Prevalence and detection of low-allele-fraction variants in clinical cancer samples

Abstract

Accurate detection of genomic alterations using high-throughput sequencing is an essential component of precision cancer medicine. We characterize the variant allele fractions (VAFs) of somatic single nucleotide variants and indels across 5095 clinical samples profiled using a custom panel, CancerSCAN. Our results demonstrate that a significant fraction of clinically actionable variants have low VAFs, often due to low tumor purity and treatment-induced mutations. The percentages of mutations under 5% VAF across hotspots in EGFR, KRAS, PIK3CA, and BRAF are 16%, 11%, 12%, and 10%, respectively, with 24% for EGFR T790M and 17% for PIK3CA E545. For clinical relevance, we describe two patients for whom targeted therapy achieved remission despite low VAF mutations. We also characterize the read depths necessary to achieve sensitivity and specificity comparable to current laboratory assays. These results show that capturing low VAF mutations at hotspots by sufficient sequencing coverage and carefully tuned algorithms is imperative for a clinical assay.

Bridging genomics and phenomics of gastric carcinoma

“…Targeted massively parallel sequencing was performed using Illumina HiSeq 2,500‐based CancerSCAN designed at Samsung Medical Center and covered 6,839 exons from 381 cancer‐related genes and introns from 23 genes in 380 GC and the list of genes are described in Supporting Information Table S2. The selected genes covered variants associated with targeted cancer therapies approved by the US FDA, genes related to clinical trials at the Precision Oncology Clinic at Samsung Medical Center, and genes reported to have an association with response to therapy in public databases and the literature . High‐quality results (mean depth >200; 99.0% > 100X; >30% on bait; duplication rate <75) were obtained in 330 cases and were further analyzed.…”

“…SNVs and indels were detected by MuTect 1.1.4 and Pindel 0.2.5a4 . For CNV, we used the same criteria as previously described …”

“…The selected genes covered variants associated with targeted cancer therapies approved by the US FDA, genes related to clinical trials at the Precision Oncology Clinic at Samsung Medical Center, and genes reported to have an association with response to therapy in public databases and the literature. 18 High-quality results (mean depth >200; 99.0% > 100X; >30% on bait; duplication rate <75) were obtained in 330 cases and were further analyzed. (Supporting Information Fig.…”

“…23 For CNV, we used the same criteria as previously described. 18 Tissue microarray construction, immunohistochemistry and in situ hybridization All H&E slides were reviewed, and representative tumor tissue samples were selected from each case. The corresponding FFPE tissue blocks were retrieved.…”

Molecular alterations and poziotinib efficacy, a pan‐HER inhibitor, in human epidermal growth factor receptor 2 (HER2)‐positive breast cancers: Combined exploratory biomarker analysis from a phase II clinical trial of poziotinib for refractory HER2‐positive breast cancer patients

“…To remove common germline polymorphisms, variants were filtered using public and in-house databases. 27 Variants were chosen if they were located in exonic regions, and synonymous variants were filtered out. SNVs with a frequency of 1% with at least five supporting reads were chosen for further analyses.…”