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

项目摘要/摘要 高危型人乳头瘤病毒株诱发口咽鳞状细胞癌的发病率 继续上升。由于HPV(+)病预示着治疗后良好的预后， 年轻患者的合并症较少，临床医生现在认识到它是与烟草不同的临床实体- 相关的HPV(-)疾病。但尽管手术和辅助放化疗提高了存活率 (CRT)，许多HPV(+)OPC患者因严重的治疗相关毒性而长期患病。 这导致了许多治疗去强化的临床试验，这些试验试图减少毒副作用，同时 保持历史上的存活率。理想情况下，高危患者将继续接受标准治疗，而低风险患者将继续接受 中等风险的患者将接受降级治疗。然而，临床上对一种 辅助主观临床评估的客观风险生物标记物：治疗前影像和术后 病理学。液体活组织检查可以提供这样的客观性；它们量化了癌细胞脱落的无细胞DNA(CfDNA)， 称为循环肿瘤DNA(CtDNA)，存在于唾液或血浆等生物体液中。此外，在HPV(+)OPC中，无细胞HPV DNA(cf-HPV)与ctDNA并列为微小残留病(MRD)的衡量标准。但血浆和唾液化验 术后缺乏检测cf-HPV MRD的敏感性。为了应对这一临床挑战，我们提供我们的新型液体 杰克逊·普拉特(JP)手术引流液(SDF)的活组织检查。我们相信，cf-hpv将丰富自卫队。 与血浆相比，因为它更靠近肿瘤切除部位和淋巴结， 在那里种植局部微转移瘤。此外，因为JP引流管还会捕获淋巴液体 从撕裂的颈部淋巴系统来看，我们还认为SDF含有信息效应白细胞 转移到转移结节和原发肿瘤的肿瘤微环境(TME)。至 为了阐明SDF的预后潜力，我们收集了成对的SDF、血浆、肿瘤活检和 转移性淋巴结样本。首先，使用聚合酶链式反应和下一代测序方法，我们将量化 配对血浆和SDF样本中的cf-hpv载量。然后我们将比较每种样本类型中的cf-hpv。 分别对风险(结外扩散和肿瘤分期)和复发的组织病理学标志物。我们 然后将跟踪从血浆和sdf中分离出来的ctDNA上与肿瘤有关的变体，以显示ctDNA水平 与cf-HPV配对，进一步验证cf-HPV是诊断MRD的良好指标。最后，我们将使用数字细胞术 使用工具和质量细胞术对SDF内的免疫细胞进行免疫表型，以确定它们是否反映 免疫反应基因在配对转移结节和肿瘤中的表达水平。如果得到证实，我们的研究已经 一种新型液体中三生物标志物分析(免疫细胞、cf-HPV和ctDNA)的可能性 分析物(SDF)可以提供精确的风险分层，以帮助进行主观的临床诊断。

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.