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Purpose: A pretherapy 124I-metaiodobenzylguanidine (MIBG) positron emission tomography (PET)∕computed tomography (CT) provides a potential method to estimate radiation dose to normal organs, as well as tumors prior to 131I-MIBG treatment of neuroblastoma or pheochromocytoma. The aim of this work was to estimate human-equivalent internal radiation dose of 124I-MIBG using PET∕CT data in a murine xenograft model. Methods: Athymic mice subcutaneously implanted with NB1691 cells that express high levels of human norepinephrine transporter (n=4) were imaged using small animal microPET∕CT over 96 h (approximate imaging time points: 0.5, 2, 24, 52, and 96 h) after intravenous administration of 3.07–4.84 MBq of 124I-MIBG via tail vein. The tumors did not accumulate 124I-MIBG to a detectable level. All four animals were considered as control and organ radiation dosimetry was performed. Volumes of interest were drawn on the coregistered CT images for thyroid, heart, lung, liver, kidney, and bladder, and transferred to PET images to obtain pharmacokinetic data. Based on tabulated organ mass distributions for both mice and adult male human, preclinical pharmacokinetic data were extrapolated to their human-equivalent values. Radiation dose estimations for different age groups were performed using the OLINDA|EXM software with modified tissue weighting factors in the recent International Commission on Radiological Protection (ICRP) Publication 103. Results: The mean effective dose from 124I-MIBG using weighting factors from ICRP 103 to the adult male was estimated at 0.25 mSv∕MBq. In different age groups, effective doses using values from ICRP 103 were estimated as follows: Adult female: 0.34, 15-yr-old: 0.39 mSv∕MBq, 10-yr-old: 0.58 mSv∕MBq, 5-yr-old: 1.03 mSv∕MBq, 1-yr-old: 1.92 mSv∕MBq, and newborn: 3.75 mSv∕MBq. For comparison, the reported effective dose equivalent of 124I-NaI for adult male (25% thyroid uptake, MIRD Dose Estimate Report No. 5) was 6.5 mSv∕MBq. Conclusions: The authors estimated human-equivalent internal radiation dose of 124I-MIBG using preclinical imaging data. As a reference, the effective dose estimation showed that 124I-MIBG would deliver less radiation dose than 124I-NaI, a radiotracer already being used in patients with thyroid cancer.

Numerous mechanism-based anticancer drugs that target the phosphatidylinositol-3-kinase (PI3K) signaling pathway are in clinical trials. However, assessment of response using traditional imaging methods remains a challenge, as it is often associated with tumor stasis. Here we show for the first time the efficacy of hyperpolarized 13C magnetic resonance spectroscopy (MRS) in detecting the effect of PI3K signaling inhibition by monitoring hyperpolarized [1-13C]-lactate levels produced from hyperpolarized [1-13C]-pyruvate through lactate dehydrogenase (LDH) activity. Studies were performed on GS-2 glioblastoma and MDA-MB-231 breast adenocarcinoma cells. Following inhibition of signaling, hyperpolarized lactate dropped significantly to 42±12% and 76±5% in GS-2 cells treated with LY294002 and everolimus respectively and to 71±15% in MDA-MB-231 cells treated with LY294002. This correlated with a drop in LDH activity to 48±4%, 63±4% and 69±12%, and was associated with a drop in LDHA mRNA levels and LDHA and HIF-1α protein levels. Taken together the data demonstrate that hyperpolarized 13C MRS of pyruvate can be used to monitor the drop in LDHA activity and expression following reduced HIF-1α levels resulting from PI3K signal inhibition. In line with these findings, in vivo studies of GS-2 subcutaneous xenografts treated with everolimus resulted in inhibition in tumor growth, which was associated with a drop in the hyperpolarized lactate-to-pyruvate ratio. In contrast an increase in the ratio was detected in controls. This works demonstrates the potential of hyperpolarized 13C MRS for noninvasive imaging of drug target modulation by treatments that modulate PI3K signaling and HIF-1α levels.

Purpose To evaluate the role of radiation therapy (RT) as a component of multimodality therapy for pediatric desmoids. Methods and Materials Twenty-one children diagnosed between 1987 and 2005 were identified. Median age at start of treatment was 13 years (range, 2–21). Primary therapy consisted of resection alone (10), resection + external beam radiation therapy (EBRT) (5), resection + chemotherapy (CT; 3), EBRT alone (1), and CT alone (2). Results The median follow-up from start of treatment is 75.7 months (range, 16–162). Examining patients with gross total resections (GTRs) (-) margins and those who had GTRs (+) margins followed by EBRT, only 2 of 7 failed primary treatment. Conversely, 13 of 14 patients with other primary treatments failed locally. Of the 15 patients who recurred, only 1 patient had a GTR (-) margins. Seven of these patients had salvage therapy that did not include RT, and of these only 2 have no evidence of disease (NED) at last follow-up. In contrast, the remaining 8 patients received RT as a component of their final salvage therapy and 7 of these are NED at last follow-up. At last follow-up, no patient has died, although toxicities of therapy have occurred. Conclusions Local control is difficult to achieve in pediatric patients with desmoids. In the setting in which negative surgical margins cannot be achieved, RT plays a key role in achieving NED status. Even after multiple recurrences, successful salvage is achievable, particularly when high-dose focal therapy is incorporated.

Purpose This open-label, prospective, single-arm, phase II study combined erlotinib with radiation therapy (XRT) and temozolomide to treat glioblastoma multiforme (GBM) and gliosarcoma. The objectives were to determine efficacy of this treatment as measured by survival and to explore the relationship between molecular markers and treatment response. Patients and Methods Sixty-five eligible adults with newly diagnosed GBM or gliosarcoma were enrolled. We intended to treat patients not currently treated with enzyme-inducing antiepileptic drugs (EIAEDs) with 100 mg/d of erlotinib during XRT and 150 mg/d after XRT. Patients receiving EIAEDs were to receive 200 mg/d of erlotinib during XRT and 300 mg/d after XRT. After XRT, the erlotinib dose was escalated until patients developed tolerable grade 2 rash or until the maximum allowed dose was reached. All patients received temozolomide during and after XRT. Molecular markers of epidermal growth factor receptor (EGFR), EGFRvIII, phosphatase and tensin homolog (PTEN), and methylation status of the promotor region of the MGMT gene were analyzed from tumor tissue. Survival was compared with outcomes from two historical phase II trials. Results Median survival was 19.3 months in the current study and 14.1 months in the combined historical control studies, with a hazard ratio for survival (treated/control) of 0.64 (95% CI, 0.45 to 0.91). Treatment was well tolerated. There was a strong positive correlation between MGMT promotor methylation and survival, as well as an association between MGMT promotor-methylated tumors and PTEN positivity shown by immunohistochemistry with improved survival. Conclusion Patients treated with the combination of erlotinib and temozolomide during and following radiotherapy had better survival than historical controls. Additional studies are warranted.

Background The components of therapy required for patients with INSS Stage 3 neuroblastoma and high-risk features remain controversial. Procedure A retrospective cohort design was used to determine if intensive chemoradiotherapy with purged autologous bone marrow rescue (ABMT) and/or 13-cis-retinoic acid (13-cis-RA) improved outcome for patients with high-risk neuroblastoma that was not metastatic to distant sites. We identified 72 patients with INSS Stage 3 neuroblastoma enrolled between 1991 and 1996 on the Phase 3 CCG-3891 randomized trial. Patients were analyzed on an intent-to-treat basis using a log-rank test. Results The 5-year event-free survival (EFS) and overall survival (OS) rates for patients with Stage 3 neuroblastoma were 55 ± 6% and 59 ± 6%, respectively (n = 72). Patients randomized to ABMT (n = 20) had 5-year EFS of 65 ± 11% and OS of 65 ± 11% compared to 41 ± 11 (P = 0.21) and 46 ± 11% (P = 0.23) for patients randomized to CC (n = 23), respectively. Patients randomized to 13-cis-RA (n = 23) had 5-year EFS of 70 ± 10% and OS of 78 ± 9% compared to 63 ± 12% (P = 0.67) and 67 ± 12% (P = 0.55) for those receiving no further therapy (n = 16), respectively. Patients randomized to both ABMT and 13-cis-RA (n = 6) had a 5-year EFS of 80 ± 11% and OS of 100%. Conclusion Patients with high-risk Stage 3 neuroblastoma have an overall poor prognosis despite aggressive chemoradiotherapy. Further studies are warranted to determine if myeloablative consolidation followed by 13-cis-RA maintenance therapy statistically significantly improves outcome. Pediatr Blood Cancer 2009;52:44–50. © 2008 Wiley-Liss, Inc.

In this study, we investigated the precursor and active forms of a p53 small-molecule inhibitor for their effects on temozolomide (TMZ) antitumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when p53 wild-type (p53wt) GBMs were cotreated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased poly(ADP-ribose) polymerase cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, which is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: TMZ + p53 inhibitor precursor cotreatment of three distinct p53wt GBM xenografts resulted in significant enhancement of TMZ antitumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (P < 0.001 for cotreatment survival benefit in each case). Mice receiving intracranial injection with p53null GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhances TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner. [Cancer Res 2008;68(24):10034–8]

To review a historical cohort of pediatric patients with supratentorial primitive neuroectodermal tumors (sPNET), to clarify the role of radiation in the treatment of these tumors.Fifteen children aged18 years with non-pineal sPNETs diagnosed between 1992 and 2006 were identified. Initial therapy consisted of surgical resection and chemotherapy in all patients and up-front radiotherapy (RT) in 5 patients. Five patients had RT at the time of progression, and 5 received no RT whatever. Kaplan-Meier estimates of overall survival were then calculated.The median follow-up from diagnosis for all patients was 31 months (range, 0.5-165 months) and for surviving patients was 49 months (range, 10-165). Of the 5 patients who received up-front RT, all were alive without evidence of disease at a median follow-up of 50 months (range, 25-165 months). Only 5 of the 10 patients who did not receive up-front RT were alive at last follow-up. There was a statistically significant difference in overall survival between the patient group that received up-front RT and the group that did not (p = 0.048). In addition, we found a trend toward a statistically significant improvement in overall survival for those patients who received gross total resections (p = 0.10).Up-front RT and gross total resection may confer a survival benefit in patients with sPNET. Local failure was the dominant pattern of recurrence. Efforts should be made to determine patients most likely to have local failure exclusively or as a first recurrence, in order to delay or eliminate craniospinal irradiation.

The phosphoinositide 3′-kinase (PI3K)–mediated signaling pathway plays a key role in fundamental cellular functions important in normal cellular homeostasis and malignant transformation. Deregulated signaling through this pathway contributes to development of gliomas and their resistance to radiation and chemotherapy. Targeting the PI3K signaling pathway has thus emerged as a promising approach to successful treatment of gliomas. We assessed the radiosensitizing potential of four small-molecule inhibitors that differ in their activities against specific isoforms of the PI3K 110-kDa catalytic subunit (p110). p110α inhibitors blocked phosphorylation of both protein kinase B/Akt and S6 in all cell lines examined, effectively decreased cellular proliferation, and produced additive cytotoxic effects in combination with radiation therapy. The p110β inhibitor exhibited limited biochemical effects and failed to decrease cellular proliferation or viability as either a single agent or in combination with radiation or rapamycin. In vivo studies examining the effects of the p110α inhibitor in combination with radiation indicated a significant reduction in tumor growth rate induced by the combined treatment compared with each treatment modality alone. This translated into a trend toward prolonged time-to-failure for mice in the combination treatment group. In conclusion, PI3K inhibitors are promising agents in the treatment of glioblastomas, especially when used in combination with ionizing radiation. [Mol Cancer Ther 2008;7(4):841–50]

Recent evidence from microarray data argues that much of the malignant potential of neoplastic cells is a result of either somatic initiating mutations or constitutional polymorphisms present in normal tissue. Consequently, we reasoned that the initiating genetic lesions seen in some of the Phakomatoses, disorders associated with predominantly benign neoplasms, may somehow fundamentally limit malignant behavior (invasion and metastasis). The function of these genes reveals a consistent pattern of antagonistic pleiotropy; they inhibit proliferation but are critical for motility. We argue that inactivating mutations in these genes (labeled RPPMs: repressors of proliferation/promoters of motility) results in predominantly benign growths because, while such mutations promote unconstrained proliferation, they limit the ability of neoplastic cells to migrate and invade. Such pleiotropy may represent an evolutionarily conserved strategy designed to limit the spread of nascent neoplasias. When not inactivated, we show how selective pressures in the tumor microenvironment would suppress mutation in these genes during progression and how wild-type RPPMs may function synergistically with oncogenes to promote malignant behavior. Finally, we explore how this model may help to explain oncogene addiction, tumor dormancy, and spontaneous regression.

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

Central neurocytomas are uncommon intraventricular neoplasms whose optimal management remains controversial due to their rarity. We assessed outcomes for a historical cohort of neurocytoma patients and evaluated effects of tumor atypia, size, resection extent, and adjuvant radiotherapy. Progression-free survival (PFS) was measured by Kaplan–Meier and Cox proportional hazards methods. A total of 28 patients (15 males, 13 females) were treated between 1995 and 2014, with a median age at diagnosis of 26 years (range 5–61). Median follow-up was 62.2 months and 3 patients were lost to follow-up postoperatively. Thirteen patients experienced recurrent/progressive disease and 2-year PFS was 75 % (95 % CI 53–88 %). Two-year PFS was 48 % for MIB-1 labeling >4 % versus 90 % for ≤4 % (HR 5.4, CI 2.2-27.8, p = 0.0026). Nine patients (32 %) had gross total resections (GTR) and 19 (68 %) had subtotal resections (STR). PFS for >80 % resection was 83 versus 67 % for ≤80 % resection (HR 0.67, CI 0.23–2.0, p = 0.47). Three STR patients (16 %) received adjuvant radiation which significantly improved overall PFS (p = 0.049). Estimated 5-year PFS was 67 % for STR with radiotherapy versus 53 % for STR without radiotherapy. Salvage therapy regimens were diverse and resulted in stable disease for 54 % of patients and additional progression for 38 %. Two patients with neuropathology-confirmed atypical neurocytomas died at 4.3 and 113.4 months after initial surgery. For central neurocytomas, MIB-1 labeling index >4 % is predictive of poorer outcome and our data suggest that adjuvant radiotherapy after STR may improve PFS. Most patients requiring salvage therapy will be stabilized and multiple modalities can be effectively utilized.

Purpose To review a historical cohort of consecutively accrued patients with high-risk neuroblastoma treated with intraoperative radiotherapy (IORT) to determine the therapeutic effect and late complications of this treatment. Methods and Materials Between 1986 and 2002, 31 patients with newly diagnosed high-risk neuroblastoma were treated with IORT as part of multimodality therapy. Their medical records were reviewed to determine the outcome and complications. Kaplan-Meier probability estimates of local control, progression-free survival, and overall survival at 36 months after diagnosis were recorded. Results Intraoperative radiotherapy to the primary site and associated lymph nodes achieved excellent local control at a median follow-up of 44 months. The 3-year estimate of the local recurrence rate was 15%, less than that of most previously published series. Only 1 of 22 patients who had undergone gross total resection developed recurrence at the primary tumor site. The 3-year estimate of local control, progression-free survival, and overall survival was 85%, 47%, and 60%, respectively. Side effects attributable to either the disease process or multimodality treatment were observed in 7 patients who developed either hypertension or vascular stenosis. These late complications resulted in the death of 2 patients. Conclusions Intraoperative radiotherapy at the time of primary resection offers effective local control in patients with high-risk neuroblastoma. Compared with historical controls, IORT achieved comparable control and survival rates while avoiding many side effects associated with external beam radiotherapy in young children. Although complications were observed, additional analysis is needed to determine the relative contributions of the disease process and specific components of the multimodality treatment to these adverse events.

Purpose We developed a co-culture system to quantitate the growth and invasion of human malignant gliomas into a background of confluent normal human astrocytes, then used this assay to assess independently the effects of irradiating both cell types on glioma invasion. Methods and Materials Enhanced green fluorescent protein (EGFP)–labeled immortalized human astrocytes, human malignant glioma cells, or transformed human astrocytes were focally plated onto a confluent layer of normal human astrocytes, and the invasiveness of EGFP-labeled cells was scored after 96 h. To address the consequences of irradiation on glioma invasion, the invasiveness of irradiated glioma cell lines and irradiated astrocytic backgrounds was assessed. Fluorescence-activated cell sorting was used to quantitate the total number of EGFP-labeled cells. Results Growth in the co-culture assay consistently reflected transformation states of the plated cells. Immortalized, but untransformed human astrocytes failed even to establish growth on confluent normal human astrocytes. In contrast, all malignant human glioma cell lines and transformed human astrocytes demonstrated various degrees of infiltration into the astrocytic bed. Irradiation failed to alter the invasiveness of U87, A172, and U373. A 1-Gy dose slightly reduced the invasiveness of U251 MG by 75% ( p post hoc Neuman-Keuls), without reducing total cell numbers. Independently irradiating the human astrocytic bed did not alter the invasiveness of nonirradiated U251, whereas the matrix metalloproteinase (MMP) inhibitor GM6001 reduced U251 invasiveness in the co-culture assay. Conclusions Growth in the co-culture assay reflects the transformation status and provides a useful in vitro model for assessing invasiveness. Human glioma invasiveness in the co-culture model responds variably to single low-dose fractions. MMP activity promotes invasiveness in the co-culture model. Reduced invasiveness in irradiated U251 appears to be mediated by MMP-independent mechanisms.

Butyroyloxymethyl-4-phenylbutyrate (AN-113) is a novel HDACI that releases potent anti-neoplastic derivatives upon intracellular hydrolysis. The precursor of AN-113, 4-phenylbutyrate has shown promising results in a Phase I study of gliomas, and we hypothesized that AN-113 offers significant advantages over the parent drug. AN-113 demonstrates selective in vitro cytotoxicity against malignant cells while sparing normal astrocytes, effective at doses over 20-fold lower than 4-phenylbutyrate. Combining AN-113 and radiation results in additive therapeutic effects. Enthusiasm is lent to this approach by the ability of AN-113 to efficiently kill glioma cells, its bioavailability and potency when administered orally, its capacity to cross the blood-brain barrier, and its effectiveness in combination with radiation.

Histone modification has emerged as a promising approach to cancer therapy. We explored the in vivo efficacy of a butyric acid derivative, pivaloyloxymethyl butyrate (AN-9), for the treatment of gliomas. Relative to control and single-modality treatments, the combination of AN-9 and radiation significantly inhibited tumor growth and prolonged time to failure in mice bearing glioma xenografts. The enhanced response to radiation was accompanied by inhibition of cellular proliferation and by increased phosphorylation of H2AX, implicating DNA double-strand breaks in the antineoplastic effects of AN-9 and radiation. The data suggest that AN-9 in combination with radiation may be an effective therapy for malignant gliomas.

Radiation (RT) is critical to the treatment of high-grade gliomas (HGGs) but cures remain elusive. The BRAF mutation V600E is critical to the pathogenesis of 10-20% of pediatric gliomas, and a small proportion of adult HGGs. Here we aim to determine whether PLX4720, a specific BRAF V600E inhibitor, enhances the activity of RT in human HGGs in vitro and in vivo. Patient-derived HGG lines harboring wild-type BRAF or BRAF V600E were assessed in vitro to determine IC50 values, cell cycle arrest, apoptosis and senescence and elucidate mechanisms of combinatorial activity. A BRAF V600E HGG intracranial xenograft mouse model was used to evaluate in vivo combinatorial efficacy of PLX4720+RT. Tumors were harvested for immunohistochemistry to quantify cell cycle arrest and apoptosis. RT+PLX4720 exhibited greater anti-tumor effects than either monotherapy in BRAF V600E but not in BRAF WT lines. In vitro studies showed increased Annexin V and decreased S phase cells in BRAF V600E gliomas treated with PLX4720+RT, but no significant changes in β-galactosidase levels. In vivo, concurrent and sequential PLX4720+RT each significantly prolonged survival compared to monotherapies, in the BRAF V600E HGG model. Immunohistochemistry of in vivo tumors demonstrated that PLX4720+RT decreased Ki-67 and phospho-MAPK, and increased γH2AX and p21 compared to control mice. BRAF V600E inhibition enhances radiation-induced cytotoxicity in BRAF V600E-mutated HGGs, in vitro and in vivo, effects likely mediated by apoptosis and cell cycle, but not senescence. These studies provide the pre-clinical rationale for clinical trials of concurrent radiotherapy and BRAF V600E inhibitors.

Medical students applying for radiation oncology residency rated “perceived quality of didactics” within the top 5 factors in ranking programs (1). However, according to a 2013 national survey, only 27% of radiation oncology clerkships included didactic components specifically for medical students (2). In 2012, a pilot radiation oncology clerkship curriculum was developed at two institutions using the six-step conceptual framework for medical education curriculum development by Kern et al (3). Eighteen students completed the pilot curriculum. Students rated the curriculum highly and reported improved comfort with their decision to pursue radiation oncology as a specialty (4). Using the cooperative group research model, the curriculum was expanded to 11 selected academic medical centers within the United States in 2013 with the goal of increasing the number of participating students to provide more robust feedback for further curriculum development. Here, we describe the structure of the curriculum with corollary student feedback that can be used by other institutions to design or enhance their medical student curriculum. The curriculum consisted of three 1-hour lectures delivered by a senior resident or faculty member; topics included: (1) an overview of radiation oncology, including a history of the specialty, types of treatments, and basic clinic flow; (2) fundamentals of radiation biology and radiation physics; and (3) practical aspects of radiation treatment simulation and planning and radiation emergencies. Ideally, one session was conducted per week with all students present. The lecture format was open, and students were encouraged to ask questions. In addition, a 1-hour hands-on radiation treatment workshop facilitated by a senior resident, faculty, physicist, or dosimetrist was implemented to teach students the fundamentals of radiation treatment planning (5). While using a radiation treatment planning workstation, each student was provided with a step-by-step guide to delineating a radiation target, selecting appropriately directed radiation beams, and modifying various beam parameters to achieve an optimal plan. Individual institutions were encouraged to adapt the lectures according to institutional treatment or practice patterns, but all institutions retained the core curriculum format (3 lectures, 1 planning session). All participating institutions obtained institutional review board exemption. A total of 94 students participated in the curriculum in the 2013 academic year, allowing both wide dissemination and robust feedback. Upon completion of the clerkship, students were invited to complete an anonymous internet-based survey to rate the curriculum components, which yielded a 73% response rate. Qualitative analysis of the evaluations was undertaken by two authors (P.M. and J.C.Y.). Evaluation-free responses were reviewed independently and divided into positive and negative “consistent” themes (Table 1). These themes reflected the students' subjective overall feeling that the course laid an excellent knowledge foundation for transition to residency and their concern that they might not retain the knowledge during the two years prior to beginning residency. Future directions for curriculum development include expansion to additional institutions while using these constructive comments to enhance the learning experience. For example, one negative theme was that lectures should be scheduled earlier in the rotation to ensure that students have a good knowledge base for the remainder of the clinical rotation. This and other constructive feedback will be considered in the future when implementing the curriculum. Table 1 Consistent positive and negative feedback themes for individual curriculum components and how the curriculum will or will not be useful for transition to residency and for the overall curriculum This successful implementation of a standardized curriculum piloted at multiple institutions provides proof-of-principle that curriculum development can follow the multi-institutional cooperative group model. In the process, we identified areas of positive and negative needs as perceived by the students, who represent the most important stakeholders. Additional data are being collected to assess the impact of this curriculum on the students' decision to apply for radiation oncology residency. In the interim, the current data can help individual departments enhance their ongoing curriculum for rotating medical students.

Pediatric low-grade gliomas (PLGG) constitute the most common group of central nervous system tumors in children. Viewed as a chronic disease, ideal therapy for PLGG should carry limited side effects that can be best achieved through selective, personalized therapy. Despite the known heterogeneity of PLGGs and characterized driver mutations most children are still treated with standard chemotherapy protocols as first line therapy. Alteration of the BRAF/MEK/MAPK pathway is the hallmark of PLGG and mTOR activation has been documented in the majority of these tumors. This study investigates combinations of MEK1/2, BRAFV600E and mTOR inhibition in PLGGs carrying specific genetic alterations of the MAPK pathway. We used human glioma cell lines containing BRAFV600E (AM38, DBTRG, BT40), or wild-type BRAF (SF188, SF9427, SF9402) and isogenic systems of KIAA1549:BRAF-expressing NIH3T3 cells and BRAFV600E-expressing murine brain tumors. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK1/2). Cell cycle distribution and apoptosis were assessed using flow cytometry; proliferation was determined using an ATP-based assay (CellTiter-Glo). In vivo activity of these inhibitors was assessed in the BT40 PLGG xenograft mouse model. In BRAFV600E tumors, the combination of MEK inhibitor with either mTOR or BRAFV600E specific inhibitor is superior to monotherapy and to combination of BRAF and mTOR inhibitors in reducing cell viability reduction and arresting cell cycle progression. In BRAFWT pediatric gliomas, everolimus + AZD6244 is superior to either agent alone. KIAA1549:BRAF-expressing tumors display marked sensitivity to MEK1/2 inhibition alone. In vivo studies in the PLGG xenograft model BT40 showed the greatest survival advantage in mice treated with combination of AZD6244 + PLX4720 or AZD6244 + everolimus compared with respective monotherapies (p < 0.01).

INTRODUCTION: First generation allosteric inhibitors of mTORC1 have shown clinical benefit for the treatment of pediatric low-grade gliomas (PLGG). MEK inhibition has shown great promise in early phase clinical trials for PLGG with the hypothesis that tumors carrying the KIAA1549:BRAF are particularly sensitive to such treatment. MLN0128, a second-generation, ATP-competitive, pan-mTOR kinase inhibitor, acts on both mTORC1 and mTORC2. Herein we investigate the effects of MLN0128 monotherapy as well as in combination with MEK inhibition in models of PLGG. METHODS: We used human glioma cell lines containing BRAFV600E (AM38), wild-type BRAF (LN229, TN98, SF188) and isogenic systems of KIAA1549:BRAF-expressing NIH3T3 cells. Signaling inhibitors included MLN0128, everolimus, BRAFV600E specific inhibitor PLX4720 and MEK specific inhibitors AZD6244 and GSK1120212. Cell proliferation was determined using an ATP-based assay. Biochemical effects were assessed using western blot analysis. In vivo activity of these inhibitors are ongoing using the BT40 PLGG xenograft mouse model. RESULTS: MLN0128 monotherapy shows stronger anti-proliferative effects compared to everolimus across cell lines independent of BRAF status. As expected, treatment with MLN0128 leads to down-regulation of p-AKT and p-4EBP1, whereas treatment with everolimus only reduces expression of p-S6. MEK inhibition with AZD6244 effectively decreases p-ERK expression in all lines; however, the addition of MLN0128 to AZD6244 causes a rebound in p-ERK expression in BRAFWT gliomas but not in gliomas expression BRAFV600E or KIAA1549:BRAF alterations. In cells carrying either the BRAFV600E or KIAA1549:BRAF alteration, combined treatment with MLN0128 and GSK1120212 or AZD6244 leads to synergistic anti-proliferative effects whereas such synergy is not present in BRAFWT gliomas. In vivo studies in the PLGG xenograft model BT40 are ongoing. CONCLUSION: MLN0128 has synergistic effects with MEK inhibition in gliomas bearing BRAFV600E or KIAA1549:BRAF. In BRAF wild-type cells such synergy is not present, perhaps due to rebound in p-ERK when MLN0128 is combined with AZD6244.

INTRODUCTION: Activating BRAF-fusion mutations occur frequently in pediatric low-grade gliomas (PLGGs). We have previously shown targeting of KIAA1549-BRAF fusion by PLX4720, the research analog of vemurafenib, results in paradoxical activation of the mitogen activated protein kinase (MAPK) signal cascade. We hypothesized that BRAF-fusions could be targeted by downstream MEK inhibitors (MEKi), and that combinatorial targeting of BRAF-fusions could also prevent acquired resistance to single agent therapy. METHODS: BRAF-fusions were screened with a selection of clinically relevant MEKi and with PLX8394, a second-generation RAF inhibitor, in vitro in cell culture and soft agar. BRAF V600E and RAS mutant cells were also screened for comparison. MEKi/PLX8394 resistant BRAF-fusion cell lines were generated in vitro and interrogated by sequencing, immunoblotting, and RNA-seq analysis to identify potential co-targets. Combinatorial therapy was then tested in vitro and in vivo to confirm efficacy. RESULTS: BRAF-fusions in PLGGs can be targeted by MEKi and by the pan-RAF inhibitor PLX8394. AZD6244, MEK162, GDC0623, and GDC0973 all inhibited downstream ERK phosphorylation; however, GSK1120212 more robustly inhibited MAPK activation in the BRAF-fusion, BRAF V600E, and RAS mutant cells. Interestingly, the MEKi/PLX8394 resistant BRAF-fusion cells all showed activation of phosphatidylinositol 3-kinase (PI3K) pathway. BRAF-fusion parental cells were screened with RAD001, a targeted mTOR inhibitor, resulting in modest downstream inhibition. Co-targeting of the parental BRAF-fusions with GSK1120212 and RAD001 resulted in robust inhibition of down-stream signaling and in significant tumor inhibition, and prevented acquired resistance to targeted inhibition. CONCLUSION: Our work demonstrates that BRAF-fusion oncogenes can be effectively targeted with GSK1120212 as single agent but even more robustly with GSK1120212 and RAD001 co-targeting. This provides preclinical rationale for treating PLGGs harboring BRAF-fusion oncogenes with combinatorial MEK and mTOR inhibitors in clinical trial.

Tumor cells rely preferentially on anaerobic glycolysis rather than on respiration for ATP generation, a phenomenon known as the Warburg effect. We explored the effects of glucose withdrawal on glioblastoma multiforme–derived cell lines and their nontransformed counterparts, normal human astrocytes. We found that glucose withdrawal induces extensive apoptosis in glioblastoma multiforme cells but not in normal astrocytes. In all cells examined, ATP levels are sustained on glucose withdrawal due to elevation of fatty acid oxidation and ensuing respiration; however, we show that oxidative stress generated in the mitochondrial respiratory chain is the direct cause of cell death in glioblastoma multiforme cells. Oxidative stress that only occurs in glioblastoma multiforme cells underlies the selective susceptibility to glucose withdrawal–induced apoptosis documented in the malignant cells. This study implicates glycolysis as a potentially efficient and selective target for glioblastoma multiforme treatment. (Mol Cancer Res 2006;4(5):319–30)

Acyloxyalkyl ester prodrugs of histone deacetylase inhibitors, a family of anti-cancer agents, are metabolized intracellularly to acids and aldehyde(s). The purpose of this study was to assess the in vitro and in vivo anticancer activity, selectivity and oral bioavailability of these prodrugs. The prodrugs exhibited a hierarchal potency of AN-193 > or = AN-7 > AN-1 and AN-9 >> AN-10 against murine lung carcinoma (3LLD122) and human breast carcinoma (MCF-7) cell lines. AN-9, and to even greater extent AN-7, displayed preferential cytotoxicity against leukemic and glioblastoma cells compared to their normal cellular counterparts-normal mononuclear and astrocytes cells, respectively. In vivo, anti-metastatic activity was evaluated in a metastatic model of lung cancer in which Lewis lung carcinoma (3LLD122) cells are injected intravenously into C57/BL mice and produce lung nodules. The prodrugs administered orally demonstrated a significant inhibition of lung-lesion formation and their hierarchal potency concurred with that observed in vitro, with the exception of AN-193 that was the least active compound. Escalating doses of AN-7 (5-100 mg/kg), administered by oral or intraperitoneal routes and displayed equivalent anti-metastatic activities, confirmed the good oral bioavailability of AN-7. Consistent with these findings, a time course study of histone acetylation in subcutaneously implanted 3LL122 tumors showed 2-4 fold increases in histone acetylation within 0.5 h of intravenous, intraperitoneal, or oral administration of AN-7 (100 mg/kg). Relative contributions of the prodrug metabolites to the anti-neoplastic activity and the best candidate for clinical studies are discussed.

Medulloblastoma is the most frequent brain tumor in children, and multimodality therapy generally includes surgery, chemotherapy, and craniospinal irradiation (CSI). Progressive improvements in treatment strategies have led to overall survival rates exceeding 80% for children with average-risk medulloblastoma, highlighting concerning long-term side effects that can include neurocognitive, growth, endocrine, hearing, and general quality of life impairments. In this issue of the International Journal of Radiation Oncology, Biology, Physics, Kennedy et al (1) report results of quality of survival and growth for patients treated on the primitive neuroectodermal tumor 4 (PNET4) European controlled trial of hyperfractionated (HFRT) versus conventional radiation therapy for standard-risk medulloblastoma. They found that HFRT had less impact on executive function than standard CSI but resulted in greater height impairment. The Behavior Rating Inventory of Executive Function (BRIEF) questionnaire aims to assess impairment of executive function for individuals aged 5-18 years. The BRIEF questionnaire involves 8 parameters and gives the clinician insight into the behavior and everyday function of the child or adolescent being testeddinformation that serves to complement performance measures. Of the 4main outcomemeasures, only executive function (BRIEF)was inferior in theHFRTarm,whereas the other 3 outcome measuresdhealth status, behavioral difficulties, and health-related quality of lifeddid not differ between arms. In addition, only a limited proportion of children in either radiation group (10%-23%) had BRIEF scores in the range of executive dysfunction. Because executive function has significant implications for everyday social and academic function, these results shed light on the cognitive, behavioral, and socialeemotional developmental consequences of treatment for our patients (2).

The epidermal growth factor receptor (EGFR) is frequently amplified, overexpressed, or mutated in glioblastomas, but only 10 to 20 percent of patients have a response to EGFR kinase inhibitors. The mechanism of responsiveness of glioblastomas to these inhibitors is unknown.We sequenced kinase domains in the EGFR and human EGFR type 2 (Her2/neu) genes and analyzed the expression of EGFR, EGFR deletion mutant variant III (EGFRvIII), and the tumor-suppressor protein PTEN in recurrent malignant gliomas from patients who had received EGFR kinase inhibitors. We determined the molecular correlates of clinical response, validated them in an independent data set, and identified effects of the molecular abnormalities in vitro.Of 49 patients with recurrent malignant glioma who were treated with EGFR kinase inhibitors, 9 had tumor shrinkage of at least 25 percent. Pretreatment tissue was available for molecular analysis from 26 patients, 7 of whom had had a response and 19 of whom had rapid progression during therapy. No mutations in EGFR or Her2/neu kinase domains were detected in the tumors. Coexpression of EGFRvIII and PTEN was significantly associated with a clinical response (P0.001; odds ratio, 51; 95 percent confidence interval, 4 to 669). These findings were validated in 33 patients who received similar treatment for glioblastoma at a different institution (P=0.001; odds ratio, 40; 95 percent confidence interval, 3 to 468). In vitro, coexpression of EGFRvIII and PTEN sensitized glioblastoma cells to erlotinib.Coexpression of EGFRvIII and PTEN by glioblastoma cells is associated with responsiveness to EGFR kinase inhibitors.

Purpose: To identify whether inhibition of phosphatidylinositol-3-kinase (PI3K) causes increased radiosensitivity through inhibition of protein kinase B (PKB), implicating PKB as an important therapeutic target in prostate cancer. Methods and Materials: The prostate cancer cell line LNCaP was treated with the PI3K inhibitor LY294002, radiation, and combinations of the two therapies. Apoptosis and survival were measured by cell cycle analysis, Western blot analysis for cleaved poly (ADP-ribose) polymerase, and clonogenic survival. To test the hypothesis that inhibition of PKB is responsible for LY294002-induced radiosensitivity, LNCaP cells expressing a constitutively active form of PKB were used. Results: The combination of PI3K inhibition and radiation caused an increase in apoptosis and a decrease in clonogenic survival when compared to either modality alone. The expression of constitutively activated PKB blocked apoptosis induced by combination of PI3K inhibition and radiation and prevented radiosensitization by LY294002. Conclusion: These data indicate that PI3K inhibition increases sensitivity of prostate cancer cell lines to ionizing radiation through inactivation of PKB. Therefore, PTEN mutations, which lead to PKB activation, may play an important role in the resistance of prostate cancer to radiation therapy. Targeted therapy against PKB could be beneficial in the management of prostate cancer patients.

Epidermal growth factor receptors (EGFRs) are amplified and overexpressed in many different human cancers, a phenomenon generally associated with poor prognosis. Inhibitors of the tyrosine kinase activity associated with this receptor have been approved for the treatment of chemotherapy-refractory nonsmall cell lung cancer, and are in clinical trials for additional tumor types. While these inhibitors, gefitinib and erlotinib, display limited response rates when assessed in cohorts that include all patients, there are subgroups, defined by patient and tumor characteristics, that preferentially respond to these agents. We recently performed an analysis of tumors obtained from a Phase I trial of erlotinib in patients with glioblastoma multiforme (GBM), the most common malignant brain tumor in adults. We showed that patients whose tumors exhibited overexpression and amplification of EGFR responded better than patients who had normal levels of this gene and protein. We also demonstrated that the phosphorylation state of PKB/Akt was an important determinant for response, with low phospho-PKB/Akt levels predicting good response to erlotinib. We discuss these findings in the context of recent molecular analyses of the placebo-controlled Phase III trials that led to approval of EGFR inhibitors. These data underscore the importance of placebo-controlled trials to distinguish between prognostic indicators of disease progression more generally and predictive markers of response to therapy. Ultimately the goal of these studies is to allow selection of patients who will preferentially respond to EGFR inhibitors.

Data on spinal ependymomas are sparse, and prognostic factors remain controversial. The primary aim of this study is to review a historical cohort, with large patient numbers and long follow-up, and provide estimates of time to progression (TTP) and survival after progression. As a secondary aim, we assess the effects of potential prognostic variables. Thirty-seven patients with spinal cord ependymomas received postoperative radiation therapy from 1955 to 2001. The influences of radiation dose, extent of resection, Karnofsky performance score, tumor location, and multifocality were assessed in univariate analyses by using the Cox proportional hazards model. The median follow-up for patients who did not fail was 121 months (range, 8–312 months). Kaplan-Meier estimates of 5-, 10-, and 15-year percentage progression free are 75% ± 7.4%, 50% ± 9.1%, and 46% ± 9.3%, respectively. Median TTP, for those who recurred, is 68 months (range, 2–324 months), with 12 of 21 failures occurring after five years. Of the prognostic factors examined, only greater extent of resection significantly correlated with longer TTP (P = 0.02). Local relapse rates for spinal ependymomas are higher than previously cited, with a large proportion of failures occurring more than five years after diagnosis. Extensive surgical resection correlates with longer time to recurrence, and we thus recommend maximal excision while avoiding surgical morbidity. The overall high rate of recurrence leads us to recommend radiation to doses of 45 to 54 Gy for all patients who do not have gross total resections, and long, close follow-up.

The phosphoinositide 3-kinase (PI3-kinase) signaling pathway is frequently aberrantly activated in glioblastoma multiforme (GM) by mutation or loss of the 3' phospholipid phosphatase PTEN. PTEN abnormalities result in inappropriate signaling to downstream molecules including protein kinase B (PKB/Akt), and mammalian target of rapamycin (mTOR). PI3-kinase activation increases resistance to radiation-induced cell death; conversely, PI3-kinase inhibition enhances the sensitivity of tumors to radiation. The effects of LY294002, a biochemical inhibitor of PI3-kinase, on the response to radiation were examined in the PTEN mutant glioma cell line U251 MG. Low doses of LY294002 sensitized U251 MG to clinically relevant doses of radiation. In contrast to LY294002, rapamycin, an inhibitor of mTOR, did not result in radiosensitization. We demonstrate that among multiple known targets of LY294002, PI3-kinase is the most likely molecule responsible for LY294002-induced radiosensitization. Furthermore, using a myristoylated PKB/Akt construct, we identified PKB/Akt as the downstream molecule that mediates the synergistic cytotoxicity between LY294002 and radiation. Thus PI3-kinase dysregulation may contribute to the notable radioresistance of GM tumors and inhibition of PKB/Akt offers an excellent target to enhance radiosensitivity.

International audience; Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the -88/-66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate.

Although human cells exposed to DNA-methylating agents undergo mismatch repair (MMR)-dependent G(2) arrest, the basis for the linkage between MMR and the G(2) checkpoint is unclear. We noted that mitogen-activated protein kinase p38alpha was activated in MMR-proficient human glioma cells exposed to the chemotherapeutic methylating agent temozolomide (TMZ) but not in paired cells made MMR deficient by expression of a short inhibitory RNA (siRNA) targeted to the MMR protein Mlh1. Furthermore, activation of p38alpha in MMR-proficient cells was associated with nuclear inactivation of the cell cycle regulator Cdc25C phosphatase and its downstream target Cdc2 and with activation of the G(2) checkpoint, actions which were suppressed by the p38alpha/beta inhibitors SB203580 and SB202590 or by expression of a p38alpha siRNA. Finally, pharmacologic or genetic inhibition of p38alpha increased the sensitivity of MMR-proficient cells to the cytotoxic actions of TMZ by increasing the percentage of cells that underwent mitotic catastrophe as a consequence of G(2) checkpoint bypass. These results suggest that p38alpha links DNA MMR to the G(2) checkpoint and to resistance to chemotherapeutic DNA-methylating agents. The p38 pathway may therefore represent a new target for the development of agents to sensitize tumor cells to chemotherapeutic methylating agents.

1 Departments of Radiation Oncology and Neurological Surgery, Helen Diller Family Comprehensive Cancer Center, UCSF Benioff Children’s Hospital, University of California San Francisco, San Francisco, CA, USA 2 Department of Radiation Oncology, University of California San Francisco, San Francisco, CA, USA 3 Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA

To evaluate the role of chemotherapy and radiation therapy in the treatment of pure germinomas of the central nervous system (CNS). We reviewed a historical cohort of 79 patients between the ages of 3-35years who received definitive treatment for newly diagnosed, pure CNS germinoma between 1985 and 2010 at the University of California, San Francisco (UCSF). Median age at diagnosis was 15years (interquartile range, IQR 12-20years) and 61 (77.2%) patients were male. Median follow-up for the cohort was 111.1months (IQR 45.7-185.1months). Five-year PFS rate was 86.4% (95% CI 76.1-92.4) and 5year OS rate was 93.0% (95% CI 84.1-97.1). Median PFS was 104.6months (IQR 41.4-170.1months). Fourteen patients progressed and 8 died of their disease. Patients who received focal irradiation (XRT) and chemotherapy had a significantly higher rate of progression compared to those who received whole brain irradiation (WBI) or whole ventricle irradiation (WVI). Three of 8 patients had a PR to chemotherapy and received focal XRT progressed whereas only 1 of 9 patients who had a CR to chemotherapy who went on to receive focal XRT progressed. Elevation of hCGβ>50mIU/ml was not significantly associated with disease progression (HR 5.64, 95% CI 0.97-32.7, p=0.054). Patients treated with WBI or WVI with or without chemotherapy achieve better disease control compared to patients treated with focal XRT+chemotherapy.

Neuroblastoma (NB) is the most frequent indication for extracranial pediatric radiotherapy. As long-term survival of high-risk localized NB has greatly improved, we reviewed treatment-related late toxicities in pediatric patients who received postoperative radiotherapy (RT) for localized NB within two French prospective clinical trials: NB90 and NB94. From 1990–2000, 610 children were enrolled. Among these, 35 were treated with induction chemotherapy, surgery, and RT. The recommended RT dose was 24 Gy at ≤ 2 years, 34 Gy at > 2 years, ± a 5 Gy boost in both age groups. The 22 patients still alive after 5 years were analyzed. The median follow-up time was 14 years (range 5–21 years). Late effects after therapy occurred in 73 % of patients (16/22), within the RT field for 50 % (11/22). The most frequent in-field effects were musculoskeletal abnormalities (n = 7) that occurred only with doses > 31 Gy/1.5 Gy fraction (p = 0.037). Other effects were endocrine in 3 patients and second malignancies in 2 patients. Four patients presented with multiple in-field late effects only with doses > 31 Gy. After a median follow-up of 14 years, late effects with multimodality treatment were frequent. The most frequent effects were musculoskeletal abnormalities and the threshold for their occurrence was 31 Gy.

Standard treatment for Glioblastoma Multiforme (GBM) consists of a combination of chemotherapy and radiation therapy followed by adjuvant chemotherapy. While the optimal dose of radiation therapy has been established, significant variability in volume of tissue irradiated exists. In this article we review the current guidelines, patterns of care, patterns of failure, imaging advances and toxicity related to radiation therapy volumes in the treatment of GBM.

Rates and characteristics of intracerebral cavernous malformations after cranial radiation therapy remain poorly understood. Herein we report on intracerebral cavernous malformations detected on follow-up imaging in pediatric cancer patients who received cranial radiation therapy at age ≤18 years from 1980 to 2009. Through chart reviews (n = 362) and phone interviews (n = 104) of a retrospective cohort, we identified 10 patients with intracerebral cavernous malformations. The median latency time for detection of intracerebral cavernous malformations after cranial radiation therapy was 12 years (range 1-24 years) at a median age of 21.4 years (interquartile range = 15-28). The cumulative incidence was 3% (95% confidence interval 1%-8%) at 10 years post cranial radiation therapy and 14% (95% confidence interval 7%-26%) at 15 years. Three patients underwent surgical resection. Two surgical specimens were pathologically similar to sporadically occurring intracerebral cavernous malformations; one was consistent with capillary telangiectasia. Intracerebral cavernous malformations are common after cranial radiation therapy and can show a spectrum of histologic features.

Purpose: To determine the clinical and functional outcomes of children undergoing limb-sparing therapy for extremity sarcomas. Methods and Materials: We retrospectively reviewed 30 patients, age ≤ 21 years, who were treated between l979 and l998 with external beam radiotherapy as a component of limb-sparing therapy for primary sarcomas of the extremity at UCSF. Included were patients for whom complete follow-up and functional outcome assessments were available. We assessed the patterns of failure, overall survival, disease-free survival, local control, and limb function. Results: At a median follow-up of 3 years, 12 of the 30 patients recurred: 3 locally, 8 distantly, and 1 with synchronous local and distant disease as site of first progression. Eighteen patients were alive with no evidence of disease. The median overall survival was 10 years, with a median disease-free survival of 8 years. Functional outcome assessment revealed 15 patients retained excellent, 12 good, 1 fair, and 2 poor limb function. Conclusion: In pediatric patients receiving limb-sparing therapy, 90% maintained excellent or good limb function without compromising survival, demonstrating the validity of limb preservation in children with extremity sarcomas.

In glioblastoma-derived cell lines, PTEN does not significantly alter apoptotic sensitivity or cause complete inhibition of DNA synthesis. However, in these cell lines PTEN regulates hypoxia- and IGF-1-induced angiogenic gene expression by regulating Akt activation of HIF-1 activity. Restoration of wild-type PTEN to glioblastoma cell lines lacking functional PTEN ablates hypoxia and IGF-1 induction of HIF-1-regulated genes. In addition, Akt activation leads to HIF-1α stabilization, whereas PTEN attenuates hypoxia-mediated HIF-1α stabilization. We propose that loss ofPTEN during malignant progression contributes to tumor expansion through the deregulation of Akt activity and HIF-1-regulated gene expression.

Purpose: Glioblastoma multiforme brain tumors (GM) are treated with a spectrum of fractionation regimens based on the clinical and anatomical characteristics of the tumor but rarely based on the molecular characteristics of the individual neoplasm. This study tests the hypothesis that the response of cell lines derived from GM to fractionated radiotherapy depends on the function of wild-type p53 (wt p53), a tumor suppressor gene frequently mutated in GM tumors. Methods & Materials: Isogenic derivatives of glioblastoma cells differing only in p53 function were prepared using a retroviral vector expressing a dominant negative mutant of p53 (mt p53). Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Apoptosis was assayed by a terminal deoxynucleotide transferase-labeling technique and chromatin morphology. Results: We have previously reported the generation of isogenic GM cell lines differing only in p53 function. U87-175.4, lacking wt p53 function, had a significantly lower α/β value than U87-LUX.8, expressing functional wt p53, leading us to hypothesize that fractionated irradiation would preferentially spare GM cells harboring mt p53 compared with those expressing functional, wt p53. Survival curves following either 2.0 Gy or 3.5 Gy/fraction demonstrated that lack of functional wt p53 was associated with resistance to fractionated irradiation. Radiation-induced apoptosis could not account for the observed differences in clonogenic survival. Rather, our data suggested that a deficit in the G1-checkpoint contributed to increased resistance to fractionated irradiation of cells expressing mutant p53. Conclusions: The effect of fractionated radiotherapy in GM may depend on the function of the tumor suppressor gene p53. A potential clinical consequence of these findings is that hyperfractionation regimens may provide a therapeutic advantage specifically for tumors expressing wt p53 whereas a radiotherapy course of fewer, larger fractions may be appropriate for the treatment of tumors carrying p53 mutations. Further studies are needed to confirm our proposal that the p53 status of GM tumors can be used to guide our choice of fractionation schemes.

BACKGROUND: High-grade gliomas (HGGs) in children consistently demonstrate poor outcome despite multi-modal therapy. ACNS0822 was a feasibility and phase II trial in newly-diagnosed pediatric HGG. METHODS: In the feasibility study, patients received vorinostat 230 mg/m2/dose daily during radiation therapy (RT). In the phase II study, patients were randomized to one of three radiosensitizing agents during RT: Arm A [vorinostat 230 mg/m2/dose on Days 1-5 weekly], Arm B [temozolomide (TMZ) 90 mg/m2/dose daily], and Arm C [bevacizumab (BEV) 10 mg/kg/dose on Days 22 and 36]. All patients received BEV (10 mg/kg/dose every 2 weeks) + TMZ (200 mg/m2/dose on Days 1-5 q 28 days) for maintenance therapy × 12 cycles. The primary objectives were to determine 1) the feasibility of administering vorinostat during RT and 2) the 1-year event-free survival (EFS) of vorinostat or BEV with RT versus TMZ with RT as the historical control followed by maintenance chemotherapy. RESULTS: Six eligible patients were enrolled on the feasibility study; none experienced a dose limiting toxicity. The 95 patients enrolled (92 eligible) on the phase II study, were randomized to Arm A (vorinostat, n = 31), Arm B (TMZ, n = 29), and Arm C (BEV, n = 32). One-year EFS for Arms A, B, and C were 36.1 ± 10.4%, 54.8± 10.2%, 38.5 ± 10.1%, respectively. All chemoradiotherapy arms, and the maintenance arm were well-tolerated. The most common grade 3/4 toxicities in maintenance therapy included thrombocytopenia (9.6%), neutropenia (7%), and leukopenia (3.6%). Intracranial hemorrhage possibly attributable to BEV was reported in 2 patients in maintenance cycles 2 [grade 3] and 7 [grade 2]. There were no therapy-related deaths. CONCLUSION: Vorinostat (230 mg/m2/dose weekly) was tolerable during RT, as was maintenance therapy with BEV + TMZ. However, neither vorinostat, nor BEV proved superior to TMZ as a radiosensitizer in pediatric HGG.

Diffuse gliomas consist of both low- and high-grade varieties, each with distinct morphological and biological features. The often extended periods of relative indolence exhibited by low-grade gliomas (LGG; WHO grade II) differ sharply from the aggressive, rapidly fatal clinical course of primary glioblastoma (GBM; WHO grade IV). Nevertheless, until recently, the molecular foundations underlying this stark biological contrast between glioma variants remained largely unknown. The discoveries of distinctive and highly recurrent genomic and epigenomic abnormalities in LGG have both informed a more accurate classification scheme and pointed to viable avenues for therapeutic development. As such, the field of neuro-oncology now seems poised to capitalize on these gains to achieve significant benefit for LGG patients. This report will briefly recount the proceedings of a workshop held in January 2013 and hosted by Accelerate Brain Cancer Cure (ABC(2)) on the subject of LGG. While much of the meeting covered recent insights into LGG biology, its focus remained on how best to advance the clinical management, whether by improved preclinical modeling, more effective targeted therapeutics and clinical trial design, or innovative imaging technology.

High-grade gliomas (HGGs) are aggressive brain tumors in children, with 5-year survival rates

Purpose To assess, in a retrospective cohort study, rates and predictors of first and recurrent stroke in patients treated with cranial irradiation (CRT) and/or cervical irradiation at ≤18 years of age. Methods and Materials We performed chart abstraction (n=383) and phone interviews (n=104) to measure first and recurrent stroke in 383 patients who received CRT and/or cervical radiation at a single institution between 1980 and 2009. Stroke was defined as a physician diagnosis and symptoms consistent with stroke. Incidence of first stroke was number of first strokes per person-years of observation after radiation. We used survival analysis techniques to determine cumulative incidence of first and recurrent stroke. Results Among 325 subjects with sufficient follow-up data, we identified 19 first strokes (13 ischemic, 4 hemorrhagic, 2 unknown subtype) occurring at a median age of 24 years (interquartile range 17-33 years) in patients treated with CRT. Imaging was reviewed when available (n=13), and the stroke was confirmed in 12. Overall rate of first stroke was 625 (95% confidence interval [CI] 378-977) per 100,000 person-years. The cumulative incidence of first stroke was 2% (95% CI 0.01%-5.3%) at 5 years and 4% (95% CI 2.0%-8.4%) at 10 years after irradiation. With each 100-cGy increase in the radiation dose, the stroke hazard increased by 5% (hazard ratio 1.05; 95% CI 1.01-1.09; P =.02). We identified 6 recurrent strokes; 5 had available imaging that confirmed the stroke. Median time to recurrence was 15 months (interquartile range 6 months-3.2 years) after first stroke. The cumulative incidence of recurrent stroke was 38% (95% CI 17%-69%) at 5 years and 59% (95% CI 27%-92%) at 10 years after first stroke. Conclusion Cranial irradiation puts childhood cancer survivors at high risk of both first and recurrent stroke. Stroke prevention strategies for these survivors are needed.

To assess poly (ADP-ribose) polymerase (PARP) inhibitor MK-4827 together with radiation for the treatment of neuroblastoma.Clonogenic survival assays were used to assess MK-4827, radiation and combination thereof in four neuroblastoma cell lines. In vivo efficacy was tested in a murine xenograft model of metastatic neuroblastoma. In vivo targeted inhibition and biological effects included measurement of cleaved caspase-3, γ-H2AX, and Ki 67 by immunohistochemistry (IHC) and poly-ADP-ribose by Enzyme-Linked Immunosorbent Assay.Treatment of neuroblastoma cell lines reduced clonogenicity and resulted in additive effects with radiation. In vivo treatment with MK-4827 and radiation prolonged survival (p0.01) compared to single modalities. In vivo superiority of MK-4827 plus radiation was further documented by significant elevations of cleaved caspase-3 and γ-H2AX in tumors from the combination group compared to single modality cohorts.Combination of MK-4827 and radiation might provide effective therapy for children with high-risk neuroblastoma.

Purpose: Loss of the p53 tumor suppressor gene has been associated with tumor progression, disease relapse, poor respone to antineoplastic therapy, and poor prognosis in many malignancies. We have investigated the contribution of p53-mediated radiation-induced apoptosis and G 1 arrest to the well described radiation resistance of glioblastoma multiforme (GM) cells. Methods and Materials: Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Isogenic derivatives of glioblastoma cells differing only in their p53 status were generated using a retroviral vector expressing a dominant negative mutant of p53. Radiation-induced apoptosis was assayed by Fluorescence-activated cell sorter (FACS) analysis, terminal deoxynucleotide transferase labeling technique, and chromatin morphology. Cells were synchronized in early G 1 phase and mitotic and labeling indices were measured. Results: Radiation-induced apoptosis of GM cells was independent of functional wild-type p53 (wt p53). Decreased susceptibility to radiation-induced apoptosis was associated with lower α values characterizing the shoulder of the clonogenic radiation survival curve. Using isogenic GM cells differing only in their p53 activity, we found that a p53-mediated function, radiation-induced G 1 arerst, could also influence the value of α and clonogenic radiation resistance. Inactivation of wt p53 function by a dominant negative mutant of p53 resulted in a significantly diminished α value with no alteration in cellular susceptibility to radiation-induced apoptosis. The clonal derivative U87-LUX.8 expressing a functional wt p53 had an α (Gy −1 ) value of 0.609, whereas the isogenic clonal derivative U87-175.4 lacking wt p53 function had an α (Gy − ) value of 0.175. Conclusion: We conclude that two distinct cellular responses to radiation, p53-independent apoptosis and p53-dependent G 1 -arrest, influence radiobiological parameters that characterize the radiation response of glioblastoma cells. Further understanding of the molecular basis of GM radiation resistance will lead to improvement in existing therapeutic modalities and to the development of novel treatment approaches.