Targeted High-Throughput Sequencing For The Routine Diagnosis Of Somatic Theranostic Mutations On Formalin-Fixed, Paraffin-Embedded Ffpe Tumor Samples: A Prospective Study Of 3366 Cancer Samples.

e22187 Background: Improving of patient care relies on tumor genetic profiling that will guide treatment strategy. This implies the ability of parallel genotyping of a growing number of mutations. By providing an increase in sequencing throughput, NGS technology is suitable for routine implementation of such diagnostic in the near future. To address this challenge, we developed a robust end-to-end production and analysis workflow for routine molecular diagnosis of FFPE samples. Methods: A custom panel of 97 amplicons covering a 5.8 kb region was designed to detect mutations in 17 key cancer genes. We used an amplicon strategy workflow for library preparation including: Access Array System (Fluidigm) for targeted amplification, and MiSeq (Illumina) for deep sequencing. A pipeline dedicated to diagnosis was developed to automate the process of variant detection from the alignment of the genomic sequences to the detection and annotation of two types of genetic variations: single nucleotide variations (SNVs) and short insertions or deletions (indels). NGS data was obtained on 3366 patients (1598 NSCLC, 1447 colorectal cancers, 230 melanomas and 91 gliomas) and compared with reference technics pyrosequencing (PS) and allele-specific PCR (ARMS) used in routine. Results: Deep sequencing results in a minimum of 500X coverage for all amplicons, and detects 826 alterations, including 770 SNVs and 56 short indels. Among theses, 822 were also found with the reference methods. NGS lead to 3 false negative mutations due to poor DNA quality. Four false positive mutations detected only by NGS suggest that NGS can detect mutations bypassed by classical methods. Considering PS and ARMS as references, deep sequencing provides a 99,6% sensitivity and a 99,9% specificity. Conclusions: We demonstrate that our deep sequencing approach can be reliably implemented as a diagnostic test for routine detection of somatic mutations. Our strategy allows the simultaneous analysis of hotspot mutations in 17 key cancer genes for 48 samples in a single assay and thus improves the molecular diagnosis of tumors in a cost and time efficient manner.