Liquid Biopsy: From Discovery to Clinical Application

Background: Early cancer detection could identify tumors at a time when outcomes are superior and treatment is less morbid. This prospective case-control sub-study (from NCT02889978 and NCT03085888 ) assessed the performance of targeted methylation analysis of circulating cell-free DNA (cfDNA) to detect and localize multiple cancer types across all stages at high specificity. Participants and methods: The 6689 participants [2482 cancer (>50 cancer types), 4207 non-cancer] were divided into training and validation sets. Plasma cfDNA underwent bisulfite sequencing targeting a panel of >100 000 informative methylation regions. A classifier was developed and validated for cancer detection and tissue of origin (TOO) localization. Results: Performance was consistent in training and validation sets. In validation, specificity was 99.3% [95% confidence interval (CI): 98.3% to 99.8%; 0.7% false-positive rate (FPR)]. Stage I–III sensitivity was 67.3% (CI: 60.7% to 73.3%) in a pre-specified set of 12 cancer types (anus, bladder, colon/rectum, esophagus, head and neck, liver/bile-duct, lung, lymphoma, ovary, pancreas, plasma cell neoplasm, stomach), which account for ~63% of US cancer deaths annually, and was 43.9% (CI: 39.4% to 48.5%) in all cancer types. Detection increased with increasing stage: in the pre-specified cancer types sensitivity was 39% (CI: 27% to 52%) in stage I, 69% (CI: 56% to 80%) in stage II, 83% (CI: 75% to 90%) in stage III, and 92% (CI: 86% to 96%) in stage IV. In all cancer types sensitivity was 18% (CI: 13% to 25%) in stage I, 43% (CI: 35% to 51%) in stage II, 81% (CI: 73% to 87%) in stage III, and 93% (CI: 87% to 96%) in stage IV. TOO was predicted in 96% of samples with cancer-like signal; of those, the TOO localization was accurate in 93%. Conclusions: cfDNA sequencing leveraging informative methylation patterns detected more than 50 cancer types across stages. Considering the potential value of early detection in deadly malignancies, further evaluation of this test is justified in prospective population-level studies.

Although programmed death-ligand 1-programmed death 1 (PD-L1-PD-1) inhibitors are broadly efficacious, improved outcomes have been observed in patients with high PD-L1 expression or high tumor mutational burden (TMB). PD-L1 testing is required for checkpoint inhibitor monotherapy in front-line non-small-cell lung cancer (NSCLC). However, obtaining adequate tumor tissue for molecular testing in patients with advanced disease can be challenging. Thus, an unmet medical need exists for diagnostic approaches that do not require tissue to identify patients who may benefit from immunotherapy. Here, we describe a novel, technically robust, blood-based assay to measure TMB in plasma (bTMB) that is distinct from tissue-based approaches. Using a retrospective analysis of two large randomized trials as test and validation studies, we show that bTMB reproducibly identifies patients who derive clinically significant improvements in progression-free survival from atezolizumab (an anti-PD-L1) in second-line and higher NSCLC. Collectively, our data show that high bTMB is a clinically actionable biomarker for atezolizumab in NSCLC.

Cell-free DNA (cfDNA) in the blood provides a noninvasive diagnostic avenue for patients with cancer 1 . However, characteristics of the origins and molecular features of cfDNA are poorly understood. We developed an approach to evaluate fragmentation patterns of cfDNA across the genome and found that cfDNA profiles of healthy individuals reflected nucleosomal patterns of white blood cells, while patients with cancer had altered fragmentation profiles. We applied this method to analyze fragmentation profiles of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric, or bile duct cancer and 245 healthy individuals. A machine learning model incorporating genome-wide fragmentation features had sensitivities of detection ranging from 57% to >99% among the seven cancer types at 98% specificity, with an overall AUC of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation based cfDNA analyses detected 91% of cancer patients. The results of these analyses highlight important properties of cfDNA and provide a proof of principle approach for screening, early detection, and monitoring of human cancer.