Although radical cystectomy surgery is the standard-of-care for muscle-invasive bladder cancer, it entails complete removal of the bladder and surrounding organs which leads to substantial loss in the quality-of-life of patients. Radiotherapy, which spares the bladder, would be a more appropriate treatment modality if we can utilize molecular markers to select patients with better response to radiation. In this study, we investigate a protein called high mobility group box protein 1 (HMGB1) as a predictive marker for radiotherapy response in bladder cancer. Our in vitro results indicate a positive correlation between higher levels of HMGB1 protein and resistance to radiation in various cell lines. Upon HMGB1 protein knockdown, highly significant (>1.5-fold) sensitization to radiotherapy was achieved. We saw that loss of HMGB1 was associated with at least two times higher (P < 0.001) DNA damage in cell lines postradiation. Our results also depicted that autophagy was inhibited more than 3-fold (P < 0.001) upon HMGB1 knockdown, implicating its role in autophagy as another cause of bladder cancer radioresistance. Further validation was done in vivo by conducting mouse tumor xenograft experiments, where HMGB1 knockdown tumors showed a significantly better (P < 0.001) response to radiotherapy and decreased autophagy (shown by P62 staining) as compared with controls. The cumulative findings of our in vitro and in vivo studies highlight the significance of HMGB1 as a radiation response marker as well as its utility in radiosensitization of bladder cancer.

Introduction Microdissection testicular sperm extraction (micro‐TESE) is a procedure commonly utilized to harvest spermatozoa in severe male factor infertility. We have developed a technique involving a mini tunica albuginea incision with superficial tissue dissection (mini‐incision micro‐TESE). The modification is designed to reduce potential tissue injury, and we studied its effectiveness to harvest spermatozoa in men with cryptozoospermia and non‐obstructive azoospermia. Materials and Methods We performed a retrospective analysis of 103 infertile men with NOA and cryptozoospermia who underwent a mini‐incision micro‐TESE between March 2015 and August 2018 at the OVO fertility clinic. We consider the mini‐incision micro‐TESE procedure successful when at least five spermatozoa are identified in the micro‐biopsies obtained from the superficial tissue exposed by the 1‐cm mini‐incision. If no spermatozoa are identified through the mini‐incision, we can easily extend the incision to the standard micro‐TESE. Results The mini‐incision procedure allowed for successful recovery of spermatozoa (intra‐operative recovery of ≥ 5 spermatozoa) in 58% of men with cryptozoospermia and 25.6% of men with NOA. Overall, a successful sperm retrieval (with conversion to conventional micro‐TESE if mini‐incision was not successful) was achieved in 89% of men with cryptozoospermia and 48% of men with NOA. A successful mini‐incision micro‐TESE was associated with a significantly lower number of biopsies than conventional micro‐TESE (8.8 vs 24.2, P < .0001). Moreover, in men undergoing a redo micro‐TESE after a previously successful micro‐TESE, 64% (9/14) had spermatozoa found and 21% (3/14) of these men only required a mini‐incision micro‐TESE. Conclusion Our data suggest that mini‐incision micro‐TESE is a useful approach in men undergoing micro‐TESE, allowing for a shorter incision and a high sperm retrieval rate in men with cryptozoospermia. Furthermore, the findings of our study offer insight into the distribution of spermatogenesis in men with cryptozoospermia and NOA.

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Purpose: Radical cystectomy is the gold standard treatment for muscle invasive bladder cancer. However, it causes a serious impact in the quality of life of the patients. Radiation therapy when combined with radio-sensitizing chemotherapy is an attractive alternative as it offers bladder preservation allowing for normal urinary and sexual functions. However, lack of local control and dose dependent toxicity are the main drawbacks of the combination therapy. As HMGB1 has been found to be associated with DNA damage response and chronic inflammation pathways of radiation resistance, we sought to investigate the role of HMGB1 in the response to radiation in bladder cancer. Methods: The expression of HMGB1 among different bladder cancer cell lines was analyzed by mRNA and Protein quantification. The mean growth inhibition to Gemcitabine was assessed by colorimetric assays of cellular viability. Knock down of HMGB1 was done using lentiviral shRNA system. To assess the effect of the combination therapy, Clonogenic assay was done. Cells were exposed to the mean growth inhibition dose of gemcitabine for 6 hours followed by varying doses of radiation (2-8 Gy). Analysis of cell cycle was done by Flow Cytometry. Western blot was utilized to assess pathways involved in cellular survival. In vivo, athymic mice were subcutaneously injected with two bladder cancer cell lines. Treatment consisted of either placebo or combined gemcitabine (1 g/m2, 6 hours prior to radiation) and fractionated IR (total 6Gy = 3Gy×2) in cells with scramble vs knockdown of HMGB1. Mice were followed over 3 weeks. Tumor weight was measured at experimental endpoint. Results: HMGB1 expression was analyzed in eight urothelial carcinoma cell lines. Amongst all, UM-UC3 and UM-UC5 had the highest expression while 253J-BV had the lowest expression of HMGB1. There was a significant correlation between the levels of HMGB1 and resistance to radiation (P value &lt;0.0001). Our results from clonogenic assays post combined treatment shows that knockdown of HMGB1 resulted in a dose modifying factor (DMF) of 2.95 for a survival fraction of 0.5. Moreover, western blot analysis revealed that high expression of HMGB1 was associated with increased and sustained activation of ERK pathway. Also, cell cycle analysis showed that upon HMGB1 knockdown, cell cycle arrest was induced at S phase post gemcitabine treatment and at G2 phase post radiation and combination therapy. To validate these results, subcutaneous tumors were induced in athymic mice and underwent two fractions of the combined treatment. Significant sensitization effect upon HMGB1 knockdown was observed, evident by tumor volume (P value &lt;0.0001) and weight (P value &lt;0.05). Conclusion: Our in vitro and in vivo results strongly suggest that investigating HMGB1 increases efficacy of combination therapy of gemcitabine and radiation. Baseline of HMGB1 could be a good marker to predict response to such therapy. Citation Format: Jose J. Mansure, Wael S. Almajed, Sanhita Shrivastava, Fabio Cury, Gerardo Ferbeyre, Marija Popovic, Jan Seuntjens, Wassim Kassouf. The role of high mobility group box 1 in the combination therapy of gemcitabine and radiation in muscle invasive bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3316. doi:10.1158/1538-7445.AM2015-3316