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

一种新型术后预后液体活检：口咽癌手术引流液中肿瘤相关 cfDNA 和白细胞分析

• 批准号: 10678080
• 负责人: Noah Jackson Earland
• 金额: $ 3.36万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678080
• 关键词: Adjuvant; Adjuvant Therapy; Aftercare; Alcohols; Benchmarking; Biological Assay; Biological Markers; Biopsy; CD8-Positive T-Lymphocytes; Cells; Cervical; Cisplatin; Clinical; Clinical Trials; Clinical assessments; Code; Cytometry; DNA; DNA analysis; Data; Decision Making; Detection; Disease; Dose; Effector Cell; Ensure; Environment; Equation; Excision; Extranodal; Future; Gene Expression; Gene Frequency; Goals; HPV-High Risk; HPV-negative head and neck cancer; Homing; Hour; Human Papillomavirus; Image; Immune; Immune Response Genes; Immunophenotyping; Incidence; Individual; Infiltration; Inflammatory; Kinetics; Laceration; Length; Leukocytes; Liquid substance; Lymph; Lymphatic System; Malignant Neoplasms; Measures; Metastatic Neoplasm to Lymph Nodes; Micrometastasis; Molecular; Morbidity - disease rate; Mutation; Neck Dissection; Nodal; Operative Surgical Procedures; Oropharyngeal Squamous Cell Carcinoma; Pathologic; Pathology; Patient risk; Patient-Focused Outcomes; Patients; Pattern; Plasma; Population; Postoperative Period; Primary Neoplasm; Proxy; Quality of life; Radiation Dose Unit; Recurrence; Recurrent disease; Regimen; Residual Neoplasm; Risk; Risk Assessment; Risk Marker; Saliva; Sampling; Signal Transduction; Site; Surgical Pathology; Survival Rate; Tobacco; Treatment outcome; Treatment-related toxicity; Tumor stage; Urine; Variant; anti-tumor immune response; cancer cell; cancer surgery; candidate selection; cell free DNA; chemoradiation; clinical diagnostics; comorbidity; digital; exome sequencing; genetic variant; high risk; human papilloma virus oropharyngeal squamous cell carcinoma; improved; leukocyte activation; liquid biopsy; lymph nodes; malignant oropharynx neoplasm; minimally invasive; mutant; neoplastic cell; next generation sequencing; novel; patient stratification; prognostic; prospective; relapse prediction; risk stratification; salivary assay; side effect; success; tool; trafficking; treatment response; tumor; tumor DNA; tumor microenvironment; tumor-immune system interactions; wound bed

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.

项目摘要/摘要 高危型（HR）HPV毒株导致的口咽鳞状细胞癌（OPC）的发病率 继续上升。由于HPV（+）疾病是良好的治疗后结局的预后， 年轻患者合并症较少，临床医生现在将其视为与烟草不同的临床实体， HPV（-）相关疾病。尽管手术和辅助放化疗后生存率有所提高， (CRT)许多HPV（+）OPC患者因严重的治疗相关毒性而长期发病。 这导致了许多治疗去强化临床试验，这些试验寻求减少毒副作用， 保持历史性的生存率。理想情况下，高风险患者将继续接受标准方案，而低风险患者将继续接受标准方案。 中度风险患者将接受降级治疗。然而，临床上非常需要 辅助主观临床评估的客观风险生物标志物：治疗前成像和术后 病理液体活检可以提供这样的客观性;它们量化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液体中。此外，在HPV（+）OPC中，无细胞HPV DNA（cf-HPV）与ctDNA平行作为微小残留病（MRD）的量度。但血浆和唾液检测 缺乏在手术后检测这种cf-HPV MRD的敏感性。为了应对这一临床挑战，我们提供了我们的新型液体 杰克逊普拉特（JP）手术引流液（SDF）的活检测定。我们相信SDF将被cf-HPV所丰富 与血浆相比，因为它更接近原发肿瘤切除部位和淋巴结， 在那里接种局部微转移。此外，由于JP引流管也捕获淋巴液， 从撕裂的颈部淋巴系统，我们还认为SDF含有信息效应白细胞 转移到转移性淋巴结和原发性肿瘤的肿瘤微环境（TME）。到 为了开始阐明SDF的预后潜力，我们收集了成对的SDF、血浆、肿瘤活检， 转移性淋巴结样本。首先，使用PCR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负荷。然后我们将比较每种样本类型中的cf-HPV 个体地与风险的组织病理学标志物（结节扩展和肿瘤阶段）和复发相关联。我们 然后将追踪从血浆和SDF中分离的ctDNA上的肿瘤信息变体，以显示ctDNA水平 与cf-HPV一致，并进一步验证cf-HPV是MRD的良好代表。最后，我们将使用数字细胞仪 工具和大量细胞计数来对SDF内的免疫细胞进行免疫表型分析，以确定它们是否反映了 配对转移性淋巴结和肿瘤中免疫应答基因表达水平。如果得到证实，我们的研究 在新型液体中证明三生物标记物分析（免疫细胞、cf-HPV和ctDNA）潜力 分析物（SDF）可以提供精确的风险分层以帮助主观临床诊断。