Abstract 2114: HPV-driven cancers show distinct methylation signatures in cell-free DNA (cfDNA)

• Published in: Cancer research (Chicago, Ill.) Ill.), 2020-08, Vol.80 (16_Supplement), p.2114-2114
• Main 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.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2020-2114

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 pre