Abstract A07: A marker to optimize BCG dosing schedules for immunotherapy
Authors: Rahmat Juwita N. , Tan Kah Wei , Ong Ting Li , Tham Sin Mun , Tsung Chong Wen , Chan Yiong Huak , Raman Lata , Chiong Edmund , Esuvaranathan Kesavan , Mahendran Ratha 
Published: August 2020

Background: BCG immunotherapy is the standard for non-muscle invasive bladder cancer (NMIBC) and there has been no change in therapy schedules, though some patients cannot tolerate repeated dosing. This study evaluates the potential of the GST theta 2 (GSTT2) gene that modulates BCG survival in bladder cancer cell lines to modulate patient response to therapy. Our hypothesis is that better intracellular BCG survival should correlate with better immune activation. GSTT2, a member of the glutathione-S-transferase family, lies on chromosome 22q. In man, a 37kb deletion removes a pseudogene (GSTT2B) and the 5' flanking region of the GSTT2 gene, resulting in low/no expression of GSTT2. This deletion is a reliable marker of GSTT2 gene expression. GSTT2B is deleted in 34% of Caucasians and 29.2% of Asians. Our aim was to genotype NMIBC patients who had received BCG immunotherapy to determine if their response correlates with their GSTT2B status and to evaluate the role of GSTT2 in immune activation.

Methods: The GSTT2B deletion and the promoter region of GSTT2 were analyzed by PCR in patients (n=201) and controls (n=150). The impact of BCG instillation on recurrence was analyzed. GSTT2 overexpression was performed in U937 macrophages (GSTT2B homozygous deleted) and bacterial survival determined. GSTT2 knockout mice were generated. Bone marrow-derived dendritic cells (DC) from GSTT2 knockout and wild-type mice were exposed to BCG, and immune activation in terms of cytokine production was examined. Data were analyzed using SPSS 23.0 and p&lt;0.05 was taken to be significant. The institutional animal care and use committee approved animal work and the institutional review board approved patient sample analysis (NHG DSRB: 2012/00475).

Results: Overexpressing GSTT2 in U937 cells decreased BCG survival at 2h (p&lt;0.05). SNPs in the GSTT2 promoter were not associated with response to BCG except for the GSTT2B deletion. Patients were segregated by the number of BCG instillations they received, namely 9 or more BCG instillations (n=135) or 8 or fewer BCG instillations (n=39). Patients with the GSTT2B homozygous deletion who received 8 or fewer BCG instillations (74% received 6 instillations), were 90% recurrence free (p&lt;0.05). For those who received 9 or more BCG instillations, the recurrence incidence was 35.5% and recurrence occurred within 2 years. Patients with GSTT2B full-length genotype did better with more BCG instillations. DC from GSTT2 knockout mice produced a 3-fold increase in IL6 production but similar TNFα and IL10 levels as wild-type DC in response to BCG.

Conclusions: GSTT2B deletion was associated with lower likelihood of recurrence for patients who received 8 or fewer BCG instillations, while for GSTT2B full-length patients, those who received 9 or more BCG instillations had reduced recurrence compared to those who received fewer instillations. GSTT2 expression modulates immune activation by modulating BCG survival. Hence, GSTT2B absence may be a marker for patients who might do well with less BCG therapy.

Citation Format: Juwita N. Rahmat, Kah Wei Tan, Ting Li Ong, Sin Mun Tham, Chong Wen Tsung, Yiong Huak Chan, Lata Raman, Edmund Chiong, Kesavan Esuvaranathan, Ratha Mahendran. A marker to optimize BCG dosing schedules for immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A07.