Login to Access Video or Poster Abstract: MP65-04
Sources of Funding: NCIS/NMRC

Introduction

In studies on human bladder cancer cell lines, BCG induced the expression of GST theta 2 (GSTT2) a member of the Glutathione-S-transferase family. The objective of this study was to investigate the cellular function of GSTT2 and its impact on the response to BCG therapy in NMIBC patients.

Methods

GSTT2B (pseudogene) deletion decreases GSTT2 expression. PCR was performed to identify GSTT2B presence or absence in human bladder cancer and macrophage cell lines, and in patients (n=139) and controls (n=150) (IRB approval, NHG DSRB: 2012/00475) GSTT2 silencing (siRNA) and overexpression (plasmid carrying GSTT2) were performed on selected cell lines. Reactive oxygen species (ROS); BCG induced cytotoxicity and intracellular BCG survival were analyzed. Patient demographic, initial disease characteristics (based on EORTC scoring system) and therapy outcomes were evaluated with respect to the GSTT2B genotypes. The impact of BCG instillation (numbers) on recurrence was analyzed in a subset of patients for whom complete 10y follow-up data was available. Analysis was performed using SPSS 23.0 and p<0.05 was taken to be significant.

Results

GSTT2 was silenced in MGH cells (GSTT2B homozygous full length (GSTT2B^fl/fl)) and overexpressed in UMUC3 and U937 cells (GSTT2B homozygous deleted (GSTT2B^del/del)). A 2h exposure to BCG resulted in decreased ROS in GSTT2 silenced cells (p<0.05) and increased ROS in GSTT2 overexpressing cells (p<0.05). There was no difference in cellular cytotoxicity to BCG with respect to GSTT2 expression. However, intracellular BCG survival increased at 2 hours when GSTT2 was silenced (p<0.05) and decreased when GSTT2 was overexpressed (p<0.05). There was no significant difference between these groups at 24h. The majority of patients with complete 10y follow-up data, completed a 6+3 BCG schedule (n=63) and n=22 had less than 8 instillations. Patients with GSTT2B^del/del genotype (n=6) who received 8 or less BCG instillations, were recurrence free (Likelihood ratio = 0.040, p=0.054). In the group that received at least 9 instillations of BCG, the GSTT2B^del/del was associated with earlier recurrence.

Conclusions

GSTT2 expression decreases cellular ROS and BCG survival. GSTT2B^del/del was associated with lower likelihood of recurrence for patients who received 8 or less BCG instillations. In contrast patients with GSTT2B^del/del who received 9 or more instillations of BCG had earlier recurrences. Hence GSTT2B^del/del could be used as a marker for patients who will do well with less BCG therapy.

Funding

NCIS/NMRC