Prehabilitation during neoadjuvant chemotherapy results in an enhanced immune response in esophageal adenocarcinoma tumors: A randomized controlled trial

Abstract

Background: For patients with locally advanced esophagogastric cancer, the standard of care in the UK is neoadjuvant chemotherapy (NAC) followed by surgery. Prehabilitation exercise training can improve physiological function and fitness. If such improvements translate to increased immune infiltration of tumors, exercise could be prescribed as an immune adjuvant during NAC and potentially improve clinical outcomes. As such, we aimed to determine whether prehabilitation increased tumor infiltrating lymphocytes (TILs).

Methods: We assessed 22 patients with locally advanced esophageal cancer on a randomized control trial comparing 16 weeks of low-to-moderate intensity twice weekly supervised and thrice weekly home-based exercise (Prehab: n = 11) to no prehabilitation (Control: n = 11). Our primary outcome was to compare tumor-immune responses between Controls and Prehab. We compared formalin-fixed paraffin-embedded tumors by high-resolution multispectral immunohistochemistry (mIHC) and NanoString spatial transcriptomics. Secondarily, we determined relationships between changes in fitness to the exercise training and tumor-immune measures. Specifically, we assessed percentage changes in peak cardiorespiratory fitness as assessed by peak oxygen uptake (V̇O_2peak) before NAC (Baseline) and after 8 weeks of NAC (Post-NAC), and changes between Baseline and following 8 weeks of NAC recovery before surgery (Pre-surgery) and correlated changes in fitness with tumor-immune responses. Finally, as an exploratory aim, we assessed clinical outcomes between groups, including survival, therapy tolerance, and tumor regrading.

Results: We observed that Prehab had significantly more CD8+ lymphocytes in their tumors (mean difference (diff.) = 1.79, 95% confidence interval (95%CI): 0.76‒2.82, p < 0.001) and their stroma (mean diff. = 1.59, 95%CI: 0.66‒2.52, p < 0.001) than the Controls. When normalized to total numbers of TILs, Prehab had higher levels of CD56+ natural killer (NK) cells (median diff. = 0.87, 95%CI: 0.25‒2.18), p = 0.0274), consisting primarily of CD56^dim NK cells (median diff. = 0.48, 95%CI: 0.03‒2.53), p = 0.0464). Evaluation of the presence and localization of tumor-associated tertiary lymphoid structures (TLS) in the esophageal tumors revealed that most TLS were in the peritumoral regions. Prehab had a higher TLS cell density (cells/mm²; median diff. = 18,959, 95%CI: 13,518‒22,635), p < 0.001) and more clearly defined germinal centers indicative of mature TLS visually. We observed that Prehab maintained their V̇O_2peak during NAC while the Controls' V̇O_2peak reduced by 9.0% ± 10.2% (mean ± SD) (Post-NAC: p = 0.018). Pre-surgery, Prehab V̇O_2peak was a clinically meaningful 3.27 ± 1.31 mL/kg/min higher than Controls (p = 0.022). Between Baseline and Post-NAC, where the Prehab maintained V̇O_2peak better than Controls, there were significant positive associations with percentage changes in V̇O_2peak and the frequencies of CD8+ TILs (r = 0.531, p = 0.016), programmed death-ligand 1+ (PDL1+) cells (r = 0.566, p = 0.009), and granzyme B+ (GrzB+) TILs (r = 0.582, p = 0.007). Similar relationships were observed for changes in V̇O_2peak from Baseline to Pre-Surgery only in the Prehab group. We observed no differences between groups regarding clinical outcomes such as survival, therapy tolerance, or tumor regrading.

Conclusion: We show that exercise training during NAC, which promotes higher levels of cardiorespiratory fitness than no exercise, is associated with increased frequencies of TILs and maturity of TLS. These data suggest that exercise during NAC enhances the immune system. Future studies are warranted to understand the clinical consequences of this.

Keywords: Esophageal cancer surgery; Exercise oncology; Prehabilitation exercise; Tertiary lymphoid structures; Tumor infiltrating lymphocytes.

Graphical abstract

Fig. 1

Prehabilitation exercise maintains cardiopulmonary fitness through neoadjuvant chemotherapy. (A) V̇O_2peak relative to body mass; (B) absolute V̇O_2peak; (C) oxygen consumption at anaerobic threshold. Data are means with standard deviation error bars. * p < 0.05 different from Baseline; ^# p < 0.05 different from Post-NAC; ^§ p < 0.05 different from Control. NAC = neoadjuvant chemotherapy; V̇O_2peak = peak oxygen uptake.

Fig. 2

Prehabilitation exercise training is associated with an increased frequency of CD8+ tumor infiltrating lymphocytes (TILs) in esophageal tumors in an aerobic capacity-mediated effect. Multiplex immunohistochemistry using an 8-plex immune panel plus DAPI was applied to the tumor tissues and imaged using Akoya’s PhenoImager HT. InForm® automated image analysis software was used to visualize and quantify the immune infiltrate. Percentages of TILs are presented relative to the total frequency of nucleated cells. (A) Representative images of tumor tissue from patients randomized to the Control (n = 11) or Prehab group (n = 11), with 2 patients from the Control group (top and bottom left) and 2 patients from the Prehab group (top and bottom right); (B) Tumor infiltrating cell marker frequencies between Prehab and Controls. (C) Stroma-infiltrating cell marker frequencies between Prehab and Controls. Relationships between the percentage change in peak oxygen consumption (mL/kg/min) from Baseline to Post-NAC and the frequency of (D) CD8+ TILs, (E) PDL1+ cells, and (F) Granzyme B+ TIL. In (A): CD8 (red), CD4 (white), CD68 (maroon), granzyme B (orange), PDL1 (green), CD57 (yellow), cytokeratin (cyan), DAPI (blue). Data are means and standard error of the mean. NAC = neoadjuvant chemotherapy.

Fig. 3

Transcriptional analyses reveal that Prehabilitation is associated with increased natural killer (NK) cell tumor infiltration. (A) We clustered gene expression values into panels to decipher and estimate the immune cell abundance for Controls (n = 8) and Prehab (n = 9). (B) Differences between groups for total tumor infiltrating lymphocytes (TIL) and log₂ transformed normalized to levels of TIL for (C) CD56+ NK cells, (D) CD56^dim NK cells, (E) CD8+ T cells, (F) cytotoxic cells, (G) exhausted T cells, (H) CD3+ T cells, (I) B cells, (J) macrophages, (K) neutrophils, (L) dendritic cells, (M) mast cells. Boxplots represent the median and interquartile range with error bars reflecting the minimum and maximum values. Prehab = prehabilitation.

Fig. 4

Pathway analysis and gene expression reveal that Prehabilitation is associated with altered tumor gene expression. (A) Clustered gene pathway analyses differences between groups. (B) Volcano plot of differential gene expression plotted showing log₂ fold expression change (X axis) vs. ‒log₁₀ (p value) (Y axis). The genes with Wald test p ≤ 0.05 are represented by blue circles (lower expression in Prehab) or red circles (higher expression in Prehab). Prehab = prehabilitation.

Fig. 5

Prehabilitation is associated with the development of more mature tertiary lymphoid structures (TLS). (A) Representative structure of a tumor associated TLS in esophageal adenocarcinoma; regions of interest containing TLS were stamped on the whole tissue scan, imaged at high resolution followed by image analysis in InForm to identify cells using machine learning algorithmic segmentation and phenotypic identification based on all stained channels. Composite fluorescent multiplex image along with single marker fluorescent images showing the phenotyping of different cell types within a TLS using InForm software (CD19, yellow; CD20, pink; CD68, orange; CD4, cyan; FOXP3, magenta; CD8, red; DCLAMP, green; Pan-CK, white; DAPI, blue). (B) Representative images of TLS from tumors from the Control vs. the Prehab cohorts. High-power field images of each of the TLS per tumor case were then used for automated trained tissue segmentation and the total cell counts within each TLS were calculated along with the TLS surface area. (C) The proportions of each of the immune cell subsets within TLS were determined for tumors from the Control and the Prehab cohorts.