Table_3_Immune changes in hilar tumor draining lymph nodes following node sparing neoadjuvant chemoradiotherapy of localized cN0 non-small cell lung cancer.docx

BackgroundWhile much progress has been accomplished in the understanding of radiation-induced immune effects in tumors, little is known regarding the mechanisms involved at the tumor draining lymph node (TDLN) level. The objective of this retrospective study was to assess the immune and biological changes arising in non-involved TDLNs upon node sparing concurrent chemoradiotherapy (CRT) of non-small cell lung cancer (NSCLC) tumors.
MethodsPatients with proven localized (cN0M0) NSCLC, treated by radical surgery plus lymph node dissection with (CRT+) or without (CRT-) neoadjuvant chemoradiotherapy, whereby radiotherapy was targeted on the primary tumor with no significant incidental irradiation of the non-involved TDLN station (stations XI), were identified. Bulk RNA sequencing of TDLNs was performed and data were analyzed based on differential gene expression (DGE) and gene sets enrichment.
ResultsSixteen patients were included and 25 TDLNs were analyzed: 6 patients in the CRT+ group (12 samples) and 10 patients in the CRT- group (13 samples). Overall, 1001 genes were differentially expressed between the two groups (CRT+ and CRT-). Analysis with g-profiler revealed that gene sets associated with antitumor immune response, inflammatory response, hypoxia, angiogenesis, epithelial mesenchymal transition and extra-cellular matrix remodeling were enriched in the CRT+ group, whereas only gene sets associated with B cells and B-cell receptor signaling were enriched in the CRT- group. Unsupervised dimensionality reduction identified two clusters of TDLNs from CRT+ patients, of which one cluster (cluster 1) exhibited higher expression of pathways identified as enriched in the overall CRT+ group in comparison to the CRT- group. In CRT+ cluster 1, 3 out of 3 patients had pathological complete response (pCR) or major pathological response (MPR) to neoadjuvant CRT, whereas only 1 out of 3 patients in the other CRT+ cluster (cluster 2) experienced MPR and none exhibited pCR.
ConclusionNeoadjuvant node sparing concurrent CRT of NSCLC patients is associated with distinct microenvironment and immunological patterns in non-involved TDLNs as compared to non-involved TDLNs from patients with non-irradiated tumors. Our data are in line with studies showing superiority of lymph node sparing irradiation of the primary tumor in the induction of antitumor immunity.

背景：尽管学界在解析肿瘤辐射诱导的免疫效应方面已取得诸多进展，但针对肿瘤引流淋巴结（Tumor Draining Lymph Node, TDLN）层面的相关作用机制，目前仍知之甚少。本回顾性研究旨在评估非小细胞肺癌（Non-Small Cell Lung Cancer, NSCLC）患者在接受淋巴结保留式同步放化疗（Concurrent Chemoradiotherapy, CRT）后，其未受累肿瘤引流淋巴结中出现的免疫与生物学变化。
方法：本研究纳入经证实的局限性（cN0M0）非小细胞肺癌患者，所有患者均接受根治性手术联合淋巴结清扫术，其中部分患者术前接受新辅助同步放化疗（CRT+组），其余患者未接受新辅助同步放化疗（CRT-组）；本研究的放疗靶区仅针对原发性肿瘤，未对未受累的肿瘤引流淋巴结站（第11组淋巴结）造成显著的意外照射。随后对肿瘤引流淋巴结样本进行批量RNA测序，并基于差异基因表达（Differential Gene Expression, DGE）与基因集富集分析对测序数据进行解读。
结果：本研究共纳入16例患者，共计分析25枚肿瘤引流淋巴结样本：CRT+组包含6例患者（12份样本），CRT-组包含10例患者（13份样本）。两组间共鉴定出1001个差异表达基因。通过g-profiler进行富集分析发现，CRT+组中富集到与抗肿瘤免疫应答、炎症应答、缺氧、血管生成、上皮间质转化以及细胞外基质重塑相关的基因集；而CRT-组仅富集到与B细胞及B细胞受体信号通路相关的基因集。无监督降维分析识别出CRT+患者的肿瘤引流淋巴结可分为两个簇：其中一个簇（簇1）相较于CRT-组，其整体富集于CRT+组的通路表达水平更高。在CRT+组的簇1中，3例患者均对新辅助同步放化疗达到病理完全缓解（pathological complete response, pCR）或主要病理缓解（major pathological response, MPR）；而在CRT+组的另一个簇（簇2）中，仅1例患者达到主要病理缓解，无患者达到病理完全缓解。
结论：相较于未接受放疗的非小细胞肺癌患者的未受累肿瘤引流淋巴结，接受淋巴结保留式新辅助同步放化疗的患者，其未受累肿瘤引流淋巴结的微环境与免疫模式存在显著差异。本研究结果与此前多项研究结论一致，即针对原发性肿瘤的淋巴结保留式放疗可更有效地诱导抗肿瘤免疫应答。