Clinical actionability enhanced through deep targeted sequencing of solid tumors

Abstract

Background: Further advances of targeted cancer therapy require comprehensive in-depth profiling of somatic mutations that are present in subpopulations of tumor cells in a clinical tumor sample. However, it is unclear to what extent such intratumor heterogeneity is present and whether it may affect clinical decision-making. To study this question, we established a deep targeted sequencing platform to identify potentially actionable DNA alterations in tumor samples.

Methods: We assayed 515 formalin-fixed paraffin-embedded (FFPE) tumor samples and matched germline DNA (475 patients) from 11 disease sites by capturing and sequencing all the exons in 201 cancer-related genes. Mutations, indels, and copy number data were reported.

Results: We obtained a 1000-fold mean sequencing depth and identified 4794 nonsynonymous mutations in the samples analyzed, of which 15.2% were present at <10% allele frequency. Most of these low level mutations occurred at known oncogenic hotspots and are likely functional. Identifying low level mutations improved identification of mutations in actionable genes in 118 (24.84%) patients, among which 47 (9.8%) otherwise would have been unactionable. In addition, acquiring ultrahigh depth also ensured a low false discovery rate (<2.2%) from FFPE samples.

Conclusions: Our results were as accurate as a commercially available CLIA-compliant hotspot panel but allowed the detection of a higher number of mutations in actionable genes. Our study reveals the critical importance of acquiring and utilizing high sequencing depth in profiling clinical tumor samples and presents a very useful platform for implementing routine sequencing in a cancer care institution.

Figure 1. Technical characterization of T200

(A) Chance of detecting (i.e., observing 2 or more reads from) a 1% frequency variant allele (Y axis) from a sequencing coverage (X axis) of 10 to 2000 folds. (B) The minimal allele frequency (Y axis) that can be confidently (FDR < 1%) detected at a given sequencing coverage (X axis). (C,D) DNA input and duplicate rates (C) and depth of coverage (D). Each dot represents one tumor sample. Areas circled in red: high duplicate rate and low coverage in samples with low DNA input; areas circled in blue and green: ideal range of DNA input to obtain low duplicate rate and desired coverage; areas circled in gray: high duplicate rate and low coverage despite the DNA input.

Figure 2. Concordance between fresh-frozen and FFPE samples

Each dot represents an SNV in a matched pair of fresh-frozen and FFPE tumor samples detected at above 500 reads (high correlation) (A) between 200 and 499 reads (medium to high correlation) (B) and below 200 reads (low correlation) (C).

Figure 3. Mutation profiling of 515 tumor samples using T200

(A) Distribution of disease sites, (B) mean coverage (Y axis) in each sample (X axis, in no particular order), (C) percentage of target bases (Y axis) that are covered by at least 200x in each sample (X axis in no particular order), (D) median mutation rate per Mb in nine major (>10 samples) cancer types, (E) the mean variant allele frequency cutoff (Y axis) applied in variant calling as a function of mean coverage (X axis) in each sample, (F) number of mutations (Y axis) detected in various allele frequency bins (X axis) for four cancer types.

Figure 4. Significantly mutated genes

Negative log10 p values (Y axis) of significantly mutated genes (X axis) in five disease categories: all diseases and breast, skin, colorectal and brain cancers.

Figure 5. Concordance with other sequencing platforms

All the samples tested in this study were also tested in the AmpliSeq46 gene panel in a CLIA accredited clinical laboratory. (A) 98.2% of mutations identified by AmpliSeq46 were also identified by T200. (B) Allele frequency of PIK3CA mutations identified in different patients by AmpliSeq46 (pink bars) and T200 (blue bars). (C) Mutations found at lower than 10% MAF by T200 in different cancer types validated by AmpliSeq46. (D) Peaks obtained from Sequenom validation of two new mutations identified by T200 (NF1_Q554* and SMARCA4_R1665*). A complete list of all sites tested on Sequenom can be found in the supplemental material.