Evaluation of HIF-1α and VEGF-A expression in radiation-induced cystitis: A case-control study

Abstract

The standard treatment for locally advanced cervical cancer (CC) is chemo-radiotherapy. Once the bladder receives part of the radiation, a typical inflammatory condition that configures radiation-induced cystitis may develop. Chronic radiation-induced cystitis is commonly characterized by the bladder new submucosal vascularization, which is typically fragile and favors hematuria. The current study aims to investigate if Hypoxia-Induced Factor (HIF-1α) and its transcriptional target Vascular Endothelial Growth Factor A (VEGF-A) could be a primary pathway leading to increased submucosal vascularization. HIF-1α and VEGF-A mRNA levels in bladder core biopsies from CC patients treated with radiotherapy versus untreated (non-irradiated) patients were analyzed using a droplet digital polymerase chain reaction technology. Gene expression results showed that HIF-1α and VEGF-A had no significant differences between bladder samples from patients previously irradiated and untreated patient samples. However, a direct relationship between the degree of late morbidity and the expression of HIF-1α and VEGF-A has been demonstrated. Despite the lack of statistical significance precludes a definitive conclusion, the data presented herein suggests that further studies investigating the role of HIF-1α in bladder neovascularization in radiation-induced cystitis are highly recommended.

Keywords: Neoplasms, Radiation-Induced; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor A.

Figure 1. Aspect of the bladder mucosa of irradiated patients with varying degrees of neovascularization comparing with normal bladder mucosa. Images were obtained during cystoscopy examinations of patients included in the study and correspond to the endoscopic findings of RTOG/EORTC late morbidity scale: A and B - normal bladder mucosa, C and D -Grade 1: slight epithelial atrophy, minor telangiectasia, E and F - Grade 2: generalized telangiectasia, G and H – Grade 3: severe generalized telangiectasia (often with petechiae).

Figure 2. Graphics of digital PCR in drops. The graphs indicate the fluorescence intensity (Ch1 Amplitude and Ch2 Amplitude for FAM or HEX, respectively) of the drops analyzed (events number). The drops are termed "positive" or "negative" based on their fluorescence amplitudes. Positive events are marked in blue (HIF-1α and VEGF-A) and green (GAPDH), while negative events in gray (drops without the DNA molecule of interest). All analyzes were performed using QuantaSoft software (Biorad). During drop formation cDNA molecules are randomly distributed in the droplets. Due to the high number of independent events, the Poisson algorithm is used to determine absolute quantitation of copy number independent of a standard curve, allowing accurate quantification of gene expression. (A) Expression of HIF1α in a control group patient (left) and in an irradiated patient (right). (B) Expression of VEGFA in a control group patient (left) and in an irradiated patient (right). (C) Expression of GAPDH in a control group patient (left) and in an irradiated patient (right).

Figure 3. Droplet digital PCR expression analysis of HIF-1α and VEGF-A mRNA. (A) and (B) Analysis of HIF-1α and VEGF-A mRNA in non-irradiated patients (control) and patients previously submitted to pelvic radiotherapy (cystitis). (C) and (D) Analysis of HIF-1α and VEGF-A mRNA expression in patients previously submitted to pelvic radiotherapy according to the late RTOG/EORTC morbidity score. (E) and (F) Analysis of HIF-1α and VEGF-A mRNA in bladder tissue samples and washing bladder samples of non-irradiated patients. The targets mRNA expression were calculated relative to a stable expressed reference gene (GAPDH). Results were expressed as median and comparison between groups was performed by Mann–Whitney U test. RTOG/EORTC: grade 1 - mild epithelial atrophy, small telangiectasia (microscopic hematuria); grade 2 -moderate frequency, generalized telangiectasia, and intermittent macroscopic hematuria.