Gene mutations and copy number alterations of egfr, kras and met

Abstract

Sequence variants and copy number variations in the EGFR, KRAS and MET genes are biomarkers for resistance to anti-EGFR therapies for cancer. This disclosure provides methods of detecting these biomarkers and using them in the diagnosis and treatment of cancer.

1 .- 25 . (canceled) 
     
     
         26 . A method comprising:
 (a) in a sample comprising nucleic acids from a cancer cell of a subject, selectively enriching for one or more nucleic acids comprising EGFR nucleotide sequences, to produce an enriched sample;   (b) sequencing the one or more nucleic acids comprising EGFR nucleotide sequences from the enriched sample to produce sequence reads;   (c) determining, among the sequence reads, a presence or an absence of at least one EGFR nucleotide sequence variant encoding an amino acid or codon sequence variant selected from the group consisting of G465V, T130A, D1083N, I491R, K467I, T211T, K1061T and V876M; and   (d) classifying the subject as having a cancer un-responsive to anti-EGFR therapy.   
     
     
         27 . The method of  claim 26 , wherein the determining comprises determining the presence or the absence of a plurality of EGFR nucleotide sequence variants. 
     
     
         28 .- 68 . (canceled) 
     
     
         69 . The method of  claim 26 , wherein the at least one EGFR nucleotide sequence variant includes one or more members selected from the group consisting of G>T G465V, A>G T130A, G>A D1083N, T>G I491R, A>T K467I, C>G T211T, A>C K1061T, and G>A V876M. 
     
     
         70 . The method of  claim 26 , wherein the subject has or is suspected of having colorectal cancer. 
     
     
         71 . The method of  claim 26 , wherein the subject has or is suspected of having colorectal cancer and has been treated with anti-EGFR therapy. 
     
     
         72 . The method of  claim 26 , wherein (c) further comprises detecting amplification of an EGFR gene. 
     
     
         73 . The method of  claim 72 , further comprising (e) administering to the subject a cancer treatment other than an anti-EGFR therapy. 
     
     
         74 . The method of  claim 73 , wherein the administering of (e) comprises administering to the subject a cancer treatment other than (i) anti-EGFR antibodies or (ii) cetuximab. 
     
     
         75 . The method of  claim 73 , wherein the cancer treatment other than anti-EGFR therapy comprises a treatment other than administration of an EGFR-directed antibody. 
     
     
         76 . The method of  claim 26 , wherein the sample comprises cell free DNA. 
     
     
         77 . The method of  claim 26 , wherein the sample is a blood sample or a tumor sample. 
     
     
         78 . The method of  claim 26 , wherein the subject has a cancer that is un-responsive to cetuximab. 
     
     
         79 . The method of  claim 26 , wherein the subject has or is suspected of having colorectal cancer or head and neck cancer. 
     
     
         80 . The method of  claim 26 , further comprising administering to the subject an antibody directed to an epitope of EGFR other than the epitope against which cetuximab is directed. 
     
     
         81 . The method of  claim 26 , wherein the subject is refractory to 5-FU-based therapy. 
     
     
         82 . The method of  claim 26 , wherein the selectively enriching comprises sequence capture of polynucleotides containing the at least one EGFR nucleotide sequence variant. 
     
     
         83 . The method of  claim 26 , wherein the determining is performed with a sensitivity of at least about 0.01%. 
     
     
         84 . The method of  claim 26 , wherein the determining is performed with a specificity of at least 99%. 
     
     
         85 . The method of  claim 26 , further comprising determining, among the sequence reads, a presence or an absence of at least one KRAS nucleotide sequence variant. 
     
     
         86 . The method of  claim 85 , wherein the at least one KRAS nucleotide sequence variant is not detected in a primary tumor of the subject or is not detected in a prior test for a presence of a KRAS nucleotide sequence variant in the subject. 
     
     
         87 . The method of  claim 85 , wherein the at least one KRAS nucleotide sequence variant is selected from the group consisting of G12C, G12R, G13D and Q61H. 
     
     
         88 . The method of  claim 26 , further comprising determining a presence of at least one sequence variant and/or at least one gene amplification in one or more genes selected from the group consisting of EGFR, KRAS, BRAF, MET, SMO, MYC, NRAS, ERBB2, ALK, Notch, PIK3CA and APC. 
     
     
         89 . A method comprising:
 (a) in a patient being treated for colorectal cancer with an anti-EGFR therapy, wherein the patent is characterized as having one or more EGFR nucleotide sequence variants encoding an amino acid selected from the group consisting of G465V, T130A, D1083N, I491R, K467I, T211T, K1061T and V876M, ceasing the anti-EGFR therapy; and   (b) commencing an alternative therapy.   
     
     
         90 . The method of  claim 89 , wherein the anti-EGFR therapy comprises administration of an EGFR tyrosine kinase inhibitor. 
     
     
         91 . The method of  claim 89 , wherein the anti-EGFR therapy comprises administration of cetuximab. 
     
     
         92 . The method of  claim 89 , wherein the alternative therapy is another, different anti-EGFR therapy. 
     
     
         93 . The method of  claim 92 , wherein the alternative therapy comprises administration of panitumumab. 
     
     
         94 . The method of  claim 89 , wherein the alternative therapy is other than an anti-EGFR therapy. 
     
     
         95 . The method of  claim 94 , wherein the alternative therapy is selected from imatinib, gefatinib, afatinib, dacomitinib, sunitinib, sorafenib, vandetanib, brivanib, cabozantib, neratinib, tivantinib, bevacizumab, cixutumumab, dalotuzumab, figitumumab, rilotumumab, onartuzumab, ganitumab, ramucirumab, ridaforolimus, tensirolimus, everolimus, BMS-690514, BMS-754807, EMD 525797, GDC-0973, GDC-0941, MK-2206, AZD6244, GSK1120212, PX-866, XL821, IMC-A12, MM-121, PF-02341066, RG7160, and Sym004. 
     
     
         96 . A method comprising:
 (a) monitoring a patient being treated for colorectal cancer with an anti-EGFR therapy for the appearance of one or more EGFR nucleotide sequence variants encoding an amino acid selected from the group consisting of G465V, T130A, D1083N, I491R, K467I, T211T, K1061T and V876M;   (b) upon appearance of any one of the one or more EGFR nucleotide sequence variants, ceasing the anti-EGFR therapy; and   (c) commencing an alternative therapy.   
     
     
         97 . The method of  claim 96  wherein the anti-EGFR therapy comprises administration of cetuximab.