Analytical validation of a multi-cancer early detection test with cancer signal origin using a cell-free DNA–based targeted methylation assay
Gregory E. Alexander, Wendy Lin, Fabian E. Ortega, Madhuvanthi Ramaiah, Byoungsok Jung, Lijuan Ji, Ekaterina Revenkova, Payal Shah, Christian Croisetière, Jennifer R. Berman, Lane Eubank, Gunjan Naik, Jacqueline M. Brooks, Andrea Mich, Seyedmehdi Shojaee, Neda Ronaghi, Hemanshi Chawla, Xinyi Hou, Qin‐Wen Liu, Christopher-James A. V. Yakym, Patriss W. Moradi, Meredith Halks‐Miller, Alexander M. Aravanis, Sonya Parpart-Li, Nathan Hunkapiller
Abstract
The analytical validation is reported for a targeted methylation-based cell-free DNA multi-cancer early detection test designed to detect cancer and predict the cancer signal origin (tissue of origin). A machine-learning classifier was used to analyze the methylation patterns of >105 genomic targets covering >1 million methylation sites. Analytical sensitivity (limit of detection [95% probability]) was characterized with respect to tumor content by expected variant allele frequency and was determined to be 0.07%-0.17% across five tumor cases and 0.51% for the lymphoid neoplasm case. Test specificity was 99.3% (95% confidence interval, 98.6-99.7%). In the reproducibility and repeatability study, results were consistent in 31/34 (91.2%) pairs with cancer and 17/17 (100%) pairs without cancer; between runs, results were concordant for 129/133 (97.0%) cancer and 37/37 (100%) non-cancer sample pairs. Across 3- to 100-ng input levels of cell-free DNA, cancer was detected in 157/182 (86.3%) cancer samples but not in any of the 62 non-cancer samples. In input titration tests, cancer signal origin was correctly predicted in all tumor samples detected as cancer. No cross-contamination events were observed. No potential interferent (hemoglobin, bilirubin, triglycerides, genomic DNA) affected performance. The results of this analytical validation study support continued clinical development of a targeted methylation cell-free DNA multi-cancer early detection test.