Analytical Validation of BRAF Mutation Testing from Circulating Free DNA Using the Amplification Refractory Mutation Testing System

Abstract

Context: In 2013, an evidence-based guideline was published by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology to set standards for the molecular analysis of lung cancers to guide treatment decisions with targeted inhibitors. New evidence has prompted an evaluation of additional laboratory technologies, targetable genes, patient populations, and tumor types for testing. Objective: To systematically review and update the 2013 guideline to affirm its validity; to assess the evidence of new genetic discoveries, technologies, and therapies; and to issue an evidence-based update. Design: The College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology convened an expert panel to develop an evidence-based guideline to help define the key questions and literature search terms, review abstracts and full articles, and draft recommendations. Results: Eighteen new recommendations were drafted. The panel also updated 3 recommendations from the 2013 guideline. Conclusions: The 2013 guideline was largely reaffirmed with updated recommendations to allow testing of cytology samples, require improved assay sensitivity, and recommend against the use of

Testing for oncogenic molecular aberrations in cell-free DNA-based liquid biopsies in the clinic: are we there yet?

“…There was 100% concordance between tissue and urinary cfDNA genotypes assessed by droplet-digital PCR assay (ddPCR) in samples from 30 treatment-naive patients. The targetable mutation BRAF V600E was also analyzed in plasma- and serum-derived cfDNA samples from 221 patients with advanced melanoma [73]. Assay sensitivity for mutation detection was 44% in serum and 52% in plasma.…”

Comparison of Next-Generation Sequencing Platforms for Clinical Testing of Non-Small Cell Lung Cancer

“…There were also significantly less off-target coverage for the PE supernatant and plasma (0.8 and 0.98%) compared to the TruSeq platform (9.3 and 3.2%). These results are very promising and application of NGS technology could extend plasma genotyping from such techniques as ARMS, 27 allele-specific PCR, COLD-PCR, and emulsion PCR to more comprehensive analysis. 8,28 Pipeline Performance Physician-friendly bioinformatics tools are crucial for successful implementation of personalized therapy selection guided by NGS.…”

“…ThyroSeq for thyroid cancer. 13 Panels can be relatively small (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) Kb capture size) that focus on well-studied mutation hotspots or much larger panels (several hundred Kb to a few Mb) that encompass mutations for which therapeutics are in the pharmaceutical pipelines as well as genomic sites that are frequently mutated and may be of clinical or pharmacodynamic relevance. 14,15 Mutation analysis of NGS raw sequence data involves a complicated series of processes that can be divided into two main parts: alignment and variant calling.…”

Plasma Circulating Tumor DNA Levels for the Monitoring of Melanoma Patients: Landscape of Available Technologies and Clinical Applications

“…The sensitivity of AS-PCR is generally not below 0.1% [3, 9, 19, 36]. However, the recent development of a novel, rapid, and inexpensive AS-PCR assay reached a sensitivity of 0.005% due to the use of a PNA designed to inhibit the amplification of the wild-type allele [18].…”

“…Plasma is a better source of ctDNA than serum [19], especially because of the large amounts of wild-type DNA released by white cells lysis during clotting. However, discordant studies have shown that higher levels of ctDNA could be recovered from paired serum samples [9].…”