Gene Copy Number Estimation From Targeted Next Generation Sequencing Of Prostate Cancer Biopsies: Analytic Validation and Clinical Qualification.

Abstract <p>Purpose</p> <p>Precise detection of copy number aberrations (CNAs) 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..</p> <p>Experimental Design</p> <p>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 fluorescent in situ hybridization (FISH) to explore the viability of this approach.</p> <p>Results</p> <p>We showed that this method produced highly reproducible CNA results (r=0.92), with the use of pooled germline DNA as a coverage reference supporting precise CNA estimation. CNA estimates from targeted next generation sequencing were comparable with WES (r=0.86) and aCGH (r=0.7); for key selected genes (BRCA2, MYC, PIK3CA, PTEN and RB1) CNA estimation correlated well with WES (r = 0.91) and aCGH (r = 0.84) results.</p> <p>The frequency of CNAs in our population was comparable to that previously described (ie. 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 intra-tumor heterogeneity and demonstrated that tumor microdissection allows NGS to provide more precise CNA estimates.</p> <p>Conclusion</p> <p>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.  

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