Supplementary Material for: Loss of DAXX/ATRX protein expression results in ischemia resistance and radiation sensitivity in pancreatic neuroendocrine tumor cells and is associated with improved response to trans-arterial radio-embolization

ntroduction: Metastatic liver pancreatic neuroendocrine tumors (PNETs) can be treated with ischemia-based (trans-arterial embolization [TAE]/trans-arterial chemo-embolization [TACE]) or radiation-based (trans-arterial radioembolization [TARE]). Guidelines for treatment selection are limited. The purpose of this study was to measure the effect of loss of DAXX/ATRX protein expression on ischemia and radiation sensitivity in Bon-1 and QGP-1 cells, and to compare TARE response in PNETs with and without a DAXX/ATRX mutation. Methods: This was a laboratory investigation and retrospective review of an institutional database of TARE-treated PNET patients. Ischemia and radiation sensitivity were tested on Bon-1 and QGP-1 cells and CRISPR-generated DAXX (C16/C45) and ATRX (QAX12/QAX24) knockouts. Post-ischemia and post-radiation cell viability, survival fraction and caspase-3 expression were measured. Local progression free survival (LPFS) was measured from time of TARE to local progression or death and estimated using Cox proportional hazards. Results: Post-ischemia DAXX (C16/C45) and ATRX (QAX12/QAX24) knockouts had increased cell viability compared with Bon-1 wild type cells (p<0.0001, days 3, 5) and QGP-1 wild type cells (p<0.0001, days 3, 5, 7). Post-radiation C16/C45 and QAX12/QAX24 had decreased survival fraction compared with respective wild type (p<0.0001, all cell lines). C16/C45 had decreased apoptotic activity post-ischemia and increased apoptotic activity post-radiation compared with wild type (p<0.0001, all cell lines). Presence of DAXX/ATRX mutation was associated with longer LPFS after TARE (p<0.001). Median LPFS after TARE was 6 months in wild type compared with 22 months in patients with DAXX/ATRX mutation. Conclusion: Loss of DAXX/ATRX protein expression is associated with ischemia resistance and radiation sensitivity in Bon-1 and QGP-1 cells and longer LPFS after TARE in PNET patients.