Abstract 2114: HPV-driven cancers show distinct methylation signatures in cell-free DNA (cfDNA)
Authors: Calef Robert , Venn Oliver , Maher M. Cyrus , Beausang John F. , Hubbell Earl , Patel Aman , Fields Alexander P. , Bredno Joerg , Jamshidi Arash , Aravanis Alexander M. 
Published: September 2020

Accurate tissue of origin (TOO) prediction is crucial for effective clinical follow-up in early cancer detection from blood. In the second substudy of the Circulating Cell-free Genome Atlas (CCGA; NCT02889978), we trained logistic regression classifiers under cross-validation to detect and localize cancer. Input features were methylation states from a targeted cfDNA assay of 2023 participants. TOO classification accuracy was 89% across 20 pre-specified prediction classes. We subsequently sought to understand the causes of the remaining TOO errors. 45% of the errors fell into clusters reflecting similarities in developmental biology, histology, or oncological drivers. Here, we analyzed tissues that may be affected by HPV-driven cancers; these accounted for 21% of TOO errors. The original classifier, which used only human epigenetic states as input, demonstrated cross-scoring between likely HPV-driven cancers of the anus (N = 14) and cervix (N = 11), as well as confirmed HPV-positive head & neck (H&N) cancers (37/62). We also observed HPV-associated vulva (N = 9) and penis (N = 1) cancers, which were not directly trained as TOO classes, were assigned high H&N scores. To test the hypothesis of HPV-driven TOO confusion, we assessed HPV cfDNA and HPV-driven methylation in human peripheral blood cfDNA, and used a specialist classifier restricted to HPV-associated cancers to resolve errors. We corroborated putative HPV-positive participants using targeted sequencing of HPV16 and HPV18 cfDNA fragments, and showed that the number of unique HPV-derived fragments in a sample matched with expected cancer localizations, HPV subtypes, and HPV status. Consistent with the literature, we found little evidence of HPV viremia in non-cancer participants despite the high prevalence of transient HPV infections in the US population. At 99.8% specificity, a cross-validated cutoff on the number of HPV cfDNA fragments in a sample achieved sensitivities of 78.6% (11/14), 36.3% (4/11), 66.6% (6/9), 100% (1/1), and 81.0% (30/37), for anus, cervix, vulva, penis, and confirmed HPV-positive H&N cancers, respectively. These sensitivities were similar to those achieved by the epigenetic classifier. Finally, we trained a cross-validated specialist classifier using the same features as the TOO classifier, but restricted to HPV-driven cancers. This improved TOO accuracy for detected anal cancers from 11% (1/9) to 100% (9/9), with little effect on other classes. These data support that HPV presence may explain observed cross-scoring patterns between H&N, cervix, and anus TOO prediction classes, which were driven by recurrent epigenomic changes in participants with HPV-positive cancers as detected by this assay. This suggests that modelling axes of shared biology across cancer types can be useful for accurate cfDNA TOO classification, which is critical to direct diagnostic work-up of diverse cancer types in a multi-cancer early detection test.

Citation Format: Robert Calef, Oliver Venn, M. Cyrus Maher, John F. Beausang, Earl Hubbell, Aman Patel, Alexander P. Fields, Joerg Bredno, Arash Jamshidi, Alexander M. Aravanis. HPV-driven cancers show distinct methylation signatures in cell-free DNA (cfDNA) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2114.