Enhanced antitumour immunity following neoadjuvant chemoradiotherapy mediates a favourable prognosis in women with resected pancreatic cancer

Abstract

Background: This study investigates sex disparities in clinical outcomes and tumour immune profiles in patients with pancreatic ductal adenocarcinoma (PDAC) who underwent upfront resection or resection preceded by gemcitabine-based neoadjuvant chemoradiotherapy (nCRT).

Methods: Patients originated from the PREOPANC randomised controlled trial. Upfront surgery was performed in 82 patients, and 66 received nCRT before resection. The impact of sex on overall survival (OS) was investigated using Cox proportional hazards models. The immunological landscape within the tumour microenvironment (TME) was mapped using transcriptomic and spatial proteomic profiling.

Results: The 5-year OS rate differed between the sexes following resection preceded by nCRT, with 43% for women compared with 22% for men. In multivariate analysis, the female sex was a favourable independent prognostic factor for OS only in the nCRT group (HR 0.19; 95% CI 0.07 to 0.52). Multivariate heterogeneous treatment effects analysis revealed a significant interaction between sex and treatment, implying increased nCRT efficacy among women with resected PDAC. The TME of women contained fewer protumoural CD163+MRC1+M2 macrophages than that of men after nCRT, as indicated by transcriptomic and validated using spatial proteomic profiling.

Conclusion: PDAC tumours of women are more sensitive to gemcitabine-based nCRT, resulting in longer OS after resection compared with men. This may be due to enhanced immunity impeding the infiltration of protumoral M2 macrophages into the TME. Our findings highlight the importance of considering sex disparities and mitigating immunosuppressive macrophage polarisation for personalised PDAC treatment.

Keywords: CHEMOTHERAPY; MACROPHAGES; PANCREATIC CANCER; PANCREATIC SURGERY; RADIOTHERAPY.

Figure 1

Schematic overview of the methodological steps The squircles illustrate the methodological steps of the study: (1) patient inclusion and clinical procedure, (2) Cox proportional hazards regression modelling, (3) gene expression profiling, and (4) digital spatial profiling (DSP). CD45, cluster of differentiation 45; FFPE, formalin-fixed paraffin-embedded; FUMA GWAS, Functional Mapping and Annotation of the Genome-Wide Association Studies; nCRT, neoadjuvant chemoradiotherapy; ORA, over-representation analysis; PanCK, Pan-cytokeratin; PDAC, pancreatic ductal adenocarcinoma; RCT, randomised controlled trial; ROI, region of interest.

Figure 2

Survival analysis stratified by treatment in patients with resected PDAC who received nCRT or upfront surgery (A) Kaplan-Meier curves and univariate Cox regression models, stratified by treatment, illustrating the significantly prolonged OS in women with resected PDAC who received nCRT compared with men. The x-axis displays the survival time (months) and the y-axis displays the survival probability (%). Cross-symbols denote censored patients. The number-at-risk table provides information on the number of patients at risk of death at each specific time point. (B) Forest plots of the stratified multivariate Cox proportional hazards models illustrate that the female sex is a favourable independent prognostic factor for OS in the nCRT group but not in the upfront surgery group. nCRT, neoadjuvant chemoradiotherapy; OS, overall survival; P.adj, P value adjusted; PDAC, pancreatic ductal adenocarcinoma; US, upfront surgery.

Figure 3

Unstratified heterogeneous treatment effect survival analysis in patients with resected PDAC who received nCRT or upfront surgery forest plots of the univariate (A) and multivariate (B) unstratified Cox proportional hazards interaction models illustrate the impact of nCRT followed by resection versus resection on OS across various subgroups. A statistically significant interaction between the sex and treatment was observed in both univariate and multivariate analysis, with an increased benefit of nCRT in women with resected PDAC. nCRT, neoadjuvant chemoradiotherapy; OS, overall survival; PDAC, pancreatic ductal adenocarcinoma; US, upfront surgery.

Figure 4

Survival and data exploration analysis in resected PDAC patients included in the transcriptomic Nanostring analysis (A) Kaplan-Meier curves and univariate Cox regression models, stratified by treatment, illustrating the preservation of the significantly prolonged OS in women with resected PDAC who received nCRT compared with men in this patient subset. The x-axis displays the survival time (months), and the y-axis displays the survival probability (%). Crosses denote censored patients. The number-at-risk table provides information on the number of patients at risk of death at each specific time point. (B) Forest plots of the stratified multivariate Cox proportional hazards models illustrate that the female sex remains a favourable independent prognostic factor for OS in the nCRT group but not in the upfront surgery group for this patient subset. (C, D) t-SNE biplots illustrating the expression of 730 immune-related genes reveal no apparent segregation of patients based on sex (C), even after stratifying by treatment (D). Each dot represents a patient, with coordinates depicting the first (x-axis) and second (y-axis) t-SNE dimensions. nCRT, neoadjuvant chemoradiotherapy; OS, overall survival; P.adj, P value adjusted for multiple testing using the Benjamini-Hochberg correction; PDAC, pancreatic ductal adenocarcinoma; t-SNE, t-Distributed stochastic neighbour embedding; US, upfront surgery.

Figure 5

Tumourous transcriptomic NanoString analysis in patients with resected PDAC who received nCRT or upfront surgery (A, B). Volcano plots illustrate the differential gene expression profiles between the sexes in the upfront surgery (A) and nCRT group (B). The x-axis displays the log2 fold of change, while the y-axis displays the −log10 P value. Each dot corresponds to a gene, with genes on the right (positive) upregulated in females compared with male PDAC patients and genes on the left (negative) upregulated in males compared with female PDAC patients. (C) Barplot illustrating the results of the over-representation analysis for pathways characterised by a gene set size of ≤100. Only pathways exhibiting a proportional overlap of ≥3% (displayed on the left side of the x-axis) and a P.adj<0.01 (displayed on the right side of the x-axis) are presented. Each bar represents a specific pathway, and the analysis revealed that the pathways showing significant enhancement were exclusively observed in females (ie, diminished in males) following the administration of gemcitabine-based nCRT. (D) Boxplots illustrating the gene alterations in response to nCRT reveal a signature with antitumourous properties in female PDAC patients. The x-axis displays enhanced (left) and diminished (right) genes in females, and the y-axis displays the log2 gene expression count. (E) Boxplots illustrating the abundance of protumourous M2 macrophages, quantified by CD163 and MRC1 expression, reveal significantly lower infiltration in the TME of female PDAC patients than males. The y-axis displays the M2 macrophage score. (F) Scatterplots illustrating Pearson’s correlations between the M2 macrophage score (y-axis) and overall survival (OS) in months (x-axis), stratified by treatment groups and sex. A significant association in females who received nCRT can be observed, where a higher number of M2 macrophages in the TME was negatively correlated with OS. In D–F, each dot represents a patient. CD163, cluster of differentiation 163; MRC1, Mannose Receptor C-Type 1; nCRT, neoadjuvant chemoradiotherapy; PDAC, pancreatic ductal adenocarcinoma; US, upfront surgery.

Figure 6

Protein-based GeoMx digital spatial profiling in patients with resected PDAC who received nCRT (A) t-SNE biplot illustrating the expression of 75 proteins reveals no apparent segregation of ROIs by sex or patient, while ROIs clearly segregate by their histological areas. Each dot represents an ROI, with coordinates depicting the first (x-axis) and second t-SNE dimensions (y-axis). (B) Boxplots illustrate the proportion of protumourous M2 macrophages, quantified by CD163 protein expression, within the total macrophage compartment, quantified by CD68 protein expression. Various compartments (x-axis) of the PDAC TME of females show significantly higher CD163 to CD68 (ie, M2 to total macrophage) ratios than males. (C) Boxplots illustrating the BAD protein expression in the PDAC TME reveal significantly higher expression in carcinoma areas of females than in males. The x-axis displays the different TME compartments, and the y-axis displays the Log2 protein expression count. (D) Scatterplots illustrating Pearson’s correlations between the CD163 to CD68 ratios (y-axis) and overall survival (OS) in months (x-axis), stratified by sex. A significant association in carcinoma areas of males as well as desmoplasia areas and immune aggregates of females can be observed, where the M2 to total macrophage ratio negatively correlates to OS. In B–D, each dot represents a patient’s ROI and immunofluorescent microscopic images below the boxplots in B and C exemplify different TME compartments (ie, histopathological areas) stained with morphological markers for tumour cells (PanCK), immune cells (CD45) and DNA (SYTO13). BAD, BCL2 associated agonist of cell death; CD, cluster of differentiation; nCRT, neoadjuvant chemoradiotherapy; P.adj, P value adjusted for multiple testing using the Benjamini-Hochberg correction; PanCK, Pan-cytokeratin; PDAC, pancreatic ductal adenocarcinoma; ROI, region of interest; TME, tumour microenvironment; t-SNE, t-distributed stochastic neighbour embedding.