HPV-Seq is Prognostic in p16-Positive Oropharyngeal Cancer (OPC): Independent Validation on a Large Prospective Cohort and a Secondary Analysis of NRG-HN002.

HPV circulating tumor DNA (ctDNA) is a liquid biopsy biomarker that could evaluate treatment response and risk stratify patients after (chemo)radiotherapy ([C]RT). However, HPV ctDNA is undetectable at baseline in up to 15% of pts and needs prospective validation. HPV next-generation sequencing (HPV-seq) offers both genotyping and ultrasensitive quantification of HPV ctDNA. We validated HPV-seq, testing pre-specified hypotheses: (1) pre-RT HPV ctDNA correlates with gross tumor volume (GTV); (2) undetectable post-RT HPV ctDNA predicts favorable 2-year locoregional control (LRC) and progression-free survival (PFS). We analyzed pre- and post-RT HPV ctDNA in 2 cohorts of non-metastatic p16+ OPC. Cohort 1 (n = 237): single-institution prospective study, standard RT/CRT; HPV ctDNA at pre-RT and ~3 mo post-RT. Cohort 2 (n = 126): secondary endpoint of NRG-HN002, a phase II trial of low-risk p16+ OPC (AJCC 7e T1-T2 N1-N2b or T3 N0-N2b, ≤10 pack-year) randomized to de-escalated RT vs. CRT to 60 Gy; HPV ctDNA at pre-RT and ~4 wk post-RT. HPV ctDNA was measured by HPV-seq, targeting 38 HPV types. Correlations were assessed by Spearman coefficient (r). Associations with LRC/PFS were assessed by Fisher exact test (2-sided α = 0.05). Pre-RT HPV ctDNA was detected in 228/237 (96.2%) of Cohort 1 and 126/126 (100%) of Cohort 2 (range = 0.2 to 408147 copies/mL). Across both cohorts, dominant HPV types were HPV-16 (87.1%), 33 (5.0%), 35 (4.8%), 18 (2.0%), 26 (0.6%), 38 (0.3%), and 59 (0.3%). Pre-RT HPV ctDNA was correlated with GTV in both Cohort 1 (r = 0.46 [0.34, 0.56]) and Cohort 2 (r = 0.25 [0.08, 0.41]). In a multivariable model, baseline HPV ctDNA was associated with N category in both Cohort 1 (p<0.001) and Cohort 2 (P = 0.0125) and not with T category or site (tonsil vs. other). At post-RT, HPV ctDNA clearance occurred in 189/237 (79.7% [74.2-84.4]) in Cohort 1 and 88/126 (69.8% [61.3, 77.2]) in Cohort 2. Pts who demonstrated post-RT HPV ctDNA clearance had better 2-yr LRC and PFS (Table) with negative predictive value (NPV) > 96% for LRC and > 94% for PFS. This study provides prospective clinical validation of the prognostic value of HPV-seq in two high quality independent cohorts. Pre-RT HPV ctDNA was detected in >96% of pts with p16+ OPC and correlated with GTV and N-category. Post-RT HPV ctDNA clearance was associated with favorable outcomes. Cohort 1 showed more clearance and stronger association with LRC and PFS, possibly due to test timing (3 vs. 1 month post-RT) and sample size. [ABSTRACT FROM AUTHOR]