US20160090633A1 - Use of fgfr mutant gene panels in identifying cancer patients that will be responsive to treatment with an fgfr inhibitor - Google Patents

Use of fgfr mutant gene panels in identifying cancer patients that will be responsive to treatment with an fgfr inhibitor Download PDF

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US20160090633A1
US20160090633A1 US14/858,627 US201514858627A US2016090633A1 US 20160090633 A1 US20160090633 A1 US 20160090633A1 US 201514858627 A US201514858627 A US 201514858627A US 2016090633 A1 US2016090633 A1 US 2016090633A1
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Suso Jesus PLATERO
Jayaprakash Karkera
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Janssen Pharmaceutica NV
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Priority to US16/136,201 priority patent/US20190078166A1/en
Priority to US16/723,975 priority patent/US20200208224A1/en
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • fibroblast growth factor receptor inhibitor Provided herein are methods of identifying a cancer patient that will be responsive to treatment with a fibroblast growth factor receptor inhibitor and methods of treating the same.
  • FGFRs Fibroblast growth factor receptors
  • FGFR fibroblast growth factor receptor
  • Also disclosed are methods of treating cancer in a patient comprising: evaluating a biological sample from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel; and treating the patient with an FGFR inhibitor if one or more FGFR mutants are present in the sample.
  • Kits and primers for identifying the presence of one or more FGFR mutant genes in a biological sample are further provided herein.
  • FIG. 1 is an illustration of exemplary FGFR fusion genes, the presence of at least one of which indicates that a patient will be responsive to treatment with an FGFR inhibitor. Also illustrated (small arrows) are exemplary primer locations for amplifying the fusion genes.
  • FIG. 2 represents Sanger sequencing results from FFPET samples positive for: A) FGFR3:TACC3 v1; B) FGFR3:TACC3 v3; C) FGFR3:TACC3 Intron; D) FGFR3:BAIAP2L1; E) FGFR2:AFF3; F) FGFR2:BICC1; G) FGFR2:CASP7; H) FGFR2:CCDC6; and I) FGFR2:OFD1.
  • FIG. 3 illustrates an exemplary strategy for SNP-specific qRT-PCR using a 3′ dideoxy wild type (WT) blocker oligonucleotide.
  • FIG. 4 illustrates an exemplary analytical validation strategy for detecting FGFR SNPs. Experiments were performed on engineered RK3E cell lines expressing the FGFR fusions and diluted into a wild type cell line harboring no FGFR3/FGFR2 fusions.
  • FIG. 5 comprising FIGS. 5A-5D , illustrates SNP-specific PCR with dideoxy WT blocker for (a) G370C, (B) Y373C, (C) S249C, and (D) R248C.
  • FIG. 6 represents efficiency standard curves for the FGFR fusion gene assays: A) FGFR3:TACC3 v1; B) FGFR3:TACC3 v3; C) FGFR3:TACC3 Intron; D) FGFR3:BAIAP2L1; E) FGFR2:AFF3; F) FGFR2:BICC1; G) FGFR2:CASP7; H) FGFR2:CCDC6; and I) FGFR2:OFD1.
  • FIG. 7 is an exemplary representation of FGFR fusion gene status in bladder (primary and metastatic), NSCLC (adenocarcinoma and squamous), ovarian, esophageal (primary and metastatic), head and neck (H&N; primary and metastatic), endometrial (metastatic), breast, and prostate cancer.
  • FIG. 8 is an exemplary representation of FGFR fusion gene and mutation status in NSCLC adenocarcinoma and squamous cell carcinoma.
  • FIG. 9 represents exemplary results from phase I patient samples.
  • Assays were performed using synthetic template assay control (ST), primers for GAPDH (quality control sample), or primers specific for: A) FGFR2:BICC1 fusions; B) FGFR3:TACC3 (exon 18:exon 1) fusions; C) FGFR2:CCDC6 fusions; or D) FGFR3:TACC3 v1, FGFR3:TACC3 v3, or FGFR2:CCDC6 fusions.
  • ST synthetic template assay control
  • B FGFR3:TACC3 (exon 18:exon 1) fusions
  • C FGFR2:CCDC6 fusions
  • D FGFR3:TACC3 v1, FGFR3:TACC3 v3, or FGFR2:CCDC6 fusions.
  • FIG. 10 represents an exemplary Phase I Study design for a First-In-Human Study of JNJ-42756493 in patients with advanced solid tumor.
  • FIG. 11 represents the maximal inhibitory percentage reduction of sum of the diameters of targeted lesions from baseline with dose level greater than or equal to 6 mg.
  • Solid tumor patients were treated with the FGFR inhibitor JNJ-42756493 at different doses administered either as a daily regimen or as an intermittent dosing regimen (7 days on-7 days off). Doses and tumor types are indicated. Reduction in tumor was measured as per the RECIST criteria. Patients whose tumors harbor FGFR gene translocations and mutations appear to be more sensitive to the FGFR inhibitor JNJ-42756493.
  • FIG. 12 illustrates the expression of various FGFR fusions in RK3E cells stably transfected with the indicated FGFR fusion.
  • FIG. 13 comprising FIGS. 13A-13B , illustrates colony formation assays in RK3E cells stably transfected with the indicated FGFR fusion.
  • A 0.1% cresyl crystal violet stained 6-well chambers and
  • B bar graph illustrating the number of colonies/100 cells plated. Results are representative of two independent experiments.
  • FIG. 14 comprising FIGS. 14A-14H , illustrates the expression of exemplary downstream targets in RK3E cells stably transfected with the indicated FGFR fusion.
  • references to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.
  • another embodiment includes from the one particular value and/or to the other particular value.
  • reference to values stated in ranges include each and every value within that range. All ranges are inclusive and combinable.
  • FGFR fibroblast growth factor receptor
  • LLOQ low limit of quantitation
  • FGFR3:TACC3 fusion between genes encoding FGFR3 and transforming acidic coiled-coil containing protein 3
  • FGFR3:BAIAP2L1 fusion between genes encoding FGFR3 and brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 1
  • FGFR2:AFF3 fusion between genes encoding FGFR2 and AF4/FMR2 family, member 3
  • FGFR2:BICC1 fusion between genes encoding FGFR2 and bicaudal C homolog 1
  • FGFR2: CASP7 fusion between genes encoding FGFR2 and caspase 7
  • FGFR2:CCDC6 fusion between genes encoding FGFR2 and coiled-coil domain containing 6
  • FGFR2:OFD1 fusion between genes encoding FGFR2 and oral-facial-digital syndrome 1
  • treating and like terms refer to reducing the severity and/or frequency of cancer symptoms, eliminating cancer symptoms and/or the underlying cause of said symptoms, reducing the frequency or likelihood of cancer symptoms and/or their underlying cause, and improving or remediating damage caused, directly or indirectly, by cancer.
  • Bio samples refers to any sample from a patient in which cancerous cells can be obtained and RNA can be isolated. Suitable biological samples include, but are not limited to, blood, lymph fluid, bone marrow, a solid tumor sample, or any combination thereof. In some embodiments, the biological sample can be FFPET.
  • pre-amplification refers to a PCR procedure that is performed prior to the amplifying step in order to increase the quantity of template cDNA for the amplification step.
  • a pre-amplification step can be performed, for example, using the TaqMan® PreAmp Master Mix (Life Technologies/Applied Biosystems® product #4391128).
  • amplifying refers to the generation of numerous identical copies of a nucleic acid sample. Suitable techniques for amplifying a nucleic acid sample include, but are not limited to, polymerase chain reaction (PCR) and real-time polymerase chain reaction (RT-PCR). In some embodiments, the amplifying step comprises RT-PCR.
  • PCR polymerase chain reaction
  • RT-PCR real-time polymerase chain reaction
  • FGFR mutant refers to a FGFR fusion gene, a FGFR single nucleotide polymorphism, or both.
  • FGFR fusion or “FGFR fusion gene” refers to a gene encoding FGFR (e.g., FGRF2 or FGFR3), or a portion thereof, and one of the herein disclosed fusion partners, or portion thereof, created by a translocation between the two genes.
  • Table 1 provides the FGFR fusion genes and the FGFR and fusion partner exons that are fused.
  • FIG. 1 provides an illustration of the various FGFR fusion genes. The sequences of the individual FGFR fusion genes are disclosed in Table 16.
  • FGFR single nucleotide polymorphism refers to a FGFR2 or FGFR3 gene in which a single nucleotide differs among individuals.
  • FGFR single nucleotide polymorphism refers to a FGFR3 gene in which a single nucleotide differs among individuals.
  • the presence of one or more of the following FGFR SNPs in a biological sample from a patient can be determined using the disclosed methods: FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, FGFR3 Y373C, or any combination thereof.
  • the sequences of the FGFR SNPs are provided in Table 2.
  • FGFR mutant gene panel includes one or more of the above listed FGFR mutants. In some embodiments, the FGFR mutant gene panel is dependent upon the patient's cancer type.
  • FGFR mutant panel that is used in the evaluating step of the disclosed methods is based, in part, on the patient's cancer type.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGER3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.
  • primers that are complementary, and bind to, a 5′ and 3′ region of the nucleic acid strand that flanks the region sought to be amplified.
  • “pair of primers” refers to the forward and reverse primers used in an amplifying step. Pairs of primers suitable for performing the disclosed methods are listed in Table 3.
  • primers having the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or any combination thereof
  • sets of primers having the sequences of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, or any combination thereof.
  • the set of primers can have the sequence of SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, the set of primers can have the sequence of SEQ ID NO:7 and SEQ ID NO:8. In some embodiments, the set of primers can have the sequence of SEQ ID NO:9 and SEQ ID NO:10. In some embodiments, the set of primers can have the sequence of SEQ ID NO:11 and SEQ ID NO:12. In some embodiments, the set of primers can have the sequence of SEQ ID NO:13 and SEQ ID NO:14. In some embodiments, the set of primers can have the sequence of SEQ ID NO:15 and SEQ ID NO:16.
  • the set of primers can have the sequence of SEQ ID NO:17 and SEQ ID NO:18. In some embodiments, the set of primers can have the sequence of SEQ ID NO:19 and SEQ ID NO:20. In some embodiments, the set of primers can have the sequence of SEQ ID NO:21 and SEQ ID NO:22. In some embodiments, the set of primers can have the sequence of SEQ ID NO:23 and SEQ ID NO:24. In some embodiments, the set of primers can have the sequence of SEQ ID NO:25 and SEQ ID NO:26. In some embodiments, the set of primers can have the sequence of SEQ ID NO:27 and SEQ ID NO:28.
  • the set of primers can have the sequence of SEQ ID NO:29 and SEQ ID NO:30. In some embodiments, the set of primers can have the sequence of SEQ ID NO:31 and SEQ ID NO:32. In some embodiments, the set of primers can have the sequence of SEQ ID NO:33 and SEQ ID NO:34. In some embodiments, the set of primers can have the sequence of SEQ ID NO:35 and SEQ ID NO:36. In some embodiments, the set of primers can have the sequence of SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, the set of primers can have the sequences of any combination of the above sets of primers.
  • FGFR inhibitors for use in the disclosed methods are provided herein.
  • the patient can be treated with a FGFR inhibitor disclosed in U.S. Publ. No. 2013/0072457 A1 (incorporated herein by reference), including any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof (suitable R groups are also disclosed in U.S. Publ. No. 2013/0072457 A1).
  • a FGFR inhibitor disclosed in U.S. Publ. No. 2013/0072457 A1 (incorporated herein by reference), including any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof (suitable R groups are also disclosed in U.S. Publ. No. 2013/0072457 A1).
  • the patient can be treated with N-(3,5-dimethoxyphenyl)-N′-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl]ethane-1,2-diamine (referred to herein as “JNJ-42756493” or “JNJ493”):
  • the patient can be treated with JNJ493 base.
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is N-[5-[2-(3,5-Dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3,5-diemthylpiperazin-1-yl)benzamide (AZD4547), as described in Gavine, P. R., et al., AZD4547: An Orally Bioavailable, Potent, and Selective Inhibitor of the Fibroblast Growth Factor Receptor Tyrosine Kinase Family, Cancer Res. Apr. 15, 2012 72; 2045:
  • any tautomeric or stereochemically isomeric form thereof including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is 3-(2,6-Dichloro-3,5-dimethoxy-phenyl)-1- ⁇ 6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimid-4-yl ⁇ -1-methyl-urea (NVP-BGJ398) as described in Int'l Publ. No. WO2006/000420:
  • any tautomeric or stereochemically isomeric form thereof including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is 4-amino-5-fluoro-3-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-quinolin-2-one (dovitinib) as described in Int't Publ. No. WO2006/127926:
  • any tautomeric or stereochemically isomeric form thereof including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is 6-(7-((1-Aminocyclopropyl)-methoxy)-6-methoxyquinolin-4-yloxy)-N-methyl-1-naphthamide (AL3810) (lucitanib; E-3810), as described in Bello, E. et al., E-3810 Is a Potent Dual Inhibitor of VEGFR and FGFR that Exerts Antitumor Activity in Multiple Preclinical Models, Cancer Res Feb. 15, 2011 71(A)1396-1405 and Int'l Publ. No. WO2008/112408:
  • any tautomeric or stereochemically isomeric form thereof including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, and a N-oxide thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof.
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is an anti-FGFR2 antibody such as that described in WO2013/076186.
  • Additional suitable FGFR inhibitors include BAY1163877 (Bayer), BAY1179470 (Bayer), TAS-120 (Taiho), ARQ087 (ArQule), ASP5878 (Astellas), FF284 (Chugai), FP-1039 (GSK/FivePrime), Blueprint, LY-2874455 (Lilly), RG-7444 (Roche), or any combination thereof, including, when chemically possible, any tautomeric or stereochemically isomeric forms thereof, N-oxides thereof, pharmaceutically acceptable salts thereof, or solvates thereof.
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is BAY1163877 (Bayer), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is BAY1179470 (Bayer), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.
  • BAY1179470 Bayer
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is TAS-120 (Taiho), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.
  • the FGFR inhibitor is TAS-120 (Taiho)
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is ARQ087 (ArQule), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.
  • ARQ087 ArQule
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is ASP5878 (Astellas), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.
  • FGFR inhibitor is ASP5878 (Astellas)
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is FF284 (Chugai), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.
  • FGFR inhibitor is FF284 (Chugai)
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is FP-1039 (GSK/FivePrime), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.
  • FGFR inhibitor is FP-1039 (GSK/FivePrime)
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is Blueprint, including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is LY-2874455 (Lilly), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.
  • FGFR inhibitor is LY-2874455 (Lilly)
  • the patient can be treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is RG-7444 (Roche), including, when chemically possible, any tautomeric or stereochemically isomeric form thereof, N-oxide thereof, pharmaceutically acceptable salt thereof, or solvate thereof.
  • FGFR inhibitor is RG-7444 (Roche)
  • Salts can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002, which is incorporated herein by reference.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.
  • the FGFR inhibitors for use in the disclosed methods may exist as mono- or di-salts depending upon the pKa of the acid from which the salt is formed.
  • Acid addition salts may be formed with a wide variety of acids, both inorganic and organic.
  • Examples of acid addition salts include salts formed with an acid including, but not limited to, acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g.
  • L-glutamic L-glutamic
  • ⁇ -oxoglutaric glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic
  • lactic e.g. (+)-L-lactic, ( ⁇ )-DL-lactic
  • lactobionic maleic, malic, ( ⁇ )-L-malic, malonic, ( ⁇ )-DL-mandelic, methanesulphonic, naphthalenesulphonic (e.g.
  • naphthalene-2-sulphonic naphthalene-1,5-disulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, pyruvic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, (+)-L-tartaric, thiocyanic, toluenesulphonic (e.g. p-toluenesulphonic), undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.
  • toluenesulphonic e.g. p-toluenesulphonic
  • undecylenic and valeric acids as well as acylated amino acids and cation exchange resins.
  • salts consist of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulphuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulphonic, toluenesulphonic, methanesulphonic (mesylate), ethanesulphonic, naphthalenesulphonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids.
  • Another group of acid addition salts includes salts formed from acetic, adipic, ascorbic, aspartic, citric, DL-Lactic, fumaric, gluconic, glucuronic, hippuric, hydrochloric, glutamic, DL-malic, methanesulphonic, sebacic, stearic, succinic and tartaric acids.
  • a salt may be formed with a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ .
  • suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ).
  • 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(CH 3 ) 4 + .
  • the compounds may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the disclosed compounds.
  • Compounds containing an amine function may also form N-oxides.
  • a reference herein to a compound that contains an amine function also includes the N-oxide.
  • one or more than one nitrogen atom may be oxidised to form an N-oxide.
  • Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle.
  • N-Oxides can be formed by treatment of the corresponding amine with an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry, by Jerry March, 4 th Edition, Wiley Interscience, pages. More particularly, N-oxides can be made by the procedure of L. W. Deady ( Syn. Comm. (1977), 7, 509-514) in which the amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent such as dichloromethane.
  • MCPBA m-chloroperoxybenzoic acid
  • solvate means a physical association of the compound with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid.
  • solvate is intended to encompass both solution-phase and isolatable solvates.
  • suitable solvates include the disclosed compounds in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, ethanolamine and the like. The compound may exert its biological effects while in solution.
  • Solvates are well known in pharmaceutical chemistry. They can be important to the processes for the preparation of a substance (e.g. in relation to their purification), the storage of the substance (e.g. its stability) and the ease of handling of the substance, and are often formed as part of the isolation or purification stages of a chemical synthesis.
  • a person skilled in the art can determine by means of standard and long used techniques whether a hydrate or other solvate has formed by the isolation conditions or purification conditions used to prepare a given compound. Examples of such techniques include thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray crystallography (e.g.
  • the compound may have one or more polymorph (crystalline) or amorphous forms.
  • the compounds include compounds with one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element.
  • a reference to hydrogen includes within its scope 1 H, 2 H (D), and 3 H (T).
  • references to carbon and oxygen include within their scope respectively 12 C, 13 C and 14 C and 16 O and 18 O.
  • the isotopes may be radioactive or non-radioactive.
  • the compounds contain no radioactive isotopes. Such compounds are preferred for therapeutic use.
  • the compound may contain one or more radioisotopes. Compounds containing such radioisotopes may be useful in a diagnostic context.
  • the patient is treated with a FGFR inhibitor if one or more FGFR mutants are present in the sample, wherein the FGFR inhibitor is N-(3,5-dimethoxyphenyl)-N′-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl]ethane-1,2-diamine (referred to herein “JNJ-42756493”), or a pharmaceutically acceptable salt thereof or a solvate thereof.
  • the FGFR inhibitor is N-(3,5-dimethoxyphenyl)-N′-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl]ethane-1,2-diamine
  • Disclosed herein are methods of treating cancer in a patient comprising: evaluating a biological sample from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel; and treating the patient with an FGFR inhibitor if one or more FGFR mutants are present in the sample.
  • the disclosed methods can be used to treat a variety of cancer types including, but not limited to, bladder cancer, metastatic bladder cancer, ovarian cancer, head and neck cancer, metastatic head and neck cancer, esophageal cancer, metastatic esophageal cancer, non-small-cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, prostate cancer, lung cancer, gastric cancer, urothelial carcinoma, small cell lung cancer, breast cancer, endometrial cancer, metastatic endometrial cancer, cholangiocarcinoma, hepatocellular carcinoma, glioblastoma, gliomas, colon carcinoma, sarcomas, solid tumors of squamous origin, and multiple myeloma.
  • 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
  • FGFR mutant panel that is used in the evaluating step is based, in part, on the patient's cancer type.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample.
  • a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having bladder cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample.
  • a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having metastatic bladder cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample.
  • a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having ovarian cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample.
  • a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having head and neck cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastic head and neck cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample.
  • a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having esophageal cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample.
  • a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample.
  • a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample.
  • a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having NSCL adenocarcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample.
  • a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample.
  • a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample.
  • a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample.
  • a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample.
  • a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having NSCL squamous cell carcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • the evaluating step comprises: isolating RNA from the biological sample; synthesizing cDNA from the isolated RNA; pre-amplifying the cDNA; and amplifying the pre-amplified cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel.
  • RNA can be isolated from the biological sample using an AllPrep DNA/RNA FFPE Kit from Qiagen (product #80234)
  • cDNA can be synthesized from isolated RNA using a High Capacity cDNA Reverse Transcriptase Kit with RNase Inhibitor from ABI (product #4374966).
  • Pre-amplification of cDNA can be performed by a number of procedures known to one skilled in the art. Amplification procedures are well known in the art.
  • cDNA can be pre-amplified using a TaqMan® PreAmp Master Mix (Life Technologies/Applied Biosystems® product #4391128).
  • the amplifying step can comprise performing real-time PCR (qRT-PCR).
  • qRT-PCR real-time PCR
  • Exemplary qRT-PCR procedures are discussed in the Example section herein.
  • the qRT-PCR can be a Taqman® Real-Time PCR assay.
  • qRT-PCR procedures can involve the use of probes to increase the specificity of the assay.
  • Suitable probes for use in the qRT-PCR assay include any of the probes disclosed herein, for example, the probes disclosed in Table 15.
  • the real-time PCR can be performed with one or more probes comprising SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55.
  • the real-time PCR can be performed with one or more probes consisting essentially of SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55.
  • the real-time PCR can be performed with one or more probes consisting of SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55.
  • the real-time PCR can be performed with one or more probes having SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55.
  • the qRT-PCR can be performed with one or more 3′ blocking oligonucleotides.
  • Exemplary qRT-PCR procedures using 3′ blocking oligonucleotides are disclosed in the Example section herein.
  • Suitable 3′ blocking oligonucleotides include, for example, those disclosed in Table 8.
  • the qRT-PCR can be performed with one or more 3′ blocking oligonucleotides comprising SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and/or SEQ ID NO: 42.
  • the qRT-PCR can be performed with one or more 3′ blocking oligonucleotides consisting essentially of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and/or SEQ ID NO: 42. In some embodiments, the qRT-PCR can be performed with one or more 3′ blocking oligonucleotides consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and/or SEQ ID NO: 42. In some embodiments, the qRT-PCR can be performed with one or more 3′ blocking oligonucleotides having SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and/or SEQ ID NO: 42.
  • Suitable pairs of primers for use in the amplifying step include those disclosed in Table 3.
  • the FGFR mutant and pair of primers can be FGFR3:TACC3 v1 and primers having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6.
  • the FGFR mutant and pair of primers can be FGFR3:TACC3 v3 and primers having the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8.
  • the FGFR mutant and pair of primers can be FGFR3:TACC3 Intron and primers having the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10.
  • the FGFR mutant and pair of primers can be FGFR3: BAIAP2L1 and primers having the amino acid sequences of SEQ ID NO:11 and SEQ ID NO:12.
  • the FGFR mutant and pair of primers can be FGFR2:BICC1 and primers having the amino acid sequences of SEQ ID NO:13 and SEQ ID NO:14.
  • the FGFR mutant and pair of primers can be FGFR2:AFF3 and primers having the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:16.
  • the FGFR mutant and pair of primers can be FGFR2:CASP7 and primers having the amino acid sequences of SEQ ID NO:17 and SEQ ID NO:18. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:CCDC6 and primers having the amino acid sequences of SEQ ID NO:19 and SEQ ID NO:20. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:OFD1 and primers having the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22.
  • the FGFR mutant and pair of primers can be R248C and primers having the amino acid sequences of SEQ ID NO:23 and SEQ ID NO:24 or SEQ ID NO:31 and SEQ ID NO:32.
  • the FGFR mutant and pair of primers can be S249C and primers having the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:33 and SEQ ID NO:34.
  • the FGFR mutant and pair of primers can be G370C and primers having the amino acid sequences of SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:35 and SEQ ID NO:36.
  • the FGFR mutant and pair of primers can be Y373C and primers having the amino acid sequences of SEQ ID NO:29 and SEQ ID NO:30 or SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, the FGFR mutant and pair of primers can be any combination of the above disclosed FGFR mutants and corresponding pair of primers.
  • the amplifying step can be performed with the following:
  • the disclosed methods comprise treating a patient if one or more FGFR mutants are present in the sample.
  • the presence of one or more FGFR mutants in the sample can be determined by, for example, sequencing the amplified cDNA.
  • Suitable FGFR inhibitors for use in the treatment methods include those previously described herein.
  • FGFR inhibitors for use in the treatment of cancer in a patient wherein the patient is identified as being responsive to treatment with the FGFR inhibitor by evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel, wherein the presence of the one or more FGFR mutants in the sample is detected.
  • FGFR inhibitors for use in the treatment of cancer in a patient wherein the patient is identified as being responsive to treatment with the FGFR inhibitor by evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel, wherein the one or more FGFR mutants are a FGFR fusion gene or a FGFR SNP, wherein the presence of the one or more FGFR mutants in the sample is detected, and wherein said evaluating comprises amplifying cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel.
  • FGFR inhibitors for use in the treatment of cancer in a patient wherein the patient is identified as being responsive to treatment with the FGFR inhibitor by evaluating a biological sample obtained from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel, wherein the FGFR mutant is a FGFR fusion gene or a FGFR SNP, wherein the presence of one or more FGFR mutants in the sample is detected, and wherein said evaluating comprises amplifying a pre-amplified cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel.
  • FGFR fibroblast growth factor receptor
  • Also provided are methods of identifying a cancer patient that is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor comprising: evaluating a biological sample from the patient for a FGFR mutant from a FGFR mutant gene panel, wherein the FGFR mutant is a FGFR fusion gene or a FGFR single nucleotide polymorphism, and wherein said evaluating comprises amplifying cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel and determining whether the one or more FGFR mutants from the gene panel are present in the sample, wherein the presence of the one or more FGFR mutants indicates that the patient is responsive to treatment with the FGFR inhibitor.
  • FGFR fibroblast growth factor receptor
  • FGFR fibroblast growth factor receptor
  • the evaluating can comprise amplifying cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel.
  • the cDNA can be pre-amplified cDNA.
  • the evaluating step comprises: isolating RNA from the biological sample and synthesizing cDNA from the isolated RNA.
  • the evaluating step can be performed on preamplified cDNA.
  • the evaluating step can further comprise pre-amplifying the cDNA prior to said amplifying step.
  • Isolating RNA from a biological sample can be performed by a number of procedures known to one skilled in the art.
  • RNA can be isolated from the biological sample using an AllPrep DNA/RNA FFPE Kit from Qiagen (for example, product #80234). Synthesizing cDNA from isolated RNA can be performed by a number of procedures known to one skilled in the art.
  • cDNA can be synthesized from isolated RNA using a High Capacity cDNA Reverse Transcriptase Kit with RNase Inhibitor from ABI (for example, product #4374966). Pre-amplification of cDNA can be performed by a number of procedures known to one skilled in the art. Amplification procedures are well known in the art. In one embodiment, cDNA can be pre-amplified using a TaqMan® PreAmp Master Mix (Life Technologies/Applied Biosystems® product #4391128).
  • FGFR fibroblast growth factor receptor
  • 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.
  • FGFR fibroblast growth factor receptor
  • the FGFR mutant panel that is used in the evaluating step is based, in part, on the patient's cancer type.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having bladder cancer.
  • the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having bladder cancer.
  • the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having bladder cancer. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants are present in a biological sample from a patient having bladder cancer.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having metastatic bladder cancer.
  • the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient having metastatic bladder cancer.
  • the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having metastatic bladder cancer. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants are present in a biological sample from a patient having metastatic bladder cancer.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having ovarian cancer.
  • the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient having ovarian cancer.
  • the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having ovarian cancer. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants is present in a biological sample from a patient having ovarian cancer.
  • a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having head and neck cancer.
  • the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having head and neck cancer.
  • the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having head and neck cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having head and neck cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having head and neck cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having head and neck cancer. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants is present in a biological sample from a patient having head and neck cancer.
  • a suitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic head and neck cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastic head and neck cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having esophageal cancer.
  • the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having esophageal cancer.
  • the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having esophageal cancer. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants is present in a biological sample from a patient having esophageal cancer.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof. Accordingly, in some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample.
  • a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastatic esophageal cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having NSCL adenocarcinoma.
  • the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3:TACC3 Intron is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient having NSCL adenocarcinoma.
  • the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having NSCL adenocarcinoma.
  • the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having NSCL adenocarcinoma. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants is present in a biological sample from a patient having NSCL adenocarcinoma.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • the evaluating step comprises determining whether FGFR3:TACC3 v1 is present in a biological sample from a patient having NSCL squamous cell carcinoma.
  • the evaluating step comprises determining whether FGFR3:TACC3 v3 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3:BAIAP2L1 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR2:BICC1 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR2:AFF3 is present in a biological sample from a patient having NSCL squamous cell carcinoma.
  • the evaluating step comprises determining whether FGFR2:CASP7 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR2:CCDC6 is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 R248C is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 S249C is present in a biological sample from a patient having NSCL squamous cell carcinoma.
  • the evaluating step comprises determining whether FGFR3 G370C is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether FGFR3 Y373C is present in a biological sample from a patient having NSCL squamous cell carcinoma. In some embodiments, the evaluating step comprises determining whether any combination of the above FGFR mutants is present in a biological sample from a patient having NSCL squamous cell carcinoma.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample.
  • a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having metastatic endometrial cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample.
  • a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having breast cancer is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:CCDC6 is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present in the sample.
  • a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present in the sample. In some embodiments, a patient having hepatocellular carcinoma is treated with an FGFR inhibitor if any combination of the above FGFR mutants is present in the sample.
  • Suitable pairs of primers for use in the amplifying step include those disclosed in Table 3.
  • the FGFR mutant and pair of primers can be FGFR3:TACC3 v1 and primers having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6.
  • the FGFR mutant and pair of primers can be FGFR3:TACC3 v3 and primers having the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8.
  • the FGFR mutant and pair of primers can be FGFR3:TACC3 Intron and primers having the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10.
  • the FGFR mutant and pair of primers can be FGFR3:BAIAP2L1 and primers having the amino acid sequences of SEQ ID NO:11 and SEQ ID NO:12.
  • the FGFR mutant and pair of primers can be FGFR2:BICC1 and primers having the amino acid sequences of SEQ ID NO:13 and SEQ ID NO:14.
  • the FGFR mutant and pair of primers can be FGFR2:AFF3 and primers having the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:16.
  • the FGFR mutant and pair of primers can be FGFR2:CASP7 and primers having the amino acid sequences of SEQ ID NO:17 and SEQ ID NO:18. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:CCDC6 and primers having the amino acid sequences of SEQ ID NO:19 and SEQ ID NO:20. In some embodiments, the FGFR mutant and pair of primers can be FGFR2:OFD1 and primers having the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22.
  • the FGFR mutant and pair of primers can be R248C and primers having the amino acid sequences of SEQ ID NO:23 and SEQ ID NO:24 or SEQ ID NO:31 and SEQ ID NO:32.
  • the FGFR mutant and pair of primers can be S249C and primers having the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:33 and SEQ ID NO:34.
  • the FGFR mutant and pair of primers can be G370C and primers having the amino acid sequences of SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:35 and SEQ ID NO:36.
  • the FGFR mutant and pair of primers can be Y373C and primers having the amino acid sequences of SEQ ID NO:29 and SEQ ID NO:30 or SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, the FGFR mutant and pair of primers can be any combination of the above disclosed FGFR mutants and corresponding pair of primers.
  • the disclosed methods comprise determining whether the one or more FGFR mutants from the gene panel are present in the sample.
  • the determining step comprises sequencing the amplified cDNA.
  • the method further comprises treating the patient with an FGFR inhibitor if the one or more FGFR mutants from the gene panel are present in the sample.
  • FGFR inhibitors for use in the treatment methods include those previously described herein, in particular JNJ-42756493.
  • kits for identifying the presence of one or more FGFR mutant genes in a biological sample comprising: pairs of primers having the sequences of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:
  • kits can further comprise one or more probes, one or more 3′ blocking oligonucleotides, or both.
  • the kits can further comprise one or more probes, for example, any one or more of the probes disclosed in Table 15.
  • the kits can further comprise one or more 3′ blocking oligonucleotides, for example, any one or more of the 3′ blocking oligonucleotides disclosed in Table 8.
  • the kits can further comprise one or more probes and one or more 3′ blocking oligonucleotides.
  • the kits can further comprise:
  • oligonucleotide probes having the sequence of any one of SEQ ID NOs:43-55.
  • the oligonucleotide probe can have the sequence of SEQ ID NO:43.
  • the oligonucleotide probe can have the sequence of SEQ ID NO:44.
  • the oligonucleotide probe can have the sequence of SEQ ID NO:45.
  • the oligonucleotide probe can have the sequence of SEQ ID NO:46.
  • the oligonucleotide probe can have the sequence of SEQ ID NO:47.
  • the oligonucleotide probe can have the sequence of SEQ ID NO:48.
  • the oligonucleotide probe can have the sequence of SEQ ID NO:49. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:50. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:51. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:52. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:53. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:54. In some embodiments, the oligonucleotide probe can have the sequence of SEQ ID NO:55.
  • oligonucleotides having the sequence of any one of SEQ ID NOs:39-42.
  • the 3′ blocking oligonucleotide can have the sequence of SEQ ID NO:39.
  • the 3′ blocking oligonucleotide can have the sequence of SEQ ID NO:40.
  • the 3′ blocking oligonucleotide can have the sequence of SEQ ID NO:41.
  • the 3′ blocking oligonucleotide can have the sequence of SEQ ID NO:42.
  • centrifuge capable of 1500 ⁇ g
  • microcentrifuge capable of 1500 ⁇ g
  • pipettors positive-displacement or air-displacement
  • vortexer Spectrophotometer
  • 37° C. shaker/incubator and an oven set to 37° C.
  • frozen bacteria were scraped off of the top of a glycerol stock tube using a sterile pipet tip, streaked onto a LB agar plate, and placed upside down in the oven at 37° C. overnight.
  • DNA plasmids were purified using Qiagen Plasmid DNA Purification protocol. Briefly, a single colony was picked from the streaked plate and incubated in a culture of 5 ml-LB medium containing 50 ⁇ g/ml Kanamycin overnight in a 37° C. shaker at approximately 300 rpm. The bacterial cells were harvested by centrifugation at 6000 ⁇ g for 15 minutes at 4° C., and the pellet was resuspended in 300 ⁇ l of buffer P1. 300 ⁇ l of buffer P2 was added, mixed by inverting the tube 4-6 times, and incubated at RT (room temperature) for 5 minutes.
  • RT room temperature
  • the DNA was precipitated by adding 0.7 volumes of isopropanol, mixed, and centrifuged immediately at 15000 ⁇ g for 30 minutes in a microcentrifuge. The supernatant was decanted, and the DNA pellet was washed in 1 ml of 70% ethanol and centrifuged at 15000 ⁇ g for 10 minutes. The supernatant was decanted. The pellet was air-dried for 5-10 minutes and the DNA was re-dissolved in 100 ⁇ l or suitable volume of nuclease-free water. Plasmid DNA was quantitated by Nanodrop and stored at ⁇ 20° C. until further use.
  • Expression vectors expressing each of the FGFR fusions were constructed. The expression vector was then transfected into normal rat kidney epithelial cells (NRK) cells. The stable cell lines were selected in media containing kanamycin following transfections. These cells were then grown and mRNA was isolated and subjected to FGFR fusion assays to confirm the presence of the specific FGFR fusions mRNA.
  • NRK normal rat kidney epithelial cells
  • 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).
  • biosafety cabinet fitted with vacuum aspiration system; CO 2 Incubator, set to 37° C. with 5% CO 2 ; ⁇ 80° C. freezer; liquid nitrogen tank; water bath, set to 37° C.; and a microscope.
  • tissue culture flasks T75 VWR #BD353136 and/or T150 VWR #15705-074
  • tissue culture 0.2 ⁇ m filtering units Thermo Scientific #566-0020
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS Fetal Bovine Serum
  • PenStrep antibiotic solution Life Technologies #15140-122
  • Trypsin-EDTA 0.25% solution Life Technologies, #25200-056
  • DPBS Dulbecco's Phosphate buffered solution, no calcium, no magnesium
  • cell freezing container for cryopreservation hand held pipetman; cell freezing media (Life Technologies, #12648-010); 15 ml conical tubes (VWR #62406-2); and cryovials (VWR #89094-800).
  • DMEM medium was prepared by combining 445 ml of DMEM, 50 ml of FBS, and 5 ml of PenStrep. The prepared media was passed through a 0.2 ⁇ m filter unit and stored at 4° C.
  • DMEM medium was warmed in the 37° C. water bath for at least 15 minutes and 15 ml of warmed medium was placed into a T75 flask.
  • Cells were removed from liquid nitrogen tank and placed immediately in a 37° C. water bath until just thawed. Cryovials were sprayed generously with 70% alcohol and the excess was wiped with paper towels. The entire content was aliquoted into the T75 flask containing DMEM. Flask was swirled gently to mix and placed in incubator for 24 hours. If the cells were not ready for splitting, the media was changed to freshly prepared DMEM to remove residual freezing media. If cells were ready to split, each cell line was propagated once the flask achieved 80% confluency (splitting ratio for each cell line was dependent upon the experimental needs).
  • the cells were removed from the culture flask and spun down in a 15 ml conical tube for 5 minutes at 1500 RPM at RT. The medium was aspirated and 6 ml of cell freezing medium was added. The cells were mixed by pipetting up and down several times, and 1 ml of cell solution was aliquoted into each of 5 cryovials. Cryovials with cells were placed in a cryofreezing container, which was stored in a ⁇ 80° C. freezer overnight, followed by long term storage in a liquid nitrogen tank.
  • centrifuge with plate adapter capable of 1500 ⁇ g
  • microcentrifuge capable of 1500 ⁇ g
  • pipettors positive-displacement or air-displacement
  • vortexer capable of incubation at 37° C., 56° C. and 80° C.
  • heating block capable of incubation at 37° C., 56° C. and 80° C.
  • pasteur pipette Pier Trans EX-FT 1.5 ml pk 500, VWR #14670-329.
  • AllPrep DNA/RNA FFPE Kit (Qiagen #80234); Absolute Ethanol (Sigma Aldrich #E7023); Isopropanol; Xylene; Nuclease Free Water (Non-DEPC treated) (from IDT or Ambion #AM9932); RNase-free Barrier (Filter) Tips; RNase-free; microtube (1.5 to 2 mL VWR #10011-724); and Qiagen AllPrep DNA/RNA FFPE Kit Handbook.
  • the sample was centrifuged for 2 minutes at maximum speed (12,000-14,000 ⁇ g) and the supernatant was discarded by aspiration. Tubes were capped immediately to avoid tissue from drying.
  • the open tubes were incubated in a heating block for 5-15 minutes at 37° C. to air dry the tissue pellet.
  • the pellet was resuspended by adding 150 ⁇ l Buffer PKD and the tube was flicked to loosen the pellet. 10 ⁇ l proteinase K was added and the tube was mixed by vortexing.
  • Tubes were incubated at 56° C. for 15 minutes, incubated on ice for 3 minutes, and centrifuged for 15 minutes at 20,000 ⁇ g.
  • the supernatant was carefully transferred without disturbing the pellet to a new 1.5 ml microcentrifuge tube for RNA purification.
  • the supernatant was incubated at 80° C. for 15 minutes.
  • the tube was briefly centrifuged to remove drops from the inside of the lid.
  • 320 ⁇ l Buffer RLT was added to adjust binding conditions, and the tube was mixed by vortexing or pipetting.
  • 1120 ⁇ l ethanol (96-100%) was added and the tube was mixed well by vortexing or pipetting.
  • Buffer FRN 350 ⁇ l Buffer FRN was added to the RNeasy MinElute spin column and centrifuged for 15 seconds at ⁇ 8000 ⁇ g ( ⁇ 10,000 rpm). Flow-through was discarded.
  • the DNase I incubation mix (80 ⁇ l) was added directly to the RNeasy MinElute spin column membrane, and placed on the benchtop (20-30° C.) for 15 minutes.
  • Buffer FRN 500 ⁇ l Buffer FRN was added to the RNeasy MinElute spin column and centrifuged for 15 seconds at ⁇ 8000 ⁇ g ( ⁇ 10,000 rpm). The flow-through was saved for use in the next step, as it contains small RNAs.
  • the RNeasy MinElute spin column was placed in a new 2 ml collection tube (supplied). The flow-through from the previous step was applied to the spin column and centrifuged for 15 seconds at ⁇ 8000 ⁇ g ( ⁇ 10,000 rpm). Flow-through was discarded.
  • the RNeasy MinElute spin column was placed in a new 2 ml collection tube and centrifuged at full speed for 5 minutes. The collection tube with the flow-through was discarded.
  • the RNeasy MinElute spin column was placed in a new 1.5 ml collection tube, 30 ⁇ l RNase-free water was added directly to the spin column membrane, incubated for 1 minute at room temperature, and centrifuged at full speed for 1 minute to elute the RNA.
  • RNA samples were immediately stored in ⁇ 80° C. freezer.
  • RT-PCR Real time PCR
  • centrifuge with plate adapter capable of 1500 ⁇ g, microcentrifuge
  • pipettors preferred single and multi-channel pipettor
  • positive-displacement or air-displacement a preferred single and multi-channel pipettor
  • vortexer a preferred single and multi-channel pipettor
  • GeneAmp® PCR System 9700 (ABI #4314879) or equivalent.
  • RNA sample tube(s) were kept on ice.
  • kit components were used to prepare 2 ⁇ Reverse Transcription (RT) Master Mix for all reactions, including 1 negative (water) control. Components were thawed on ice for approximately 15 minutes, gently inverted to mix and centrifuged briefly to bring down the solution. All reagents were returned to the ice. Tubes were not vortexed.
  • RT Reverse Transcription
  • One Master Mix was prepared on ice in a 1.5 ml tube for the appropriate number of reactions (#reactions+10%, per 20- ⁇ L reaction) by combining the following amount of reagent per one reaction: 2 ⁇ l 10 ⁇ RT Buffer Mix; 0.8 ⁇ l 25 ⁇ dNTP Mix; 2 ⁇ l 10 ⁇ RT Random Primers; 1 ⁇ l 50 U/ ⁇ L MultiScribe Reverse Transcriptase; 1 ⁇ l RNase inhibitor; and 3.2 ⁇ l Nuclease/RNase free H 2 O.
  • the Master Mix was vortexed several times (5 to 10) to mix and centrifuged briefly (1500 ⁇ g, 5 to 10 seconds). 10 ⁇ l of the reaction mix was added to the appropriate wells of a 96-well plate.
  • RNA samples were diluted to a concentration of 20 ng/ ⁇ l. 10 ⁇ L of each RNA sample was added, including the water negative control, to the appropriate corresponding wells of the 96-well plate to a final reaction volume of 20 ⁇ L. The wells were mixed gently by pipetting up and down 3 times, sealed with a plate seal, and centrifuged briefly (1500 ⁇ g for 60 seconds). Plates were kept on ice until ready to load in thermocycler.
  • reaction plate was loaded into ABI 9700 Thermal Cycler in Clean Lab or Workstation and run using the following reverse-transcription program with a reaction volume of 20 82 l:
  • Synthesized cDNA was stored at ⁇ 20° C. for next step of Pre-amplification.
  • the preamp assay pool mixture associated with the FFPET SNP Assay Pre-amplification Protocol was prepared as described below.
  • microcentrifuge microcentrifuge
  • pipettors positive-displacement or air-displacement
  • vortexer vortexer
  • the 0.2 ⁇ PreAmp Assay Pool was vortexed briefly to mix (5 to 10 seconds) and centrifuged briefly (1500 ⁇ g, 5-10 seconds). 100 ⁇ L of PreAmp Primer Pool was aliquoted into 1.5 ml tubes and stored at ⁇ 20° C.
  • centrifuge with plate adapter capable of 1500 ⁇ g
  • microcentrifuge capable of 1500 ⁇ g
  • pipettors positive-displacement or air-displacement
  • vortexer GeneAmp® PCR System 9700 (ABI #4314879) or equivalent.
  • Samples were prepared by placing the cDNA and 0.2 ⁇ assay mix pool on ice to thaw, approximately 5 minutes, and centrifuging the plate briefly (1500 ⁇ g for 5 to 10 seconds).
  • kit components were used to prepare 2 ⁇ PreAmp Master Mix.
  • the kit components were allowed to thaw on ice for approximately 5 minutes. After all reagents were thawed, the tubes were gently inverted to mix and briefly centrifuged to bring down the solution. All reagents were returned to the ice. The tubes were not vortexed.
  • each Master Mix was prepared for the appropriate number of reactions on ice by combining the required volumes of reagents as indicated Table 5 below (#reactions+10%):
  • Each Master Mix was vortexed several times (5 to 10) to mix, followed by a brief centrifuge (1500 ⁇ g, 5 to 10 seconds). 18.75 ⁇ L of each Master Mix was aliquoted to the appropriate wells in a 96-well reaction plate. 6.25 ⁇ L of each cDNA samples, including water negative control well, was transferred into the appropriate wells in the Master Mix reaction plate for each preamp reaction. The sample was mixed gently by pipetting up and down 3 times and the cap was closed. The plate was briefly centrifuged (1500 ⁇ g for 60 seconds) and kept on ice until ready to load in thermocycler.
  • reaction plate ABI 9700 Thermal Cycler was loaded and run using the following program:
  • Max Mode was Selected and Ramp Rate was set at 77%.
  • the PreAmp reaction plate was centrifuged briefly (1500 ⁇ g for 60 seconds) after PreAmp completion. 100 ⁇ l of IDTE was added to the appropriate wells of a new deep 96-well plate and 25 ⁇ L of each PreAmp product was transferred to the corresponding wells to have final dilution volume of 125 ⁇ L. The each well was mixed by pipetting up and down 3 times, the plate was sealed with foil adhesive, the plate was centrifuged briefly (1500 ⁇ g for 5 to 10 seconds), and the PreAmp product was stored at ⁇ 20° C. until further use.
  • centrifuge with plate adapter capable of 1500 ⁇ g
  • microcentrifuge capable of 1500 ⁇ g
  • pipettors preferred single and multi-channel pipettor
  • positive-displacement or air-displacement a preferred single and multi-channel pipettor
  • vortexer a preferred single and multi-channel pipettor
  • ABI ViiA 7 real time PCR instrument Life Technologies.
  • Table 15 lists the sequences of the probes used during the Real Time PCR assays.
  • SNP assays were placed on ice to thaw for approximately 5 minutes. All reagents protected from light, to protect exposure of the fluorescent probes. Diluted PreAmp plates were placed on ice to thaw in a Dirty Lab or Workstation after preparing Genotyping Master Mix.
  • Genotyping Master Mix was thawed on ice for approximately 5 minutes.
  • the Master Mix (MM) was prepared in the required number of tubes on ice.
  • the required volumes of reagents were combined in the appropriate labeled tubes as indicated in Table 6 below (# reactions+10%):
  • the Master Mix was vortexed several times (5 to 10) to mix and then centrifuged briefly (1500 ⁇ g, 5 to 10 seconds). 15 ⁇ l of each Master Mix was added to the appropriate wells of a MicroAmpTM Optical 384-Well Reaction Plates. The reaction plates were sealed with optical adhesive film.
  • the plate with 1:5 diluted PreAmp product was placed on ice for approximately 5-10 minutes to thaw. Using a multi-channel pipettor, 5 82 L of each diluted PreAmp product was transferred to the appropriate corresponding wells. The reaction plate was sealed with optical adhesive film and centrifuged briefly (1500 ⁇ g for 60 seconds). Plates were kept on ice until ready to load in thermocycler.
  • RNA based SNP detection assay combined with the pre-amplification step in the assay, boosts the low or the rare mutant signals.
  • FIG. 3 An exemplary strategy for SNP-specific qRT-PCR using a 3′ dideoxy WT blocker oligonucleotide is shown in FIG. 3 , and an exemplary FFPE sample validation strategy is illustrated in FIG. 4 .
  • qRT-PCR was performed using the FGFR SNP primers in the presence of a 3′ dideoxy WT blocker oligonucleotide, which was complementary to, and contained a short stretch of nucleotides flanking, the WT allele. Binding of the blocker oligonucleotide to the WT allele prevented applification of the WT allele, while the FGFR SNP primers bound to and specifically amplified the FGFR SNP.
  • the 3′ dideoxy WT blocker oligonucleotides used in the FGFR SNP-specific qRT-PCR are shown in Table 8.
  • the FGFR SNP primers used in the FGFR SNP-specific qRT-PCR were: SEQ ID NO:31 and SEQ ID NO:32 (FGFR3 R248C); SEQ ID NO:33 and SEQ ID NO:34 (FGFR3 S249C); SEQ ID NO:35 and SEQ ID NO:36 (FGFR3 G370C); and SEQ ID NO:37 and SEQ ID NO:38 (FGFR3 Y373).
  • Table 15 lists the sequences of the probes used during the real time PCR assays.
  • FIGS. 5A-5D Exemplary validation data of the SNP-specific qRT-PCR using a 3′ dideoxy WT blocker oligonucleotide for FGFR3 G370C, FGFR3 Y373, FGFR3 S249C, and FGFR3 R248C is illustrated in FIGS. 5A-5D , respectively.
  • Raw Ct (cycle threshold) data for the FFPE samples with SNP-specific qRT-PCR with 3′ dideoxy WT blocker oligonucleotides are shown in Table 10.
  • the data derived from DNA and RNA using different platforms/techniques suggests that SNP-specific PCR with the 3′ blocking nucleotide is a robust, reliable and a sensitive assay.
  • the validation data suggests that one mutant allele/SNP can be detected in a large excess of WT-bearing genomic DNA, thus emphasizing the sensitivity and the specificity of each assay.
  • FGFR fusion “synthetic mini-genes,” plasmids encoding FGFR fusions, and stable cell lines containing FGFR fusions were generated. Briefly, Synthetic mini genes were artificially constructed by linking a series of nucleotides, of about 100 base pairs, to each other corresponding to the target DNA sequence of the gene of interest. Plasmids encoding FGFR fusions were generated by cloning cDNA encoding the various FGFR fusion genes into an expression vector. Stable cell lines containing FGFR fusions were generated by transfecting plasmids encoding FGFR genes into normal rat kidney epithelial cells (NRK cells). The stable cell lines were selected under the G418 antibiotic.
  • the FGFR fusion Taqman assay was performed on the total RNA isolated from these cell lines to confirm the successful generation of stable cell line(s) expressing the FGFR fusion(s).
  • the stable cell lines expressing FGFR fusions are used a positive control.
  • Table 15 lists the sequences of the probes used during the real time PCR assays.
  • FGFR fusion products were generated by TaqMan PCR (as described in Example 4) and confirmed by Sanger Sequencing ( FIG. 2 ). 100 pg of fusion positive DNA was mixed with normal human cDNA (confirmed fusion-negative), serially diluted 1:10, and analyzed using the Applied Biosystems ViiA7 Software v1.1. Efficiency standard curves are shown in FIG. 6 . FGFR fusion LLOQ and efficiency are shown in Table 11.
  • FGFR fusion gene assay was next validated in fusion gene-positive cell lines.
  • serial dilutions were prepared by spiking fusion protein-positive cells lines into a fusion protein-negative cell line. For example, a 1:2 serial dilution was prepared for both FGFR3:TACC3v1 and FGFR3:BAIAP2L1 and spiked into 1 million BAF cells.
  • RNA was isolated (using Qiagen Rneasy kit), followed by RT-PCR, preamplification of cDNA, and TaqMan Real Time PCR for the targeted FGFR fusion gene.
  • both the FGFR3:TACC3v1 and FGFR3:BAIAP2L1 Fusion Gene TaqMan assays are able to detect the fusion target in 31 out of 1 million fusion-negative cells (sensitivity of 0.003%).
  • the R248C, S249C, and Y373C SNPs were observed in approximately 8%, approximately 61%, and approximately 19% of bladder cancer samples tested, respectively.
  • Table 13 shows the FGFR fusion prevalence in different cancers.
  • FGFR fusions detected in FFPE samples from different cancers such as bladder (primary and metastatic), NSCLC (adenocarcinoma and squamous), ovarian, esophageal (primary and metastatic), head and neck (H&N; primary and metastatic), endometrial (metastatic), breast, and prostate cancer using the qRT-PCR method. All FGFR fusions tested were negative for prostate cancer samples.
  • FGFR3:TACC3intron fusion was negative in bladder (primary), NSCLC (squamous), ovarian and esophageal (primary), H&N (primary and metastatic) and breast.
  • FGFR2:OFD1 fusion was negative in bladder (primary and metastatic), NSCLC (adenocarcinoma), ovarian and esophageal (primary and metastatic).
  • FGFR2:CCDC6 fusion was negative in bladder (primary and metastatic), NSCLC (adenocarcinoma), ovarian and esophageal (primary) and H&N (primary and metastatic)
  • FIG. 8 is an exemplary representation of FGFR fusion gene and mutation status in NSCLC adenocarcinoma and squamous cell carcinoma.
  • FGFR fusion positive NSCLC adenocarcinoma samples 3/17 samples were positive for EGFR mutation, 3/17 samples were positive for KRAS mutation, and 1/17 samples were positive for cMET mutation. No EGFR, KRAS, or cMET mutations, however, were observed in FGFR fusion positive NSCLC squamous cell carcinoma samples.
  • FIG. 9 illustrates exemplary results from phase I patient samples, in which FGFR fusions in Phase I JNJ-427493 (EDI10001) trial samples were detected using the qRT-PCR assay. All FGFR fusion assays were run simultaneously with positive controls (ST) and GAPDH for quality control assessment of the RNA.
  • FIG. 10 represents an exemplary Phase I Study design for a First-In-Human Study of JNJ-42756493 in patients with advanced solid tumor. Shown is a graphical depiction of a traditional 3+3 design dose escalation method for the phase I clinical trial.
  • the dose escalation phase aimed to establish the maximum tolerated dose (MTD) and recommended Phase II dose (RPD).
  • the Part 1 arm was used to determine the intermittent dosing schedule, i.e., 7 days on and seven days off (10 mg/kg and 12 mg/kg).
  • the Part 2 arm was used to determine the PD biomarkers (pharmacodynamics biomarkers; makers examined to link the effect of the drug to the target and biological tumor response) wherein the biopsy and blood sample were tested.
  • the Part 3 arm was used the dose expansion cohort and included accrual of additional patients in specific indications (NSCLC,SCLC, breast and solid tumors) with different eligibility criteria (FGFR aberrations: translocation/mutation/amplifications) to further characterize the toxicity profiles of the JNJ493.
  • RK3E rat kidney epithelial cells
  • ATCC Manassas, Va., USA
  • DMEM fetal bovine serum
  • antibiotics Invitrogen, Grand Island, N.Y., USA
  • FGFR fusion gene constructs were designed and cloned into the pReceiver expression vector (Genecopoeia, Rockville, Md., USA), which contains an HA-tag.
  • Clones were transfected into RK3E cells using the Amaxa Cell Line Nucleofector (Lonza, Basel, Switzerland) following the manufacturer's protocol.
  • the stably transfected cells were selected in complete medium with 800 ug/ml of G418 (Invitrogen).
  • FIG. 12 Overexpression of the fusions in the stably transfected cells was confirmed by real-time PCR and immunoblotting using an anti-pFGFR antibody ( FIG. 12 ). As shown in FIG. 12 , the stable cell lines showed expression of active FGFR fusion kinases, as exhibited by the expression of phosphorylation of FGFR.
  • Anchorage-independent growth of the FGFR fusion stably transfected RK3E cells was tested. 1 ml culture medium with 0.8% low melting point agarose was first plated into each of three wells of a six-well plate. After the agar solidified, each well received another 1 ml of 0.4% agar in culture medium containing 100 cells. After 14 days, colonies were fixed and stained with 0.1% cresyl crystal violet. The number of colonies was determined microscopically by manual counting from triplicate wells for each cell line. A representative view of each fusion-overexpressing cell line is shown in FIG. 13A . Anchorage-independent cell growth in soft-agar could be detected in the FGFR fusion stably transfected cells, but not in the empty vector control.
  • FIG. 13B represents a quantitative analysis of colonies in soft agar for the FGFR fusion stably transfected RK3E cells and empty vector control. All experiments were carried out in duplicate and the results are expressed as colonies/100 cells plated. All of the FGFR fusions tested induced anchorage independent growth, highlighting their transforming ability
  • FGFR fusion stably transfected RK3E cells were plated in complete growth medium, serum starved overnight, then re-fed with 0.5% FBS growth media. Cells were treated with 1 ⁇ M of JNJ-42756493, AZD4547 or NVP-BGJ398 in the presence of ligands for 1 hour. For immunoblotting, whole cell lysates were collected in RIPA buffer (Thermo Scientific, Waltham, Mass., USA) and sample protein concentration was assayed using BCA Protein Assay (Thermo Scientific). Equal amounts of protein (30 pg per lane) were loaded onto on 4-12% Bis-Tris gels (Invitrogen) before an SDS-page was performed.
  • Proteins were transferred to nitrocellulose membranes and probed with antibodies against p-FGFR, total-FGFR2, p-MAPK, total-MAPK, p-S6, total S6, B-actin (Cell Signaling Technology, Danvers, Mass., USA), and total-FGFR3 (Santa Cruz, Dallas, Tex., USA).
  • the membranes were blocked with Odyssey blocking buffer for 1 h at room temperature and incubated overnight at 4° C. in a primary antibody solution diluted in Odyssey blocking buffer (1:1000).
  • FGFR fusion stably transfected RK3E cells were seeded into 96 well plates (1000 cells/well) in triplicates in complete growth medium plus and the ligands FGF-1 and FGF-2. After 24 hours, cells were serum starved overnight, then re-fed with 0.5% FBS growth media. 72 hours after plating, cells were treated with various concentrations of an 18 point 1:3 dilution series, starting at 10 ⁇ M, of JNJ493, AZD4547 (AZD), and NVP-BGJ398 (NVS).
  • the Microtiter plates were then incubated for 72 hours and assayed for adenosine triphosphate (ATP; a marker of metabolically active cells) content using the Cell Titer-Glo® Luminescent Cell Viability assay (Promega Corp., Madison, Wis., USA) following the manufacturer's instructions, with modifications. Briefly, cells were allowed to equilibrate to room temperature, at which time a 1:1 mixture of Cell Titer-Glo® reagent was added. Cells were then placed on an orbital shaker for 2 minutes and incubated for 10 minutes at room temperature to stabilize the luminescent signal. The luminescence was quantified and measurements were conducted using an Envision Multilabel plate reader (Perkin Elmer; Waltham, Mass., USA).
  • ATP adenosine triphosphate
  • IC 50 values (shown in Table 14) were calculated using GraphPad Prism 5.0. As shown in Table 14, cells harboring the FGFR fusions showed sensitivity to the FGFR inhibitor JNJ-42756493, AZD4547 and NVP-BGJ398 in vitro, with JNJ-42756493 exhibiting enhanced sensitity (nanomolar concentration range) when compared to AZD4547 and NVP-BGJ398, whereas the empty vector control did not.
  • the nucleotide sequences for the FGFR fusion cDNA that were engineered into expression vectors is provided in Table 16.
  • the underlined sequences correspond to either FGFR3 or FGFR2, the sequences in normal font represent the fusion partners and the sequence in italic fonts represent the intron sequence of the FGFR3 gene.

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US11707463B2 (en) 2017-06-02 2023-07-25 Janssen Pharmaceutica Nv FGFR2 inhibitors for the treatment of cholangiocarcinoma
US20220054483A1 (en) * 2018-09-21 2022-02-24 Janssen Pharmaceutica Nv Treatment of cholangiocarcinoma
WO2021050100A1 (fr) * 2019-09-09 2021-03-18 Zepto Life Technology, LLC Systèmes et procédés de détection de variation génétique dans des acides nucléiques améliorée
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