PO.TB10.02 · 肿瘤生物学
HPV阴性头颈癌术后淋巴渗出液的多组学研究
Multi-omicsinpostoperative lymphatic exudate in HPV-negative head and neck cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
我们团队已证明，通过手术引流管收集的淋巴渗出液（“淋巴液”）是一种富含ctDNA的近端生物体液，可预测HPV非依赖性头颈癌（HPV-HNSCC）的复发。淋巴液中还富含蛋白质、代谢物和细胞等其他分析物。为理解肿瘤切除后即刻的液态肿瘤微环境（TME），我们对HNSCC中24小时的淋巴液进行了表征。我们开展了多组学调查，证明淋巴液不同于血浆，代表着一个在很大程度上未被探索的、与免疫调节和转移相关的生物标志物来源。对淋巴液上清液和血浆进行代谢组学和蛋白质组学分析。蛋白质组学筛查通过Olink进行，并归一化至内标以进行定量分析。代谢组学样本通过LC-MS检测并归一化。16s文库利用Illumina测序并在QIIME中分析。对6例患者的16s测序显示术后淋巴液中存在一系列细菌属，且在各患者间具有一致性。分布主要由口腔原生微生物组成，提示术后淋巴液可能反映手术前的原生微生物组。在部分患者中观察到病原体暴发。正在进行的分析将把微生物组数据与癌症和手术结局相关联。对4例患者（包括3例在术后1年内复发者）的术后淋巴液和配对血浆进行了代谢物谱分析。所有4例患者淋巴液中的代谢物谱比血浆更多样，每毫升的代谢物丰度更高。13%的已鉴定代谢物在淋巴液中富集>2倍。复发患者的淋巴液显示出高水平的饱和及单不饱和脂肪酸，据报道其与转移相关。术后淋巴液具有独特且在很大程度上未经探索的代谢组，可用于挖掘与复发相关的生物标志物。对44例HPV-HNSCC患者的淋巴液和血浆蛋白质组进行了比较。该队列包括25例复发（REC）患者和19例随访>1年无疾病证据（NED）的患者。淋巴液和血浆显示出高度不重叠的蛋白质谱。平均绝对火山图评分为25.2，表明淋巴液与血浆之间存在强烈的整体差异。淋巴液显著富集91种蛋白质（p值范围4.1e-47至0.037），其中许多与炎症相关。与NED相比，REC淋巴液还富集炎症标志物，包括IL-1a、TNF和TSLP（p值分别为0.002、0.015、0.003），这些标志物与癌症转移、侵袭和血管生成有关。淋巴液的命中蛋白与血浆大多不重叠，表明在这种体液中存在新型生物标志物发现的机会。
对术后淋巴液的微生物组、代谢组和蛋白质组的分析揭示了一种丰富且有差异性的肿瘤邻近生物体液，可用于研究与转移和TME相关的生物标志物。
查看英文原文 English abstract
Our team has demonstrated that lymphatic exudate collected via surgical drains (“lymph”) is a proximal biofluid rich in ctDNA and prognostic of recurrence in HPV-independent Head and Neck Cancer (HPV- HNSCC). Other analytes such as proteins, metabolites, and cells are also abundant in lymph. To understand the liquid tumor microenvironment (TME) immediately after tumor resection, we characterized lymph at 24 hrs in HNSCC. We performed a multi-omic survey, demonstrating that lymph is distinct from plasma and represents a largely unexplored source of biomarkers relevant to immune modulation and metastasis.Metabolomics and proteomics were run on lymph supernatant and plasma. Proteomic screening was performed via Olink and normalized to internal standards for quantitative analysis. Metabolomic samples were assayed via LC-MS and normalized. 16s libraries were sequenced utilizing Illumina and analyzed in QIIME. 16s sequencing on 6 patients indicated an array of bacterial genera present in post-surgical lymph with consistency across patients. The distribution consisted primarily of microbes native to the oral cavity, suggesting post-surgical lymph may reflect the native microbiome prior to surgery. Pathogenic blooms were observed in a subset of patients. Ongoing analysis will correlate microbiome data with both cancer and surgical outcomes.Metabolite profiling was performed on post-surgical lymph and matched plasma from 4 patients, including 3 who recurred within 1 year of surgery. The metabolite profile in lymph of all 4 patients was more diverse than plasma with higher metabolite abundance per mL. 13% of identified metabolites were >2-fold enriched in lymph. Lymph from recurred patients showed high levels of saturated and monounsaturated fatty acids, reported to be associated with metastasis. Post-surgical lymph has a unique and largely uncharted metabolome to mine for biomarkers associated with recurrence.Proteomes were compared between lymph and plasma from 44 HPV-HNSCC patients. This cohort consists of 25 recurred (REC) and 19 patients with no evidence of disease (NED) with >1 year of follow up. Lymph and plasma displayed highly non-overlapping protein repertoires. The mean absolute volcano score was 25.2, indicating a strong global difference between lymph and plasma. Lymph was significantly enriched in 91 proteins (p range 4.1e-47 to 0.037), many associated with inflammation. REC lymph was also enriched in inflammatory markers compared to NED, including IL-1a, TNF, and TSLP (p = 0.002, 0.015, 0.003, respectively), which have been implicated in cancer metastasis, invasion, and angiogenesis. Lymph hits were largely non-overlapping with plasma, indicating the opportunity for novel biomarker discovery in this fluid.
Analysis of the microbiome, metabolome, and proteome of post-surgical lymph reveals a rich and differentiated tumor-adjacent biofluid to study biomarkers relevant to metastasis and the TME.
利益披露 Disclosure
Z. Costliow, None.