COLD-PCR

Abstract: Plasma of cancer patients contains cell-free tumor DNA that carries information on tumor mutations and tumor burden. Individual mutations have been probed using allele-specific assays, but sequencing of entire genes to detect cancer mutations in circulating DNA has not been demonstrated. We developed a method for tagged-amplicon deep sequencing (TAm-Seq) and screened 5995 genomic bases for low-frequency mutations. Using this method, we identified cancer mutations present in circulating DNA at allele frequencies as low as 2%, with sensitivity and specificity of >97%. We identified mutations throughout the tumor suppressor gene TP53 in circulating DNA from 46 plasma samples of advanced ovarian cancer patients. We demonstrated use of TAm-Seq to noninvasively identify the origin of metastatic relapse in a patient with multiple primary tumors. In another case, we identified in plasma an EGFR mutation not found in an initial ovarian biopsy. We further used TAm-Seq to monitor tumor dynamics, and tracked 10 concomitant mutations in plasma of a metastatic breast cancer patient over 16 months. This low-cost, high-throughput method could facilitate analysis of circulating DNA as a noninvasive "liquid biopsy" for personalized cancer genomics.

Abstract: Inherited loss-of-function mutations in the tumor suppressor genes BRCA1, BRCA2, and multiple other genes predispose to high risks of breast and/or ovarian cancer. Cancer-associated inherited mutations in these genes are collectively quite common, but individually rare or even private. Genetic testing for BRCA1 and BRCA2 mutations has become an integral part of clinical practice, but testing is generally limited to these two genes and to women with severe family histories of breast or ovarian cancer. To determine whether massively parallel, “next-generation” sequencing would enable accurate, thorough, and cost-effective identification of inherited mutations for breast and ovarian cancer, we developed a genomic assay to capture, sequence, and detect all mutations in 21 genes, including BRCA1 and BRCA2, with inherited mutations that predispose to breast or ovarian cancer. Constitutional genomic DNA from subjects with known inherited mutations, ranging in size from 1 to >100,000 bp, was hybridized to custom oligonucleotides and then sequenced using a genome analyzer. Analysis was carried out blind to the mutation in each sample. Average coverage was >1200 reads per base pair. After filtering sequences for quality and number of reads, all single-nucleotide substitutions, small insertion and deletion mutations, and large genomic duplications and deletions were detected. There were zero false-positive calls of nonsense mutations, frameshift mutations, or genomic rearrangements for any gene in any of the test samples. This approach enables widespread genetic testing and personalized risk assessment for breast and ovarian cancer.