Zhao, Dong

Abstract Purpose: We recently reported the development of a cell-free DNA (cfDNA) targeted methylation (TM)-based sequencing approach for a multi-cancer early detection (MCED) test that includes cancer signal origin prediction. Here, we evaluated the prognostic significance of cancer detection by the MCED test using longitudinal follow-up data. Experimental Design: As part of a Circulating Cell-free Genome Atlas (CCGA) substudy, plasma cfDNA samples were sequenced using a TM approach, and machine learning classifiers predicted cancer status and cancer signal origin. Overall survival (OS) of cancer participants in the first 3 years of follow-up was evaluated in relation to cancer detection by the MCED test and clinical characteristics. Results: Cancers not detected by the MCED test had significantly better OS (P < 0.0001) than cancers detected, even after accounting for other covariates, including clinical stage and method of clinical diagnosis (i.e., standard-of-care screening or clinical presentation with signs/symptoms). Additionally, cancers not detected by the MCED test had better OS than was expected when data were adjusted for age, stage, and cancer type from the Surveillance, Epidemiology, and End Results (SEER) program. In cancers with current screening options, the MCED test also differentiated more aggressive cancers from less aggressive cancers (P < 0.0001). Conclusions: Cancer detection by the MCED test was prognostic beyond clinical stage and method of diagnosis. Cancers not detected by the MCED test had better prognosis than cancers detected and SEER-based expected survival. Cancer detection and prognosis may be linked by the underlying biological factor of tumor fraction in cfDNA.

Abstract Background The Circulating Cell-free Genome Atlas (CCGA; NCT02889978) study is a prospective, multi-center, observational, case-control study with longitudinal follow-up to support the development of a plasma cfDNA-based multi-cancer early detection assay. We previously reported that the fraction of tumor-derived cfDNA fragments in plasma (estimated tumor fraction [TF]) is associated with detection of multiple cancer types using a prototype whole-genome bisulfite sequencing (WGBS) assay in a pre-specified substudy. In addition, detectability of breast cancer varied by hormone-receptor (HR) status: HR negative (HR-) breast cancer had a higher detection rate than the overall breast cancer cohort. Here, we performed a planned in-depth analysis of the breast cancer cohort to understand the main clinical and biological determinants of detectability using our WGBS assay. Methods Blood samples were prospectively collected from participants (pts) with newly-diagnosed, untreated breast cancer. In a pre-specified CCGA substudy, 511 (31.4%) of 1,628 pts had a clinicopathologic diagnosis of breast cancer (any stage, excluding stage 0) and a WGBS result. Pre-specified biological and clinical factors were assessed for correlation with TF: clinical stage, pathologic N stage, lesion size (by imaging), T stage, histologic grade, hormone receptor (HR) status, and proliferative rate (Ki-67 by IHC [MIB-1 clone]) via univariate and multivariate analyses. Ki-67 was obtained from pathology reports provided by enrollment sites and also assessed centrally (Mayo Clinic [Rochester, MN]). Stage-specific differences in detection rates between HR positive (HR+) and HR- breast cancers due to biological differences such as histologic grade and/or proliferative rate was also assessed. Results Of 511 pts, 94 (18.4%; 48.9% HR- and 12.3% in HR+) had WGBS-detected cancer. Higher TF was significantly associated with cancer detection (p<0.001). In univariate analyses, the variables significantly associated with TF were clinical stage, pathological N stage, and histologic grade (p<0.001 each). Primary lesion size was modestly correlated with TF (all stages combined: Spearman's correlation coefficient rs=0.4, p<0.001), and when grouped by early and late stages (stages I/II: rs=0.27, p<0.01; stages III/IV: rs=0.50, p<0.01); clinical T stage was significantly associated with TF only when all stages were combined (p<0.001). Proliferative rate (Ki-67) was positively correlated with TF at stages I/II (rs=0.29, p<0.05) but not III/IV (rs=-0.24, p=0.34); Ki-67 results obtained through Mayo were similar to that from enrollment sites (Pearson’s correlation coefficient rp=0.68, p<0.001). HR- breast cancers had significantly higher TF versus HR+ breast cancers only at stage II (p<0.001). In addition, compared to HR+ breast cancers, HR- breast cancers had a significantly higher proportion of high histologic grade (p<0.001 at stages I and II) and proliferative rate at earlier stages (p<0.05 at stages I, II, and III). In the multivariate analysis, clinical stage (stage III vs I and IV vs I) and HR status remained the most significantly associated with TF (p<0.001 each). Conclusions Using a prototype methylation-based plasma cfDNA cancer detection assay, TF was found to be associated with detection, and with clinical and biological features of breast cancer. Features routinely used to assess clinical aggressiveness (eg, HR- status and histologic grade) also demonstrated stage-specific associations with TF. Given the known relationship between TF and detection, these data suggest that clinical and biological cancer features may also provide insight into the variability of cancer detection using plasma-based cfDNA tests. Citation Format: Minetta C. Liu, Jodi M. Carter, Daniel W. Visscher, Karla Kopp, Rita Shaknovich, Xiaoji Chen, Kathryn N. Kurtzman, Shilpen Patel, Jacqueline D. Brooks, Carlo R. Cosenza, Jafi A. Lipson, Donald A. Richards, Fergus J. Couch, Zhao Dong, Hai Liu, Oliver Venn, Joerg Bredno, Eric T. Fung, Anne-Renee Hartman. Blood-based cancer detection in plasma cell-free DNA (cfDNA): Evaluating clinical and pathologic tumor characteristics in participants with breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-01.

1545 Background: Screening tests for early cancer detection are often criticized due to risk of overdiagnosis—detection of good prognosis cancers which may not require immediate treatment. We recently reported development of cfDNA sequencing approaches for cancer detection; longitudinal follow-up (F/U) data were utilized here to evaluate prognostic significance of cancer detection using cfDNA. Methods: Plasma cfDNA samples were subjected to whole-genome bisulfite sequencing (WGBS, 30X) as part of a previously-reported Circulating Cell-free Genome Atlas (CCGA; NCT02889978) substudy. This exploratory analysis evaluated the overall survival (OS) of training and test set participants (pts) with cancer (20 cancer types, any stage I-IV). Combining train and test set pts, univariate and multivariate analyses (Cox proportional hazards) assessed OS association with WGBS result (cancer detected vs not detected, set at 98% specificity), clinical stage (IV vs I-III), diagnostic method (symptom- vs screen-detected), sex, age, and histologic grade. Results: Of 827 pts from the training set with F/U (median 12.2 mo), 334 (40.4%) had WGBS-detected cancer. Among 127 (15.4%) pts with cancer that died during F/U, cancer was detected in 104 (81.9%). Results were similar in the test set. In univariate analyses all variables were associated with prognosis, including WGBS result (HR 7.7 p<0.001). In multivariate analyses accounting for other covariates, the three variables that most significantly remained prognostic were WGBS (HR 3.0, p<0.001), clinical stage (HR 3.3, p<0.001), and diagnostic method (HR 3.0, p<0.001). Validation of these findings is ongoing in an independent cohort of ~5,000 cancer pts from CCGA using an optimized assay; updated performance results will be reported. Conclusions: Cancers detected using WGBS of cfDNA had a worse prognosis than cancers not detected. WGBS cancer detection carried comparable prognostic significance as clinical stage. By preferentially detecting higher risk cancers, cancer detection using plasma cfDNA may avoid some of the overdiagnosis that has been seen with some existing cancer screening methods. Clinical trial information: NCT02889978.