Abstract A45: HPV sequencing facilitates ultrasensitive detection of HPV circulating tumor DNA
Authors: Leung Eric , Han Kathy , Zou Jinfeng , Zhao Zhen , Zheng Yangqiao , Wang Ting Ting , Siu Lillian L. , Pugh Trevor J. , Bratman Scott V. 
Published: June 2020

Abstract
Background: Human papillomavirus (HPV)-associated cancers often present with locoregionally confined disease and are treated with curative intent. An emerging treatment paradigm includes radical therapy (i.e., chemoradiotherapy or surgery) followed by adjuvant treatment. Accurate detection of minimal residual disease (MRD) following radical therapy could enable personalized use of adjuvant treatment. The HPV genome offers a convenient circulating tumor (ct)DNA marker for HPV-associated cancers, but current methods such as digital (d)PCR provide insufficient accuracy for accurate MRD detection and for other clinical applications in patients with low disease burden. We asked whether a next-generation sequencing approach (HPV-seq) could provide quantitative and qualitative assessment of HPV ctDNA in low-disease-burden settings.
Methods: We conducted preclinical technical validation studies on HPV-seq using cervix cancer cell lines. We then applied HPV-seq retrospectively to a prospective multicenter cohort study of locally advanced cervix cancer patients accrued from 2015 to 2016 (NCT02388698). Patients were treated with radical chemoradiotherapy with blood obtained at baseline, end of treatment, and 3 months post-treatment. Median follow-up was 27.5 months. In 38 plasma samples from 17 patients, HPV-seq results were compared with dPCR. The primary outcome was progression-free survival according to end-of-treatment HPV ctDNA detectability.
Results: HPV-seq achieved reproducible detection of HPV DNA at levels &lt;0.01%. HPV-seq and dPCR results were highly correlated (R=0.98, p=1.8E-22) with HPV-seq detecting ctDNA at levels down to 0.001% in dPCR-negative post-treatment samples. Detectable HPV ctDNA at the end-of-treatment timepoint was associated with inferior progression-free survival (log-rank p=0.05) with 100% sensitivity and 67% specificity for recurrence. Accurate HPV genotyping was successful from 100% of pretreatment plasma samples. HPV ctDNA fragment sizes were consistently shorter than non-cancer-derived cell-free DNA fragments (median fragment size difference: 22 bp) regardless of HPV genotype or clinical setting.
Conclusions: HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR. HPV-seq also reveals qualitative information about ctDNA fragments such as HPV genotype and ctDNA fragment length distribution. Our findings will have implications for MRD detection in HPV-related cancers. Future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates.
Citation Format: Eric Leung, Kathy Han, Jinfeng Zou, Zhen Zhao, Yangqiao Zheng, Ting Ting Wang, Lillian L. Siu, Trevor J. Pugh, Scott V. Bratman. HPV sequencing facilitates ultrasensitive detection of HPV circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A45.