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

Abstract

Background: While 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.

Methods: Patients 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.

Results: Sixteen 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.

Conclusion: Neoadjuvant 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.

Keywords: immune changes; lymph nodes sparing irradiation; non small cell lung cancer (NSCLC); radiotherapy; tumor draining lymph node (TDLN).

Figure 1

TDLNs from patients receiving neoadjuvant CRT and those treated with upfront surgery exhibit distinct molecular signatures. (A) Differentially expressed genes between CRT⁺ and CRT^- groups. In red, are represented genes with an adjusted P-value ≤ 0.05 and a log2 fold-change [Log2 (FC)] ≤ -1 or ≥ 1. All other genes are in grey. (B) CRT⁺ enriched pathways found with GSEA. (C) Enriched pathways found with g-profiler. Each pathway is colored depending on the broad biological processes in which it is involved. (D) CRT^- enriched pathways found with GSEA. n.a., not applicable.

Figure 2

Unsupervised analysis shows two clusters in the CRT⁺ group. (A) t-SNE representation of TDLNs, colored depending on treatment received by patients. Differentially expressed genes between (B) CRT⁺ cluster 1 and all CRT^- samples, (C) CRT⁺ cluster 2 and all CRT^- samples and (D) CRT⁺ cluster 1 and CRT+ cluster 2. In red, are represented genes with an adjusted P-value ≤ 0.05 and a log2 fold-change [Log2 (FC)] ≤ -1 or ≥ 1. All other genes are in grey.

Figure 3

Enriched pathways in the two CRT⁺ clusters compared to CRT^- samples. Heatmap representation of the scaled expression (Z-score) of the differentially expressed genes, associated to the enriched pathways found with g-profiler (A) in the CRT⁺ cluster 1 and CRT^- comparison; (B) in the CRT⁺ cluster 2 and CRT^- comparison and (C) in the CRT⁺ cluster 1 and CRT⁺ cluster 1 comparison. Log2 (FC): log2 fold-change.