Enhanced specificity of high sensitivity somatic variant profiling in cell-free DNA via paired normal sequencing: design, validation, and clinical experience of the MSK-ACCESS liquid biopsy assay

Abstract

<jats:title>Abstract</jats:title><jats:p>Circulating cell-free DNA (cfDNA) from blood plasma of cancer patients can be used to interrogate somatic tumor alterations non-invasively or when adequate tissue is unavailable. We have developed and clinically implemented MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), an NGS assay for detection of very low frequency somatic alterations in select exons and introns of 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 98% for <jats:italic>a priori</jats:italic> mutation profiling. To evaluate the performance and utility of MSK-ACCESS, we report results from the first 681 prospective blood samples (617 patients) that underwent clinical analysis to guide patient management. Somatic mutations, copy number, and/or structural variants were detected in 73% of the samples, and 56% of these circulating-tumor DNA (ctDNA) positive samples had clinically actionable alterations. The utilization of matched white blood cell sequencing allowed retention of somatic alterations while filtering out over 10,000 germline and clonal hematopoiesis variants, thereby greatly enhancing the specificity of the assay. Taken together, our experience illustrates the importance of analyzing a matched normal sample when interpreting cfDNA results and highlights the potential of cfDNA profiling to guide treatment selection, monitor treatment response, and identify mechanisms of treatment resistance.</jats:p>

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology

“…FoundationACT ™ showed ~99% sensitivity for SNVs with VAF > 0.5%, ~95% for 0.25%–0.5% VAF and ~70% for 0.125–0.25% VAF 13 . MSK-ACCESS ™ showed ~98% sensitivity for SNVs with VAF > 0.5%, declining to ~74% for 0.1%–0.5% VAF 12 . Validation of the amplicon-based InVisionFirst ™ assay, suggested this may have superior LOD to the hybrid-capture assays above, with ~99% sensitivity for SNVs as low as 0.25–0.35% VAF 24 .…”

“…Among 859 patients tested with FoundationACT ™, half of all variants detected had VAFs below ~1.3% and a third below ~0.5% 13 . Among 435 patients tested with MSK-ACCESS ™, >5% of all mutations detected were missed by NGS but identified by more sensitive genotyping methods, with these having a median VAF of ~0.08% 12 . These results, which are generally based on the analysis of patients with advanced disease, demonstrate the tendency for ctDNA mutations to be represented at very low frequencies.…”

“…The reliability of ctDNA sequencing assays is commonly assessed by measuring concordance between alternative assays or assay replicates 27 , parallel ctDNA and tumor-biopsy tests 25 , 26 , 40 , or orthogonal analysis of plasma samples with non-NGS based techniques, such is ddPCR 23 . These approaches have been applied across large cohorts of clinical specimens to inform assay development and validation 12 , 23 , 40 .…”

“…Patient plasma samples vary widely in mutational burden and cell-free DNA yields, with no single sample representing the full diversity of mutation types and frequencies that should be considered in rigorous proficiency testing. The use of different clinical cohorts, cell-free DNA input quantities and test materials makes it difficult to draw reliable comparisons between validation studies performed by different ctDNA sequencing providers 12 , 13 , 23 , 24 .…”

“…In spite of these challenges, ctDNA assays of increasing resolution have been developed 12 – 24 . With clinical adoption already underway, it is critical for the community to understand the sensitivity, accuracy and reproducibility of ctDNA assays, and the variables that impact analytical performance.…”