An Eight-Gene Hypoxia Signature Predicts Survival in Pancreatic Cancer and Is Associated With an Immunosuppressed Tumor Microenvironment

Abstract

Intratumoral hypoxia is a widely established element of the pancreatic tumor microenvironment (TME) promoting immune escape, tumor invasion, and progression, while contributing to treatment resistance and poor survival. Despite this critical role, hypoxia is underrepresented in molecular signatures of pancreatic ductal adenocarcinoma (PDA) and concurrent investigations into the hypoxia-immune status are lacking. In this work a literature-based approach was applied to derive an eight-gene hypoxia signature that was validated in fourteen cancer cell lines and in a cohort of PDA. The eight-gene hypoxia signature was significantly associated with overall survival in two distinct PDA datasets and showed independent prognostic value in multivariate analysis. Comparative analysis of tumors according to their hypoxia score (high versus low) determined that tumors with high hypoxia were significantly less enriched in cytotoxic T-cells, and cytolytic activity. In addition, they had lower expression of cytokines and tumor inflammatory markers, pointing to the signature's ability to discern an immune "cold", hypoxic TME. Combining the signature with an immune metric highlighted a worse survival probability in patients with high hypoxia and low immune reactivity, indicating that this approach could further refine survival estimates. Hypoxia as determined by our signature, was significantly associated with certain immune checkpoint inhibitors (ICI) biomarkers, suggesting that the signature reflects an aspect of the TME that is worth pursuing in future clinical trials. This is the first work of its kind in PDA, and our findings on the hypoxia-immune tumor contexture are not only relevant for ICI but could also guide combinatorial hypoxia-mediated therapeutic strategies in this cancer type.

Keywords: hypoxia; immunosuppresive; immunotharapy; microenvironment; pancreatic cancer; prognostic; signature; tumor inflammation.

Figure 1

Gene expression fold change in fourteen cancer cell lines: LogFC (fold change) of gene expression in hypoxia (1% O₂) relative to normoxia (21% O₂) reported as mean ± SEM (standard error of mean) of three independent experiments. Absence of a FC value indicates absence of gene expression or negligible expression in normoxia, preventing normalization and calculation of FC ( Supplementary Figure S1B ).

Figure 2

Prognostic and predictive value of hypoxia signature in pancreatic cancer: Heatmaps of the expression levels of the eight genes in the signature in hypoxia score (HS) high (in orange) and HS low (in blue) tumors in PAAD Data-1 (A) and PAAD Data-2 (B). (C) Kaplan-Meier survival plots with log-rank test, comparing the groups divided based on hypoxia score in PAAD Data-1 and PAAD Data-2. (D) ROC curves depicting the predictive efficiency of the hypoxia signature on OS considering the 1-, 2-, and 3- years survival rates. (E) Multivariate COX PH forest plots showing hazard ratio of OS considering the 8-gene HS and other clinical covariates. P-value < 0.05 considered statistically significant; OS, overall survival; AUC, area under the curve; FP, false positive (1 - specificity); TP, true positive (sensitivity).

Figure 3

Hypoxia and immune markers in pancreatic cancer: (A) Mean SEM of hypoxia scores in tumors based on their molecular classification. (B) Mean SEM of immune scores (IS) based on the eighteen-gene tumor inflammation signature in HS high and low tumors. Spearman correlation plots between expression levels of the hypoxia signature and the tumor inflammation signature gene sets, as well as the immune score (IS) and hypoxia score (HS) in PAAD Data-1 (C) and PAAD Data-2 (D). Strength of correlation reflected in the size of the colored square and white squares indicate no correlation (C, D). Number of samples in each group is indicated above the error bars (A, B). Significance determined by ordinary one-way ANOVA with Bonferroni correction for multiple testing (A) and two-tailed unpaired t-test analysis (B). P-value < 0.05 considered statistically significant. *p-value > 0.01; **p-value < 0.0021; ***p-value < 0.0002; ****p-value<0.0001.

Figure 4

Hypoxia-immune landscape of pancreatic cancer: (A) Violin plots of immune cell fractions determined by CIBERSORTx in hypoxia score (HS) low and high tumors. (B) Mean SEM of Cytolytic (Cyt) index in HS high and low tumors and in IS high and low tumors. Number of samples in each group is indicated above the error bars. Significance determined by ordinary one-way ANOVA with Bonferroni correction for multiple testing (A) and two-tailed unpaired t-test analysis (B). P-value < 0.05 considered statistically significant. *p-value > 0.01; **p-value < 0.0021; ***p-value < 0.0002; ****p-value < 0.0001.

Figure 5

Prognostic and predictive efficiency of a combined hypoxia and immune classification in pancreatic cancer: (A) Kaplan-Meier survival plots with log-rank test, comparing the groups divided based on both hypoxia score and immune score. (B) Kaplan-Meier survival plots with log-rank test, comparing the two groups of Hypoxia Low/Immune Low and Hypoxia High/Immune Low. (C) ROC curves depicting the predictive efficiency of the hypoxia signature on OS considering the 1-, 2-, and 3- years survival rates in the two groups. (D) Multivariate COX PH forest plots showing hazard ratio of OS considering the 8-gene HS and other clinical covariates in the two groups. P-values < 0.05 considered statistically significant. OS, overall survival; AUC, area under the curve; FP, false positive (1 - specificity); TP, true positive (sensitivity).

Figure 6

Hypoxia score and indicators of ICI in pancreatic cancer: (A) Mean SEM of hypoxia scores in tumors classified according to the four-chemokine signature. (B) Mean SEM of PDL-1 protein abundance levels based on RPPA in HS high and low tumors. (C) Mean SEM of MSI and Mutation count in HS high and low tumors. MSI scores are based on the MSI-calling tool, MANTIS. Significance determined by ordinary one-way ANOVA with Bonferroni correction for multiple testing (A) and two-tailed unpaired t-test analysis (B, C). P-value < 0.05 considered statistically significant. Number of samples in each group is indicated above the error bars. *p-value > 0.01; **p-value < 0.0021; ***p-value < 0.0002; ****p-value < 0.0001.