Pre-Treatment Circulating Tumor Human Papillomavirus (HPV) DNA and Recurrence Risk in HPV Associated Malignancy: Oropharyngeal, Anal Canal, and Cervical Cancer

Human papillomavirus (HPV) is a primary oncogenic driver of cancers of the anal canal (AC), uterine cervix (UC), and oropharynx (OPX). Circulating tumor HPV DNA (ctHPVDNA) may assist in cancer detection, risk stratification, treatment assessment, and surveillance. The purpose of this study was to determine pre-treatment detectability of HPV ctDNA in OPX, AC, and UC cancer patients and to investigate the association of pretreatment ctHPVDNA quantity with clinical and pathologic factors and disease recurrence. Patients with newly diagnosed cancers of the AC, UC, and OPX referred for definitive chemoradiotherapy (CRT) participated in a prospectively collected IRB-approved serum biobank. Serum was collected prior to the initiation of CRT and was analyzed in a blinded fashion for HPV (serotypes 16, 18, 31, and 33) E6 and E7 ctDNA using a digital droplet PCR multiplex assay. Univariate associations between pre-treatment ctHPVDNA level and pre-treatment patient characteristics or disease recurrence were assessed using Fisher’s exact test or Wilcoxon rank sum test and a univariate Fine and Gray Cox regression model, respectively. Seventy-one patients were included. Distribution of primary site was 18 OPX, 33 AC, and 20 UC. Forty-one (58%) of the 71 patients were confirmed to be p16+ or HPV+, 3% were HPV-negative, and 39% were unknown. ctHPVDNA was detectable in 89% of OPX, 85% anal canal, and 60% of cervical cancer patients. Of 41 patients with known p16+ or HPV+ status, 28 patients (68%) had detectable ctHPVDNA. Median copy number per mL was 41.7 in the total cohort, 1054.2 (range 17.5-161083.3) in OPX, 479.2 (range 22.9-38500.0) in AC, and 39.2 (range 12.9-39666.7) in UC patients. There was no association between ctHPVDNA detectability and age at diagnosis, sex, smoking history, T stage, N stage, or M stage. Among patients with cervical cancer, ctHPVDNA was detectable in 80% of patients with squamous or adenosquamous histology vs 25% of patients with adenocarcinoma (p = 0.0314). The median follow-up for all patients was 27 months (IQR 17.9-34.3 months). Recurrence at any site occurred in 14 of 71 patients (19.7%). Compared to patients with undetectable ctHPVDNA, detectable ctHPVDNA was not associated with disease recurrence (hazard ratio 0.925, 95% CI 0.266-3.217, p = 0.902). There was no association between ctHPVDNA copy number and recurrence when stratifying by <200 copies per mL versus >200 copies per mL. We demonstrate the feasibility of pre-treatment ctHPVDNA detectability in patients with squamous or adenosquamous carcinoma of the oropharynx, anal canal, and uterine cervix. This is the first validation of a ctHPVDNA assay in anal canal and cervical cancer. Further investigation of ctHPVDNA levels pre-treatment, post-treatment, and during surveillance in these malignancies is warranted.