Deciphering CHFR Role in Pancreatic Ductal Adenocarcinoma

Abstract

Checkpoint with forkhead-associated and ring finger domains (CHFR) has been proposed as a predictive and prognosis biomarker for different tumor types, but its role in pancreatic ductal adenocarcinoma (PDAC) remains unknown. The aim of this study was two-pronged: to review the role of CHFR in PDAC and evaluating CHFR as a potential predictive biomarker in this disease. For this purpose, we first explored the CHFR messenger (m)RNA expression and promoter methylation through the TCGA database. Secondly, the CHFR expression and promoter methylation were prospectively evaluated in a cohort of patients diagnosed with borderline (n = 19) or resectable (n = 16) PDAC by immunohistochemistry (IHC), methylation specific-PCR (MSP), and pyrosequencing. The results from the TCGA database showed significant differences in terms of progression-free survival (PFS) and overall survival (OS) based on the CHFR mRNA expression, which was likely independent from the promoter methylation. Importantly, our results showed that in primarily resected patients and also the entire cohort, a higher CHFR expression as indicated by the higher IHC staining intensity might identify patients with longer disease-free survival (DFS) and OS, respectively. Similarly, in the same cohorts, patients with lower methylation levels by pyrosequencing showed significantly longer OS than patients without this pattern. Both, the CHFR expression intensity and its promoter methylation were established as independent prognostic factors for PFS and OS in the entire cohort. In contrast, no significant differences were found between different methylation patterns for CHFR and the response to taxane-based neoadjuvant treatment. These results suggest the potential role of the higher expression of CHFR and the methylation pattern of its promoter as potential prognostic biomarkers in PDAC, thus warranting further comprehensive studies to extend and confirm our preliminary findings.

Keywords: DNA methylation; checkpoint with forkhead and ring finger domains (CHFR); immunohistochemistry (IHC); methylation; pancreatic ductal adenocarcinoma (PDAC).

Figure 1

CHFR expression analysis. IHC pictures with differential expression, strong and weak stain (A,B) H-Score values from quantitative analysis performed in QuPath software with differential expression of CHFR (C). Kaplan-Meier curves representing progression-free survival and overall survival for borderline cohort (15 patients with moderate stain vs. 4 strong stain) (D,E). Kaplan-Meier curves representing disease-free survival and overall survival for resectable cohort (14 patients with moderate stain vs. 2 strong stain) (F,G, respectively). *Statistically significant differences (p < 0.05) and ns no significant differences (p > 0.05).

Figure 2

CHFR methylation analysis of human samples, borderline (A–F) and resectable cohort (G–L). Methylation-Specific PCR (left) and Pyrosequencing Assay (right). T (tumoral tissue) and A (Adjacent tissue). *Statistically significant differences (p < 0.05). Columns represents mean and error bars correspond to standard deviations.

Figure 3

Correlation of CHFR methylation with clinical outcomes in resectable patients. Kaplan-meier curve representing OS for resectable cohort and dichotomized by CpG-2 median methylation percentage.