Sequencing formalin fixed paraffin embedded samples
Personalized medicine involves the use of a patient's molecular markers to guide treatment regimens for the patient. The scientific literature provides multiple examples of correlations between drug treatment efficacy and the presence or absence of molecular markers in a patient sample. Methods are provided herein that permit efficient dissemination of scientific findings regarding treatment efficacy and molecular markers found in patient tumors to health care providers.
1 .- 75 . (canceled)
76 . A method of determining a status of a plurality of molecular markers in a sample, the method comprising:
a. obtaining a sample from a subject, wherein the sample comprises a formalin-fixed paraffin embedded (FFPE) tumor biopsy, wherein the FFPE tumor biopsy comprises nucleic acid;
b. amplifying nucleic acid from the FFPE tumor biopsy; and
c. determining a status of a plurality of molecular markers in the sample, wherein the determining comprises de novo DNA sequencing of the amplified nucleic acids from the FFPE tumor biopsy.
77 . The method of claim 76 , further comprising comparing the status of the plurality of molecular markers determined in step c) with the status of the plurality of molecular markers in a historic FFPE sample from the subject.
78 . The method of claim 76 , wherein the de novo sequencing does not comprise pyrosequencing.
79 . The method of claim 76 , wherein the de novo DNA sequencing comprises bridge amplification of DNA.
80 . The method of claim 76 , wherein the de novo DNA sequencing comprises use of reversible terminating nucleotides.
81 . The method of claim 76 , wherein the de novo DNA sequencing comprises pyrosequencing.
82 . The method of claim 76 , wherein the de novo DNA sequencing comprises attaching the nucleic acid to a solid surface.
83 . The method of claim 82 , wherein the solid surface is a bead.
84 . The method of claim 76 , wherein the de novo DNA sequencing comprises sequential hybridization and ligation of random oligonucleotides.
85 . The method of claim 76 , wherein the de novo DNA sequencing comprises nanopore sequencing.
86 . The method of claim 76 , wherein the de novo DNA sequencing comprises measuring a change in current after incorporation of one or more triphosphates into a new nucleic acid strand.
87 . The method of claim 76 , wherein the amplifying comprises polymerase chain reaction.
88 . The method of claim 76 , wherein the amplifying comprises quantitative polymerase chain reaction.
89 . The method of claim 76 , wherein the tumor biopsy comprises a cell from a colon cancer, a bone cancer, a breast cancer, a central nervous system cancer, a gastric cancer, a cervical cancer, a blood cancer, an esophageal cancer, a head and neck cancer, a kidney cancer, a skin cancer, a lung cancer, or a carcinoma.
90 . The method of claim 76 , wherein the determining comprises determining an absence of one or more mutations or a presence of the one or more mutations in the molecular markers.
91 . The method of claim 90 , wherein the one or more mutations comprise a de novo mutation, nonsense mutation, missense mutation, silent mutation, frameshift mutation, insertion, substitution, point mutation, single nucleotide polymorphism (SNP), deletion, rearrangement, amplification, chromosomal translocation, interstitial deletion, chromosomal inversion, loss of heterozygosity, loss of function mutation, gain of function mutation, dominant negative mutation, or lethal mutation.
92 . The method of claim 76 , wherein the molecular markers comprise c-kit, Kras, BRAF, microsatellite sequence, EGFR, chromosome 18q, thymidylate synthase, and/or Topo1.
93 . The method of claim 92 , wherein the c-kit has an activating mutation.
94 . The method of claim 92 , wherein the Kras has a mutation in exon 2.
95 . The method of claim 92 , wherein the BRAF sequence encodes a V600E mutation.