ROR1 and ROR2 expression in pancreatic cancer

Abstract

Background: The Wnt receptors ROR1 and ROR2 are generating increased interest as cancer therapeutic targets but remain understudied in pancreatic ductal adenocarcinoma (PDAC). Compared to canonical Wnt/ β-catenin signalling, the role of noncanonical Wnt signalling in PDAC remains largely unknown. Only one study has investigated the prognostic significance of the noncanonical Wnt signalling receptor, ROR2 in PDAC. No studies have investigated the prognostic role of ROR1 in PDAC.

Methods: Here, we performed analysis of ROR1 and ROR2 mRNA expression in three publicly available datasets ICGC-PACA-AU (n = 81), TCGA-PAAD (n = 150) and CPTAC-PDAC (n = 137). ROR1 and ROR2 protein expression from the CPTAC-PDAC discovery cohort were also analysed. Immunohistochemistry (IHC) using the validated anti ROR1 monoclonal antibody (4A5) was performed on the Australian Pancreatic Cancer Genome Initiative (APGI) cohort of PDAC samples (n = 152). Association between ROR1 cytoplasmic staining intensity and clinicopathological parameters including stage, grade and overall survival (OS) was investigated.

Results: High ROR1 mRNA expression levels correlated with a favourable OS outcome in all of the ICGC-PACA-AU, TCGA-PAAD and CPTAC-PDAC cohorts. ROR1 protein expression was not associated with stage, grade or OS in the APGI cohort.

Conclusion: ROR1 and ROR2 have potential as prognostic markers when measured at the mRNA level in PDAC. Our IHC cohort did not support ROR1 protein expression in predicting OS, and highlighted the discrepancy of prognostic biomarkers when measured by MS, IHC and RNAseq.

Fig. 1

Association between ROR2 mRNA/protein expression level and clinicopathological parameters in the ICGC-PACA-AU,TCGA-PAAD and CPTAC-PDAC cohorts. A. No significant difference in ROR2 expression was observed between low and high tumour grade. B. ROR2 mRNA expression was not associated with stage, ROR2 protein expression was significantly different between low and high stage (p = 0.010). C. High ROR2 mRNA expression level was associated with better overall survival (p = 0.023) in the ICGC-PACA-AU cohort. D. High ROR2 mRNA expression level was associated with better overall survival (p = 0.008) in the TCGA-PAAD cohort. E. ROR2 mRNA was not associated with overall survival in the CPTAC-PDAC cohort. F. ROR2 protein was not associate with overall survival in the CPTAC-PDAC cohort.*Significance at p < 0.05 level

Fig. 2

Association between ROR1 mRNA/protein expression level and clinicopathological parameters in the ICGC-PACA-AU, TCGA-PAAD and CPTAC-PDAC cohorts. A. No significant difference of ROR1 expression was observed between low and high tumour grade. B. ROR1 expression was associated with stage in ICGC-PACA-AU and TCGA-PAAD cohorts in opposite directions (p = 0.033 and 0.036 respectively). No significant correlation between ROR1 mRNA and stage was observed in CPTAC-PDAC cohort. C. High ROR1 mRNA expression level was associated with better overall survival (p < 0.001) in the ICGC-PACA-AU cohort. D. High ROR1 mRNA expression was associated with better overall survival (p = 0.045) in the TCGA-PAAD cohort. E. High ROR1 mRNA expression was associated with better overall survival (p = 0.026) in the CPTAC-PDAC cohort. F. ROR1 protein expression was not associated with overall survival in the CPTAC-PDAC cohort. *Significance at p < 0.05 level

Fig. 3

Representative immunohistochemistry images of ROR1 staining. Representative images of score 0 (absence; A,B), 1 (low; C,D) and 2 (high; E,F) for ROR1 expression measured by immunohistochemistry. Original magnification × 8 (scale bar = 300 μm) for A, C, E; magnification × 30 (scale bar = 80 μm) for B, D, F

Fig. 4

Correlation between ROR1 protein expression and tumour grade, stage, tumour size and overall survival in the APGI clinical cohort. A-C. Expression of ROR1 stratified by tumour grade, stage and tumour size. D. Kaplan-Meier analysis for ROR1 stratified by low/absent (score 0,1) and high (score 2). E. Multivariable overall survival analysis of ROR1 incorporating gender, tumour stage and grade