Background The Pediatric Preclinical Testing Program (PPTP) has been successfully used to determine the efficacy of novel agents against solid tumors by testing them within a mouse-flank in vivo model. To date, radiation therapy has not been applied to this system. We report on the feasibility and biologic outcomes of a pilot study using alveolar and embryonal rhabdomyosarcoma xeno-graft lines. Procedures We developed a high-throughput mouse-flank irradiation device that allows the safe delivery of radiotherapy in clinically relevant doses. For our pilot study, two rhabdomyosarcoma xenograft lines from the PPTP, Rh30 (alveolar) and Rh18 (embryonal) were selected. Using established methods, xenografts were implanted, grown to appropriate volumes, and were subjected to fractionated radiotherapy. Tumor response-rates, growth kinetics, and event-free survival time were measured. Results Once optimized, the rate of acute toxicity requiring early removal from study in 93 mice was only 3%. During the optimization phase, it was observed that the alveolar Rh30 xenograft line demonstrated a significantly greater radiation resistance than embryonal Rh18 in vivo. This finding was validated within the standardized 30 Gy treatment phase, resulting in overall treatment failure rates of 10% versus 60% for the embryonal versus alveolar subtype, respectively. Conclusions Our pilot study demonstrated the feasibility of our device which enables safe, clinically relevant focal radiation delivery to immunocompromised mice. It further recapitulated the expected clinical radiobiology.

Purpose Alveolar rhabdomyosarcoma that harbors the PAX3/FOXO1 fusion gene (t-ARMS) is a common and lethal subtype of this childhood malignancy. Improvement in clinical outcomes in this disease is predicated upon the identification of novel therapeutic targets. Experimental Design Robust mouse models were used for in vivo analysis, and molecular studies were performed on xenografts treated in parallel. Two independent patient sets (n=101 and 124) of clinically-annotated tumor specimens were used for analysis of FANCD2 levels and its association with clinical and molecular characteristics and outcomes. Results Our xenograft studies reveal a selective suppression of FANCD2 by m-TOR kinase inhibition and radiosensitization of the t-ARMS line only. In the initial patient set, we show FANCD2 transcript levels are prognostic in univariate analysis, and are significantly associated with metastatic disease and that the co-presence of the translocation and high expression of FANCD2 is independently prognostic. We also demonstrate a significant and non-random enrichment of mTOR-associated genes that correlate with FANCD2 gene expression within the t-ARMS samples, but not within other cases. In the second patient set, we show that on a protein level, FANCD2 expression correlates with PAX3/FOXO1 fusion gene and is strongly associated with phospho-P70S6K expression in cases with the fusion gene. Conclusions Our data demonstrate that FANCD2 may have a significant role in the radiation resistance and virulence of t-ARMS. Indirectly targeting this DNA repair protein, through mTOR inhibition, may represent a novel and selective treatment strategy.

Purpose. Alveolar rhabdomyosarcoma that harbors the PAX3/FOXO1 translocation (t-ARMS) is the most common and lethal subtype of this childhood malignancy. In this report we identify FANCD2 as a potential mediator of radiation resistance and biomarker of increased virulence in t-ARMS. Methods. After the observation that knockdown of mTOR resulted in a loss of FANCD2 expression, we postulated that pharmacologic inhibition of TORC1/TORC2 would result in a similar suppression of this DNA-repair protein and increase cell-kill after radiotherapy. Therefore, an initial set of mice bearing t-ARMS and embryonal rhabdomyosarcoma (ERMS) xenografts were subjected to a dose escalation study, followed by a standard dosing study which tested the concurrent administration of AZD8055 (an mTOR kinase inhibitor) and radiotherapy versus either treatment alone. Molecular studies, focusing on drug action and resultant FANCD2 expression and function were also performed on xenografts treated in parallel. We also performed clonogenic survival assays in RMS cell lines with and without FANCD2 knockdown to determine the impact of this protein on radiation sensitivity in these lines. Lastly, we tested the prognostic significance of FANCD2 in the tumors from two independent patient sets. Results. In a dose escalation study of 30 mice, we first observed the selective radiosensitization within the t-ARMS xenograft line versus the ERMS line. In a second set of 80 mice, subjected to standardized treatment arms, validated this selective radiosensitization. To verify the desired drug action, we analyzed xenografts from mice treated in parallel (collected at various points in treatment) and found that FANCD2 expression and activity is significantly suppressed by the addition of the drug in the sensitized, t-ARMS, but much less so in the ERMS xenograft line. We further observed that direct FANCD2 suppression (via siRNA knockdown) confers sensitivity to radiation therapy in both of these lines in vitro. In the initial patient set (n=108), we show that, on a protein level, FANCD2 expression correlates with PAX3/FOXO1 translocation and that the presence of this marker is additive to the poor prognosis of a translocated status. The co-existence of these two biomarkers is independently prognostic from other known clinical variables and the PAX3/FOXO1 translocation itself. In our second patient set (n=101), we show that based on the mRNA expression of FANCD2, that quartile-FANCD2 levels are prognostic in a univariate analysis, significantly associated with metastatic (Stage 4) disease and that, again, the co-presence of the translocation and high expression of FANCD2 is independently prognostic. Conclusion. Our data demonstrate that FANCD2 may have a significant role in the radiation resistance and virulence of t-ARMS, and indirectly targeting this DNA repair protein, through mTOR inhibition, may represent a novel and selective treatment strategy. Citation Format: Mamata Singh, Justin Leasure, Christopher Chronowski, Ning Li, Kathryn Bondra, Wenrui Duan, Miguel Villalona, Anthony Vergis, Lauren Hensley, Rita Kaplon, Brian Geier, Raushan Kurmasheva, Gary Woods, Sue Hammond, Peter Houghton, Christopher E. Pelloski. Targeting FANCD2 as a radiosensitizer in pediatric rhabdomyosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4439. doi:10.1158/1538-7445.AM2013-4439