Background: Anal squamous cell carcinoma (SCC) is a rare gastrointestinal malignancy associated with human papillomavirus (HPV) in more than 90% of cases. HPV circulating tumor (ct)-DNA is an emerging biomarker for monitoring some HPV-driven cancers, however, its role in anal cancer remains undefined.

Methods: We conducted a multicenter, single-arm phase II clinical trial of pembrolizumab in patients with advanced anal SCC (n=32). Enrolled patients had metastatic anal SCC and received pembrolizumab 200 mg IV every three weeks. The primary endpoint was objective response rate (ORR) by RECIST v 1.1, and exploratory objectives included analysis of TTMV-HPV DNA (NavDx^TM, Naveris, Waltham, MA) using serially collected plasma samples. Clinical benefit rate (CBR) was defined as best response of complete response (CR), partial response (PR), or stable disease (SD) ≥ 6 months. Progression-free survival (PFS) was defined as time from first dose of therapy to progression or death. The date of second plasma blood draw was used as the landmark initial timepoint for PFS of change in TTMV-HPV DNA score.

Results: The investigator-assessed ORR was 9.4% (95% CI: 2.0–25.0) including 1 CR and 2 PR. The CBR was 21.9% (95% CI: 9.3-40.0). Median PFS was 2.2 months (95% CI: 1.9-4.1). TTMV-HPV DNA was detected in 86.2% (25/29) of patients with a baseline sample available (score range: 4-2,462,500). Pre-treatment TTMV-HPV DNA scores positively correlated with greater tumor burden, as defined by baseline RECIST measurements (Pearson coefficient, r=0.71, p<0.0001) and were significantly associated with clinical benefit (median score 372 in those with clinical benefit versus 35,046 in patients without benefit, p=0.01). The percentage change in TTMV-HPV DNA score from baseline to cycle 3 (6 weeks) was significantly associated with clinical benefit to pembrolizumab (median 72% decrease in TTMV-HPV DNA score with clinical benefit versus 70% increase of TTMV-HPV DNA score without benefit, p=0.01). Patients who had a decreasing TTMV-HPV DNA score at 6 weeks had a median PFS of 3.0 months (95% CI: 0.66-11.9) versus 0.7 months (95% CI: 0.6-2.5) in patients with a rising TTMV-HPV score (HR: 0.37; 95% CI: 0.14–0.99, log-rank p=0.04). The patient with a CR had a detectable TTMV-HPV DNA score at baseline which became undetectable by 18 weeks of treatment (26 weeks prior to radiographic resolution of disease). The patient completed two years of pembrolizumab and continues to have no evidence of disease 5.3 years after enrollment.

Conclusions: Pembrolizumab is efficacious in a subgroup of patients with anal SCC, and TTMV-HPV DNA appears to be a circulating biomarker of immunotherapy response, representing a dynamic monitoring tool with the potential to enable rapid assessment of therapeutic response ahead of radiographic assessment of disease.

James Cleary

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