Multi-cancer early detection (MCED) in circulating tumor DNA (ctDNA) using an ultrasensitive mutation-based amplicon next-generation sequencing (NGS) assay.
Authors: Jonathan Poh, Yong Chai Ang, Jia Min Ho, Hao Chen, Chae Yin Cher, Michelle Pek, Mei Mei Chang, Sau Yeen Loke, Pannapa Pinweha, Ee Wern Kwong, Yee Fang Hum, Chwee Ming Lim, Min-Han Tan
Journal: Journal of Clinical Oncology (2024)

e15042 Background: Cancer remains a leading cause of mortality globally. For most cancers, routine screening options are still unavailable. Blood-based MCED has emerged as a promising strategy to bridge this gap. Mutation-based NGS in ctDNA detects highly specific cancer signals, an attribute critical for MCED assays. We developed an ultrasensitive mutation-based NGS assay optimized to detect cancer signals in 50 cancers and predict tissue-of-origin (TOO) for up to 21 cancer classes. Methods: An amplicon-based NGS assay incorporating mirror barcodes for error correction was designed to detect cancer-associated alterations (CAAs), including single nucleotide variants (SNVs), insertion-deletion mutations (indels), and viral DNA (EBV and 20 genotypes of HPV), in 50 cancers. Analytical validation was performed using reference standards and clinical validation was performed in a retrospective case-control cohort of 601 subjects comprising cell-free DNA (cfDNA) from 313 self-declared healthy donors and 288 cancer patients (30.9% early-stage, 50.3% late-stage and 9.4% hematolymphoid cancers). Tissue-of-origin (TOO) was predicted for 21 cancer classes using a weighted sum model. Where relevant, matched buffy coat genomic DNA (gDNA) was analyzed to rule out clonal hematopoiesis. Results: Analytical validation demonstrated a limit of detection (LoD) of 0.05% variant allele frequency (VAF) for indels and 0.07% VAF for SNVs in cfDNA. LoD was matched in gDNA (0.04% and 0.05% VAF for indels and SNVs respectively). In clinical validation, the assay demonstrated a specificity of 99.0% (310/313) and an overall sensitivity of 80.9% (233/288), with sensitivities of 64.0%, 91.7%, and 85.2% in early-stage, late-stage, and hematolymphoid cancers respectively. The mean number of CAAs detected in early-stage, late-stage, and hematolymphoid CAA-positive cancers was 2.1, 3.1, and 4.6. Inclusion of viral targets aided the detection of nasopharyngeal (NPC), cervical, and p16+ oropharyngeal (oPSCC) cancers. EBV DNA was detected in 91.7% of NPC cases. In cervical cancer and p16+ oPSCC, HPV DNA (genotypes 16, 18, 33 and 39) was detected in 71.4% and 90.9% of cases respectively. Conversely, none of the p16- oPSCC cancers had detectable HPV DNA. Across the 21 TOO classes, a high confidence prediction could be made in 62.4% (mean 73.6%, range 26.7%-100%) of cases with an overall accuracy of 87.7% (mean 84.9%, range 33.3%-100%). Overall TOO prediction performance was modeled in a simulated cohort based on cancer incidence in Singapore and Hong Kong, giving an accuracy of 87.3% and 88.7% respectively. Conclusions: The mutation-based NGS assay sensitively detects cancer signals and accurately predicts the tumor origin for early-stage, late-stage, and hematolymphoid cancers. Inclusion of viral targets additionally enables early detection of virus-associated cancers.