High-Throughput Detection of Clinically Relevant Mutations in Archived Tumor Samples by Multiplexed PCR and Next-Generation Sequencing

Abstract

Purpose: Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false-positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high-content genetic profiling in archived clinical samples.Experimental Design: We first validated the technical performance of MMP-seq in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin-embedded (FFPE) clinical samples using matched fresh-frozen tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 patients with endometrial cancer.Results: We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false-positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K (phosphoinositide 3-kinase) and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA.Conclusions: MMP-seq provides a robust solution for comprehensive, reliable, and high-throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice. Clin Cancer Res; 20(8); 2080-91. Ó2014 AACR.

Consensus Statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas

“…DNA from the tumour should be processed and assessed for quality according to standard protocols 29,31,32 . Over the past few years, protocols for DNA, and even RNA, isolation from FFPE samples have considerably improved, and technical adaptations for handling potential artefacts generated from these materials have yielded increasingly reliable sequencing data, which has expanded the use of FFPE in clinical settings 35 . Tumour tissue, when available, can provide valuable information that will aid interpretation of results from germ line samples.…”

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

“…17 We custom designed a lung adenocarcinoma research assay to query the entire coding region of STK11, TP53, PTEN, KRAS, and NRAS, exons 11 and 15 of BRAF, exons 18 to 21 of EGFR, and other clinically relevant coding portions of MET, PIK3CA, and ERBB2. Briefly, DNA aliquots from 13 lung tumor specimens used for the TST assay were made available for the Access Array library preparation and Illumina MiSeq sequencing.…”

“…Duplicate libraries were prepared for the Access Array, according to the manufacturer's protocol, from each tumor specimen using approximately 4000 functional DNA copies (AE1000 copies) estimated using real-time PCR, as described. 17 The libraries were sequenced (2 Â 150, 300 cycles) in the same Illumina MiSeq sequencing run using the version 2 reagent kit. Barcoded specimens we demultiplexed and FASTQ reads were processed into annotated variant calls using a custom Galaxy workflow (M.R.R., unpublished data) that used Bowtie-FreeBayes/VarScan-ANNOVAR.…”

Upregulation of Periostin and Reactive Stroma Is Associated with Primary Chemoresistance and Predicts Clinical Outcomes in Epithelial Ovarian Cancer

“…We performed deep sequencing on all exons and exon-intron junctions of the entire TP53 gene using a previously developed MMP-Seq targeted cancer panel (12). Quality of the FFPE DNA samples was quantified as number of functional copies using a TRAK2 qPCR "ruler assay" (12).…”

“…Quality of the FFPE DNA samples was quantified as number of functional copies using a TRAK2 qPCR "ruler assay" (12). Five thousand functional copies of DNA from each sample were used as the input for target enrichment and library construction using Fluidigm Access Array followed by deep sequencing on an Illumina MiSeq sequencer.…”