Abstract 5119: Customizable all-in-one cancer panels to detect copy number variation, DNA rearrangement and mutations by targeted NGS
Authors: Vadapalli Arjun , Ashutosh Ashutosh , Tao Heather , Forsmark Linus , Cocq Christian Le , Khare Akansha , Arezi Bahram , Amparo Gilbert , Ruvolo Michael , Pabon Carlos , Ghosh Jayati , Jin Jimmy , Liu Tracy , Roberts Douglas 
Published: August 2019

Abstract
Background: The current standard of genomic profiling of cancer tissues relies upon multiple separate technologies to aid in tumor characterization. Here we describe a customizable NGS design and analysis workflow for cancer samples that can detect single nucleotide polymorphisms (SNPs), insertions and deletions (INDEL), somatic copy number alterations (SCNAs), and translocations (TLs) in a single assay.
Methods: We have created a library of strategically placed target-enrichment probes to provide for specific detection of SNP/INDEL, SCNAs, and translocation in genes identified as important in several cancer subtypes. The probes are optimized with the SureSelect XTHS protocol, which enables extremely sensitive detection of rare exonic mutation events within a heterogeneous sample. SCNA detection is further enhanced with additional probes targeting high minor allele frequency SNPs within the target gene boundary. Translocation detection is enabled by targeting intronic regions of fusion gene candidates. The data analysis is performed by the software SureCall that calls SNPs across all regions covered by probes. SCNA detection utilizes either a matched normal or standard control DNA and aberrant regions are identified by jointly segmenting the read depth ratios and SNP allele frequencies. The clonal structure is discerned using two Bayesian cancer models in a hierarchical manner. Translocation are identified by analyzing split reads across putative fusion breakpoints that are supported by both high- and low-confidence reads that pass quality filters.
Results: We demonstrate rare allele detection in cancer cells down to 1%. We also show detection of 3 copies of MET at 20% tumor fraction and 16 copies of ERBB2 at 2% tumor fraction. For translocations, DNA level gene fusions were detected for ALK and ROS fusion genes at &lt;=3% tumor fraction. In 39 ERBB2 FISH-positive FFPE samples, ERBB2 CNV detected by AIO panel was 97.4% concordant with FISH (scored according to standard ASCO/CAP convention). Using FISH as the gold standard, the false negative rate was 2.6% and false positive rate was 2.4%. In 43 ALK FISH-positive FFPE samples, ALK fusion was detected using a small AIO lung panel which includes probes spanning introns 18/19 of the ALK gene and is 90.7% concordant with FISH (Agilent SureFISH ALK BA). Using FISH as the gold standard, the false negative rate was 9.3% and false positive rate was 2.3%.
Conclusions: This work represents an important advancement in the development of a single assay to detect copy number variation, DNA rearrangement and mutations.
Citation Format: Arjun Vadapalli, Ashutosh Ashutosh, Heather Tao, Linus Forsmark, Christian Le Cocq, Akansha Khare, Bahram Arezi, Gilbert Amparo, Michael Ruvolo, Carlos Pabon, Jayati Ghosh, Jimmy Jin, Tracy Liu, Douglas Roberts. Customizable all-in-one cancer panels to detect copy number variation, DNA rearrangement and mutations by targeted NGS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5119.