Abstract 3638: A rapid, multiplexed, and highly accurate next-generation sequencing RAS Panel for FFPE colorectal samples reporting on the absence or presence of low frequency somatic variants
Authors: Dunn Tamsen , Iyer Anita , Porter Margaret , Haigis Robert , Smith Shannon , Lee Desiree , Udar Nitin 
Published: July 2016

Abstract
Introduction: Next generation sequencing (NGS) is a highly sensitive method for detecting somatic mutations. Mutations in NRAS and KRAS may affect up to 50% of patients with colorectal cancer (CRC). Recent data suggest the clinical benefit of panitumumab is restricted to patients who have no tumor mutations in RAS, defined as codons 12, 13, 59, 61, 117 and 146 of KRAS and NRAS genes. An Extended RAS panel* targets these codons as a single multiplex assay. A dual strand approach distinguishes true mutations from artifacts commonly found in DNA from Formalin Fixed Paraffin Embedded (FFPE) tissue. Analytical performance of this panel was characterized.
Methods: FFPE tissue was examined for tumor content and area. DNA was extracted from FFPE tissue, cell lines and plasmids. The DNA quality and quantity were simultaneously determined through a quantitative PCR measurement of amplifiability. TruSeq Custom Amplicon technology targeting each strand of DNA was used to construct NGS libraries. Sequencing was performed on the MiSeqDx, and the Illumina Somatic Variant Caller was used to call somatic variants.
Results: The dual-strand amplicon approach results in high accuracy by eliminating false positives observed on one strand of DNA typical of an FFPE artifact, by requiring every variant to be detected on both strands. We evaluated performance on 500 FFPE CRC samples processed with the Extended RAS panel. Concordance against Qiagen TheraScreen and Sanger Sequencing results was evaluated. The somatic variant caller yields a specificity of 0.98 and sensitivity of 0.72 for variants in the 5%-10% range, and a specificity of 0.96 and sensitivity of 0.98, overall, where we take the alternate methods as truth. Cumulative FFPE tissue area influences performance of the RAS panel. FFPE of a range of tissue area were tested using 1, 3, 5, and 8 sections per sample. 80mm2 minimum (240mm2 recommended) cumulative tissue area using 5uM slices was optimal for performance and accuracy. DNA quantity and quality also influence test accuracy and sensitivity. A study with 40 FFPE specimens assayed over multiple operators and sequencers produced average coverage of 32,000 reads per target per sample, and over 93% alignable reads per sample. Reproducibility of mutations detected over multiple operators and instruments was 100%. The Extended RAS Panel demonstrates robust performance across many sources of variation, including FFPE DNA extraction method, DNA storage time, recommended DNA input range, qPCR/PCR thermocyclers with little to no impact on sample pass rate and agreement with Sanger sequencing. We also demonstrate detection of 56 distinct mutations in a single run.
Conclusion: This multiplex assay achieves high accuracy for detection of somatic mutations from DNA extracted from FFPE colorectal tissue samples.
*In development For Research Use Only. Not for use in diagnostic procedures.
Citation Format: Tamsen Dunn, Anita Iyer, Margaret Porter, Robert Haigis, Shannon Smith, Desiree Lee, Nitin Udar. A rapid, multiplexed, and highly accurate next-generation sequencing RAS Panel for FFPE colorectal samples reporting on the absence or presence of low frequency somatic variants. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3638.