Abstract 3359: Sensitive detection of locoregional MRD after head and neck cancer surgery
Authors: Earland Noah , Ramirez Ricardo J. , Gerndt Sophie P. , Harris Peter K. , Hearn Andrew I. , Inkman Matthew , Szymanski Jeffrey J. , Semenkovich Nick , Wahle Benjamin M. , Xu Zhongping , Chen Kevin , Zhang Jin , Zevallos Jose P. , Chaudhuri Aadel A. 
Published: April 2023

Abstract
Background: Plasma cell-free DNA (cfDNA) has low sensitivity to detect locoregional molecular residual disease (MRD) immediately after surgery for limited-stage head and neck cancer. To enhance locoregional MRD sensitivity, we utilize a novel biofluid proximal to the primary tumor: the serosanguinous fluid collected by surgical drains inserted into the wound bed following regional lymph node dissection. We demonstrate that cancer-associated cfDNA in the surgical drain fluid (SDF) is a sensitive biomarker of locoregional MRD detection, risk stratification, and prognostication.
Methods: We extracted DNA from 168 SDFs, 48 paired plasmas, and 44 tumor specimens collected 24 hours after surgery from 90 patients: 74 HPV (+) oropharyngeal cancers (OPC), 8 HPV (-) oral cancers, 2 thyroid cancers, and 6 benign neck pathologies. SDFs were also collected at 0, 6, 12, and 24 hours after surgery from 11 HPV (+) patients. We applied TaqMan qPCR to our specimens to quantify cell-free HPV (cf-HPV), a proxy for cancer-associated cfDNA in HPV (+) malignancies. We then measured the association between cf-HPV and extranodal extension (ENE), AJCC 8 staging, and established clinical and pathological risk criteria from 4 HPV (+) OPC clinical trials. We validated our findings using HPV next-generation sequencing. We also compared the predictive value of high-risk pathology with cf-HPV MRD for HPV (+) OPC recurrences. Lastly, we developed a second assay for cell-free BRAF V600E (cf-V600E) in thyroid cancer SDF.
Results: Serially collected SDF revealed cf-HPV decreased continuously from 0 to 24 hours postoperative, and that the 24-hour timepoint stratified detectable versus undetectable persistent residual disease. At 24 hours, SDF was significantly more enriched with cf-HPV compared to plasma (P &lt; 0.0001). In a subset of 9 patients, HPV genotype and detection in SDF were 100% concordant between NGS and PCR. Strikingly, SDF cf-HPV was 11-fold higher in pN2 patients versus pN1/N0 (P = 0.02) and 15-fold higher in cases with ENE (P = 0.003), suggesting SDF reflects aggressive nodal pathology and captures locoregional MRD. When we classified our patients according to established clinical and pathological risk criteria, we found significantly higher SDF cf-HPV among high-risk patients (AUC = 0.77). Crucially, in HPV (+) patients treated with surgery alone, SDF MRD detected 100% of locoregional recurrences, while plasma MRD and high-risk pathology detected 0%. Lastly, as proof of concept, we demonstrated SDF cf-V600E detection in the context of high-risk thyroid cancer.
Conclusions: Surgical drain fluid cancer-associated cfDNA is a novel minimally invasive biomarker. Our SDF assay was strongly associated with high-risk criteria and detected locoregional relapse with 100% sensitivity compared to 0% in plasma. Our data suggest that SDF liquid biopsy is ultra-sensitive and has the potential to inform postoperative molecular-based risk stratification.
Citation Format: Noah Earland, Ricardo J. Ramirez, Sophie P. Gerndt, Peter K. Harris, Andrew I. Hearn, Matthew Inkman, Jeffrey J. Szymanski, Nick Semenkovich, Benjamin M. Wahle, Zhongping Xu, Kevin Chen, Jin Zhang, Jose P. Zevallos, Aadel A. Chaudhuri. Sensitive detection of locoregional MRD after head and neck cancer surgery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3359.