861P Combined patient specific tumor and HPV sequencing enables high sensitivity detection of ctDNA in HPV-associated oropharyngeal carcinoma patients

HPV-associated oropharyngeal carcinoma (OPC) patients shed viral DNA which has been employed to monitor treatment response. The sensitivity of this approach, however, appears to be limited in OPC patients with low-burden disease. We hypothesized that circulating tumor DNA (ctDNA) detection through personalized tumor-specific alterations in combination with HPV ctDNA offers a more sensitive yet specific approach in early-stage HPV OPC patients. Cell-free DNA (cfDNA) from HPV+ OPC patients enrolled in a phase II chemoradiation (CRT) de-escalation trial (NCT03323563) was collected. All patients underwent resection of the primary tumor before CRT and a baseline plasma was drawn at this time. DNA from the tumor and matched normal underwent whole-exome sequencing to identify somatic variants and for HPV typing. A dual MRD-HPV assay was developed based on the Invitae’s Personalized Cancer MonitoringTM (PCM) assay including personalized tumor-specific and custom HPV-specific primers targeting high-risk HPV types. The MRD-HPV assay was applied to plasma cfDNA samples. 158 patients with T0-2/N1-N2c HPV-OPC were enrolled. The MRD-HPV assay was successfully designed for 111 patients targeting a median of 50 mutations per patient (range: 26-50). 64.0% (71/111) of patients had HPV16-related tumors and available baseline plasma, and all subsequent analyses were conducted on this group. While ctDNA detection by HPV testing resulted in sensitivity of 85.9% (61/71), ctDNA detection by MRD increased sensitivity to 90.1% (64/71). Furthermore, ctDNA detection by either HPV and/or MRD increased sensitivity to 95.8% (68/71). The 10 patients with negative HPV ctDNA tests harbored tumors with lower tumor volume and lower levels of genome instability than HPV ctDNA positive patients. Two of the three patients with negative baseline MRD-HPV had detectable ctDNA within 1 month from the start of treatment. The combination of PCM MRD and HPV ctDNA assays provide a highly sensitive approach for detection of low disease burden in early HPV-associated OPC patients.