Gene Copy Number Estimation from Targeted Next-Generation Sequencing of Prostate Cancer Biopsies: Analytic Validation and Clinical Qualification

Abstract

Purpose: Precise detection of copy number aberrations (CNA) from tumor biopsies is critically important to the treatment of metastatic prostate cancer. The use of targeted panel next-generation sequencing (NGS) is inexpensive, high throughput, and easily feasible, allowing single-nucleotide variant calls, but CNA estimation from this remains challenging.Experimental Design: We evaluated CNVkit for CNA identification from amplicon-based targeted NGS in a cohort of 110 fresh castration-resistant prostate cancer biopsies and used capture-based whole-exome sequencing (WES), array comparative genomic hybridization (aCGH), and FISH to explore the viability of this approach.Results: We showed that this method produced highly reproducible CNA results (r ¼ 0.92), with the use of pooled germline DNA as a coverage reference supporting precise CNA estimation.CNA estimates from targeted NGS were comparable with WES (r ¼ 0.86) and aCGH (r ¼ 0.7); for key selected genes (BRCA2, MYC, PIK3CA, PTEN, and RB1), CNA estimation correlated well with WES (r ¼ 0.91) and aCGH (r ¼ 0.84) results. The frequency of CNAs in our population was comparable with that previously described (i.e., deep deletions: BRCA2 4.5%; RB1 8.2%; PTEN 15.5%; amplification: AR 45.5%; gain: MYC 31.8%). We also showed, utilizing FISH, that CNA estimation can be impacted by intratumor heterogeneity and demonstrated that tumor microdissection allows NGS to provide more precise CNA estimates.Conclusions: Targeted NGS and CNVkit-based analyses provide a robust, precise, high-throughput, and cost-effective method for CNA estimation for the delivery of more precise patient care.

Diagnostic Targeted Sequencing Panel for Hepatocellular Carcinoma Genomic Screening

“…A recent study investigated the use of an amplicon-based sequencing strategy that targeted all exons of 113 genes related to DNA repair. 25 The researchers demonstrated that, with an appropriate analysis strategy and quality control, amplicon-based sequencing strategy is feasible and cost-effective for CNA profiling in FFPE samples. 25 In the present study, the strategy of computing and centering the log 2 ratios for the primer two pools separately, before merging and segmentation proved to be an effective strategy in resolving issues associated with variable amplification efficiencies, with 86% of the genes showing concordant copy number states.…”

“…Of the 10 fresh-frozen pairs sequenced by using the HCC panel, one was excluded for excessive residual copy number log 2 ratio (segment interquartile range, >0.8). 25 For the nine evaluable samples, a correlation of r Z 0.80 (r 2 Z 0.64) was found between the copy number log 2 ratio of the two platforms ( Figure 5A). When the copy number profiles of the 34 evaluable FFPE tumors were compared with the matched profiles from WES, a correlation of r Z 0.73 (r 2 Z 0.54) was observed between the copy number log 2 ratios ( Figure 5A).…”

“…Samples with excessive residual copy number log 2 ratio (segment interquartile range >0.8) were excluded, as previously described. 25 For each tumor/nontumor pair, log 2 ratio was computed for each amplicon, separately for the two amplicon pools by using Varscan2 version 2.4.3 (https://github.com/dkoboldt/ varscan). 26 Log 2 ratios for the two pools were separately centered then merged for segmentation by using circular binary segmentation.…”

“…It has previously been reported that tumor purity had an impact on the ability to make CNA calls. 25,34 The impact of tumor purity on CNA analysis was therefore evaluated by using an in silico simulation on 12 cases (six fresh-frozen and six FFPE, selected on the basis of the presence of gene amplification/high gain or deep deletion), by replacing tumor reads with reads sampled from the normal samples to simulate tumor content 5%, 10%, 20% up to the actual tumor content for the samples. It was observed that amplifications/high gains were readily detected at 5% tumor content in many cases and at 20% in all cases (Supplemental Figure S5).…”

Genetic manipulation of LKB1 elicits lethal metastatic prostate cancer

“…Libraries for next-generation targeted sequencing were constructed from 40 ng of DNA using a customized panel (Generead DNaseq Mix-n-Match Panel v2; Qiagen) as previously described (Mateo et al, 2015). CNVs were assessed from the aligned sequence files (.bam) using CNVkit v0.7.3 (Seed et al, 2017; Talevich et al, 2016; Turajlic et al, 2018).…”