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Background Atypical and malignant meningiomas can recur despite resection and radiation. Objective To determine outcomes of patients with recurrent atypical or malignant meningioma treated with repeat resection and permanent iodine-125 ( 125 I) brachy-therapy. Methods Charts of patients who underwent surgical resection and 125 I brachyther-apy implantation for atypical and malignant meningiomas between 1988 and 2013 were retrospectively reviewed. The Kaplan-Meier actuarial method was used to calculate progression-free and overall survival. The log-rank test was used to compare groups. Significance was set at P Results Forty-two patients underwent 50 resections with 125 I brachytherapy im-plantations. All patients had undergone previous resections and 85% had previously undergone radiation. Median follow-up was 7.5 years after diagnosis and 2.3 years after brachytherapy. Median time to progression after resection with 125 I brachytherapy was 20.9 months for atypical meningioma, 11.4 months for malignant meningioma, and 11.4 months for the combined groups. Median survival after re-resection and 125 I brachytherapy was 3.5 years for atypical meningioma, 2.3 years for malignant menin-gioma, and 3.3 years for all subjects. Median overall survival after diagnosis was 11.1 years for atypical meningioma, 9.1 years for malignant meningioma, and 9.4 years for all subjects. Complications occurred in 17 patients and included radiation necrosis (n = 8, 16%), wound breakdown (n = 6, 12%), hydrocephalus (n = 4, 8%), infection (n = 3, 6%), and a pseudomeningocele (n = 2, 5%). Conclusion This is the largest experience with adjuvant 125 I brachytherapy for recurrent high-grade meningiomas. The outcomes support the use of adjuvant brachytherapy as an option for these aggressive tumors.

The apical damage kinase, ATR, is activated by replication stress (RS) both in response to DNA damage and during normal S-phase. Loss of function studies indicates that ATR acts to stabilize replication forks, block cell cycle progression and promote replication restart. Although checkpoint failure and replication fork collapse can result in cell death, no direct cytotoxic pathway downstream of ATR has previously been described. Here, we show that ATR directly reduces survival by inducing phosphorylation of the p50 (NF-κB1, p105) subunit of NF-кB and moreover, that this response is necessary for genome maintenance independent of checkpoint activity. Cell free and in vivo studies demonstrate that RS induces phosphorylation of p50 in an ATR-dependent but DNA damage-independent manner that acts to modulate NF-кB activity without affecting p50/p65 nuclear translocation. This response, evident in human and murine cells, occurs not only in response to exogenous RS but also during the unperturbed S-phase. Functionally, the p50 response results in inhibition of anti-apoptotic gene expression that acts to sensitize cells to DNA strand breaks independent of damage repair. Ultimately, loss of this pathway causes genomic instability due to the accumulation of chromosomal breaks. Together, the data indicate that during S-phase ATR acts via p50 to ensure that cells with elevated levels of replication-associated DNA damage are eliminated.

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

Intraventricular neurocytomas (IVNs) are rare tumors of neuronal differentiation, accounting for 0.1% to 0.5% of all primary brain tumors. Complete surgical resection is highly recommended, but at least one-third to one-half are subtotally resected. Stereotactic radiosurgery (SRS) and conventional radiotherapy have been used for management of residual and recurrent tumors, but the optimal treatment following subtotal surgical resection is less clear. Although there are trends toward higher local tumor control and survival rates with lower complications among IVNs treated with SRS, higher quality data are required to confirm these findings.

Purpose Students applying to radiation oncology residency programs complete 1 or more radiation oncology clerkships. This study assesses student experiences and perspectives during radiation oncology clerkships. The impact of didactic components and number of clerkship experiences in relation to confidence in clinical competency and preparation to function as a first-year radiation oncology resident are evaluated. Methods and Materials An anonymous, Internet-based survey was sent via direct e-mail to all applicants to a single radiation oncology residency program during the 2012-2013 academic year. The survey was composed of 3 main sections including questions regarding baseline demographic information and prior radiation oncology experience, rotation experiences, and ideal clerkship curriculum content. Results The survey response rate was 37% (70 of 188). Respondents reported 191 unique clerkship experiences. Of the respondents, 27% (19 of 70) completed at least 1 clerkship with a didactic component geared towards their level of training. Completing a clerkship with a didactic component was significantly associated with a respondent's confidence to function as a first-year radiation oncology resident (Wilcoxon rank–sum P =.03). However, the total number of clerkships completed did not correlate with confidence to pursue radiation oncology as a specialty (Spearman ρ P =.48) or confidence to function as a first year resident (Spearman ρ P =.43). Conclusions Based on responses to this survey, rotating students perceive that the majority of radiation oncology clerkships do not have formal didactic curricula. Survey respondents who completed a clerkship with a didactic curriculum reported feeling more prepared to function as a radiation oncology resident. However, completing an increasing number of clerkships does not appear to improve confidence in the decision to pursue radiation oncology as a career or to function as a radiation oncology resident. These results support further development of structured didactic curricula for the radiation oncology clerkship.

Occludin loss enhances paracellular macromolecular permeability (radius up to ∼62.5 Å) and is necessary for TNF-induced barrier loss. The latter requires the C-terminal OCEL domain, which stabilizes tight junction–associated occludin and regulates trafficking. Thus OCEL-mediated interactions are critical regulators of macromolecular paracellular flux., In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ∼62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)–deficient enhanced green fluorescent protein (EGFP)–occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludinΔOCEL. Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1–binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK–binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.

The cellular reaction to genomic instability includes a network of signal transduction pathways collectively referred to as the DNA damage response (DDR). Activated by a variety of DNA lesions, the DDR orchestrates cell cycle arrest and DNA repair, and initiates apoptosis in instances where damage cannot be repaired. As such, disruption of the DDR increases the prevalence of DNA damage secondary to incomplete repair, and in doing so, enhances radiation-induced cytotoxicity. This article describes the molecular agents and their targets within DDR pathways that sensitize cells to radiation. Moreover, it reviews the therapeutic implications of these compounds, provides an overview of relevant clinical trials and offers a viewpoint on the evolution of the field in the years to come.

Genomic damage triggers a signal transduction network that modulates gene expression and protein stability to repair DNA and influence cell survival. Inhibition of the DNA damage response protein PARP-1 is the subject of

microRNAs (miRNAs) are endogenous short RNAs that mediate vast networks of post-transcriptional gene regulation. Although computational searches and experimental profiling provide evidence for hundreds of functional targets for individual miRNAs, such data rarely provide clear insight into the phenotypic consequences of manipulating miRNAs in vivo. We describe a genome-wide collection of 165 Drosophila miRNA transgenes and find that a majority induced specific developmental defects, including phenocopies of mutants in myriad cell-signaling and patterning genes. Such connections allowed us to validate several likely targets for miRNA-induced phenotypes. Importantly, few of these phenotypes could be predicted from computationally predicted target lists, thus highlighting the value of whole-animal readouts of miRNA activities. Finally, we provide an example of the relevance of these data to miRNA loss-of-function conditions. Whereas misexpression of several K box miRNAs inhibited Notch pathway activity, reciprocal genetic interaction tests with miRNA sponges demonstrated endogenous roles of the K box miRNA family in restricting Notch signaling. In summary, we provide extensive evidence that misexpression of individual miRNAs often induces specific mutant phenotypes that can guide their functional study. By extension, these data suggest that the deregulation of individual miRNAs in other animals may frequently yield relatively specific phenotypes during disease conditions.

Fourth-year medical students (MS4s) participate in clerkships to familiarize themselves with the field of radiation oncology and to evaluate and audition for residency programs. However, MS4s’ perceptions of their radiation oncology clerkship experiences have never been formally evaluated. This study aimed to characterize the MS4 perception of the radiation oncology clerkship experience from the current nationwide pool of applicants.

Purpose/Objective(s): Pituitary tumors are relatively uncommon. Primary goals of treatment include normalization of hormonal level and improving visual or neurological symptoms. Surgery, medical treatment, irradiation, or combination of these modalities is used to achieve these goals. The purpose of this analysis is to determine the clinical outcome of pituitary adenoma treated with fractionated external beam radiation therapy. Materials/Methods: This is a retrospective analysis of pituitary adenoma treated at our center between 2006 and 2014. A total of 88 patients with biopsy-proven adenomas were identified. Median age at diagnosis was 38 years (18e79 years) and 49 patients (56%) were males. Majority of tumors were marcoadenoma (87%). Presenting symptoms were mainly hormonal disturbance in 43 patients (49%), visual defects in 29 patients (33%) and headache in 21 patients (23%). Prolactinomas were the most common secretory adenoma in 22 patients (51%), followed by growth hormone secreting adenomas in 15 patients (35%) and ACTH-secreting tumors in 5 patients (12%), while one patient had TSH-secreting adenoma. The cohort follow up time reached 87 months (mean; 32 months). The primary endpoints are clinical and biochemical control. Results: Thirteen patients lost to follow up, therefore 75 patients were included in the final analysis. Surgery was the primary treatment modality in 70 patients (93%).While medical treatment was used for inoperable or hormonal secreting adenoma in 30 patients (40%). Fractionated radiation therapy was delivered to 28 patients (37%), indications for radiation were symptomatic, incompletely resected tumors (16 patients) or persistent hypersecretion (12 patients). For secretory tumors the median radiation dose was 52Gy (50-54Gy), and lower dose was delivered for non-secretory adenoma; mean 50Gy (45-52Gy). For patients received radiation therapy, the tumor control rate is 75% for non-functioning adenomas, and 69% for functioning adenomas. Conclusion: Pituitary adenoma is a slow growing benign tumor; however devastating effect can result from neurological deficits or hormonal imbalance. Our results reveal that functioning adenomas carry worse prognosis. Fractionated radiation therapy, as an adjunct to surgery and medical treatment, offers good clinical and biochemical control for pituitary adenoma. More aggressive multimodal approach and incorporation of new radiation technique may help to achieve better clinical outcome. Author Disclosure: R.A. Abu-Hijlih: None. Y. Ismael: None. H. Ghatasheh: None. A. Fawzi: None. A. Alsharbaji: None. M. Elyan: None. B. Al-Haj: None. A. Almousa: None.

The functional significance of the signaling pathway induced by O(6)-methylguanine (O(6)-MeG) lesions is poorly understood. Here, we identify the p50 subunit of NF-κB as a central target in the response to O(6)-MeG and demonstrate that p50 is required for S(N)1-methylator-induced cytotoxicity. In response to S(N)1-methylation, p50 facilitates the inhibition of NF-κB-regulated antiapoptotic gene expression. Inhibition of NF-κB activity is noted to be an S phase-specific phenomenon that requires the formation of O(6)-MeG:T mismatches. Chk1 associates with p50 following S(N)1-methylation, and phosphorylation of p50 by Chk1 results in the inhibition of NF-κB DNA binding. Expression of an unphosphorylatable p50 mutant blocks inhibition of NF-κB-regulated antiapoptotic gene expression and attenuates S(N)1-methylator-induced cytotoxicity. While O(6)-MeG:T-induced, p50-dependent signaling is not sufficient to induce cell death, this pathway sensitizes cells to the cytotoxic effects of DNA breaks.

Tissue barriers that restrict passage of liquids, ions, and larger solutes are essential for the development of multicellular organisms. In simple organisms this allows distinct cell types to interface with the external environment. In more complex species, the diversity of cell types capable of forming barriers increases dramatically. Although the plasma membranes of these barrier-forming cells prevent flux of most hydrophilic solutes, the paracellular, or shunt, pathway between cells must also be sealed. This function is accomplished in vertebrates by the zonula occludens, or tight junction. The tight junction barrier is not absolute but is selectively permeable and is able to discriminate between solutes on the basis of size and charge. Many tight junction components have been identified over the past 20 years, and recent progress has provided new insights into the proteins and interactions that regulate structure and function. This review presents these data in a historical context and proposes an integrated model in which dynamic regulation of tight junction protein interactions determines barrier function.

The perijunctional actomyosin ring contributes to myosin light chain kinase (MLCK)-dependent tight junction regulation. However, the specific protein interactions involved in this process are unknown. To test the hypothesis that molecular remodeling contributes to barrier regulation, tight junction protein dynamic behavior was assessed by fluorescence recovery after photobleaching (FRAP). MLCK inhibition increased barrier function and stabilized ZO-1 at the tight junction but did not affect claudin-1, occludin, or actin exchange in vitro. Pharmacologic MLCK inhibition also blocked in vivo ZO-1 exchange in wild-type, but not long MLCK −/− , mice. Conversely, ZO-1 exchange was accelerated in transgenic mice expressing constitutively active MLCK. In vitro, ZO-1 lacking the actin binding region (ABR) was not stabilized by MLCK inhibition, either in the presence or absence of endogenous ZO-1. Moreover, the free ABR interfered with full-length ZO-1 exchange and reduced basal barrier function. The free ABR also prevented increases in barrier function following MLCK inhibition in a manner that required endogenous ZO-1 expression. In silico modeling of the FRAP data suggests that tight junction-associated ZO-1 exists in three pools, two of which exchange with cytosolic ZO-1. Transport of the ABR-anchored exchangeable pool is regulated by MLCK. These data demonstrate a critical role for the ZO-1 ABR in barrier function and suggest that MLCK-dependent ZO-1 exchange is essential to this mechanism of barrier regulation.

Intestinal barrier function is reduced in inflammatory bowel disease (IBD). Tumor necrosis factor (TNF) and interleukin (IL)-13, which are up-regulated in IBD, induce barrier defects that are associated with myosin light chain kinase (MLCK) activation and increased claudin-2 expression, respectively, in cultured intestinal epithelial monolayers. Here we report that these independent signaling pathways have distinct effects on tight junction barrier properties and interact in vivo. MLCK activation alters size selectivity to enhance paracellular flux of uncharged macromolecules without affecting charge selectivity and can be rapidly reversed by MLCK inhibition. In contrast, IL-13-dependent claudin-2 expression increases paracellular cation flux in vitro and in vivo without altering tight junction size selectivity but is unaffected by MLCK inhibition in vitro. In vivo, MLCK activation increases paracellular flux of uncharged macromolecules and also triggers IL-13 expression, claudin-2 synthesis, and increased paracellular cation flux. We conclude that reversible, MLCK-dependent permeability increases cause mucosal immune activation that, in turn, feeds back on the tight junction to establish long-lasting barrier defects. Interactions between these otherwise distinct tight junction regulatory pathways may contribute to IBD pathogenesis.

Purpose/Objective(s): To report the long-term clinical outcomes of patients with high-risk adenocarcinoma of the prostate treated with pelvic radiation therapy followed by high-dose-rate (HDR) brachytherapy boost. Materials/Methods: 115 consecutive patients with high-risk prostate cancer ( cT3, Gleason (GS) 8-10, or PSA 20) treated between July 1997 and November 2005 were included in this study. All patients underwent whole pelvic radiation therapy to 45 Gy followed by HDR brachytherapy boost. HDR brachytherapy boost consisted of 6 Gy x 3 (38 patients) or 9.5 Gy x 2 (77 patients) to the prostate and seminal vesicles. 47%, 48%, and 5 % patients received long-term (>18 mos), short-term (4-6 mos), or no androgen deprivation therapy, respectively. Results: The median age at diagnosis was 64.8 yrs. 50 (43%), 90 (78.2%), and 24 (20.8%) patients were diagnosed with GS 8-10, cT3 disease, and PSA 20, respectively. Mean PSA was 14.94 (range 0.21-99.3). 21 patients (18.2%) had seminal vesicle invasion and 42 patients (37%) had at least 2 high-risk features. With a median follow-up time of 94 months, biochemical free survival as defined by the Phoenix definition was 78.2%. 4 patients (3.5%) failed locally as determined by biopsy without metastatic disease. Overall survival and disease specific survival were 84.3% and 95.7%, respectively. Six secondary malignancies (bladder 1 year posttreatment, colon, lung, melanoma, hepatocellular carcinoma) developed. One patient developed grade 3 ureteral stricture causing hydronephrosis and required stent placement. There were no other grade 3 or greater genitourinary or gastrointestinal toxicity. Conclusions: Pelvic radiation therapy followed by HDR brachytherapy boost is an effective treatment for high-risk prostate cancer with excellent long-term disease control and minimal toxicity. Author Disclosure: A.J. Chang: None. M. Roach: None. A. Gottschalk: None. A.J. Cunha: None. Z.A. Seymour: None. J.A. Johnson: None. D. Raleigh: None. K. Shinohara: None. I.J. Hsu: None.

and 6 hr of NFK-b, Nrf2 and TGF-b. MnSOD tet/tet cells, maintained in DOX, did show at 6 hr after irradiation a 50% increase in expression of Nrf2. Conclusions: MnSOD tet/tet cell lines are radiosensitive, as reflected in decreased irradiation induced gene expression which is only partially reversed by DOX. These novel mice and derived cell lines should prove valuable for understanding the link between antioxidant levels and irradiation responsive genes mediating both acute and chronic normal tissue damage effects. Acknowledgements: This project was supported by NIAID U191A168021. Author Disclosure: R. Kalash: None. J.R. Chaillet: None. F. Houghton: None. X. Zhang: None. M. Epperly: None. S. Cao: None. J.S. Greenberger: None.

Background: O6-methylguanine (O6-MeG) is the primary cytotoxic lesion formed by SN1-methylating agents such as temozolomide. Cytotoxicity is induced following the mispairing of O6-MeG with deoxythymidine (T) residues and the subsequent activation of a mismatch repair (MMR)-mediated process. While it is generally recognized that MMR-induced DNA double strand breaks (DSBs) are central to O6-MeG-induced cell death, our previous work demonstrates that signaling via the p50 subunit of nuclear factor-κB (NF-κB) is also necessary for efficient SN1-methylation-induced cytotoxicity. In the current study, we isolated O6-MeG:T mismatches using a series of oligonucleotide duplexes and examined the hypothesis that O6-MeG:T mismatches induce a p50-dependent signaling pathway that sensitizes cells to DNA strand break-mediated cell death. Methods: A series of 40bp oligonucleotide duplex substrates bearing a unique central O6-MeG:T, G:T, O6-MeG:C, or G:C base pair were used. Cell lines included both human glioma cells and mouse embryonic fibroblasts. Loss of function was performed with shRNA-mediated p50 knockdown and targeted p50 knockout. Techniques used include EMSA, NF-κB-dependent luciferase reporter assays, in-vitro kinase assay, indirect immunofluorescence, annexin V binding and colony formation assays. Results: When administered to nuclear extracts, O6-MeG:T substrate inhibits NF-κB DNA binding substantially more than control duplex, and, when used in live cells, inhibition of NF-kB-dependent activity is seen only in response to O6-MeG:T substrate. The inhibition of NF-kB by O6-MeG:T substrate is noted only in p50-proficient cells. O6-MeG:T, but not control substrate, induces Chk1-mediated phosphorylation of p50 at a novel serine residue, a pathway that is shown to be necessary for inhibition of NF-kB DNA binding. O6-MeG:T substrate, used at a concentration sufficient to induce MMR- and p50-mediated signaling, does not increase apoptosis or decrease clonogenic survival relative to control duplexes. However, O6-MeG:T, but not control duplex, sensitizes p50-proficient, but not p50-deficent, cells to non-cytotoxic doses of ionizing radiation. O6-MeG:T-induced sensitization to cytotoxicity occurs without changing the level of IR-induced DSB and is only seen when duplex substrate is given prior to, but not after, IR. Conclusion: While an O6-MeG-induced signaling pathway has been previously described, the role of such a pathway to the damage response is disputed. The above findings suggest that O6-MeG:T-induced, p50-dependent signaling sensitizes cells to cytotoxicity that is ultimately induced by DNA DSBs. Such a model is temporally consistent with the response to O6-MeG in that MMR-induced signaling is induced after the first S-phase following O6-MeG formation while DSBs are formed after the second S-phase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 262. doi:1538-7445.AM2012-262

SN1-methylating agents form O6-methylguanine (O6-MeG) lesions that induce apoptosis following mis-pairing with thymine (T) and activation of the mismatch repair (MMR) system. Previous work has shown that O6-MeG inhibits the activity of the transcription factor, nuclear factor-κB (NF-κB), by a mechanism involving MMR-dependent inhibition of NF-κB DNA-binding. In the current study, we set out to determine the mechanism by which methylating agents block NF-κB as a prerequisite to understanding the role of NF-κB in the response to O6-MeG. We hypothesize that a single NF-κB subunit is the target that is essential for mediating both inhibition of NF-κB and cytotoxicity in response to methylation damage. Methods: Temozolomide and methyl-nitrosourea were used as model methylators and both human glioma cells and mouse embryonic fibroblasts were used. Loss of function was performed with siRNA-mediated NF-κB knockdown and targeted NF-κB subunit knockout. Techniques used include EMSA, ChIP assay, NF-κB- and Gal4-dependent luciferase reporter assays, quantitative real time-PCR, single cell gel electrophoresis (comet) assay, flow cytometric analysis of annexin V binding, MTS short term cell killing assay and long-term colony formation assay. Results: The NF-κB subunits affected by SN1-methylators include p65 and p50 suggesting that one or both of these subunits mediates the response to SN1-methylation. Studies demonstrate that while p65 is dispensable for methylation-induced inhibition of NF-κB, p50 is required for inhibition of NF-κB. Furthermore, p50 is necessary for methylation-induced inhibition of endogenous NF-κB-dependent anti-apoptotic gene expression. The specific role of O6-MeG: T mismatches to inhibition of NF-κB was confirmed using oligonucleotide duplexes bearing a central O6-MeG: T, G: T, O6-MeG: C, or G: C base pair and observing that only O6-MeG: T mismatches inhibit NF-κB, in a p50-dependent manner. Because of the importance O6-MeG and MMR to inhibition of NF-κB, the requirement of p50 for methylator-induced cytotoxicity was examined. p50-deficient cells are resistant to methylation-induced apoptosis and also show increased clonal viability following methylator treatment compared to wild-type (wt) cells. The resistance to killing is specific to MMR-mediated cytotoxicity as p50 deficient cells are as sensitive as wt cells to ionizing radiation and doxorubicin. Importantly, there is equivalent damage induction following methylator treatment in p50 deficient cells relative to wt cells as assessed by comet assay. The specificity of p50 to methylation-induced killing was further confirmed by demonstrating that deletion of the p65 subunit increases methylation-induced cytotoxicity. Conclusion: The p50 NF-κB subunit acts downstream of MMR to enable apoptosis in response to SN1-methylation damage. This study sets the groundwork for evaluation of a novel intermediate, p50, in the MMR-directed cytotoxic response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3659.