Retrospective dosimetric analysis of brain lesions planned in Pinnacle 9.8 via a HDMLC linac.

The University of Toledo Medical Center's Eleanor N. Dana Cancer Center located in northwest Ohio currently utilizes the Edge Radiosurgery System (Varian Medical Systems Inc., Palo Alto, CA) to deliver stereotactic radiosurgery for the treatment of brain lesions. The purpose of this study is to determine the quality of conformal arc radiotherapy in treating patients with brain lesions at The University of Toledo Medical Center and to provide more data for conformity and gradient indices (due to a lack of current data) to hopefully improve national standards by allowing centers to compare among each other. Treatment plans were assessed using the Pinnacle3 v9.8 Radiation Therapy Planning System (Philips Healthcare, Amsterdam, Netherlands). For patients (n = 41) presenting with small brain lesions (n = 82) and treated with conformal arc radiotherapy via the Edge Radiosurgery System, the RTOG conformity index, Paddick conformity index, conformity gradient index, gradient index, and dose gradient index were determined for each plan. This study additionally provides data to suggest the more accurate method of volume derivation provided by the Pinnacle3 v9.8 software. Using this method, average values for each of the following indices were calculated: RTOG conformity index = 1.36 ± 0.29; Paddick conformity index = 0.72 ± 0.12; conformity gradient index = 214.67 ± 12.35; gradient index = 3.64 ± 1.09; dose gradient index = -0.11 ± 0.16. Thus, The University of Toledo Medical Center provides favorable conformity of dose to intracranial target lesions.

Inhibition of MEK confers hypersensitivity to X-radiation in the context of BRAF mutation in a model of childhood astrocytoma.

PURPOSE: Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low-grade tumors or anaplastic astrocytoma may receive radiation treatment; however, the long-term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic astrocytoma. EXPERIMENTAL DESIGN: The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant glioblastoma cell lines. The effect of MEK inhibition (selumetinib), XRT (total dose 10 Gy as 2 Gy daily fractions), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts. RESULTS: Inhibition of MEK signaling by selumetinib suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of FANCD2 and conferred hypersensitivity to XRT in BT-40 xenografts without increasing local skin toxicity. CONCLUSIONS: Selumetinib suppressed TORC1 signaling in the context of BRAF mutation. Selumetinib caused a rapid downregulation of FANCD2 and markedly potentiated the effect of XRT. These data suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF or maintaining tumor control at lower doses of XRT that would decrease long-term sequelae.

Inhibition of MDM2 by RG7388 confers hypersensitivity to X-radiation in xenograft models of childhood sarcoma.

BACKGROUND: Curative therapy for childhood sarcoma presents challenges when complete resection is not possible. Ionizing radiation (XRT) is used as a standard modality at diagnosis or recurrence for childhood sarcoma; however, local recurrence is still problematic. Most childhood sarcomas are TP53 wild type at diagnosis, although approximately 5-10% have MDM2 amplification or overexpression. PROCEDURES: The MDM2 inhibitor, RG7388, was examined alone or in combination with XRT (20Gy given in 2 Gy daily fractions) to immune-deficient mice bearing Rh18 (embryonal) or a total of 30 Gy in 2 Gy fractions to mice bearing Rh30 (alveolar) rhabdomyosarcoma xenografts. RG7388 was administered by oral gavage using two schedules (daily ×5; schedule 1 or once weekly; schedule 2). TP53-responsive gene products (p21, PUMA, DDB2, and MIC1) as well as markers of apoptosis were analyzed. RESULTS: RG7388 showed no significant single agent antitumor activity. Twenty Grays XRT induced complete regressions (CR) of Rh18 with 100 percent tumor regrowth by week 7, but no tumor regrowth at 20 weeks when combined with RG7388. RG7388 enhanced time to recurrence combined with XRT in Rh30 xenografts compared to 30 Gy XRT alone. RG7388 did not enhance XRT-induced local skin toxicity. Combination treatments induced TP53 responsive genes more rapidly and to a greater magnitude than single agent treatments. CONCLUSIONS: RG7388 enhanced the activity of XRT in both rhabdomyosarcoma models without increasing local XRT-induced skin toxicity. Changes in TP53-responsive genes were consistent with the synergistic activity of RG7388 and XRT in the Rh18 model.

Radiation therapy may increase metastatic potential in alveolar rhabdomyosarcoma.

BACKGROUND: We previously determined that radiation could be safely administered using a mouse-flank in vivo model to both alveolar (Rh30) and embryonal (Rh18) rhabdomyosarcoma xenografts. Mice from both tumor lines in this experiment developed metastases, an event not previously described with these models. We sought to determine if radiation-induced changes in gene expression underlie an increase in the metastatic behavior of these tumor models. PROCEDURE: Parental Rh18 and Rh30 xenografts, as well as tumor that recurred locally after radiotherapy (Rh18RT and Rh30RT), were grown subcutaneously in the flanks of SCID mice and then subjected to either fractionated radiotherapy or survival surgery alone. Metastasis formation was monitored and recorded. Gene expression profiling was also performed on RNA extracted from parental, recurrent, and metastatic tissue of both tumor lines. RESULTS: Rh30 and Rh30RT xenografts demonstrated metastases only if they were exposed to fractionated radiotherapy, whereas Rh18 and Rh18RT xenografts experienced significantly fewer metastatic events when treated with fractionated radiotherapy compared to survival surgery alone. Mean time to metastasis formation was 40 days in the recurrent tumors and 73 days in the parental xenografts. Gene expression profiling noted clustering of Rh30 recurrent and metastatic tissue that was independent of the parental Rh30 tissue. Rh18RT xenografts lost radiosensitivity compared to parental Rh18. CONCLUSION: Radiation therapy can significantly decrease the formation of metastases in radio-sensitive tumors (Rh18) and may induce a more pro-metastatic phenotype in radio-resistant lines (Rh30).

FANCD2 is a potential therapeutic target and biomarker in alveolar rhabdomyosarcoma harboring the PAX3-FOXO1 fusion gene.

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 that FANCD2 transcript levels are prognostic in univariate analysis, and are significantly associated with metastatic disease and that the copresence of the translocation and high expression of FANCD2 is independently prognostic. We also demonstrate a significant and nonrandom 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.

Using NanoDot dosimetry to study the RS 2000 X-ray biological irradiator.

PURPOSE: To use NanoDot dosimeters to study the RS 2000 X-ray Biological Irradiator dosimetry characteristics and perform in vivo dosimetry for cell or small animal experiments. METHODS AND MATERIALS: We first calibrated the Landauer NanoDot(™) Reader by irradiating some NanoDot dosimeters with a set of known doses at specific positions defined by the irradiator. A group of five NanoDot dosimeters were placed at five specific positions where the dose rates were known and provided by the irradiator. Each group was irradiated for a set of times respectively. By correlating the readings of dosimeters with the given irradiated doses, we established the dose-reading relationship for the irradiator under the specific running condition. The established calibration curve was validated by exposing arbitrary known doses to a set of dosimeters, using the Landauer NanoDot(™) Reader to measure the doses, and then making the comparison between the two doses. To study the dose gradient of the X-ray inside the irradiated target (dose variation/cm), we placed dosimeters under different thicknesses of water-equivalent bolus and irradiated them, then measured the doses to determine the dose gradient. RESULTS: Using the method described above, we were able to calibrate the Landauer InLight NanoDot(™) Reader and use NanoDot dosimeters to measure the actual doses delivered to the targets for the cell/small animal experiments that use the RS 2000 X-ray Biological Irradiator. CONCLUSIONS: NanoDots are ideal dosimeters to use for in vivo dosimetry for cell/small animal irradiation experiments. The dose decrease inside the animal tissue is about 20% per cm.

The application of radiation therapy to the Pediatric Preclinical Testing Program (PPTP): results of a pilot study in rhabdomyosarcoma.

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 xenograft 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.