Correlation between messenger RNA expression and protein expression of immune checkpoint-associated molecules in bladder urothelial carcinoma: A retrospective study

Abstract

Objectives: Immunotherapy for bladder cancer seems to have promising results. Here, we evaluated the association between messenger RNA (mRNA) and protein levels and possible prognostic value of the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) immune checkpoint pathways during bladder carcinogenesis.

Methods and materials: Tumor samples were obtained from 155 patients (84 with muscle-invasive bladder cancer [MIBC], and 71 non-muscle-invasive bladder cancer [NMIBC]) and normal bladder tissue from 15 patients. We evaluated the mRNA expression of 3 genes in the PD-1 pathway (PD-1, PD-L1, and PD-L2) and 4 in the CTLA4 pathway (CTLA4, CD28, CD80, and CD86) in normal and tumoral human bladder samples by quantitative real-time reverse transcription polymerase chain reaction, with immunohistochemistry used to evaluate the protein expression of PD-1 and PD-L1 in tumor and immune cells. Results of molecular analyses were compared with survival analyses.

Results: As compared with normal bladder tissue, MIBC tissue showed PD-1, PD-L1, CTLA4, and CD80 overexpression (59.5%, 60.7%, 84.5%, and 92.9%, respectively), whereas overexpression was lower in NMIBC tissue (22.5%, 4.2%, 35.2%, and 46.5%, respectively). The results of reverse transcription polymerase chain reaction analysis were confirmed by immunohistochemistry, with a high correlation between mRNA and protein expression. On multivariate analyses, overexpression of the studied genes was not associated with prognosis in relapse or progression of NMIBC or in recurrence-free and overall survival of MIBC.

Conclusions: The CTLA4 pathway appears to be deregulated along with the PD-1/PD-L1 pathway in bladder carcinogenesis, with good correlation between mRNA and protein expression endorsing the useful role of immune checkpoints, especially for a large subgroup of MIBC.

Keywords: Bladder cancer; CTLA4; Immune checkpoints; Immunohistochemistry; Molecular marker; PD-1; RT-PCR.