Increased CD4/CD8 Lymphocyte ratio predicts favourable neoadjuvant treatment response in gastric cancer: A prospective pilot study

Abstract

Background: Despite optimal neoadjuvant chemotherapy only 40% of gastric cancer tumours achieve complete or partial treatment response. In the absence of treatment response, neoadjuvant chemotherapy in gastric cancer contributes to adverse events without additional survival benefit compared to adjuvant treatment or surgery alone. Additional strategies and methods are required to optimize the allocation of existing treatment regimens such as FLOT chemotherapy (5-Fluorouracil, Leucovorin, Oxaliplatin and Docetaxel). Predictive biomarkers detected using immunohistochemistry (IHC) methods may provide useful data regarding treatment response.

Aim: To investigate the utility of CD4, CD8, Galectin-3 and E-cadherin in predicting neoadjuvant FLOT chemotherapy tumour response in gastric adenocarcinoma.

Methods: Forty-three adult patients with gastric adenocarcinoma, of which 18 underwent neoadjuvant chemotherapy, were included in a prospective clinical cohort. Endoscopic biopsies were obtained from gastric cancer and normal adjacent gastric mucosa. Differences in expression of Galectin-3, E-cadherin, CD4⁺ and CD8⁺ molecules between tumours with and without treatment response to neoadjuvant chemotherapy were assessed with IHC. Treatment response was graded by clinical pathologists using the Tumour Regression Score according to the College of American Pathologists criteria. Treatment response was defined as complete or near complete tumour response, whereas partial or poor/no response was defined as incomplete. Digital IHC images were annotated and quantitatively assessed using QuPath 0.3.1. Biomarker expression between responsive and incomplete response tumours was assessed using a two-sided Wilcoxon test. Biomarker expression was also compared between normal and cancer tissue and between 15 paired tumour samples before and after chemotherapy. We performed a preliminary multivariate analysis and power analysis to guide future study. Statistical analyses were completed using R 4.1.2.

Results: The ratio between CD4⁺ and CD8⁺ lymphocytes was significantly greater in treatment responsive tumours (Wilcoxon, P = 0.03). In univariate models, CD4⁺/CD8⁺ ratio was the only biomarker that significantly predicted favourable treatment response (Accuracy 86%, P < 0.001). Using a glmnet multivariate model, high CD4⁺/CD8⁺ ratio and low Galectin-3 expression were the most influential variables in predicting a favourable treatment response. Analyses of paired samples found that FLOT chemotherapy also results in increased expression of CD4⁺ and CD8⁺ tumour infiltrating lymphocytes (Paired Wilcoxon, P = 0.002 and P = 0.008, respectively). Our power analysis suggests future study requires at least 35 patients in each treatment response group for CD8 and Galectin-3 molecules, whereas 80 patients in each treatment response group are required to assess CD4 and E-cadherin biomarkers.

Conclusion: We demonstrate that an elevated CD4⁺/CD8⁺ Ratio is a promising IHC-based biomarker to predict favourable treatment response to FLOT neoadjuvant chemotherapy in locally advanced gastric cancer.

Keywords: CD4; CD8; Galectin-3; Gastric cancer; Neoadjuvant chemotherapy; Treatment response.