Defining innate immune training potential as a predictor of Bacillus Calmette-Guérin immunotherapy response in nonmuscle-invasive bladder cancer

522 Background: Intravesical instillation of the live attenuated mycobacterium Bacillus Calmette-Guerin (BCG) remains the most effective therapy for the treatment of non-muscle invasive bladder cancer. The precise mechanism of BCG is thought to involve phagocytosis, induction of inflammation, activation of the innate immune apparatus, and an effective cell-mediated T cell response. An increasing body of evidence suggests that the innate immune system has characteristics that involve a heterologous memory of past insults through a process thought to involve epigenetic reprogramming, termed trained immunity. We seek to investigate the potential for innate immune training as a predictor of the anti-tumor effects of BCG. Methods: A prospective biospecimen collection was performed on venous blood from non-muscle invasive bladder cancer patients prior to receiving intravesical BCG therapy. To investigate the role of trained immunity in primary monocytes, cells were isolated from peripheral blood mononuclear cells (PBMC) by plate adhesion. 2-5 x 105 cells/well were assayed in 96-well format, cultured in RPMI 1640 supplemented with 10% Fetal Bovine Serum and 2mM L-glutamine and stimulated with 10:1 MOI BCG for 24 hours. Cells were then washed in media and allowed to rest 6 days. Media was changed on day 3 of rest. Subsequently, cells were stimulated with 25 ng/ml lipopolysaccharide (a TLR4 agonist) for 24 hours. Cell free supernatant was measured for TNF-alpha production by enzyme-linked immunosorbent assay. Results: In vitro training of primary monocytes induced an increase in proinflammatory cytokine production upon re-stimulation. This phenotype was dose dependent on stimulation with LPS, however, the relative change between trained and untrained cells was diminished at higher concentrations of agonist. In our in vitro training experiments, there was demonstrable interpatient variability seen pre-BCG exposure in a cohort of NMIBC patients. These results will be correlated with future long term BCG response in this prospective cohort. Conclusions: We present data on a trained innate immune phenotype on primary monocytes collected from patient samples. These data serve as proof of principle for the ability to induce a trained phenotype in vitro. These findings will be correlated with prospective clinical data on BCG responsiveness and recurrence free survival to allow us to garner further insight into the role of trained immunity in BCG immunotherapy. Such insight will have potential clinical impact on the development of biomarkers and clinical assays that could predict immune responsiveness to BCG a priori, as well as identify possible immunological adjuvants that could enhance the effect of intravesical BCG.