Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing of Plasma DNA

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.

Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens

“…6,18e21 However, to date there have been no rigorous studies comparing sequence data obtained from routine clinical FFPE tissue to paired fresh frozen tissue. A recent study by Forshew et al 18 found a similar background frequency of nonreference base changes in 47 FFPE samples enriched by multiplex PCR. In another study, Kerick et al 21 reported a false-positive rate of 0.98% for SNV calls from low coverage (Â20) NGS data from FFPE tissue; the authors note, however, that the false-positive rate can be reduced with increased (> Â80) coverage.…”

“…18 We therefore conducted additional analysis to determine whether fixation-induced DNA artifacts could be detected at low levels. To enrich for true base changes in the parent library molecules and avoid sequencing errors, only discrepancies in high-quality read alignments with a minimum mapping quality score of 20 were considered, and discrepant bases were required to have base qualities of at least 20; positions called as SNVs by a standard variant identification method were also excluded.…”

Cancer biomarkers: Written in blood

“…Researchers soon found similar results for other types of cancer. Rosenfeld and his Cancer Research UK colleagues James Brenton and Carlos Caldas showed that ctDNA provides a precise portrait of advanced ovarian and breast cancers 8 . And in the largest study yet, Diaz and other members of the Johns Hopkins group detected ctDNA in at least 75% of patients with advanced tumours, in organs as diverse as the pancreas, bladder, skin, stomach, oesophagus, liver and head and neck 9 .…”

PyClone: statistical inference of clonal population structure in cancer

“…The advances we present have practical implications for inference of clonal populations, and show measurable reductions in spurious inference relative to current approaches. As the practice of measuring allelic prevalences during the treatment cycle 15,16 or through retrospective analysis of multiple samplings increases 10, 14, 17 , we suggest PyClone will contribute a robust statistical inference approach for studying selection patterns underpinning disease progression in cancer.…”