Performance study of an amplification-based NGS test on clinical FFPE specimens in China’s first multi-center study.

e13112 Background: Next Generation Sequencing (NGS) assays provide a comprehensive view of clinically actionable variations in patients. The increasing application of NGS holds new promise towards accurate diagnosis, personalized treatment and precision medicine. Formalin-fixed Paraffin-embedded (FFPE) treatment remains to be the most popular format of tissue preservation. To further standardize the whole process of the application of NGS assays on FFPE tissues, the Chinese Medical Association Pathology Division organized a multi-center performance study of a NGS assay on more than 1,000 clinical FFPE specimens by inviting top pathology departments in the country. Methods: OncoAim Tumor Mutation and PharmGx Detection Kit (Singlera Genomics, Shanghai) was used for this study. Reference samples with known mutations and allele frequencies together with 1045 clinical FFPE specimens from 11 participating hospitals were tested. Among them, 615 Colorectal Cancer (CRC) and 430 Non-Small Cell Lung Cancer (NSCLC) FFPE samples were processed and sequenced on Illumina Miseq platform and Thermo ion torrent PGM platform, respectively. Results: The results on reference materials show 100% (Confidence interval = 100%) analytical sensitivity for mutations with MAF ≥ 10% when median coverage is ≥ 500X.Positive predictive value of > 99% was observed for mutations with MAF at 5% (data not shown). The median depth for CRC and NSCLC samples were 977Xand 828X, respectively. Among all CRC samples, 564 (91.7%) samples were found containing at least 1 clinical hotspot alteration. The top mutated genes were TP53, KRAS, APC, PIK3CA, SMAD4, BRAF, FBXW7, NRAS, PTEN, and ERBB2. For all the NSCLC samples, 304 (70.69%) were detected containing at least 1 clinical hotspot mutation. The top 10 mutated genes were EGFR, TP53, KRAS, PIK3CA PTEN, VHL, ERBB2, SMAD4, NFE2L2 and CTNNB1. Conclusions: In this study, we showed that the amplification-based NGS assay can achieve very high sensitivity and specificity for samples with median depth over 500X. The samples containing at least 1 clinical hotspot mutation were about 83% on average, suggesting NGS could be of great use for further characterization of the tissues.