Next Generation Sequencing Assay for Detection of Circulating HPV DNA (cHPV-DNA) in Patients Undergoing Radical (Chemo)Radiotherapy in Anal Squamous Cell Carcinoma (ASCC)

Abstract

Background: Following chemo-radiotherapy (CRT) for human papilloma virus positive (HPV+) anal squamous cell carcinoma (ASCC), detection of residual/recurrent disease is challenging. Patients frequently undergo unnecessary repeated biopsies for abnormal MRI/clinical findings. In a pilot study we assessed the role of circulating HPV-DNA in identifying "true" residual disease. Methods: We prospectively collected plasma samples at baseline (n = 21) and 12 weeks post-CRT (n = 17). Circulating HPV-DNA (cHPV DNA) was measured using a novel next generation sequencing (NGS) assay, panHPV-detect, comprising of two primer pools covering distinct regions of eight high-risk HPV genomes (16, 18, 31, 33, 35, 45, 52, and 58) to detect circulating HPV-DNA (cHPV DNA). cHPV-DNA levels post-CRT were correlated to disease response. Results: In pre-CRT samples, panHPV-detect demonstrated 100% sensitivity and specificity for HPV associated ASCC. PanHPV-detect was able to demonstrate cHPV-DNA in 100% (9/9) patients with T1/T2N0 cancers. cHPV-DNA was detectable 12 weeks post CRT in just 2/17 patients, both of whom relapsed. 1/16 patients who had a clinical complete response (CR) at 3 months post-CRT but relapsed at 9 months and 1/1 patient with a partial response (PR). PanHPV-detect demonstrated 100% sensitivity and specificity in predicting response to CRT. Conclusion: We demonstrate that panHPV-detect, an NSG assay is a highly sensitive and specific test for the identification of cHPV-DNA in plasma at diagnosis. cHPV-DNA post-treatment may predict clinical response to CRT.

Keywords: Plasma HPV-DNA; anal cancer; chemo-radiation; locally advanced; response prediction.

Copyright © 2020 Lee, Cutts, White, Augustin, Garcia-Murillas, Fenwick, Matthews, Turner, Harrington, Gilbert and Bhide.

Figures

Figure 1

Plasma HPV-DNA levels at baseline and 12 weeks post-CRT. 14/15 patients had above threshold levels of cHPV-DNA at baseline. At 12 weeks post- CRT, cHPV-DNA levels dropped to below threshold in all 14 patients. (A) 11 patients had complete response on DRE/MRI and cHPV-DNA below threshold. (B) 4 patients with partial response on DRE/MRI at 12 weeks and below threshold cHPV-DNA; further DRE/MRI at 6/12 months showed no evidence of relapse The dashed line on the Y-axis denotes the panHPV-detect threshold at 5.5 amplicons with more than 10 reads.

Figure 2

cHPV DNA levels in 2 patients with either a partial response or relapse using panHPV-detect. (A) In patient A03 cHPV-DNA was below threshold levels at 6 weeks, cHPV-DNA levels then increased above threshold at 12 weeks with residual disease on DRE/MRI (B) Above threshold levels of cHPV-DNA in patient A14 with complete response on DRE/MRI, distal relapse was detected at 9 months post-CRT The dashed line on the Y-axis denotes the panHPV-detect threshold at 5.5 amplicons with more than 10 reads.