Intratumoral regulatory T cells are associated with treatment response to neoadjuvant chemotherapy and prognosis in gastroesophageal adenocarcinoma

Abstract

Gastroesophageal junction (GEJ) adenocarcinoma is an increasingly common cancer with complex biology and poor prognosis. The treatment strategy for locally advanced tumors involves multimodal treatment with perioperative chemotherapy. However, survival rates remain low, especially for advanced disease. Here, formalin-fixed paraffin-embedded tumor sections from 72 patients with GEJ I and II adenocarcinoma who underwent primary resection or perioperative standard-of-care FLOT treatment were analyzed for their intratumoral T cell composition using multiplex immunohistochemistry. The proportions of T cells and their influence on survival were evaluated using Mann-Whitney U and log rank analyses. A comparison of short- and long-term survivors revealed significant differences in the infiltration of regulatory T cells (Tregs). Tumors after neoadjuvant FLOT treatment presented increased proportions of CD8⁺ T cells with reduced Granzyme B expression, indicating an altered immune response. Overall survival analysis revealed that high infiltration of Tregs was associated with poor survival. Notably, responders to FLOT therapy had a greater T cell frequency and improved survival, whereas nonresponders presented higher levels of Tregs and CD8⁺ T cells expressing TIM-3. Overall, GEJ cancer patients had increased CD8⁺ T cells after neoadjuvant chemotherapy with FLOT, and Tregs were associated with treatment response and reduced survival.

Keywords: GEJ adenocarcinoma; Multiplex immunohistochemistry; intratumoral T cells; neoadjuvant chemotherapy.

Figure 1.

Differential abundance of tumor-infiltrating CD8⁺ T cells between primary resected and neoadjuvant treated gastroesophageal junction adenocarcinomas. (A) Representative mIHC image of the T cell markers CD3 (green), CD4 (red), Tbet (purple), GATA3 (yellow), RORγt (orange), and FOXP3 (cyan) counterstained with DAPI (blue). The image is from a tumor section from a treatment-naïve patient (PR). (B) Representative mIHC image of the T cell markers CD8 (green), LAG−3 (red), GzmB (purple), PD−1 (yellow), TIM−3 (orange), and Ki−67 (cyan) and counterstaining with DAPI (blue). The image is from a tumor section from a patient who received neoadjuvant chemotherapy prior to surgery (NAT). Scale bar in overview image: 100 μm, scale bar in magnifications: 10 µm for A and B. Violin plots of the median (C) proportions of CD3⁺CD4⁺ T cells alone, or coexpressing (D) Tbet⁺, (E) GATA3⁺, (F) RORγt⁺, (G) FOXP3⁺, (H) FOXP3⁻, and (I) CD8⁺ T cells, co-expressing (J) Ki−67, (K) GzmB, (L) PD−1, (M) LAG−3, and (N) TIM−3 of PR patients (n = 26; in blue) and NAT patients (n = 46; in orange). P values were calculated using the unpaired t test (Mann‒Whitney U, MWU). *P ≤ 0.05.

Figure 2.

A low proportion of intratumoral Tregs is associated with improved overall survival after neoadjuvant chemotherapy. (A) Representative mIHC image of high- and low-frequency CD3⁺CD4⁺ Th cells and CD8⁺ cytotoxic T cells, both of which were counterstained with DAPI. Scale bar: 50 μm. Images of CD3⁺CD4⁺ and high-CD8⁺ cells were obtained from NATs, whereas images of low-CD8⁺ cells were obtained from a primary resected tumor. Kaplan‒Meier analysis of overall survival of patients with GEJ, stratified by the median of the indicated proportions of (B) CD3⁺CD4⁺FOXP3⁺, (C) CD3⁺CD4⁺FOXP3⁻, and (D) CD8⁺ T cells of PR and NAT patients. P values were calculated using the log-rank test. **P ≤ 0.01.

Figure 3.

Nonresponders to neoadjuvant chemotherapy display increased proportions of Treg cells and TIM−3-expressing CD8⁺ T cells. (A) Kaplan‒Meier analysis of the overall survival of patients with GEJ according to treatment response to FLOT according to the pathological assessment. The responders (R) included major or moderate responses (TRG1/2, n = 27), and the nonresponders (NR) included minor responses (TRG3, n = 19). The P value was calculated using the log-rank test; ***P ≤ 0.001. Violin plots of the median proportions of intratumoral conventional T cells CD3⁺CD4⁺ alone (B) or coexpressing FOXP3⁺, (C) FOXP3⁻, and (D) cytotoxic T cells CD8⁺, or coexpressing (E) Ki−67, (F) GzmB, (G) PD−1, (H) LAG−3 or (I) TIM−3, according to response status. Mann‒Whitney U test. **P ≤ 0.01, *P ≤ 0.05.

Figure 4.

Increased intratumoral Treg cells predict short-term survival. Patients were stratified into short-term (<2y, n = 36) and long-term (≥2y, n = 36) survivors based on their overall survival. Violin plots of GEJ patients depict the median proportions of intratumoral T helper cells (A) CD3⁺CD4⁺ alone, or coexpressing (B) Tbet⁺, (C) GATA3⁺, (D) RORγT⁺, (E) FOXP3⁺, (F) FOXP3⁻, and (G) cytotoxic CD8⁺ T cells alone, or co-expressing (H) Ki−67, (I) GzmB, (J) PD−1, (K) LAG−3, and (L) TIM−3 compared between short-term (violet) and long-term (green) survivors. Mann‒Whitney U test. **P ≤ 0.01.