggagattaca gcttccccag actacctgga gatagccatt tactgcatag gggtcttctt aatcgcctgt atggtggtaa cagtcatcct gtgccgaatg aagaacacga ccaagaagcc agacttcagc agccagccgg ctgtgcacaa gctgaccaaa cgtatccccc tgcggagaca ggtaacagtt tcggctgagt ccagctcctc catgaactcc aacaccccgc tggtgaggat aacaacacgc ctctcttcaa cggcagacac ccccatgctg gcaggggtct ccgagtatga acttccagag gacccaaaat gggagtttcc aagagataag ctgacactgg gcaagcccct gggagaaggt tgctttgggc aagtggtcat ggcggaagca gtgggaattg acaaagacaa gcccaaggag gcggtcaccg tggccgtgaa gatgttgaaa gatgatgcca cagagaaaga cctttctgat ctggtgtcag agatggagat gatgaagatg attgggaac acaagaat cataaatctt cttggagcct gcacacagga tgggcctctc tatgtcatag ttgagtatgc ctctaaaggc aacctccgag aatacctccg agcccggagg 1740 ccacccggga tggagtactc ctatgacatt aaccgtgttc ctgaggagca gatgaccttc aggacttgg tgtcatgcac ctaccagctg gccagaggca tggagtactt ggcttcccaa 1860 aaatgtattc atcgagattt agcagccaga aatgttttgg taacagaaaa caatgtgatg 1920 aaaatagcag actttggact cgccagagat atcaacaata tagactatta caaaaagacc 1980 accaatgggc ggcttccagt caagtggatg gctccagaag ccctgtttga tagtatac 2040 actcatcaga gtgatgtctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 2100 ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 2160 agaatggata agccagccaa ctgcaccaac gaactgtaca tgatgatgag ggactgttgg 2220 catgcagtgc cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt 2280 ctcactctca caaccaatga gacacaactt cgaaaccagc taattcatga gttgatgcac 2340 cctgtattga gtggagaact gcagcctcgg tccatttcag tagaagggag ctccctctta 2400 ataggcgcct ctaactcttt agtggcagat cacttacaaa gatgtggcta tgaatattca 2460 ctttctgttt tctttccaga aagtggtttg gcaaaagaaa aggtatttac tatgcaggat 2520 ctattacaac tcattaaaat caaccctact tccagtctct acaaatcact ggtttcagga 2580 tctgataaag aaaatcaaaa aggttttctt atgcattttt taaaagaatt ggcagaatat 2640 catcaagcta aagagagttg tatatggaa actcagacaa gttcgacatt taacagagat 2700 tctctggctg agaagcttca gcttattgat gatcagtttg cagatgctta ccctcagcgt 2760 atcaagttcg aatctttaga aataaagcta aatgagtata agagagaaat agaagagcaa 2820 cttcgggcag aaatgtgtca aaagttgaag ttttttaaag ataccgagat agcaaaaatt 2880 aaaatggaag caaaaaaaaa gtatgaaaag gagttaacca tgttccagaa tgattttgaa 2940 aaagcttgtc aagcaaaatc tgaagctctc gttcttcggg aaaagagtac ccttgaaaga 3000 attcacaagc accaagagat tgaaacaaaa gaaatttatg ctcaaaggca acttttacta 3060 aaagatatgg atttgctaag aggaagagaa gcagagctga agcaaagagt tgaagcttttt 3120 gaattgaacc agaagctcca ggaagaaaaa cataaaagca taactgaggc acttaggaga 3180 caggagcaga atataaagag ttttgaggag acctatgacc gaaagctcaa gaatgaactt 3240 ctaaagtac aacttgaact gaaggatgac tacatcatta gaactaatcg actgattgaa 3300 gatgaaagga agaataaga aaagctgtt cattgcaag aggagctcat agctattaat 3360 tcaaaaagg aggaactca tcaatctgta aatcgtgtga aagaacttga gcttgaatta 3420 gagtctgtca aagcccagtc ttggcaata aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagct gatgaaaag 3480 gttaaagaga tgagtgatta ttcacta aaagagaga aactggagct tctggcacaa 3540 aaaattac ttaacaaca actggaagg agtagaatg aaaacctgcg tctcctaaac 3600 cgcctagctc agccggctcc tgaacttgca gtctttcaga agaactacg gaagccgaa 3660 aaggctatag tggttgagca tgaggagttc gaagctgca ggcaagctct gcacaacaa 3720 ctgcaagacg aaattgagca ttctgcacag ctgaggccc agatctagg ttacaaagct 3780 tctgtaaga gtttaactac tcaggttgcc gatttaaaat tgcaactgaa gcaactcag 3840 acagccctag agaatgaagt gtactgcaat ccaagcagt ctgtgatcga tcgttctctc 3900 aatggattaa taatggcaa tgtggtgcct tgcaatggtg agatagtgg ggatttcttg 3960 aacaatcctt ttaaacagga aaacgttcta gcacgtatgg ttgcatcaag gatcacaaat 4020 tatccaactg catgggtgga gggtagttcc cctgattctg accttgagtt tgtagccaat 4080 actaaggcaa gggtcaaaga gcttcagcaa gaggccgaac gcttggaaaa ggctttcaga 4140 agttaccatc ggagagtcat taaaaaactct gccaaaagcc cactagcagc aaagagccca 4200 ccatctctgc acttgctgga agccttcaaa aacattactt ccagttcccc ggaaagacat 4260 atttttggag aggacagagt tgtctctgag cagcctcaag tgggcacact tgaagaaagg 4320 aatgacgtcg tggaagcact gacaggcagt gcagcctcga ggctccgcgg gggcacttcc 4380 tccagacgcc tctcttccac accccttcca aaagcaaaaa gaagcctcga aagtgaaatg 4440 tatctggaag gtctgggcag atcacacatt gcttccccca gtccttgtcc tgacagaatg 4500 cccctaccat caccactga gtctaggcac agcctctcca tccctcctgt ctccagccct 4560 ccggagcaga aagtgggtct ttatcgaaga caaactgaac ttcaagacaa aagtgaattt 4620 tcagatgtgg acaagctagc ttttaaggat aatgaggat ttgaatcatc ttttgaatct 4680 gcagggaaca tgccaaggca gttggaaatg ggcgggcttt ctcctgccgg ggatatgtct 4740 catgtggacg ctgctgcagc tgctgtgccc ctctcatatc agcacccaag tgtagatcag 4800 aaacaaattg aagaacaaaa ggaagaagaa aaaatacggg aacagcaagt gaaagaacga 4860 aggcagagag aagaaagaag gcagagtaac ctacaagaag ttttagaaag ggaacgaaga 4920 gaactagaaa aactgtatca ggaaaggaag atgattgaag aatcactgaa gattaaaata 4980 aaaaaggaat tagaaatgga aaatgaatta gaaatgagta atcaagaaat aaaagacaaa 5040 tctgctcaca gtgaaaatcc tttagagaaa tacatgaaaa tcatccagca ggagcaagac 5100 caggagtcgg cagataagag ctcaaaaaag atggtccaag aaggctccct agtggacacg 5160 ctgcaatcta gtgacaaagt cgaaagttta acaggctttt ctcatgaaga actagacgac 5220 tcttggtaa 5229 <210> 65 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <400> 65 tccaccgacg taaaggcg 18 <210> 66 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 66 taccgtgacg tccaccga 18 <210> 67 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 67 gccttctggc ccaggtgc 18 <210> 68 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 68 tccaccgaca atgttatg 18 <210> 69 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 69 accaatgagg agagtaga 18 <210> 70 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 70 accaatgaga tcatggag 18 <210> 71 <211> 16 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 71 accaatgaga tggcag 16 <210> 72 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 72 accaatgagc aagccagg 18 <210> 73 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <400> 73 accaatgaga cacaactt 18

Claims

1. Use of primer pairs for amplifying one or more FGFR mutant genes, including an FGFR3 fusion, in the preparation of a kit for identifying urothelial carcinoma patients responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor, wherein the FGFR inhibitor is a compound of formula (I). (I) The FGFR3 fusion comprises the FGFR3 exon 18-TACC3 exon 11 fusion of SEQ ID NO: 56, the FGFR3 exon 18-TACC3 exon 10 fusion of SEQ ID NO: 57, or the FGFR2 exon 19-BICC1 exon 3 fusion of SEQ ID NO: 60, or a combination thereof, and the identification comprises assessing the presence of the one or more FGFR mutant genes from the FGFR mutant genome in a biological sample from the patient; wherein the determination of the presence of the FGFR3 exon 18-TACC3 exon 11 fusion, the FGFR3 exon 18-TACC3 exon 10 fusion, or the FGFR2 exon 19-BICC1 exon 3 fusion, or a combination thereof, indicates that the patient has responded to treatment with the FGFR inhibitor.

2. The use according to claim 1, wherein the evaluation comprises amplifying cDNA with primer pairs and determining the presence of one or more FGFR mutant genes in the sample.

3. The use according to claim 1 or 2, wherein the urothelial carcinoma is metastatic.

4. The use according to claim 2, wherein the cDNA is pre-amplified cDNA.

5. The use according to any one of claims 1-4, wherein the one or more FGFR mutant genes and primer pairs are: FGFR3 exon 18-TACC3 exon 11 fusion complex and primers having sequences of SEQ ID NO:5 and SEQ ID NO:6; FGFR3 exon 18-TACC3 exon 10 fusion and primers having sequences of SEQ ID NO:7 and SEQ ID NO:8; or FGFR2 exon 19-BICC1 exon 3 fusion complex and primers having sequences of SEQ ID NO:13 and SEQ ID NO:14; Or any combination thereof.

6. The use according to any one of claims 1-4, wherein the evaluation comprises: RNA is isolated from the biological sample, and cDNA is synthesized from the isolated RNA and amplified.

7. The use according to claim 6 further includes pre-amplifying the cDNA prior to the amplification step.

8. The use according to any one of claims 6-7, wherein the amplification step comprises performing real-time PCR.

9. The use according to claim 8, wherein the real-time PCR is performed using one or more probes comprising SEQ ID NO: 43, SEQ ID NO: 44 and / or SEQ ID NO:

45.

10. The use according to any one of claims 6-9, wherein determining the presence of one or more FGFR mutant genes comprises sequencing the amplified cDNA.

11. The use according to any one of claims 1-10, further comprising treating the urothelial carcinoma patient with the compound of formula (I) when the presence of one or more FGFR mutant genes is identified.

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