Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA LiuMC OxnardGR KleinEA SwantonC SeidenMV ConsortiumCCGA CummingsSteven R AbsalanFarnaz AlexanderGregory AllenBrian AminiHamed AravanisAlexander M BagariaSiddhartha BazarganLeila BeausangJohn F BermanJennifer BettsCraig BlockerAlexander BrednoJoerg CalefRobert CannGordon CarterJeremy ChangChristopher ChawlaHemanshi ChenXiaoji ChienTom C CivelloDaniel DavydovKonstantin DemasVasiliki DesaiMohini DongZhao FayzullinaSaniya FieldsAlexander P FilippovaDarya FreesePeter FungEric T GnerreSante GrossSamuel Halks-MillerMeredith HallMegan P HartmanAnne-Renee HouChenlu HubbellEarl HunkapillerNathan JagadeeshKarthik JamshidiArash JiangRoger JungByoungsok KimTaeHyung KlausnerRichard D KurtzmanKathryn N LeeMark LinWendy LipsonJafi LiuHai LiuQinwen LopatinMargarita MaddalaTara MaherM Cyrus MeltonCollin MichAndrea NautiyalShivani NewmanJonathan NewmanJoshua NiculaVirgil NicolaouCosmos NikolicOngjen PanWenying PatelShilpen PrinsSarah A RavaRichard RonaghiNeda SakaryaOnur SatyaRavi Vijaya SchellenbergerJan ScottEric SehnertAmy J ShaknovichRita ShanmugamAvinash ShashidharKC ShenLing ShenoyArchana ShojaeeSeyedmehdi SinghPranav SteffenKristan K TangSusan ToungJonathan M ValouevAnton VennOliver WilliamsRichard T WuTony XuHui H YakymChristopher YangXiao YeciesJessica YipAlexander S YoungrenJack YueJeanne ZhangJingyang ZhangLily ZhangLori Quan ZhangNan CurtisChristina BerryDonald A 2020 © 2020 The Authors 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.