Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy)

Abstract

CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5–1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles. In the mCRC cohort, comparison of mutation results obtained by testing FFPE to those obtained by testing cfDNA by ddPCR resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV). For BEAMing, we observed 93% sensitivity, 69% specificity, 78% PPV and 90% NPV. Finally, sensitivity of NGS was 73%, specificity was 77%, PPV 79% and NPV 71%.Our study highlights the complementarity of different diagnostic approaches and variability of results between OncoBEAM™-RAS-CRC and NGS assays. While the NGS assay provided a larger breadth of coverage of the major targetable alterations of 56 genes in one run, its performance for specific alterations was frequently confirmed by ddPCR results.

Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma

“…This sample and three other samples only showed mutations in loci not covered by the KRAS Screening Multiplex Kit used for ddPCR ( KRAS codon 61, BRAF and TP53 ). Nonetheless, we observed good agreement between ddPCR and NGS, which was also reported in other studies …”

Profiling of circulating tumor DNA in plasma of non‐small cell lung cancer patients, monitoring of epidermal growth factor receptor p.T790M mutated allelic fraction using beads, emulsion, amplification, and magnetics companion assay and evaluation in future application in mimicking circulating tumor cells

“…All experiments were conducted at the Lyon Universitary Hospital. Briefly, as described previously, the cfDNA sample was diluted in 123 µL of AVE buffer (Qiagen). A PCR Master Mix, specifically targeting p.T790M was mixed with the cfDNA samples and split into six replicate of 65 µL reactions in the initial target‐specific spanning PCR.…”

“…After breaking the emulsion PCR reaction, WT and mutant‐specific probes were hybridized and flow cytometry analysis was conducted using a cytometer (BD Bioscience, Erembodegem, Belgium). The threshold of positivity is defined by two parameters: the MAF had to exceed 0.02% and the absolute number of mutant beads had to be greater than 50 . All samples with lower mutated bead counts were considered negative, even if the MAF exceeded 0.02%, this is most likely due to a low cfDNA input amount, typically below 2 ng.…”

“…Altogether, these limitations prompt the need for accurate and sensitive mutation detection technologies that do not rely on invasive tumor tissue sampling. Sensitive detection technologies capable of accurately detecting mutations in plasma samples obtained from blood provide a minimally invasive alternative to tissue‐based methods . This clinical need has prompted the development and regulatory approval in the United States and Europe of plasma EGFR companion diagnostic tests based on either real‐time or conventional polymerase chain reaction (PCR) .…”

“…Thus, with conventional PCR if a rare mutant molecule is present and provides a weak signal, it may not be detected in the sample, because the signal is lost among the abundant nontumor signal, as well as signal from tumor DNA unrelated to the mutation. To this end, highly sensitive and selective technologies have been developed to overcome the inherent challenge of obtaining a strong signal from the very low fraction of tumor‐derived DNA in comparison to the wild‐type (WT) fraction . In this regard, BEAMing (beads, emulsion, amplification, and magnetics), a technology based on digital PCR, which entails partitioning the PCR process into many individual reactions in order to provide higher resolution for the less frequently encountered DNA sequences (ie mutated DNA), appears to be a highly promising technique.…”