The combination of patient-specific tumor and HPV sequencing to enable high-sensitivity detection of ctDNA in patients with HPV-associated oropharyngeal carcinoma.

6058 Background: Detection of Human papillomavirus (HPV) in plasma has been used to monitor treatment response in HPV-associated oropharyngeal carcinoma (OPC) but was found to have limited sensitivity in patients with low disease burden. We hypothesized that the combined detection of personalized tumor-specific alterations and HPV could provide a more sensitive approach for detecting circulating tumor DNA (ctDNA) and for disease monitoring in early-stage HPV-associated OPC patients. Methods: Patients with HPV-associated OPC (T0-2/N1-N2c) enrolled in a phase II chemoradiation (CRT) de-escalation trial (NCT03323563) were chosen for this study. The patients underwent resection of the primary tumor prior to CRT and a baseline plasma was drawn at this time. Additional plasma samples were collected weekly throughout CRT. Whole-exome and targeted HPV sequencing was performed on the primary resection to build a dictionary of somatic variants and for HPV typing, respectively. A dual PCM-HPV assay was developed based on the Invitae Personalized Cancer MonitoringTM (PCM) platform and used to evaluate the plasma samples. Tumor volume was measured by T2 MRI using a region of interest outlining the largest lymph node in the undissected neck. Results: 158 patients with T0-2/N1-N2c HPV-OPC were enrolled on the trial and the dual PCM-HPV assay was successfully designed for 111 patients targeting a median of 50 patient-specific mutations (range: 26-50). Preliminary analyses were performed on 71/111 patients (64.0%) with HPV16-associated tumors and available baseline plasma. 64/71 (90.1%) and 61/71 (85.9%) were ctDNA positive by PCM testing and HPV testing alone, respectively. Combining HPV plasma testing with PCM (PCM-HPV) improved the sensitivity to 68/71 (95.7%). This increased sensitivity was maintained when analyzing plasma samples collected weekly following CRT. The 3 patients with double negative HPV and PCM results at baseline had a lower tumor volume (p=0.05) and lower levels of genome instability (p=0.02) than those with ctDNA or PCM positive baseline plasma. HPV cfDNA levels are weakly correlated with tumor HPV genome copy number (r=0.16; p=0.001). Conclusions: The combined detection of personalized tumor-specific alterations and HPV provides a highly sensitive approach for detecting low disease burden in early stage HPV-associated OPC patients before and during treatment. Table: see text