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.
Highlights
RAS proto-oncogenes (HRAS, KRAS and NRAS) encode a family of GDP/GTP-regulated proteins critical for signal transduction mediating cell growth and survival
In the metastatic colorectal cancer (mCRC) cohort, comparison of mutation results obtained by testing Formalin-Fixed Paraffin-Embedded (FFPE) to those obtained by testing circulating-free DNA (cfDNA) by droplet-digital PCR (ddPCR) resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV)
We evaluated the comparison of results between the molecular profile of FFPE tissues and cfDNA samples, and reported on the sensitivity and specificity of each of the cfDNA technologies, which included ddPCR (Biorad), BEAMing (Sysmex Inostics), and next-generation sequencing (NGS) using the targeted SWIFT56G panel (Swift Biosciences)
Summary
RAS proto-oncogenes (HRAS, KRAS and NRAS) encode a family of GDP/GTP-regulated proteins critical for signal transduction mediating cell growth and survival. The carboxyl-terminus of the proteins contains a hyper-variable region, which diverges radically in primary sequence from the remainder of these genes. This region of the gene product is susceptible to many post-translational modifications which confer major differences in trafficking and intracellular localization of the mature protein. RAS family members are frequently found in their mutated, oncogenic forms in human tumors. Mutations in KRAS, mainly in exons 2, 3 and 4 (codons 12, 13, 59, 61, 117, 146), account for nearly 85% of all RAS mutations found in human tumors, whereas NRAS contributes to ~15%, and HRAS to less than 1% [3]. Mutations in RAS are prominent drivers of colon, pancreatic and lung cancers
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