Multiplex Cell-Free DNA Reference Materials for Quality Control of Next-Generation Sequencing-Based In Vitro Diagnostic Tests of Colorectal Cancer Tolerance

Background: Liquid biopsies based on next-generation sequencing (NGS) assays are confronted with more opportunities and challenges. Widespread clinical implementation of NGS-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials (RMs) which could provide full control of the process from nucleic acid extraction to test report generation. Quality control based on cell-free DNA (cfDNA) RMs is especially important for liquid biopsies. Methods: Here, we used genomic DNA from thirteen cell lines to establish four negative cfDNA RMs (N1-N4) and four multiplex cfDNA RMs (L1-L4) at serial allelic frequencies ranging from approximately 2% to 0.1%. All the cfDNA RMs were quantified and validated via both droplet digital polymerase chain reaction (ddPCR) and NGS. These RMs were distributed to eight domestic manufacturers to collaboratively evaluate the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton, and BGISEQ). Results: Each multiplex RM has eleven colorectal cancer-related mutations, including six KRAS mutations (G12S, G12C, G12D, G12A, G12V, and G13D), three NRAS mutations (G12D, Q61R, and Q61K), one PIK3CA mutation (H1047R), and one BRAF mutation (V600E). Each mutation in the cfDNA RMs was quantified and validated via both ddPCR and NGS, showing the good relevance of mutant allelic frequency. These RMs were distributed to eight domestic manufacturers for collaborative evaluation. All eight manufacturers provided similar results by domestic NGS-based cancer IVDs, except for manufacturer #5. The coefficient of variation (CV) was increased with decreasing mutant allelic frequency, and poor repetition occurred when the allelic frequency was lower than 0.5%. Conclusions: These results indicated that these cfDNA RMs would be pivotal for NGS-based cancer IVDs, especially for liquid biopsies of colorectal cancer-related mutations and would guide the further development of RMs covering more onco-related mutations.