Application Of A Comprehensive Platform Technology For Detection Of Cancer-Associated Genomic Alterations And Viral Dna In Liquid Biopsies

Background: Targeted detection of somatic cancer-associated variants by next generation sequencing technology have demonstrated clinical utility. Microsatellite instability, copy number variants (CNVs), structural rearrangements and cancer-associated viral DNA are other key targets with clinical relevance. We report here the enhancement of an ultra-sensitive sequencing platform technology (AmpliMARK) for the simultaneous detection of multiple classes of genomic alterations in liquid biopsies, extending to common cancers such as breast, colorectal, lung and nasopharyngeal cancers (NPC). Methods: A primer-based target capture panel covering 50 genes, 6 microsatellite loci and 2 viral targets was validated using biologically relevant reference materials and clinical samples (cfDNA) from breast, lung, colorectal and NPC (n = 117). Orthogonal validation was done with PCR-based EGFR detection, EBV BamHI-W CpG DNA, and MSI fragment analysis, and to matched tumor tissue where available (n = 65). Results: The lower limits of detection (LOD) for variant alleles, MSI, viral DNA and CNVs were 0.1%, 5%, 50 IU/mL and 1.5-fold, respectively. Concordance with PCR-based detection of EGFR mutations was high with 100% PPV and 95.2% NPV. Cancer-associated viral DNA detection by AmpliMARK had 89% concordance with PCR in NPC. For MSI detection in cfDNA, PPV was 100% with NPV of 93.5% compared to tissue-based testing. For EGFR, KRAS and BRAF mutations, matched tissue testing had a combined 100% PPV and 65% NPV. CNVs were detected in cfDNA from 5 of 9 known ERBB2 (HER2)-amplified breast cancer cases. ALK, MET, EGFR CNVs ranging from 1.89- to 35-fold were also confirmed in lung cancer samples including in pleural effusion and cerebrospinal fluid. Among lung cancer cases with no EGFR mutations, BRAF, NRAS, MET exon 14 skipping, ERBB2 exon 20 insertion (n = 1 each) and KRAS (n = 6) were confirmed by the platform. Structural rearrangements resulting in fusion products of ALK, ROS1 and RET were detectable by the platform in reference material. Conclusions: We report the enhancement of a technology platform for comprehensive genomic profiling of liquid biopsies. Excellent concordance to standard methods and tissue results is demonstrated. Legal entity responsible for the study: Lucence Diagnostics Pte. Ltd. Funding: Lucence Diagnostics Pte. Ltd. Disclosure: Y. Choudhury, H. Chen, F. Fadhlullah, D.K.H. Tan, K.T. Ng, W.M. Phyo, M.H. Tan: Employee: Lucence Diagnostics Pte. Ltd.