A Novel Postoperative Prognostic Liquid Biopsy: Tumor-Associated cfDNA and Leukocyte Analysis in Oropharyngeal Cancer Surgical Drain Fluid

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PROJECT SUMMARY/ABSTRACT The incidence of oropharyngeal squamous cell carcinoma (OPC) driven by high-risk (HR) HPV strains continues to rise. As HPV (+) disease is prognostic for good post-treatment outcomes and arises in relatively young patients with fewer co-morbidities, clinicians now recognize it as a distinct clinical entity from tobacco- associated HPV (-) disease. But despite improved survival following surgery and adjuvant chemoradiation (CRT), many HPV (+) OPC patients suffer prolonged morbidity from severe treatment-associated toxicities. This has led to many treatment de-intensification clinical trials, which seek to reduce toxic side effects while maintaining historic survival rates. Ideally, high-risk patients would remain on standard regimens while low to intermediate-risk patients would receive de-escalated therapy. However, there is a great clinical need for an objective biomarker of risk to aid the subjective clinical assessments: pre-treatment imaging and postoperative pathology. Liquid biopsies can offer such objectivity; they quantify cell-free DNA (cfDNA) shed by cancer cells, called circulating tumor DNA (ctDNA), in biofluids like saliva or plasma. Further, in HPV (+) OPC, cell-free HPV DNA (cf-HPV) parallels ctDNA as a measure of minimal residual disease (MRD). But plasma and saliva assays lack sensitivity to detect this cf-HPV MRD after surgery. To this clinical challenge, we offer our novel liquid biopsy assay of Jackson Pratt (JP) surgical drain fluid (SDF). We believe SDF will be enriched with cf-HPV compared to plasma because it's more proximal to the primary tumor resection site and to the lymph nodes, where locoregional micrometastases are seeded. Additionally, because the JP drains also capture lymph fluid from the lacerated cervical lymphatic system, we also believe the SDF contains informative effector leukocytes that were in transit to the tumor microenvironments (TMEs) of metastatic nodes and the primary tumor. To begin to elucidate the prognostic potential of SDF, we have collected paired SDF, plasma, tumor biopsy, and metastatic lymph node samples. First, using PCR and next-generation sequencing approaches we will quantify the cf-HPV burden in paired plasma and SDF samples. Then we will compare cf-HPV in each sample type individually to histopathological markers of risk (extranodal extension and tumor stage) and recurrences. We will then track tumor-informed variants on ctDNA, isolated from plasma and SDF, to show that ctDNA levels align with cf-HPV and further validate that cf-HPV is a good proxy for MRD. Lastly, we will use digital cytometry tools and mass cytometry to immunophenotype the immune cells within the SDF to determine if they reflect immune response gene expression levels in paired metastatic nodes and tumors. If confirmed, our study has the potential to demonstrate that tri-biomarker analysis (immune cell, cf-HPV, and ctDNA) in a novel liquid analyte (SDF) can provide precision risk-stratification to aid subjective clinical diagnostics.