BR122024005360A2 - METHODS FOR IDENTIFYING A PATIENT WITH BLADDER CANCER WHO IS RESPONSIVE TO TREATMENT WITH AN FGFR INHIBITOR AND KIT - Google Patents

Abstract

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

Description

REFERENCE TO RELATED ORDERS

[001] This application claims priority to U.S. provisional application No. 62/056,159, filed on September 26, 2014, the description of which is incorporated herein in its entirety by reference.

SEQUENCE LISTING

[002] This application contains a listing of sequences that was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The referenced ASCII copy, created on Thursday, August 6, 2015, is named 103693.000782_SL.txt and has a size of 66,185 bytes.

FIELD OF TECHNIQUE

[003] Presented here are methods for identifying a patient with cancer that is responsive to treatment with a fibroblast growth factor receptor inhibitor and methods of treating the same.

BACKGROUND

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

SUMMARY

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

[006] Methods for treating cancer in a patient are also presented, comprising: evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from the panel of mutant FGFR genes; and treating the patient with an FGFR inhibitor if one or more FGFR mutants are present in the sample.

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

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0012] Figure 4 illustrates an exemplary analytical validation strategy for detecting FGFR SNPs. Experiments were performed in engineered RK3E cell lines expressing FGFR fusions and diluted in a wild-type cell line containing no FGFR3/FGFR2 fusions.

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

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

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

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

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

[0018] Figure 10 represents an exemplary phase I study design for a pioneering human study of JNJ-42756493 in patients with advanced solid tumors.

[0019] Figure 11 represents the percentage of maximum inhibitory reduction of the sum of target lesion diameters from baseline with a dose level greater than or equal to 6 mg. Patients with solid tumors were treated with FGFR inhibitor JNJ-42756493 at different doses administered as a daily regimen or as an intermittent dosing regimen (7 days on-7 days off). Doses and tumor types are indicated. Tumor shrinkage was measured according to RECIST criteria. Patients whose tumors carry FGFR gene translocations and mutations appear to be more sensitive to the FGFR inhibitor JNJ-42756493.

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

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

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

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

[0023] The present methods, kits and primers can be more readily understood by reference to the following detailed description considered in conjunction with the attached figures, which form part of this description. It should be understood that the methods, kits and primers disclosed are not limited to the methods, kits and primers described and/or shown herein, and that the terminology used in the present invention is intended to describe particular embodiments by way of example only and not is intended to limit the claimed invention.

[0024] Reference to a specific numerical value includes at least that specific value, unless the context clearly indicates otherwise. When a range of values is expressed, another modality includes from a specific value and/or to another specific value. Additionally, reference to the values indicated in the ranges includes each and every value within that range. All tracks are inclusive and combinable.

[0025] It should be considered that certain features of the presented methods, kits and primers that are, for greater clarity, described herein in the context of separate modalities, may also be provided in combination in a single modality. Conversely, various features of the presented methods, kits and primers that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0026] For use in the present invention, the singular forms "a", "an", "the" and "a" include the plural forms.

[0027] The following abbreviations are used throughout the specification. FGFR (fibroblast growth factor receptor); LLOQ (lower limit of quantification); FGFR3:TACC3 (fusion between the genes encoding FGFR3 and the acidic coil-containing transformation protein 3); FGFR3: BAIAP2L1 (fusion between genes encoding FGFR3 and brain-specific angiogenesis inhibitor 1-associated protein 2-like protein); FGFR2: AFF3 (fusion between the genes encoding FGFR2 and the AF4/FMR2 family, element 3); FGFR2: BICC1 (fusion between the genes encoding FGFR2 and two-tailed C 1 homologue); FGFR2: CASP7 (fusion between the genes encoding FGFR2 and caspase 7); FGFR2: CCDC6 (fusion between the genes encoding FGFR2 and the coiled-coil domain-containing protein 6); FGFR2: OFD1 (fusion between the genes encoding FGFR2 and oral-facial-digital syndrome protein 1); FFPET (formalin-fixed paraffin-embedded tissue); SNP (single nucleotide polymorphism); NSCLC (non-small cell lung cancer), ct (cycle threshold).

[0028] For use in the present invention, "treatment" and similar terms refer to reducing the severity and/or frequency of cancer symptoms, eliminating cancer symptoms and/or the underlying cause of said symptoms, reducing the frequency or the likelihood of cancer symptoms and/or their underlying cause and improvement or repair of damage caused directly or indirectly by cancer.

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

[0030] For use in the present invention, "pre-amplification" refers to a PCR procedure that is performed before the amplification step in order to increase the amount of template cDNA for the amplification step. A pre-amplification step can be performed, for example, using TaqMan® PreAmp master mix (Life Technologies/Applied Biosystems® product no. 4391128).

[0031] For use in the present invention, "amplification" and "amplify," and similar terms, refer to the generation of several identical copies of a nucleic acid sample. Suitable techniques for amplification of a nucleic acid sample include, but are not limited to, polymerase chain reaction (PCR) and real-time polymerase chain reaction (RT-PCR). In some embodiments, the amplification step comprises RT-PCR.

FGFR MUTANTS

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

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

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

[0035] The sequences correspond to nucleotides 920 to 1510 of FGFR3 (Genebank ID no. NM_000142.4).

[0036] The underlined and bold nucleotides represent the SNP. • Sometimes mistakenly called Y375C in literature.

[0037] As used herein, "mutant FGFR gene panel" includes one or more of the above-mentioned FGFR mutants. In some embodiments, the panel of mutant FGFR genes depends on the patient's cancer type.

[0038] The panel of FGFR mutants that is used in the evaluation stage of the presented methods is based, in part, on the type of cancer in the patient. For patients with bladder cancer, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0039] For patients with metastatic bladder cancer, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C , FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[0040] For patients with ovarian cancer, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[0041] For patients with head and neck cancer, a panel of mutant FGFR genes may comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[0042] For patients with metastatic head and neck cancer, a panel of mutant FGFR genes may comprise FGFR3:BAIAP2L1, FGFR2:CASP7 or FGFR2:OFD1, or any combination thereof.

[0043] For patients with esophageal cancer, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[0044] For patients with metastatic esophageal cancer, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6 or FGFR2:OFD1, or any combination thereof.

[0045] For patients with non-small cell lung adenocarcinoma, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[0046] For patients with non-small cell lung squamous cell carcinoma, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7 , FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[0047] For patients with metastatic endometrial cancer, a suitable mutant FGFR gene panel may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6 or FGFR2:OFD1, or any combination thereof.

[0048] For breast cancer patients, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2 :CCD6 or FGFR2:OFD1, or any combination thereof.

INITIATORS TO AMPLIFY FGFR MUTANTS

[0049] Those skilled in the art know that amplification of nucleic acids requires primers that are complementary, and bind to a 5' and a 3' region of the nucleic acid strand that flanks the region to be amplified. As used herein, "primer pair" refers to the sense and reverse primers used in an amplification step. Suitable primer pairs to perform the presented methods are listed in Table 3. TABLE 3

[0050] Primers are presented here that have the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 , SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO : 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 , SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or any combination thereof.

[0051] Also presented here are sets of primers with the sequences of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, or any combination thereof.

[0052] In some embodiments, the primer set may have the sequence of SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the primer set may have the sequence of SEQ ID NO: 7 and SEQ ID NO : 8. In some embodiments, the primer set may have the sequence of SEQ ID NO: 9 and SEQ ID NO: 10. In some embodiments, the primer set may have the sequence of SEQ ID NO: 11 and SEQ ID NO: : 12. In some embodiments, the primer set may have the sequence of SEQ ID NO: 13 and SEQ ID NO: 14. In some embodiments, the primer set may have the sequence of SEQ ID NO: 15 and SEQ ID NO : 16. In some embodiments, the primer set may have the sequence of SEQ ID NO: 17 and SEQ ID NO: 18. In some embodiments, the primer set may have the sequence of SEQ ID NO: 19 and SEQ ID NO: : 20. In some embodiments, the primer set may have the sequence of SEQ ID NO: 21 and SEQ ID NO: 22. In some embodiments, the primer set may have the sequence of SEQ ID NO: 23 and SEQ ID NO: : 24. In some embodiments, the primer set may have the sequence of SEQ ID NO: 25 and SEQ ID NO: 26. In some embodiments, the primer set may have the sequence of SEQ ID NO: 27 and SEQ ID NO : 28. In some embodiments, the primer set may have the sequence of SEQ ID NO: 29 and SEQ ID NO: 30. In some embodiments, the primer set may have the sequence of SEQ ID NO: 31 and SEQ ID NO: : 32. In some embodiments, the primer set may have the sequence of SEQ ID NO: 33 and SEQ ID NO: 34. In some embodiments, the primer set may have the sequence of SEQ ID NO: 35 and SEQ ID NO : 36. In some embodiments, the primer set may have the sequence of SEQ ID NO: 37 and SEQ ID NO: 38. In some embodiments, the primer set may have the sequences of any combination of the above primer sets.

FGFR INHIBITORS FOR USE IN THE PRESENTED METHODS

[0053] FGFR inhibitors suitable for use in the methods presented are provided herein.

[0054] In some embodiments, if one or more FGFR mutants are present in the sample, the patient can be treated with an FGFR inhibitor described in pub. US No. 2013/0072457 A1 (incorporated herein by reference), including any tautomeric or stereochemically isomeric form thereof, and an N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof (suitable R groups are also presented in US pub. no. 2013/0072457 A1). In some aspects, for example, the patient may be treated with N-(3,5-dimethoxy-phenyl)-N'-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4- il)quinoxalin-6-yl]ethane-1,2-diamine (referred to herein as "JNJ-42756493" or "JNJ493"): including an N-oxide thereof, a pharmaceutically acceptable salt thereof or a solvate thereof. In some aspects, the pharmaceutically acceptable salt is the HCl salt. In some aspects, the patient can be treated with a JNJ493 base.

[0055] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, where 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 in Gavine, PR, et al., AZD4547: An Orally Bioavailable, Potent, and Selective Inhibitor of the Fibroblast Growth Factor Receptor Tyrosine Kinase Family, Cancer Res. April 15, 2012 72; 2045: including, where chemically possible, any tautomeric or stereochemically isomeric form thereof, an N-oxide thereof, a pharmaceutically acceptable salt thereof or a solvate thereof.

[0056] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, where the FGFR inhibitor is 3-(2,6-dichloro-3,5-dimethoxy -phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimid-4-yl}-1-methyl-urea (NVP-BGJ398), as described in Publ. Inter. No. WO2006/000420: including, where chemically possible, any tautomeric or stereochemically isomeric form thereof, an N-oxide thereof, a pharmaceutically acceptable salt thereof or a solvate thereof.

[0057] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, where the FGFR inhibitor is 4-amino-5-fluoro-3-[6-( 4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-quinolin-2-one (dovitinib), as described in international publication No. WO2006/127926: including, where chemically possible, any tautomeric or stereochemically isomeric form thereof, an N-oxide thereof, a pharmaceutically acceptable salt thereof or a solvate thereof.

[0058] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, where the FGFR inhibitor is 6-(7-((1-aminocyclopropyl)-methoxy) -6-methoxyquinolin-4-yloxy)-N-methyl-1-naphthamide (AL3810) (lucitanib; E-3810), as described in Bello, E. et al., E-3810 Is a Potent Dual Inhibitor of VEGFR and FGFR that Exerts Antitumor Activity in Multiple Preclinical Models, Cancer Res February 15, 2011 71(A)1396-1405 and in international publication no. WO2008/112408: including, where chemically possible, any tautomeric or stereochemically isomeric form thereof, an N-oxide thereof, a pharmaceutically acceptable salt thereof or a solvate thereof.

[0059] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being an anti-FGFR2 antibody as described in WO2013/076186.

[0060] Additional suitable FGFR inhibitors include BAY1163877 (Bayer), BAY1179470 (Bayer), TAS-120 (Taiho), ARQ087 (ArQule), ASP5878 (Astellas), FF284 (Chugai), FP-1039 (GSK/FivePrime) , Blueprint, LY-2874455 (Lilly), RG-7444 (Roche) or any combination thereof, including, where chemically possible, any tautomeric or stereochemically isomeric forms thereof, N-oxides thereof, pharmaceutically acceptable salts thereof or solvates of the same.

[0061] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being BAY1163877 (Bayer), including, when chemically possible, any tautomeric form or stereochemically isomeric thereof, N-oxide thereof, a pharmaceutically acceptable salt thereof or solvate thereof.

[0062] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being BAY1179470 (Bayer), including, when chemically possible, any tautomeric form or stereochemically isomeric thereof, N-oxide thereof, a pharmaceutically acceptable salt thereof or solvate thereof.

[0063] In some embodiments, the patient may be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being TAS-120 (Taiho), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, a pharmaceutically acceptable salt thereof or solvate thereof.

[0064] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being ARQ087 (ArQule), including, when chemically possible, any tautomeric form or stereochemically isomeric thereof, N-oxide thereof, a pharmaceutically acceptable salt thereof or solvate thereof.

[0065] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being ASP5878 (Astellas), including, when chemically possible, any tautomeric form or stereochemically isomeric thereof, N-oxide thereof, a pharmaceutically acceptable salt thereof or solvate thereof.

[0066] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being FF284 (Chugai), including, when chemically possible, any tautomeric form or stereochemically isomeric thereof, N-oxide thereof, a pharmaceutically acceptable salt thereof or solvate thereof.

[0067] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being FP-1039 (GSK/FivePrime), including, when chemically possible , any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, a pharmaceutically acceptable salt thereof or solvate thereof.

[0068] In some embodiments, the patient may be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being Blueprint, including, when chemically possible, any tautomeric or stereochemically isomeric form. thereof, N-oxide thereof, a pharmaceutically acceptable salt thereof or solvate thereof.

[0069] In some embodiments, the patient may be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being LY-2874455 (Lilly), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, a pharmaceutically acceptable salt thereof or solvate thereof.

[0070] In some embodiments, the patient can be treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, the FGFR inhibitor being RG-7444 (Roche), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, a pharmaceutically acceptable salt thereof or solvate thereof.

[0071] Salts can be synthesized from an original compound that contains an acidic or basic moiety by conventional chemical methods such as the methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, hardcover, 388 pages, August 2002, which is incorporated herein by reference. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or an organic solvent, or a mixture of the two; Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are used. FGFR inhibitors for use in the methods presented may exist as mono- or disalts, depending on the pKa of the acid from which the salt is formed.

[0072] Acid addition salts can be formed with a wide variety of inorganic and organic acids. Examples of acid addition salts include salts formed with an acid, including, but not limited to, acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g., L-ascorbic), L-aspartic, benzenesulfonic acid. , benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphorsulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2- disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic , hydrochloric, hydriodic, isethionic, lactic acid (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL - mandelic, methanesulfonic, naphthalenesulfonic (e.g. naphthalene-2-sulfonic), naphthalene-1,5-disulfonic, 1-hydroxy 2-naphtoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, pyruvic, salicylic, 4-aminosalicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, toluenesulfonic (e.g. p-toluenesulfonic), undecylenic and valeric, as well as acylated amino acids and cation exchange resins.

[0073] A particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, methanesulfonic (mesylate), ethanesulfonic acid , naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic. Another group of acid addition salts includes salts formed from acetic, adipic, ascorbic, aspartic, citric, DL-lactic, fumaric, gluconic, glucuronic, hippuric, hydrochloric, glutamic, DL-malic, methanesulfonic, sebacic, stearic, succinic acids. and tartaric.

[0074] If the compound is anionic, or has a functional group that can be anionic (for example, -COOH can be -COO-), then a salt can be formed with 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 Ca2+ and Mg2+, and other cations such as Al3+. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4+) and substituted ammonium ions (e.g., NH3R+, NH2R2+, NHR3+, NR4+).

[0075] Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.

[0076] When compounds contain an amine function, they can form quaternary ammonium salts, for example, by reaction with an alkylating agent according to methods well known to those skilled in the art. These quaternary ammonium compounds are within the scope of the compounds presented. Compounds containing an amine function can also form N-oxides. A reference herein to a compound containing an amine function also includes the N-oxide. When a compound contains multiple amine functions, one or more than one nitrogen atom can be oxidized to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle. N-oxides can be formed by treating the corresponding amine with an oxidizing agent such as hydrogen peroxide or a peroxyacid (e.g., a peroxycarboxylic acid), see, for example, Advanced Organic Chemistry, by Jerry March, 4th edition, Wiley Interscience , pages More particularly, N-oxides can be prepared by the procedure of L. W. Deady (Syn. Comm. (1977), 7, 509-514) with the amine compound reacting with m-chloroperoxybenzoic acid (MCPBA), e.g. , in an inert solvent, such as dichloromethane.

[0077] As used here, the term "solvate" means a physical association of the compound with one or more solvent molecules. This physical association involves different degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. The term "solvate" is intended to encompass isolable and solution-phase solvates. Some non-limiting examples of suitable solvates include the compounds shown in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, ethanolamine and the like. The compound can exert its biological effects while in solution.

[0078] Solvates are well known in pharmaceutical chemistry. They can be important to the processes for the preparation of a substance (e.g. in relation to its purification), for the storage of the substance (e.g. its stability) and for the ease of handling of the substance, and are often formed as part of the isolation or purification stage of a chemical synthesis. A person skilled in the art can determine, using standard and long-used techniques, whether a hydrate or other solvate was formed by the isolation conditions or the purification conditions used to prepare a given compound. Examples of such techniques include thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray crystallography (e.g. single crystal X-ray crystallography or powder X-ray diffraction) and state NMR. solid (SS-NMR, also known as "Magic Angle Spinning NMR" or "MAS-NMR"). Such techniques are a part of the standard analytical toolkit of the knowledgeable chemist such as NMR, IR, HPLC and MS. Alternatively, one skilled in the art can deliberately form a solvate using crystallization conditions that include an amount of a solvent necessary for the specific solvate. Then, the standard methods described can be used to establish whether solvates have formed. Also covered are any complexes (e.g., inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals) of the FGFR inhibitor.

[0079] Furthermore, the compound may have one or more polymorphic (crystalline) or amorphous forms.

[0080] Compounds include compounds with one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element. For example, a reference to hydrogen includes in its scope 1H, 2H (D), and 3H (T). Similarly, references to carbon and oxygen include in their scope, respectively, 12C, 13C and 14C, and 16O and 18O. Isotopes can be radioactive or non-radioactive. In one embodiment, the compounds do not contain radioactive isotopes. Such compounds are preferred for therapeutic use. In another embodiment, however, the compound may contain one or more radioisotopes. Compounds containing such radioisotopes may be useful in a diagnostic context.

[0081] In some embodiments, the patient is treated with an FGFR inhibitor if one or more FGFR mutants are present in the sample, where the FGFR inhibitor is N-(3,5-dimethoxy-phenyl)-N'- (1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl]ethane-1,2-diamine (called herein "JNJ-42756493"), or a salt pharmaceutically acceptable thereof, or a solvate thereof.

CANCER TREATMENT METHODS IN A PATIENT

[0082] Presented here are methods for treating cancer in a patient comprising: evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from the panel of mutant FGFR genes; and treating the patient with an FGFR inhibitor if one or more FGFR mutants are present in the sample.

[0083] The presented methods can be used to treat a variety of cancer types including, but not limited to, bladder cancer, metastatic bladder cancer, ovarian cancer, head and neck cancer, metastatic head and neck cancer, esophageal cancer, metastatic esophageal cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, prostate cancer, lung cancer, gastric cancer, urothelial carcinoma, small cell lung cancer, cancer breast cancer, endometrial cancer, metastatic endometrial cancer, cholangiocarcinoma, hepatocellular carcinoma, glioblastoma, gliomas, colon carcinoma, sarcomas, solid tumors of squamous origin and multiple myeloma.

[0084] The panel of FGFR mutants that is used in the evaluation stage is based, in part, on the patient's type of cancer. For patients with bladder cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient who has bladder cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0085] For patients with metastatic bladder cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C , FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient who has metastatic bladder cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0086] For patients with ovarian cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient who has ovarian cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0087] For patients with head and neck cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient who has head and neck cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has head and neck cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has head and neck cancer is treated with an FGFR FGFR3 inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient who has head and neck cancer is treated with an FGFR inhibitor if FGFR3:S249C is present in the sample. In some embodiments, a patient who has head and neck cancer is treated with an FGFR inhibitor if FGFR3:G370C is present in the sample. In some embodiments, a patient who has head and neck cancer is treated with an FGFR inhibitor if FGFR3:Y373C is present in the sample. In some embodiments, a patient who has head and neck cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0088] For patients with metastatic head and neck cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:BAIAP2L1, FGFR2:CASP7 or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient who has metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has metastatic head and neck cancer is treated with an FGFR inhibitor if FRFR2:CASP7 is present in the sample. In some embodiments, a patient who has metastatic head and neck cancer is treated with an FGFR inhibitor if FRFR2:OFD1 is present in the sample. In some embodiments, a patient who has metastatic head and neck cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0089] For patients with esophageal cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C , or any combination thereof. Accordingly, in some embodiments, a patient who has esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has esophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient who has esophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has esophageal cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient who has esophageal cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient who has esophageal cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient who has esophageal cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient who has esophageal cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0090] For patients with metastatic esophageal cancer, for example, a panel of mutant FGFR genes, may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6 or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 intron is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0091] For patients with non-small cell lung adenocarcinoma (NSCL), for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2: AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 intron is present in the sample. In some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient who has NSCL adenocarcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0092] For patients with non-small cell lung squamous cell carcinoma (NSCL), a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient who has NSCL squamous cell carcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0093] For patients with metastatic endometrial cancer, for example, a suitable mutant FGFR gene panel may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6 or FGFR2 :OFD1, or any combination thereof. Accordingly, in some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 intron is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0094] For breast cancer patients, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2: CASP7, FGFR2:CCD6 or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient who has breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 intron is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient who has breast cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0095] For patients with hepatocellular carcinoma, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7 , FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 intron is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[0096] In some embodiments, the evaluation step comprises: isolating the RNA from the biological sample; synthesize cDNA from isolated RNA; pre-amplify the cDNA; and amplifying the pre-amplified cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the panel of mutant FGFR genes.

[0097] The isolation of RNA from the biological sample can be carried out by various procedures known to those skilled in the art. In one embodiment, RNA can be isolated from the biological sample using a Qiagen AllPrep DNA/RNA FFPE kit (product no. 80234).

[0098] The synthesis of cDNA from isolated RNA can be carried out by a variety of 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 with RNase inhibitor from ABI (product no. 4374966).

[0099] Pre-amplification of cDNA can be performed by a variety of procedures known to those skilled in the art. Amplification procedures are well known in the art. In one embodiment, the cDNA can be pre-amplified using a TaqMan® PreAmp master mix (Life Technologies/Applied Biosystems® product no. 4391128).

[00100] In some embodiments, the amplification step may comprise performing real-time PCR (qRT-PCR). Exemplary qRT-PCR procedures are discussed in the examples section of the present invention. In some aspects, qRT-PCR may be a Taqman® real-time PCR assay. qRT-PCR procedures may involve the use of probes to increase assay specificity. Suitable probes for use in the qRT-PCR assay include any of the probes set forth herein, e.g., the probes disclosed in Table 15. In some embodiments, for example, real-time PCR may be performed on one or more probes that comprise 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 with one or more probes consisting of essentially in 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 with one or more probes that consist of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and/or SEQ ID NO: 55. In other embodiments, real-time PCR can be performed with one or more probes that has 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.

[00101] qRT-PCR can be performed with one or more 3' blocking oligonucleotides. Exemplary qRT-PCR procedures using 3' blocking oligonucleotides are presented in the examples section of the present invention. Suitable 3' blocking oligonucleotides include, for example, those set forth in Table 8. In some embodiments, qRT-PCR can be performed with one or more 3' blocking oligonucleotides comprising SEQ ID NO: 39, SEQ ID NO: 40 , SEQ ID NO: 41 and/or SEQ ID NO: 42. In some embodiments, qRT-PCR can be performed with one or more 3' blocking oligonucleotides consisting essentially of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and/or SEQ ID NO: 42. In some embodiments, qRT-PCR can be performed with one or more 3' blocking oligonucleotides consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and/or SEQ ID NO: 42. In some embodiments, qRT-PCR can be performed with one or more 3' blocking oligonucleotides that have SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and/or SEQ ID NO: 42.

[00102] Suitable pairs of primers for use in the amplification step include those presented in Table 3. For example, in some embodiments, the FGFR mutant and the primer pair may be FGFR3:TACC3 v1 and the primers having the sequences 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 the 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 intron and the primers having the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10. In some embodiments , the FGFR mutant and the primer pair may be FGFR3: BAIAP2L1 and the primers having the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 12. In some embodiments, the FGFR mutant and the primer pair may be FGFR2:BICC1 and the primers having the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14. In some embodiments, the FGFR mutant and the primer pair may be FGFR2:AFF3 and the 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 the 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 the primers having the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20. In some embodiments , the FGFR mutant and the primer pair may be FGFR2:OFD1 and the primers having the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 22. In some embodiments, the FGFR mutant and the 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 the primer pair may be S249C and the 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 the primer pair may be G370C and the 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 the primer pair may be Y373C and the 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 the primer pair it can be any combination of the above FGFR mutants and the corresponding pair of primers.

[00103] In some embodiments, the amplification step can be performed as follows: a. the primer pair has the sequences SEQ ID NO: 5 and SEQ ID NO: 6 and the probe has the sequence of SEQ ID NO: 43; B. the primer pair has the sequences SEQ ID NO:7 and SEQ ID NO:8 and the probe has the sequence SEQ ID NO: 44; w. the primer pair has the sequences SEQ ID NO: 9 and SEQ ID NO: 10 and the probe has the sequence of SEQ ID NO: 46; d. the primer pair has the sequences SEQ ID NO: 11 and SEQ ID NO: 12 and the probe has the sequence of SEQ ID NO: 47; It is. the primer pair has the sequences SEQ ID NO: 13 and SEQ ID NO: 14 and the probe has the sequence of SEQ ID NO: 45; f. the primer pair has the sequences SEQ ID NO: 15 and SEQ ID NO: 16 and the probe has the sequence of SEQ ID NO: 48; g. the primer pair has the sequences SEQ ID NO: 17 and SEQ ID NO: 18 and the probe has the sequence of SEQ ID NO: 49; H. the primer pair has the sequences SEQ ID NO: 19 and SEQ ID NO: 20 and the probe has the sequence of SEQ ID NO: 50; i. the primer pair has the sequences SEQ ID NO: 21 and SEQ ID NO: 22 and the probe has the sequence of SEQ ID NO: 51; j. the primer pair has the sequences SEQ ID NO: 23 and SEQ ID NO: 24 and the probe has the sequence of SEQ ID NO: 52; k. the primer pair has the sequences SEQ ID NO: 25 and SEQ ID NO: 26 and the probe has the sequence of SEQ ID NO: 53; l. the primer pair has the sequences SEQ ID NO: 27 and SEQ ID NO: 28 and the probe has the sequence of SEQ ID NO: 54; m. the primer pair has the sequences SEQ ID NO: 29 and SEQ ID NO: 30 and the probe has the sequence of SEQ ID NO: 55; n. the primer pair has the sequences SEQ ID NO: 31 and SEQ ID NO: 32 and the probe has the sequence of SEQ ID NO: 52; and the 3' blocking oligonucleotide has the sequence of SEQ ID NO: 39; O. the primer pair has the sequences SEQ ID NO: 33 and SEQ ID NO: 34 and the probe has the sequence of SEQ ID NO: 53; and the 3' blocking oligonucleotide has the sequence of SEQ ID NO: 40; P. the primer pair has the sequences SEQ ID NO: 35 and SEQ ID NO: 36 and the probe has the sequence of SEQ ID NO: 54; and the 3' blocking oligonucleotide has the sequence of SEQ ID NO: 41; q. the primer pair has the sequences SEQ ID NO: 37 and SEQ ID NO: 38 and the probe has the sequence of SEQ ID NO: 55; and the 3' blocking oligonucleotide has the sequence of SEQ ID NO: 42; or r. and combinations thereof.

[00104] The methods presented comprise treating a patient if one or more FGFR mutants are present in the sample. The presence of one or more FGFR mutants in the sample can be determined, for example, by sequencing the amplified cDNA.

[00105] FGFR inhibitors suitable for use in treatment methods include those previously described in the present invention.

[00106] FGFR inhibitors are also disclosed for use in the treatment of cancer in a patient, with the patient being identified as responsive to treatment with the FGFR inhibitor by evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from a panel of mutant FGFR genes, and the presence of one or more FGFR mutants in the sample is detected.

[00107] FGFR inhibitors are also presented for use in the treatment of cancer in a patient wherein the patient is identified as responsive to treatment with the FGFR inhibitor by evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from the panel of mutant FGFR genes, wherein the one or more FGFR mutants are an FGFR fusion gene or FGFR SNP, wherein the presence of one or more FGFR mutants in the sample is detected, and said evaluation comprising amplifying the cDNA with a pair of primers that bind and amplify one or more FGFR mutants from the panel of mutant FGFR genes.

[00108] FGFR inhibitors are also presented for use in the treatment of cancer in a patient, with the patient being identified as responsive to treatment with the FGFR inhibitor by evaluating a biological sample obtained from the patient for the presence of a or more FGFR mutants from the panel of mutant FGFR genes, 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 said evaluation comprising amplifying a pre-amplified cDNA with a pair of primers that bind to one or more FGFR mutants and amplifying one or more FGFR mutants from the panel of mutant FGFR genes.

METHODS OF IDENTIFYING A CANCER PATIENT WHO WILL BE RESPONSIVE TO TREATMENT WITH A FIBROBLAST GROWTH FACTOR RECEPTOR (FGFR) INHIBITOR

[00109] Presented in the present invention are methods for identifying a patient with cancer that is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor, comprising: evaluating a biological sample obtained from the patient for an FGFR mutant a from a panel of mutant FGFR genes, wherein the FGFR mutant is an FGFR fusion gene or a single nucleotide polymorphism of FGFR, and wherein said evaluation comprises amplifying cDNA which with a pair of primers which bind to and amplify one or more FGFR mutants from the panel of mutant FGFR genes and determine whether the one or more FGFR mutants from the gene panel are present in the sample, the presence of the one or more FGFR mutants indicating that the patient will be responsive to treatment with the FGFR inhibitor.

[00110] Methods for identifying a patient with cancer that is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor are also provided, comprising: evaluating a biological sample obtained from the patient for an FGFR mutant from a panel of mutant FGFR genes, wherein the FGFR mutant is an FGFR fusion gene or an FGFR single nucleotide polymorphism, and said assessment comprising amplifying cDNA that with a pair of primers that bind and amplify one or more FGFR mutants from the panel of FGFR mutant genes and determine whether the one or more FGFR mutants from the panel of genes are present in the sample, wherein the presence of the one or more FGFR mutants indicates that the patient is responsive to FGFR inhibitor treatment.

[00111] Methods are also presented for identifying a patient with cancer that is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor, which comprises evaluating a biological sample from the patient for the presence of one or more FGFR mutants from a panel of mutant FGFR genes, wherein the FGFR mutant is an FGFR fusion gene or an FGFR single nucleotide polymorphism, with the presence of one or more FGFR mutants indicating that the patient is responsive to FGFR inhibitor treatment.

[00112] In some embodiments, the evaluation may comprise amplifying cDNA with a pair of primers that bind and amplify one or more FGFR mutants from the panel of mutant FGFR genes. In some embodiments, the cDNA may be pre-amplified cDNA.

[00113] In some embodiments, the evaluation step comprises: isolating the RNA from the biological sample and synthesizing cDNA from the isolated RNA. In some aspects, the evaluation step can be performed on pre-amplified cDNA. In this way, the evaluation step can also include the pre-amplification of the cDNA before said amplification step. Isolation of RNA from a biological sample can be accomplished by a variety of procedures known to those skilled in the art. In one embodiment, RNA can be isolated from the biological sample using a Qiagen AllPrep DNA/RNA FFPE kit (e.g., product no. 80234). The synthesis of cDNA from isolated RNA can be carried out by a variety of 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 with RNase inhibitor from ABI (e.g., product no. 4374966). Pre-amplification of cDNA can be performed by a variety of procedures known to those skilled in the art. Amplification procedures are well known in the art. In one embodiment, the cDNA can be pre-amplified using a TaqMan® PreAmp master mix (Life Technologies/Applied Biosystems® product no. 4391128).

[00114] The methods presented can be used to identify patients with several different types of cancer that will be responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor, including, but not limited to, bladder cancer, metastatic bladder cancer, ovarian cancer, head and neck cancer, esophageal cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, prostate cancer, lung cancer, gastric cancer, urothelial carcinoma , small cell lung cancer, breast cancer, endometrial cancer, cholangiocarcinoma, hepatocellular carcinoma, glioblastoma, gliomas, colon carcinoma, sarcomas, solid tumors of squamous origin, and multiple myeloma.

[00115] The panel of FGFR mutants that is used in the evaluation stage is based, in part, on the patient's type of cancer. For patients with bladder cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient who has bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample obtained from a patient who has bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample obtained from a patient who has bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:BICC1 is present in a biological sample obtained from a patient who has bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:AFF3 is present in a biological sample obtained from a patient who has bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:CASP7 is present in a biological sample obtained from a patient who has bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 R248C is present in a biological sample obtained from a patient who has bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 S249C is present in a biological sample obtained from a patient who has bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 G370C is present in a biological sample obtained from a patient who has bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 Y373C is present in a biological sample obtained from a patient who has bladder cancer. In some embodiments, the evaluation step comprises determining whether any combination of the above FGFR mutants is present in a biological sample obtained from a patient who has bladder cancer.

[00116] For patients with metastatic bladder cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C , FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient who has metastatic bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample obtained from a patient who has metastatic bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample obtained from a patient who has metastatic bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:BICC1 is present in a biological sample obtained from a patient who has metastatic bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:AFF3 is present in a biological sample obtained from a patient who has metastatic bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:CASP7 is present in a biological sample obtained from a patient who has metastatic bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 R248C is present in a biological sample obtained from a patient who has metastatic bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 S249C is present in a biological sample obtained from a patient who has metastatic bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 G370C is present in a biological sample obtained from a patient who has metastatic bladder cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 Y373C is present in a biological sample obtained from a patient who has metastatic bladder cancer. In some embodiments, the evaluation step comprises determining whether any combination of the above FGFR mutants is present in a biological sample obtained from a patient who has metastatic bladder cancer.

[00117] For patients with ovarian cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient who has ovarian cancer. In some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample obtained from a patient who has ovarian cancer. In some embodiments, the evaluation step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample obtained from a patient who has ovarian cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:BICC1 is present in a biological sample obtained from a patient who has ovarian cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:AFF3 is present in a biological sample obtained from a patient who has ovarian cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:CASP7 is present in a biological sample obtained from a patient who has ovarian cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 R248C is present in a biological sample obtained from a patient who has ovarian cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 S249C is present in a biological sample obtained from a patient who has ovarian cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 G370C is present in a biological sample obtained from a patient who has ovarian cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 Y373C is present in a biological sample obtained from a patient who has ovarian cancer. In some embodiments, the evaluation step comprises determining whether any combination of the above FGFR mutants is present in a biological sample obtained from a patient who has ovarian cancer.

[00118] For patients with head and neck cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluation step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient who has head and neck cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient who has head and neck cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 R248C is present in a biological sample from a patient who has head and neck cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 S249C is present in a biological sample from a patient who has head and neck cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 G370C is present in a biological sample from a patient who has head and neck cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient who has head and neck cancer. In some embodiments, the evaluation step comprises determining whether any combination of the above FGFR mutants is present in a biological sample obtained from a patient who has head and neck cancer.

[00119] For patients with metastatic head and neck cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:BAIAP2L1, FGFR2:CASP7 or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient who has metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient who has metastatic head and neck cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[00120] For patients with esophageal cancer, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C , or any combination thereof. Accordingly, in some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient who has esophageal cancer. In some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample obtained from a patient who has esophageal cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:BICC1 is present in a biological sample obtained from a patient who has esophageal cancer. In some embodiments, the evaluation step comprises determining whether FGFR2:CASP7 is present in a biological sample obtained from a patient who has esophageal cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 R248C is present in a biological sample obtained from a patient who has esophageal cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 S249C is present in a biological sample obtained from a patient who has esophageal cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 G370C is present in a biological sample obtained from a patient who has esophageal cancer. In some embodiments, the evaluation step comprises determining whether FGFR3 Y373C is present in a biological sample obtained from a patient who has esophageal cancer. In some embodiments, the evaluation step comprises determining whether any combination of the above FGFR mutants is present in a biological sample obtained from a patient who has esophageal cancer.

[00121] For patients with metastatic esophageal cancer, for example, a panel of mutant FGFR genes, may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6 or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 intron is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient who has metastatic esophageal cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[00122] For patients with non-small cell lung adenocarcinoma (NSCL), for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2: AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient who has NSCL adenocarcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient who has NSCL adenocarcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 intron is present in a biological sample from a patient who has NSCL adenocarcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient who has NSCL adenocarcinoma. In some embodiments, the evaluation step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient who has NSCL adenocarcinoma. In some embodiments, the evaluation step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient who has NSCL adenocarcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3 R248C is present in a biological sample from a patient who has NSCL adenocarcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3 S249C is present in a biological sample from a patient who has NSCL adenocarcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3 G370C is present in a biological sample from a patient who has NSCL adenocarcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient who has NSCL adenocarcinoma. In some embodiments, the evaluation step comprises determining whether any combination of the above FGFR mutants is present in a biological sample obtained from a patient who has NSCL adenocarcinoma.

[00123] For patients with non-small cell lung squamous cell carcinoma (NSCL), a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether FGFR2:CCDC6 is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3 R248C is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3 S249C is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3 G370C is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient who has NSCL squamous cell carcinoma. In some embodiments, the evaluation step comprises determining whether any combination of the above FGFR mutants are present in a biological sample obtained from a patient who has squamous cell carcinoma.

[00124] For patients with metastatic endometrial cancer, for example, a suitable mutant FGFR gene panel may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6 or FGFR2 :OFD1, or any combination thereof. Accordingly, in some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 intron is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient who has metastatic endometrial cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[00125] For breast cancer patients, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2: CASP7, FGFR2:CCD6 or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient who has breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 intron is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a breast cancer patient is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient who has breast cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[00126] For patients with hepatocellular carcinoma, for example, a panel of mutant FGFR genes may comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7 , FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 intron is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient who has hepatocellular carcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.

[00127] Suitable pairs of primers for use in the amplification step include those presented in Table 3. For example, in some embodiments, the FGFR mutant and the primer pair may be FGFR3:TACC3 v1 and the primers having the sequences 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 the primers having the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8. In some embodiments, the FGFR mutant and the primer pair may be FGFR3:TACC3 intron and the primers having the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10. In some embodiments, the FGFR mutant and the primer pair may be FGFR3:BAIAP2L1 and the primers having the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 12. In some embodiments, the FGFR mutant and the primer pair may be FGFR2:BICC1 and the primers having the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14. In some embodiments, the FGFR mutant and the primer pair may be FGFR2:AFF3 and the 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 the primers having the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 18 In some embodiments, the FGFR mutant and the primer pair may be FGFR2:CCDC6 and the primers having the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20. In some embodiments, the FGFR mutant and. the primer pair may be FGFR2:OFD1 and the primers having the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 22. In some embodiments, the FGFR mutant and the primer pair may be R248C and the primers which have 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 the primer pair may be S249C and the primers which have 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 the primer pair may be G370C and the primers which have 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 the primer pair may be Y373C and the primers that have 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 the primer pair can be any combination of the mutants of FGFR presented above and the corresponding pair of primers.

[00128] The methods presented comprise determining whether one or more FGFR mutants from the gene panel are present in the sample. In some embodiments, the determination step involves sequencing the amplified cDNA.

[00129] In some embodiments, the method further comprises treating the patient with an FGFR inhibitor if one or more FGFR mutants from the gene panel is present in the sample. Suitable FGFR inhibitors for use in treatment methods include those previously described in the present invention, in particular, JNJ-42756493.

KITS TO IDENTIFY THE PRESENCE OF FGFR MUTANT GENES

[00130] Kits are also presented to identify the presence of one or more FGFR mutant genes in a biological sample comprising primer pairs with the sequences of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO :7 and SEQ ID NO:8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO : 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, or any combination thereof; and instructions for performing an assay to detect one or more mutant FGFR genes.

[00131] Kits may further comprise one or more probes, one or more 3' blocking oligonucleotides, or both. In some embodiments, the kits may further comprise one or more probes, e.g., any one or more of the probes set forth in Table 15. In some embodiments, the kits may additionally comprise one or more 3' blocking oligonucleotides, e.g. , any one or more of the 3' blocking oligonucleotides shown in table 8. In some embodiments, the kits may further comprise one or more probes and one or more 3' blocking oligonucleotides. For example, in some embodiments, the kits may additionally comprise: a. the primer pair having the sequences SEQ ID NO: 5 and SEQ ID NO: 6 and the probe having the sequence SEQ ID NO: 43; B. the primer pair having the sequences SEQ ID NO:7 and SEQ ID NO:8 and the probe having the sequence SEQ ID NO: 44; w. the primer pair having the sequences SEQ ID NO: 9 and SEQ ID NO: 10 and the probe having the sequence SEQ ID NO: 46; d. the primer pair having the sequences SEQ ID NO: 11 and SEQ ID NO: 12 and the probe having the sequence SEQ ID NO: 47; It is. the primer pair having the sequences SEQ ID NO: 13 and SEQ ID NO: 14 and the probe having the sequence SEQ ID NO: 45; f. the primer pair having the sequences SEQ ID NO: 15 and SEQ ID NO: 16 and the probe having the sequence SEQ ID NO: 48; g. the primer pair having the sequences SEQ ID NO: 17 and SEQ ID NO: 18 and the probe having the sequence SEQ ID NO: 49; H. the pair of primers having the sequences SEQ ID NO: 19 and SEQ ID NO: 20 and the probe having the sequence of SEQ ID NO: 50; i. the primer pair having the sequences SEQ ID NO: 21 and SEQ ID NO: 22 and the probe having the sequence SEQ ID NO: 51; j. the primer pair having the sequences SEQ ID NO: 23 and SEQ ID NO: 24 and the probe having the sequence SEQ ID NO: 52; k. the primer pair having the sequences SEQ ID NO: 25 and SEQ ID NO: 26 and the probe having the sequence SEQ ID NO: 53; l. the primer pair having the sequences SEQ ID NO: 27 and SEQ ID NO: 28 and the probe having the sequence SEQ ID NO: 54; m. the primer pair having the sequences SEQ ID NO: 29 and SEQ ID NO: 30 and the probe having the sequence SEQ ID NO: 55; n. the pair of primers having the sequences SEQ ID NO: 31 and SEQ ID NO: 32 and the probe having the sequence of SEQ ID NO: 52; and the 3' blocking oligonucleotide having the sequence of SEQ ID NO: 39; O. the primer pair having the sequences SEQ ID NO: 33 and SEQ ID NO: 34 and the probe having the sequence SEQ ID NO: 53; and the 3' blocking oligonucleotide having the sequence of SEQ ID NO: 40; P. the primer pair having the sequences SEQ ID NO: 35 and SEQ ID NO: 36 and the probe having the sequence SEQ ID NO: 54; and the 3' blocking oligonucleotide having the sequence of SEQ ID NO: 41; q. the pair of primers having the sequences SEQ ID NO: 37 and SEQ ID NO: 38 and the probe having the sequence SEQ ID NO: 55; and the 3' blocking oligonucleotide having the sequence of SEQ ID NO: 42; or r. and combinations thereof.

OLIGONUCLEOTIDE PROBES

[00132] Oligonucleotide probes with the sequence of any of SEQ ID NOs: 43-55 are also presented. 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 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 embodiments, the oligonucleotide probe may have the sequence of SEQ ID NO: 55.

3' BLOCKING OLIGONUCLEOTIDES

[00133] Also presented in the present invention are oligonucleotides with the sequence of any of SEQ ID NOs: 39-42. In some embodiments, the 3' blocking oligonucleotides may have the sequence of SEQ ID NO: 39. In some embodiments, the 3' blocking oligonucleotides may have the sequence of SEQ ID NO: 40. In some embodiments, the blocking oligonucleotides may have the sequence of SEQ ID NO: 40. 3' may have the sequence of SEQ ID NO: 41. In some embodiments, the 3' blocking oligonucleotides may have the sequence of SEQ ID NO: 42.

EXAMPLES EXAMPLE 1- ISOLATION AND PURIFICATION OF PLASMID DNA

[00134] Below is an example procedure for preparing FGFR fusion plasmid DNA.

[00135] Required equipment: centrifuge, with a capacity of 1500 x g; microcentrifuge; pipettors, positive displacement or air displacement; vortexer; nanodrop spectrophotometer; shaker/incubator at 37°C; and an oven set to 37°C.

[00136] Required materials: bacterial stock solution frozen in glycerol containing plasmid DNA; LB agar plates with kanamycin (Teknova no. L1155); LB broth (Life Technologies no. 10855-021); Kanamycin (Sigma no. K0254); plasmid purification kit (Qiagen no. 12123); Absolute ethanol (Sigma Aldrich no. E7023); isopropanol (Sigma Aldrich no. W292907); Nuclease-free water (not treated with DEPC) (from IDT or Ambion no. AM9932); Tips with RNAse-free barrier (filter); RNase-free microtube (1.5 to 2 mL, VWR no. 10011-724); serological pipettes; and 14 mL round-bottom tubes (VWR no. 352057).

[00137] To recover bacteria from the glycerol stock, frozen bacteria were scraped from the top of a glycerol stock tube using a sterile pipette tip, streaked onto an LB agar plate and placed upside down. in the oven at 37°C overnight.

[00138] The DNA plasmids were purified using the Qiagen plasmid DNA purification protocol. Briefly, a single colony was picked from the streak plate and incubated in a 5 mL culture of LB medium containing 50 μg/mL kanamycin overnight on a shaker at 37°C at approximately 300 rpm. Bacterial cells were harvested by centrifugation at 6000 X g for 15 minutes at 4°C, and the pellet was resuspended in 300 mL of buffer P1. 300 μL of buffer P2 was added, mixed by inverting the tube 4 to 6 times, and incubated at room temperature (RT) for 5 minutes. 300 μL of chilled buffer P3 was added, immediately mixed by inversion 4 to 6 times, incubated on ice for 5 minutes, and centrifuged at maximum speed for 10 minutes. The supernatant containing the plasmid DNA was quickly removed. One Qiagen tip was equilibrated by applying 1 mL of QBT buffer and allowed to empty by gravity flow. The supernatant was applied to the Qiagen 20 tip and allowed to enter the resin by gravity flow. The Qiagen 20 tip was washed with 2 X 2 mL of QC buffer and the DNA was eluted with 800 μL of QF buffer, and the eluate was collected in a 1.5 mL Eppendorf tube. DNA was precipitated by adding 0.7 volume of isopropanol, mixed and centrifuged immediately at 15,000 x g for 30 minutes in a microcentrifuge. The supernatant was decanted and the DNA pellet was washed in 1 mL of 70% ethanol and centrifuged at 15,000 x g for 10 minutes. The supernatant was decanted. The pellet was air-dried for 5 to 10 minutes and the DNA was redissolved in 100 μL of appropriate volume of nuclease-free water. Plasmid DNA was quantified by Nanodrop and stored at −20 °C until further use.

EXAMPLE 2 - GENERATION OF NRK CELL LINES

[00139] Expression vectors expressing each of the FGFR fusions were constructed. The expression vector was then transfected into normal rat kidney epithelial cells (NRK). Stable cell lines were selected in medium containing kanamycin after transfections. These cells were then cultured and mRNA was isolated and subjected to FGFR fusion assays to confirm the presence of mRNA specific to FGFR fusions.

EXAMPLE 3 - MAINTENANCE OF FGFR FUSION CELL LINEAGE

[00140] The protocol below describes an exemplary procedure for cultivating and maintaining cell lines that overexpress the NRK FGFR fusion. Cell lines include, but are not limited to: NRK/FGFR3:TACC3v1, NRK/FGFR3:TACC3 v3, NRK/FGFR3:BAIAP2L1, NRK/FGFR2:BICC1, NRK/FGFR2:CASP7, NRK/FGFR2:CCDC6, NRK/ FGFR2:AFF3, NRK/FGFR2:OFD1, and NRK/EMPTY VECTOR (plasmid control).

[00141] Required equipment: Biosafety cabinet, equipped with a vacuum aspiration system; CO2 incubator set at 37°C with 5% CO2 freezer at -80°C; liquid nitrogen tank; water bath, set to 37°C; and a microscope.

[00142] Required materials: serological pipettes; tissue culture flasks (T75 VWR no. BD353136 and/or T150 VWR no. 15705 074); tissue culture 0.2 μm filtration units (Thermo Scientific no. 566-0020); DMEM cell culture medium (Dulbecco's modified Eagle's medium) (Life Technologies, catalog no. 11965-084); fetal bovine serum (FBS), certified, heat inactivated (Life Technologies, no. 10082147); PenStrep antibiotic solution (Life Technologies no. 15140-122); 0.25% trypsin-EDTA solution (Life Technologies, no. 25200-056); DPBS (phosphate-buffered Dulbecco's solution, without calcium and without magnesium) (Life Technologies, no. 14190136); cell freezing container for cryopreservation; portable pipetman; cell freezing medium (Life Technologies, no. 12648-010); 15 mL conical tubes (VWR no. 62406-2); and cryovials (VWR n°89094-800).

[00143] To prepare the cell culture medium, DMEM medium was prepared by combining 445 mL of DMEM, 50 mL of SFB, and 5 mL of PenStrep. The prepared media were passed through a 0.2 μm filter unit and stored at 4°C.

[00144] To thaw the frozen cells, the prepared DMEM medium was heated in the water bath at 37°C for at least 15 minutes, and 15 mL of warmed medium was placed in a T75 flask. Cells were removed from the liquid nitrogen tank and immediately placed in a 37°C water bath until thawed. Cryoflasks were sprayed generously with 70% alcohol and excess alcohol was wiped off with paper towels. The entire contents were aliquoted into the T75 flask containing DMEM. The vial was gently rotated to mix and placed in an incubator for 24 hours. If cells were not ready for division, the medium was changed to freshly prepared DMEM to remove residual freezing medium. If cells were ready for division, each cell line was propagated when the flask reached 80% confluence (the division rate for each cell line was dependent on experimental needs).

[00145] To freeze the cell lines, the cells were removed from the culture flask and centrifuged in a 15 mL conical tube for 5 minutes at 1500 rpm at room temperature. The medium was aspirated and 6 mL of cell freezing medium was added. The cells were mixed by pipetting up and down several times, and 1 mL of the cell solution was aliquoted into each of 5 cryovials. The cryovials with the cells were placed in a cryofreezing container, which was stored at -80°C overnight in a freezer, followed by long-term storage in a liquid nitrogen tank.

EXAMPLE 4 - FFPET SNP ASSAY

[00146] An exemplary workflow and protocol for performing an FFPET SNP assay is described below. A similar procedure is performed for FFPET fusion tests, the results of which are shown in Figure 2.

FFPET DEPARAFINIZATION

[00147] The slides were subjected to increasing amounts of xylene, followed by treatment with alcohol to remove the paraffin.

FFPET RNA EXTRACTION

[00148] The procedure for extracting RNA from formalin-fixed paraffin-embedded breast cancer tissue samples for downstream expression assay is described below.

[00149] Required equipment: centrifuge with plate adapter, with a capacity of 1500 x g; microcentrifuge; pipettors, positive displacement or air displacement; vortexer; NanoDrop 8000; heating block with incubation capacity at 37°C, 56°C and 80°C; and Pasteur pipette (Pipet Trans EX-FT 1.5 mL pk 500, VWR n°14670-329).

[00150] Required materials: AllPrep DNA/RNA FFPE Kit (Qiagen no. 80234); Absolute ethanol (Sigma Aldrich no. E7023); Isopropanol xylene Nuclease-free water (not treated with DEPC) (from IDT or Ambion no. AM9932); Tips with RNAse-free barrier (filter); no RNase; microtube (1.5 to 2 mL, VWR no. 10011-724); and Qiagen AllPrep DNA/RNA FFPE kit manual.

[00151] RNA was extracted using the AllPrep DNA/RNA FFPE kit. Briefly, a 1 to 10 μm section was placed in a 1.5 mL reaction tube and 800 μL of HemoDe or Xylene was added. The sample was vortexed for 4 seconds 3 times, incubated for 2 minutes, centrifuged for 4 seconds 3 times and incubated for 5 minutes.

[00152] The sample was centrifuged for 2 minutes at maximum speed (12,000 to 14,000 x g) and the supernatant was discarded through aspiration. The tubes were capped immediately to prevent the tissue from drying.

[00153] The above steps were repeated.

[00154] 800 μL of abs ethanol. were added and the tube was impacted to release the pellet, vortexed for 4 seconds, 3 times, centrifuged for 2 minutes at maximum speed (12,000 to 14,000 x g) and the supernatant was discarded through aspiration.

[00155] 800 μL of 70% ethanol was added and the tube was impacted to release the pellet, vortexed for 4 seconds, 3 times, centrifuged for 2 minutes at maximum speed and the supernatant was discarded through aspiration. After removing the 70% ethanol, the tube was recentrifuged for 10 to 20 seconds and the residual liquid was carefully removed with a fine-bore pipette.

[00156] The open tubes were incubated in a heating block for 5 to 15 minutes at 37°C to air dry the pellet.

[00157] The pellet was resuspended in buffer by adding 150 μL of PKD buffer and the tube was impacted to release the pellet. 10 μL of proteinase K was added and the tube was mixed by vortexing.

[00158] The tubes were incubated at 56°C for 15 minutes, incubated on ice for 3 minutes, and centrifuged for 15 minutes at 20,000 x g.

[00159] The supernatant was carefully transferred without shaking the pellet to a new 1.5 mL microcentrifuge tube for RNA purification. The supernatant was incubated at 80°C for 15 minutes. The tube was briefly centrifuged to remove drops from the inside of the cap. 320 μL of RLT buffer was added to adjust binding conditions and the tube was mixed by vortexing or pipetting. 1120 μL of ethanol (96 to 100%) was added and the tube was carefully mixed by vortexing or pipetting.

[00160] 700 μL of the sample, including any precipitate that may have formed, was transferred to an RNeasy MinElute centrifuge column placed in a 2 mL collection tube, and centrifuged for 15 seconds at > 8000 x g (> 0.000 rpm) . The pass-through flow has been discarded. This step was repeated until the entire sample passed through the RNeasy MinElute centrifuge column.

[00161] 350 μL of FRN buffer was added to the RNeasy MinElute centrifuge column and centrifuged for 15 seconds at > 8000 x g (> 10,000 rpm). The pass-through flow has been discarded.

[00162] 10 μL of DNAse stock solution was added to 70 μL of RDD buffer, mixed gently by inverting the tube, and centrifuged quickly to collect residual liquid from the sides of the tube.

[00163] The DNAse I incubation mixture (80 μL) was added directly to the membrane of the RNeasy MinElute centrifuge column, and placed on the bench (20-30°C) for 15 minutes.

[00164] 500 μL of FRN buffer was added to the RNeasy MinElute centrifuge column and centrifuged for 15 seconds at > 8000 x g (> 10,000 rpm). The flow-through was reserved for use in the next step as it contains small RNAs.

[00165] The RNeasy MinElute centrifuge column was placed in a new 2 mL collection tube (supplied). The flow-through from the previous step was applied to the centrifuge column and centrifuged for 15 seconds at > 8000 x g (> 10,000 rpm). The pass-through flow has been discarded.

[00166] 500 μL of RPE buffer was added to the RNeasy MinElute spin column and centrifuged for 15 seconds at >8000 x g (>10,000 rpm) to wash the spin column membrane. The pass-through flow has been discarded.

[00167] 500 μL of RPE buffer was added to the RNeasy MinElute spin column and centrifuged for 15 seconds at >8000 x g (>10,000 rpm) to wash the spin column membrane. The collection tube with the passing flow was discarded.

[00168] The RNeasy MinElute centrifuge column was placed in a new 2 mL collection tube and centrifuged at maximum speed for 5 minutes. The collection tube with the passing flow was discarded.

[00169] The RNeasy MinElute centrifuge column was placed in a new 1.5 mL collection tube, 30 μL RNase-free water was added directly to the centrifuge column membrane, incubated for 1 hour at room temperature, and centrifuged at speed maximum for 1 minute to elute the RNA.

[00170] The RNA samples were immediately stored in a freezer at -80°C.

cDNA SYNTHESIS

[00171] Presented below is a process for cDNA synthesis for FFPET SNP assays using real-time PCR (RT-PCR) analysis.

[00172] Necessary equipment: centrifuge with plate adapter, with a capacity of 1500 x g, microcentrifuge; pipettors (single-channel and multi-channel pipettor preferred), positive displacement or air displacement; vortexer; and GeneAmp® PCR 9700 PCR System (ABI no. 4314879) or equivalent.

[00173] Required materials: High-capacity cDNA reverse transcriptase kit with an RNase inhibitor, 200 reactions (ABI no. 4374966); Nuclease-free (non-DEPC treated) water (from IDT) or equivalent; Tips with RNAse-free barrier (filter); RNase-free microtube (1.5 to 2 mL, VWR no. 10011-724); MicroAmp™ optical 96-well reaction plates (Life Technologies, no. 4306736); and sealing film (VWR n°60941-072).

[00174] After RNA extraction (presented above) the RNA sample tube(s) were kept on ice.

[00175] The kit components were used to prepare the 2x reverse transcription (RT) master mix for all reactions, including 1 negative control (water). Components were thawed on ice for approximately 15 minutes, gently inverted to mix, and centrifuged briefly to decant the solution. All reagents were returned to ice. The tubes were not vortexed.

[00176] A master mix was prepared on ice in a 1.5 mL tube for the appropriate number of reactions (no. of reactions + 10%, per 20 μL of reaction) by combining the following amounts of reagent for one reaction : 2 μL of 10X RT buffer mix; 0.8 μL of 25X dNTP mix; 2 μL of 10X RT random primers; 1 μL 50U/μL MultiScribe Reverse Transcriptase; 1 μL of RNase inhibitor; and 3.2 μL of nuclease/RNase-free H2O.

[00177] The master mixture was vortexed several times (5 to 10) and the mixture was centrifuged briefly (1500 x g, 5 to 10 seconds). 10 microliters of the reaction mixture was added to the appropriate wells of a 96-well plate.

[00178] The RNA samples were diluted to a concentration of 20 ng/μL. 10 microliters of each RNA sample was added including the water negative control to the appropriate corresponding wells of the 96-well plate in a final reaction volume of 20 μL. The wells were carefully mixed by pipetting up and down 3 times, sealed with a plate seal, and centrifuged briefly (1500 x g for 60 seconds). The plates were kept on ice until loading into the thermocycler.

[00179] The reaction plate was loaded into the ABI 9700 thermocycler at a clean workstation or laboratory and operated using the following reverse transcription program with a reaction volume of 20 μL: Step 1: 25°C for 10 minutes Step 2: 37°C for 120 minutes Step 3: 85°C for 5 seconds Step 4: Maintain at 4°C until infinity

[00180] The synthesized cDNA was stored at -20°C for the next step of pre-amplification.

PREPARATION OF THE PRE-AMPLIFIATION TEST SET MIXTURE

[00181] The pre-amplification assay set mix associated with the FFPET SNP assay pre-amplification protocol was prepared as described below.

[00182] Necessary equipment: microcentrifuge; pipettors, positive displacement or air displacement; and vortexer.

[00183] Required materials: Nuclease-free water (not treated with DEPC) (from IDT) or equivalent; IDTE pH 8.0 (1X TE solution) (IDT Technologies); Tips with RNAse-free barrier (filter); and RNase-free tubes (1.5 to 2 mL, VWR n°10011-724).

[00184] All TaqMan SNP assays were purchased from Applied Biosystems, Life Technologies, Inc.

[00185] 100 μL of 20X SNP assays were prepared.

[00186] To prepare the 0.2X PreAmp assay set, all assays were thawed on ice for approximately 15 minutes. The following volume of components was added to a 1.5 mL tube: TABLE 4

[00187] Note: The volumes above are for preparing 200 μL of 0.2X pre-amplification assay set. Volumes can be adjusted accordingly depending on the number of samples to be tested.

[00188] The 0.2X pre-amplification assay set was vortexed briefly to mix (5 to 10 seconds) and briefly centrifuged (1500 x g, 5 to 10 seconds). 100 μL of the pre-amplification primer set was aliquoted into 1.5 mL tubes and stored at -20°C.

PRE-AMPLIFIATION FOR SNP ASSAY OF FORMALIN-FIXED PARAFFIN-EMBEDDED BREAST CANCER TISSUE USING REAL-TIME PCR (RT-PCR) ANALYSIS

[00189] Required equipment: centrifuge with plate adapter, with a capacity of 1500 x g; microcentrifuge; pipettors, positive displacement or air displacement; vortexer; GeneAmp® 9700 PCR System (ABI n°4314879) or equivalent.

[00190] Required materials: TaqMan® PreAmp master mix (2X) (Life Technologies n°4391128); Pooled assay mix 0.2X (see assay preparation and handling protocol); 1X IDTE Buffer (10 mM Tris/0.1 mM EDTA, pH7.5, from IDT) or equivalent; Nuclease-free (non-DEPC treated) water (from IDT) or equivalent; Tips with RNAse-free barrier (filter); RNase-free microtube (1.5 to 2 mL, VWR no. 10011-724); MicroAmp™ optical 96-well reaction plates (Life Technologies, no. 4306736); Microamp® optical adhesive film (Applied Biosystems PN 4311971); deep well plates (VWR n°47734-788); sealing sheets (VWR n°60941-126).

[00191] Samples were prepared by placing the cDNA and the 0.2X assay mix pool on ice to thaw, approximately 5 minutes, and centrifuging the plate briefly (1500 x g for 5 to 10 seconds).

[00192] The kit components were used to prepare the Pre-Amp 2x master mix. The kit components were allowed to thaw on ice for approximately 5 minutes. After all reagents were thawed, the tubes were gently inverted to mix and briefly centrifuged to decant the solution. All reagents were returned to ice. The tubes were not vortexed.

[00193] In a clean biosafety hood or laboratory, each master mix was prepared for the appropriate number of reactions on ice by combining the required volumes of reagents as indicated below in Table 5 (# of reactions + 10%): TABLE 5

[00194] Assay kits contain primers and probes.

[00195] To avoid cross-initialization of the SNP assays, all 5 assays were divided into 3 pre-amplification reactions per sample.

[00196] Each master mix was centrifuged several times (5 to 10) to mix, followed by brief centrifugation (1500 x g, 5 to 10 seconds). 18.75 μL of each master mix was aliquoted into the appropriate wells in a 96-well reaction plate. 6.25 μL of each cDNA sample, including a negative control well with water, was transferred to the appropriate wells in the master mix reaction plate for each preamplification reaction. The sample was mixed gently by pipetting up and down 3 times and the lid was closed. The plate was centrifuged briefly (1500 x g for 60 seconds) and kept on ice until loading into the thermocycler.

[00197] The thermocycler with ABI 9700 reaction plate was loaded and run using the following program: Step 1: 95°C for 10 minutes Step 2: 95°C for 15 seconds Step 3: 60°C for 4 minutes Step 4: Keep step 2-3 for 10 cycles

[00198] If a block of gold or silver was used, the maximum mode was selected and the rise rate was set at 77%. If an aluminum block was used, standard mode (no speed change) was selected. Step 5: Maintain at 4°C to infinity

[00199] The reaction volume was adjusted to 25 μL.

[00200] The pre-amplification reaction plate was centrifuged briefly (1500 x g for 60 seconds) after the end of pre-amplification. 100 μL of IDTE was added to the appropriate wells of a new deep 96-well plate and 25 μL of each preamplification product was transferred to the corresponding wells to have a final dilution volume of 125 μL. Each well was mixed by pipetting up and down 3 times, the plate was sealed with adhesive foil, the plate was centrifuged briefly (1500 x g for 5 to 10 seconds), and the preamplification product was stored at -20°C. °C until further use.

FFPET SNP ASSAY - REAL-TIME PCR.

[00201] The following is the procedure for SNP assay of formalin-fixed paraffin-embedded tissue using real-time PCR analysis.

[00202] Required equipment: centrifuge with plate adapter, with a capacity of 1500 x g; microcentrifuge; pipettors (single-channel and multi-channel pipettor preferred), positive displacement or air displacement; vortexer; and the ABI ViiA 7 real-time PCR instrument (Life Technologies).

[00203] Required materials: Taqman genotyping master mix (Life Technologies no. 4371355); SNP assays; Nuclease-free water (not treated with DEPC, from IDT) or equivalent; Tips with RNAse-free barrier (filter); RNase-free microtube (1.5 to 2 mL, VWR no. 10011-724); Microamp® optical adhesive film (Applied Biosystems PN 4311971); and MicroAmp™ optical 384-well reaction plates.

[00204] Table 15 lists the sequences of the probes used during real-time PCR assays.

[00205] To prepare the samples, at a workstation or laboratory, the SNP assays were placed on ice to thaw for approximately 5 minutes. All reagents were protected from light to protect from exposure to fluorescent probes. Diluted preamplification plates were placed on ice to thaw on a dirty workstation or laboratory after master mix preparation for genotyping.

[00206] To prepare master mix for genotyping, the master mix for genotyping was thawed on ice for approximately 5 minutes. The master mix (MM) was prepared in the required number of tubes on ice. The required volumes of reagents were combined in the appropriate labeled tubes as indicated in table 6 below (no. of reactions + 10%): TABLE 6

[00207] The 20X SNP master mix contains primers, probes and blocking oligos.

[00208] The master mix was vortexed several times (5 to 10) to mix and then centrifuged briefly (1500 x g, 5 to 10 seconds). 15 μL of each master mix was added to the appropriate wells of Microamp™ 384-well optical reaction plates. The reaction plates were sealed with optical adhesive film.

[00209] The plate with pre-amplification product diluted 1:5 was placed on ice for approximately 5 to 10 minutes to thaw. Using a multi-channel pipettor, 5 μL of each diluted preamplification product was transferred into the appropriate corresponding wells. The reaction plate was sealed with an adhesive optical film and centrifuged briefly (1500 x g for 60 seconds). The plates were kept on ice until loading into the thermocycler.

[00210] The following conditions were performed using the viiA 7 software with the volume adjusted to 20 μL: TABLE 7

qRT-PCR specific for FGFR SNP

[00211] Detection of rare somatic mutations in an excess of wild-type alleles is increasingly important in cancer diagnosis. When mutations of interest are close to each other, detection becomes difficult. To aid in the identification of FGFR SNPs, a SNP-specific qRT-PCR assay was developed, in which SNP-specific amplification using MGB Taqman probes combined with 3'dideoxy wild-type allele blocks was used. The assay prevented nonspecific binding from occurring, improved the amount of on-target amplification, reduced false-positive signals from wild-type alleles, and increased the sensitivity of the assay. This RNA-based SNP detection assay, combined with the pre-amplification step in the assay, amplifies low or rare mutant signals.

[00212] An example strategy for SNP-specific qRT-PCR using a wild-type 3'dideoxy blocking oligonucleotide is shown in Figure 3, an example FFPE sample validation strategy is illustrated in Figure 4. Briefly, qRT-PCR was performed using FGFR SNP primers in the presence of a 3'dideoxy blocking oligonucleotide, which was complementary to, and contained a short stretch of flanking nucleotides, the WT (wild-type) allele. Binding of the blocking oligonucleotide to the WT prevented amplification of the WT allele, whereas the FGFR SNP primers specifically bound and amplified the FGFR SNP. The WT 3'dideoxy blocking oligonucleotides used in the FGFR SNP-specific qRT-PCR are shown in Table 8. The FGFR SNP primers used in the FGFR SNP-specific qRT-PCR were: SEQ ID NO: 31 and SEQ ID NO: 32 (FGFR3 R248C); SEQ ID NO: 33 and SEQ ID NO: 34 (FGFR3 S249C); SEQ ID NO: 35 and SEQ ID NO: 36 (FGFR3 G370C); and SEQ ID NO: 37 and SEQ ID NO: 38 (FGFR3 Y373). Table 15 lists the probe sequences used during real-time PCR assays. TABLE 8 *Y can be T or C. The WT 3' blocking oligo will have 50% T and 50% C at that particular position during synthesis (purified by the manufacturer to provide T or C at that particular position).

[00213] Samples for validation studies were prepared as shown in Table 9. Examples of SNP-specific qRT-PCR validation data using a WT 3'dideoxy blocking oligonucleotide for FGFR3 G370C, FGFR3 Y373, FGFR3 S249C, and FGFR3 R248C is illustrated in Figures 5A to 5D, respectively. The raw Ct (cycle threshold) data for the FFPE samples with SNP-specific qRT-PCR with WT 3'dideoxy blocking oligonucleotides are shown in Table 10. The data derived from DNA and RNA using different platforms/techniques suggests that SNP-specific PCR with the 3' blocking nucleotide is a robust, reliable and sensitive assay. Validation data suggest that a mutant allele/SNP can be detected in a large excess of WT-containing genomic DNA, thus emphasizing the sensitivity and specificity of each assay. TABLE 9

[00214] RNA from stable cell lines that express each FGFR3 SNPs (R248C, S249C, G370C, Y373C) and FGFR3 WT TABLE 10

[00215] FMI/NGS = Next generation sequencing technique in which DNA is used as a template to identify mutations (no 3' blocking oligonucleotide); Janssen R&D = performed on RNA template (without the 3' blocking oligonucleotide); SNP-specific PCR performed on the RNA template with the 3' blocking nucleotide.

EXAMPLE 5 - VALIDATION OF THE CUSTOM FGFR FUSION GENE DETECTION ASSAY GENERATION OF POSITIVE CONTROLS FOR FGFR FUSION ASSAYS

[00216] FGFR fusion "synthetic mini-genes", plasmids encoding FGFR fusions, and stable cell lines containing FGFR fusions have been generated. Briefly, synthetic minigenes were artificially constructed by linking a series of nucleotides, about 100 base pairs long, to each other, corresponding to the target DNA sequence of interest. Plasmids encoding FGFR fusions were generated by cloning the cDNA encoding the various FGFR fusion genes into an expression vector. Stable cell lines containing FGFR fusions were generated by transfection of plasmids encoding FGFR genes into normal rat kidney epithelial cells (NRK cells). Stable cell lines were selected with the antibiotic G418. The FGFR Taqman fusion assay was performed on total RNA isolated from these cell lines to confirm the successful generation of stable cell lines expressing the FGFR fusion(s). Stable cell lines expressing FGFR fusions are used as a positive control. Table 15 lists the probe sequences used during real-time PCR assays.

ANALYSIS OF THE LOWER LIMIT OF QUANTIFICATION AND EFFICIENCY OF FGFR FUSION ASSAYS

[00217] To determine the lower limit of quantification (LLOQ) and efficiency of FGFR fusion gene assays, FGFR fusion products were generated by Taqman (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 as fusion-negative), serially diluted 1:10, and analyzed using Applied Biosystems ViiA7 v1.1 software. Efficiency standard curves are shown in Figure 6. FGFR fusion LLOQ and efficiency are shown in Table 11. TABLE 11

[00218] The FGFR fusion gene was then validated in cell lines positive for the fusion gene. FGFR fusion gene expression serial dilutions were prepared by adding fusion protein-positive cells into a fusion protein-negative cell line. For example, a 1:2 serial dilution was prepared for FGFR3:TACC3v1 and for FGFR3:BAIAP2L1 and was spiked into 1 million BAF cells. RNA was isolated (using the Qiagen Rneasy kit), followed by RT-PCR, cDNA preamplification, and Taqman real-time PCR for the target FGFR fusion gene. As shown in Table 12, both Taqman assays for the FGFR3:TACC3v1 and FGFR3:BAIAP2L1 fusion gene are capable of detecting the fusion target in 31 out of 1 million fusion-negative cells (0.003% sensitivity). TABLE 12

[00219] RT112 and SW780 = commercially available bladder cancer cell lines that carry FGFR fusions (from the American Type Culture Collection).

EXAMPLE 6 - VALIDATION OF THE CUSTOM FGFR SNP DETECTION ASSAY EVALUATION OF FGFR3 MUTATIONS IN BLADDER CANCER

[00220] The R248C, S249C, and Y373C SNPs were observed in approximately 8%, approximately 61%, and approximately 19%, of the bladder cancer samples tested, respectively.

EXAMPLE 7 - ANALYSIS OF CANCER SAMPLES

[00221] Samples were analyzed using the procedure described in Example 4. The results are presented in Table 13 and Figure 7. Table 13 shows the prevalence of FGFR fusion in different cancers. FGFR fusions detected in FFPE samples from different cancers such as bladder (primary and metastatic), NSCLC (adenocarcinoma and squamous), ovarian, esophagus (primary and metastatic), head and neck (H&N; primary and metastatic), endometrial (metastatic ), breast, and prostate using the qRT-PCR method. All FGFR fusions tested were negative for prostate cancer samples. The FGFR3:TACC3 intron fusion was negative in bladder (primary), NSCLC (squamous), ovarian and esophageal (primary), H&N (primary and metastatic), and breast cancers. The FGFR2:OFD1 fusion was negative in bladder (primary and metastatic), NSCLC (adenocarcinoma), ovarian and esophageal (primary and metastatic) cancers. FGFR2:CCDC6 fusion was negative in bladder cancer (primary and metastatic), NSCLC (adenocarcinoma), ovarian and esophageal (primary), and H&N (primary and metastatic)

[00222] Figure 8 is an example representation of the FGFR fusion gene and mutation status in NSCL adenocarcinoma and squamous cell carcinoma. In FGFR fusion-positive NSCL adenocarcinoma samples, 3/17 samples were positive for the EGFR mutation, 3/17 samples were positive for the KRAS mutation, and 1/17 samples were positive for the cMET mutation. No EGFR, KRAS, or cMET mutations, however, were observed in the FGFR fusion-positive NSCLC squamous cell carcinoma samples.

EXAMPLE 8 - TREATMENT OF PATIENTS WITH ADVANCED SOLID TUMORS

[00223] A clinical trial was conducted in which patients with various solid tumors expressing the FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:CCDC6 and FGFR2:BICC1 fusion genes were treated with JNJ-42756493. Figure 9 illustrates exemplary results from phase I patient samples, in which FGFR fusions in phase I test samples JNJ-427493 (EDI10001) were detected using the qRT-PCR assay. All FGFR fusion assays were run simultaneously with positive controls (ST) and GAPDH to assess RNA quality control. A) Graphical representation of qRT-PCR data generated for pt no. 1000081: positive only for the FGFR2:BICC1 fusion (inset shows details of Ct values for the FGFR2:BICC1 fusion, for the positive control ST and GAPDH). B) Graphical representation of qRT-PCR data generated for pt no. 33000158: positive only for the FGFR3:TACC3v1 fusion (inset shows details of Ct values for the FGFR3:TACC3v1 fusion, for the positive control ST and GAPDH). C) Graphical representation of qRT-PCR data generated for pt no. 34000123: positive only for the FGFR2:CCDC6 fusion (inset shows details of Ct values for the FGFR2:CCDC6 fusion, for the positive control ST and GAPDH). D) Graphical representation of qRT-PCR data generated for pt no. 340000115: positive for FGFR3:TACC3v1, FGFR3:TACC#v3 and FGFR2:CCDC6 fusions (inset shows details of Ct values for FGFR fusions, controls ST and GAPDH positive).

[00224] Figure 10 represents an exemplary phase study design for a first-in-human study of JNJ-42756493 in patients with advanced solid tumors. A graphical representation of a traditional 3+3 design dose escalation method for phase I clinical testing is shown. The dose escalation phase aims to establish the maximum tolerated dose (MTD) and recommended phase II dose (DFR). The part 1 arm was used to determine the intermittent dosing schedule, i.e., 7 days on and 7 days off (10 mg/kg and 12 mg/kg). The part 2 arm was used to determine PD biomarkers (pharmacodynamic biomarkers; markers examined to link drug effect to target and biological tumor response) and biopsy and blood samples were tested. The part 3 arm was the dose expansion cohort used and included accrual of additional patients in specific indications (NSCLC, SCLC, and solid breast tumors) with different eligibility criteria (FGFR aberrations: translocation/mutation/amplifications) to further characterize the toxicity profiles of JNJ493.

EVALUATION OF CLINICAL ACTIVITY

[00225] [0214] Significant clinical responses (RECIST) were observed on administration of 9 mg once daily (QD), 12 mg QD, and 12 mg 7 days on/off in patients with the FGFR fusion genes. (Figure 11; represents all dosage regimens).

EXAMPLE 9 - GENERATION OF RK3E CELLS STABLELY TRANSFECTED WITH FGFR FUSION CELL LINES THAT OVEREXPRESS FGFR FUSION

[00226] 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). FGFR fusion gene constructs were designed and cloned into the pReceiver expression vector (Genecopoeia, Rockville, MD, USA), which contains an HA tag. Clones were transfected into RK3E cells using the Amaxa Cell Line Nucleofector (Lonza, Basel, Switzerland) according to the manufacturer's protocol. Stably transfected cells were selected in complete medium with 800 ug/mL G418 (Invitrogen). Overexpression of the fusions in stably transfected cells was confirmed by real-time PCR and immunoblotting using an anti-pFGFR antibody (Figure 12). As shown in Figure 12, stable cell lines showed expression of active FGFR fusion kinases, as shown by expression of FGFR phosphorylation.

COLOGY FORMATION ASSAY

[00227] Anchorage-independent growth of RK3E cells stably transfected with the FGFR fusion was tested. One mL of 0.8% low-melting point agarose culture medium was plated into each of three wells of a six-well plate. After the agar had solidified, each well received an additional 1 mL of 0.4% agar in a culture medium containing 100 cells. After 14 days, colonies were fixed and stained with 0.1% cresyl crystal violet. The number of colonies was determined microscopically by manual counting of wells in triplicate for each cell line. A representative view of each cell line that overexpresses the fusion is shown in Figure 13A. Anchorage-independent cell growth in soft agar could be detected in cells stably transfected with the FGFR fusion, but not in the empty control vector. Figure 13B represents a quantitative soft agar colony analysis for RK3E cells stably transfected with the FGFR fusion and the control empty vector. All experiments were performed in duplicate and results are expressed as colonies/100 cells plated. All FGFR fusions tested induced anchorage-independent growth, emphasizing their transforming capacity.

DOWNSTREAM TARGET EXPRESSION

[00228] RK3E cells stably transfected with the FGFR fusion were plated in complete culture medium, serum starved overnight, then refed with 0.5% FBS growth medium. Cells were treated with 1 μM JNJ-42756493, AZD4547, or NVP-BGJ398 in the presence of ligands for 1 hour. For immunoblotting, whole cell lysates were collected in RIPA buffer (Thermo Scientific, Waltham, MA, USA) and sample protein concentration was assessed using the BCA protein assay (Thermo Scientific). Equal amounts of protein (30 μg per lane) were loaded onto 4 to 12% Bis-Tris gels (Invitrogen) before an SDS-Page was performed. Proteins were transferred to nitrocellulose membranes and probed with antibodies against p-FGFR, total FGFR2, p-MAPK, total MAPK, p-S6, total S6, B-actin (Cell Signaling Technology, Danvers, MA, USA), and Total FGFR3 (Santa Cruz, Dallas, TX, USA). Membranes were blocked with Odyssey blocking buffer for 1 h at room temperature and incubated overnight at 4°C in a primary antibody solution diluted in Odyssey blocking buffer (1:1000). After three washes in Tris-buffered saline and 0.1% Tween (TBST), membranes were probed with IR 670 or 800cw goat anti-mouse or donkey anti-rabbit dye-labeled secondary antisera in blocking buffer. Odyssey for 1 h at room temperature. Washes were repeated after secondary labeling and membranes were visualized using a LiCor Odyssey scanner and Odyssey 3.0 analytical software (LiCor, Lincoln, NE, USA). The effects of JNJ-42756493 were compared with AZD4547 and NVP-BGJ398. As shown in Figures 14A to 14H, treatment with JNJ-42756493, AZD4547, and NVP-BGJ398 (columns 2 to 4 on each blot) inhibited the phosphorylation of 14A-14H and downstream targets, i.e., MAPK and S6.

DRUG RESPONSE TESTING FOR CELL LINES THAT OVEREXPRESS FGFR FUSION

[00229] RK3E cells stably transfected with the FGFR fusion were seeded in 96-well plates (1000 cells/well) in triplicates in complete culture medium plus FGF-1 and FGF-2 ligands. After 24 hours, the cells were serum starved overnight, then refed with growth medium with 0.5% FBS. 72 hours after plating, cells were treated with various concentrations of an 18-point 1:3 serial dilution, starting at 10 μM, of JNJ493, AZD4547 (AZD), and NVP-BGJ398 (NVS). Microtiter plates were incubated for 72 hours and tested for adenosine triphosphate (ATP; a marker of metabolically active cells) content using the Cell Titer-Glo® Luminescent cell viability kit (Promega Corp., Madison, WI, USA) from according to the manufacturer's instructions, with modifications. Briefly, the cells were allowed to equilibrate to room temperature and at this time a 1:1 mixture of Cell Titer-Glo® reagent was added. The cells were then placed on an orbital shaker for 2 minutes and incubated for 10 minutes at room temperature to stabilize the luminescent signal. Luminescence was quantified and measurements were conducted using an Envision Multilabel plate reader (Perkin Elmer; Waltham, MA, USA). IC50 values (shown in table 14) were calculated using GraphPad Prism 5.0. As shown in Table 14, cells containing the FGFR fusions showed sensitivity to the inhibitor of JNJ-42756493, AZD4547, and NVP-BGJ398 in vitro, with JNJ-42756493 showing enhanced sensitivity (nanomolar concentration range) compared to AZD4547 and NVP - BGJ398 while the control empty vector does not. TABLE 14

[00230] Those skilled in the art will appreciate that various changes and modifications can be made to preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is therefore intended to cover in the attached claims all such changes and modifications, as they fall within the true spirit and scope of the invention.

[00231] The descriptions of each patent, patent application, and publication cited or described in this document are incorporated herein by reference in their entirety. NUCLEOTIDE SEQUENCE OF FGFR FUSION GENES

[00232] The nucleotide sequences for the FGFR fusion cDNA that were engineered into expression vectors are provided in table 16. The underlined sequences correspond to FGFR3 or FGFR2, the sequences in normal font represent the fusion partners and the sequence in italics represent the intron sequence of the FGFR3 gene. TABLE 16

MODALITIES

[00233] The following list of embodiments is intended to complement, rather than displace, or replace, the previous descriptions.

[00234] Embodiment 1. A method for identifying a patient with cancer that is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor comprising: evaluating a biological sample from the patient for an FGFR mutant a from a panel of mutant FGFR genes, the FGFR mutant being an FGFR fusion gene or an FGFR single nucleotide polymorphism, and said evaluation comprising amplifying the cDNA with a pair of primers that bind and amplify one or more FGFR mutants from the panel of mutant FGFR genes; and and determining whether the one or more FGFR mutants from the panel of mutant FGFR genes is present in the sample, with the presence of the one or more FGFR mutants indicating that the patient is responsive to treatment with the FGFR inhibitor.

[00235] Embodiment 2. A method for identifying a patient with cancer that is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor comprising: evaluating a biological sample obtained from the patient for the presence of a or more FGFR mutants from a panel of mutant FGFR genes, wherein the FGFR mutant is an FGFR fusion gene or an FGFR single nucleotide polymorphism, wherein the presence of the one or more FGFR mutants indicates that the patient is responsive to treatment with the FGFR inhibitor.

[00236] Modality 3. The method of modality 1 or 2, wherein the FGFR fusion gene comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6 or FGFR2:OFD1, or any combination thereof.

[00237] Modality 4. The method of modality 1 or 2, wherein the FGFR single nucleotide polymorphism comprises R248C, S249C, G370C or Y373C, or any combination thereof.

[00238] Modality 5. The method of modality 1 or 2, where the cancer is bladder cancer and the panel of mutant FGFR genes comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2: AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[00239] Modality 6. The method of modality 1 or 2, characterized by the fact that the cancer is metastatic bladder cancer and the panel of mutant FGFR genes comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2: BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[00240] Modality 7. The method of modality 1 or 2, characterized by the fact that the cancer is ovarian cancer and the panel of mutant FGFR genes comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1 , FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[00241] Modality 8. The method of modality 1 or 2, characterized by the fact that the cancer is head and neck cancer and the panel of mutant FGFR genes comprises FGFR3: BAIAP2L1, FGFR2: CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[00242] Modality 9. The method of modality 1 or 2, wherein the cancer is metastatic head and neck cancer and the panel of mutant FGFR genes comprises: FGFR3:BAIAP2L1, FGFR2:CASP7 or FGFR2:OFD1, or any combination thereof same.

[00243] Modality 10. The method of modality 1 or 2, where the cancer is esophageal cancer and the panel of mutant FGFR genes comprises: FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C , FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

[00244] Modality 11. The method of modality 1 or 2, wherein the cancer is metastatic esophageal cancer and the panel of mutant FGFR genes comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6 or FGFR2:OFD1, or any combination thereof.

[00245] Modality 12. The method of modality 1 or 2, where the cancer is small cell lung carcinoma, adenocarcinoma and the panel of mutant FGFR genes comprises: FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron , FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[00246] Modality 13. The method of modality 1 or 2, where the cancer is small non-cell lung carcinoma, squamous cell carcinoma and the panel of mutant FGFR genes comprises: FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[00247] Modality 14. The method of modality 1 or 2, where the cancer is metastatic endometrial cancer and the panel of mutant FGFR genes comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2 :CASP7, FGFR2:CCDC6 or FGFR2:OFD1, or any combination thereof.

[00248] Modality 15. The method of modality 1 or 2, where the cancer is breast cancer and the panel of mutant FGFR genes comprises: FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7 or any combination thereof.

[00249] Modality 16. The method of modality 1 or 2, where the cancer is hepatocellular carcinoma and the panel of mutant FGFR genes comprises: FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 intron, FGFR3:BAIAP2L1, FGFR2 :BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C or FGFR3 Y373C, or any combination thereof.

[00250] Modality 17. Method, according to any one of modalities 2 to 16, wherein the evaluation comprises amplifying cDNA that with a pair of primers binds and amplifies one or more FGFR mutants from the panel of mutant FGFR genes.

[00251] Modality 18. The method of modality 17, wherein the cDNA is pre-amplified cDNA.

[00252] Modality 19. Method, according to any of the previous modalities, with the FGFR mutant and the primer pair being:

[00253] FGFR3:TACC3 v1 and primers having the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6;

[00254] FGFR3:TACC3 v3 and primers having the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8;

[00255] FGFR3: TACC3 intron and primers having the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10;

[00256] FGFR3:BAIAP2L1 and primers having the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 12;

[00257] FGFR2:BICC1 and the primers having the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14;

[00258] FGFR2:AFF3 and the primers having the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 16;

[00259] FGFR2:CASP7 and the primers having the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 18;

[00260] FGFR2:CCDC6 and the primers having the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20;

[00261] FGFR2:OFD1 and the primers having the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 22;

[00262] R248C and primers that have the amino acid sequences of SEQ ID NO: 23 and SEQ ID NO: 24 or SEQ ID NO: 31 and SEQ ID NO: 32;

[00263] 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;

[00264] 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;

[00265] Y373C and primers having the amino acid sequences of SEQ ID NO: 29 and SEQ ID NO: 30 or SEQ ID NO: 37 and SEQ ID NO: 38; or any combination thereof.

[00266] Modality 20. Method, according to any of the previous modalities, with the evaluation comprising: isolating the RNA from the biological sample and synthesizing cDNA from the isolated RNA.

[00267] Modality 21. The method of modality 20, which further comprises pre-amplifying the cDNA before the amplification step.

[00268] Modality 22. Method, according to any of modalities 1 or 3 to 21, with the cDNA being pre-amplified.

[00269] Modality 23. Method, according to any of modalities 1 or 3 to 22, with the amplification step comprising execution of real-time PCR.

[00270] Modality 24. The method of modality 23, wherein real-time PCR is performed with one or more probes comprising SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and /or SEQ ID NO: 55.

[00271] Modality 25. Method, according to modality 23 or 24, wherein real-time PCR is performed with one or more 3' blocking oligonucleotides comprising SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and/or SEQ ID NO: 42.

[00272] Modality 26. Method, according to any of modalities 1 or 3 to 25, said determination step comprising sequencing the amplified cDNA.

[00273] Embodiment 27. A kit for identifying the presence of one or more mutant FGFR genes in a biological sample comprising: pairs of primers having the sequences of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, or any combination thereof; and instructions for performing an assay to detect one or more mutant FGFR genes.

[00274] Modality 28. Kit of modality 27, which further comprises one or more probes, one or more 3' blocking oligonucleotides, or both.

[00275] Modality 29. The kit of modality 28, where: a. the primer pair having the sequences SEQ ID NO: 5 and SEQ ID NO: 6 and the probe having the sequence SEQ ID NO: 43; B. the primer pair having the sequences SEQ ID NO:7 and SEQ ID NO:8 and the probe having the sequence SEQ ID NO: 44; w. the primer pair having the sequences SEQ ID NO: 9 and SEQ ID NO: 10 and the probe having the sequence SEQ ID NO: 46; d. the primer pair having the sequences SEQ ID NO: 11 and SEQ ID NO: 12 and the probe having the sequence SEQ ID NO: 47; It is. the primer pair having the sequences SEQ ID NO: 13 and SEQ ID NO: 14 and the probe having the sequence SEQ ID NO: 45; f. the primer pair having the sequences SEQ ID NO: 15 and SEQ ID NO: 16 and the probe having the sequence SEQ ID NO: 48; g. the primer pair having the sequences SEQ ID NO: 17 and SEQ ID NO: 18 and the probe having the sequence SEQ ID NO: 49; H. the primer pair having the sequences SEQ ID NO: 19 and SEQ ID NO: 20 and the probe having the sequence SEQ ID NO: 50; i. the primer pair having the sequences SEQ ID NO: 21 and SEQ ID NO: 22 and the probe having the sequence SEQ ID NO: 51; j. the primer pair having the sequences SEQ ID NO: 23 and SEQ ID NO: 24 and the probe having the sequence SEQ ID NO: 52; k. the primer pair having the sequences SEQ ID NO: 25 and SEQ ID NO: 26 and the probe having the sequence SEQ ID NO: 53; l. the primer pair having the sequences SEQ ID NO: 27 and SEQ ID NO: 28 and the probe having the sequence SEQ ID NO: 54; m. the primer pair having the sequences SEQ ID NO: 29 and SEQ ID NO: 30 and the probe having the sequence SEQ ID NO: 55; n. the pair of primers having the sequences SEQ ID NO: 31 and SEQ ID NO: 32 and the probe having the sequence of SEQ ID NO: 52; and the 3' blocking oligonucleotide having the sequence of SEQ ID NO: 39; O. the primer pair having the sequences SEQ ID NO: 33 and SEQ ID NO: 34 and the probe having the sequence SEQ ID NO: 53; and the 3' blocking oligonucleotide having the sequence of SEQ ID NO: 40; P. the primer pair having the sequences SEQ ID NO: 35 and SEQ ID NO: 36 and the probe having the sequence SEQ ID NO: 54; and the 3' blocking oligonucleotide having the sequence of SEQ ID NO: 41; q. the primer pair having the sequences SEQ ID NO: 37 and SEQ ID NO: 38 and the probe having the sequence SEQ ID NO: 55; and the 3' blocking oligonucleotide having the sequence of SEQ ID NO: 42; or r. and combinations thereof.

[00276] Embodiment 30. A primer has the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or any combination thereof.

[00277] Embodiment 31. A set of primers, which has the sequences of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, or any combination thereof.

[00278] Embodiment 32. An oligonucleotide probe that has the sequence of any of SEQ ID NOs: 43-55, or any combination thereof.

[00279] Embodiment 33. An oligonucleotide that has the sequence of any of SEQ ID NOs: 39-42, or any combination thereof.

Claims (15)

1. Method for identifying a patient with bladder cancer who is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor, wherein the FGFR inhibitor is N-(3,5-dimethoxy-phenyl )-N'-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl]ethane-1,2-diamine, an N-oxide thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, characterized in that it comprises: (a) evaluating a patient biological sample for one or more FGFR mutants from a panel of FGFR mutant genes comprising FGFR3 FGFR3-TACC3 v1, FGFR3-TACC3 v3, or FGFR3-BAIAP2L1, and wherein said evaluation comprises amplifying a cDNA with a pair of primers that amplify the one or more FGFR mutants from the panel of FGFR mutant genes; and determining whether the one or more FGFR mutants from the panel of FGFR mutant genes are present in the sample, wherein the presence of the one or more FGFR mutants indicates that the patient is responsive to treatment with the FGFR inhibitor; or (b) evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from a panel of FGFR mutant genes comprising FGFR3 FGFR3-TACC3 v1, FGFR3-TACC3 v3, or FGFR3-BAIAP2L1, wherein the presence of one or more FGFR mutants indicates that the patient is responsive to treatment with the FGFR inhibitor. 2. Method according to claim 1, characterized by the fact that the bladder cancer is metastatic bladder cancer. 3. Method according to claim 1 or 2, characterized in that the evaluation comprises amplifying the cDNA with a pair of primers that bind and amplify one or more FGFR mutants from the panel of FGFR mutant genes. 4. Method according to claim 3, characterized by the fact that the cDNA is a pre-amplified cDNA. 5. Method according to any one of claims 1 to 7, characterized by the fact that the FGFR3 mutant and the primer pair are: FGFR3:TACC3 v1 and primers having the sequences of SEQ ID NO: 5 and SEQ ID NO : 6; or FGFR3:TACC3 v3 and primers having the sequences of SEQ ID NO:7 and SEQ ID NO:8; or FGFR3:BAIAP2L1 and primers having the sequences of SEQ ID NO: 11 and SEQ ID NO: 12. 6. Method, according to any one of claims 1 to 5, characterized by the fact that the evaluation comprises: isolating an RNA from the biological sample and synthesizing cDNA from the isolated RNA. 7. Method, according to claim 6, characterized by the fact that it further comprises pre-amplifying the cDNA before the amplification step. 8. Method according to any one of claims 1 to 7, characterized in that the cDNA is pre-amplified. 9. Method according to any one of claims 1 to 8, characterized by the fact that the amplification step comprises executing a real-time PCR. 10. Method according to claim 9, characterized by the fact that real-time PCR is performed with one or more probes comprising SEQ ID NO: 43 or SEQ ID NO: 44, or SEQ ID NO: 47. 11. Method according to any one of claims 1 to 10, characterized by the fact that said determination step comprises sequencing the amplified cDNA. 12. Kit for carrying out the method as defined in claim 1, characterized by the fact that it comprises: pairs of primers with the sequences of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or SEQ ID NO: 11 and SEQ ID NO: 12; and instructions for performing an assay to detect one or more FGFR mutant genes. 13. Kit according to claim 2, characterized by the fact that it further comprises one or more probes, one or more 3' blocking oligonucleotides, or both. 14. Kit, according to claim 13, characterized by the fact that: a. the primer pair has the sequences SEQ ID NO: 5 and SEQ ID NO: 6 and the probe has the sequence of SEQ ID NO: 43; B. the primer pair has the sequences SEQ ID NO:7 and SEQ ID NO:8 and the probe has the sequence SEQ ID NO: 44; w. the primer pair has the sequences SEQ ID NO: 11 and SEQ ID NO: 12 and the probe has the sequence of SEQ ID NO: 47; or d. any combination thereof. 15. Method according to any one of claims 1 to 11, characterized in that the FGFR inhibitor must be administered to the patient identified as responsive to treatment with the FGFR inhibitor.