Introduction

Background: Genetic alterations within ERCC2 correlate with extraordinary responses to neoadjuvant cisplatin-based chemotherapy and bladder-sparing ionizing radiation (IR) in muscle-invasive bladder cancer (MIBC). Two studies correlated ERCC2 mutations with pathologic response to chemotherapy and improved disease-free and bladder intact survival following trimodality therapy. We sought to characterize the biological significance of these mutations in bladder cancer cells using CRISPR/Cas9-mediated ablation of ERCC2 function.

Methods

Methods: The ERCC2 T484A/M point mutation was the most common alteration (4 of 36 patients) within a prospectively collected cohort of 299 bladder cancer patients sequenced at our institution. We infected the ERCC2 wild-type KU19-19 bladder cancer cell line with a CRISPR/Cas9 lentivirus targeting residues 481-487 of ERCC2 and identified a cell clone harboring an ERCC2 in-frame deletion (M483_T484del). Following exposure to cisplatin and IR, cell viability was examined using Cell-titer Glo and clonogenic assays. Apoptosis was gauged by subG1 cell fraction measurement by flow cytometry.

Results

Results: ERCC2 mutant cells exhibited significantly increased cisplatin sensitivity compared to parental cells (IC50 0.3uM vs 2.0uM, p<0.0001). Cisplatin treatment after 48 hours resulted in increased apoptosis in ERCC2 mutant vs parental cells (sub-G1 fraction 52% vs 16%, p=0.01). ERCC2 mutant cells were more sensitive to combined IR (2 Gy) and cisplatin (1uM) compared to parental cells (SF2Gy (surviving fraction 17 % vs 60%).

Conclusions

Conclusions: ERCC2 mutations enhance cisplatin and IR sensitivity in a bladder cancer cell line model. ERCC2 alterations are likely the genetic basis for extraordinary response to cisplatin chemotherapy and IR in MIBC patients. Prospective genetic sequencing may help select ERCC2 mutant MIBC patients who are most likely to respond to chemotherapy or trimodality bladder-sparing therapy.

Funding

Supported in part by funds from T32 CA082088