The Clinical Utility of a Next-Generation Sequencing-Based Approach to Detecting Circulating HPV DNA in Patients with Advanced Anal Cancer.

Background: To extend the practicality of liquid biopsy beyond the historical HPV circulating tumor DNA (ctDNA) assays, we evaluated the clinical relevance of a novel next-generation sequencing HPV ctDNA assay in patients with locally advanced and metastatic squamous cell cancer of the anal canal (mSCCA).

Methods: ctDNA isolated from the plasma of patients with mSCCA was sequenced using a 1.4 Mb hybrid-capture target-enrichment panel covering the whole genome sequences of all 193 HPV types. The HPV type, copy number (CN), and integration sites were determined using a bioinformatic pipeline.

Results: A total of 77 plasma samples from 28 patients with HPV-related SCCA were retrospectively analyzed. HPV ctDNA was detected in 26 cases (93%) (including uncommon subtypes). The median HPV CN was higher in metastatic versus locally recurrent/unresectable SCCA (p = 0.043). Changes in the HPV CN were concordant with the radiographic response (p = 0.027). An integration event was detected in 23 patients (82%), with presumed episomal HPV DNA present in the remaining patients. Higher HPV integration (a mean of ≥1 integration across samples) was associated with a worse overall survival from the start of immunotherapy (13.6 months versus 36.0 months; p = 0.003).

Conclusions: Using HPV-informed next-generation sequencing of the ctDNA, we found changes in the HPV CN correlated with the treatment response and that HPV integration detected in the ctDNA is an unfavorable prognostic biomarker.