Medulloblastomas overexpress the p53-inactivating oncogene WIP1/PPM1D.

Medulloblastoma is the most common malignant brain tumor of childhood. Despite numerous advances, clinical challenges range from recurrent and progressive disease to long-term toxicities in survivors. The lack of more effective, less toxic therapies results from our limited understanding of medulloblastoma growth. Although TP53 is the most commonly altered gene in cancers, it is rarely mutated in medulloblastoma. Accumulating evidence, however, indicates that TP53 pathways are disrupted in medulloblastoma. Wild-type p53-induced phosphatase 1 (WIP1 or PPM1D) encodes a negative regulator of p53. WIP1 amplification (17q22-q23) and its overexpression have been reported in diverse cancer types. We examined primary medulloblastoma specimens and cell lines, and detected WIP1 copy gain and amplification prevalent among but not exclusively in the tumors with 17q gain and isochromosome 17q (i17q), which are among the most common cytogenetic lesions in medulloblastoma. WIP1 RNA levels were significantly higher in the tumors with 17q gain or i17q. Immunoblots confirmed significant WIP1 protein in primary tumors, generally higher in those with 17q gain or i17q. Under basal growth conditions and in response to the chemotherapeutic agent, etoposide, WIP1 antagonized p53-mediated apoptosis in medulloblastoma cell lines. These results indicate that medulloblastoma express significant levels of WIP1 that modulate genotoxic responsiveness by negatively regulating p53.

Computer simulation of glioma growth and morphology.

Despite major advances in the study of glioma, the quantitative links between intra-tumor molecular/cellular properties, clinically observable properties such as morphology, and critical tumor behaviors such as growth and invasiveness remain unclear, hampering more effective coupling of tumor physical characteristics with implications for prognosis and therapy. Although molecular biology, histopathology, and radiological imaging are employed in this endeavor, studies are severely challenged by the multitude of different physical scales involved in tumor growth, i.e., from molecular nanoscale to cell microscale and finally to tissue centimeter scale. Consequently, it is often difficult to determine the underlying dynamics across dimensions. New techniques are needed to tackle these issues. Here, we address this multi-scalar problem by employing a novel predictive three-dimensional mathematical and computational model based on first-principle equations (conservation laws of physics) that describe mathematically the diffusion of cell substrates and other processes determining tumor mass growth and invasion. The model uses conserved variables to represent known determinants of glioma behavior, e.g., cell density and oxygen concentration, as well as biological functional relationships and parameters linking phenomena at different scales whose specific forms and values are hypothesized and calculated based on in vitro and in vivo experiments and from histopathology of tissue specimens from human gliomas. This model enables correlation of glioma morphology to tumor growth by quantifying interdependence of tumor mass on the microenvironment (e.g., hypoxia, tissue disruption) and on the cellular phenotypes (e.g., mitosis and apoptosis rates, cell adhesion strength). Once functional relationships between variables and associated parameter values have been informed, e.g., from histopathology or intra-operative analysis, this model can be used for disease diagnosis/prognosis, hypothesis testing, and to guide surgery and therapy. In particular, this tool identifies and quantifies the effects of vascularization and other cell-scale glioma morphological characteristics as predictors of tumor-scale growth and invasion.

LOSS OF GPRC5A ENHANCES SURVIVAL IN NORMAL AND MALIGNANT LUNG EPITHELIAL CELLS BY ELICITING PERSISTENT STAT3 ACTIVATION INDUCED BY AUTOCRINE LIF

Signal transduction and activator of transcription 3 (Stat3) is activated by cytokines and growth factors in many cancers. Persistent activation of Stat3 plays important role in cell growth, survival, and transformation through regulating its targeted genes. Previously, we found that mice with a deletion of the G protein-coupled receptor, family C, group 5, member a (Gprc5a) gene develop lung tumors indicating that Gprc5a is a tumor suppressor. In the present study, we examined he mechanism of Gprc5a-mediated tumor suppression. We found that epithelial cells from Gprc5a knockout mouse lung (Gprc5a-/- cells) survive better in vitro in medium deprived of exogenous growth factors and form more colonies in semi-solid medium than their counterparts from wildtype mice (Gprc5a+/+ cells). The phosphorylation of tyrosine 705 on Stat3 and the expression of Stat3-regulated anti-apoptotic genes Bcl-XL, Cryab, Hapa1a, and Mcl1 were higher in the Gprc5a-/- than in Gprc5a+/+ cells. In addition, their responses to Lif were different; Stat3 activation was persistent by Lif treatment in the Gprc5a-/- cells, but was transient in the Gprc5a+/+ cells. The persistent activation of Stat3 by Lif in Gprc5a-/- cells is due to a decreased level of Socs3 protein, a negative inhibitor of the Lif-Stat3 signaling. Restoration of Socs3 inhibited the persistent Stat3 activation in Gprc5a-/- cells. Lung adenocarcinoma cells isolated from Gprc5a-/- mice also exhibited autocrine Lif-mediated Stat3 activation. Treatment of Gprc5a-/- cells isolated from normal and tumor tissue with AG490, a Stat3 signaling inhibitor, or with dominant negative Stat3(Y705F) increased starvation-induced apoptosis and inhibited anchorage-independent growth. These results suggest that persistent Stat3 activation increased the survival and transformation of Gprc5a-/- lung cells. Thus, the tumor suppressive effects of Gprc5a are mediated, at least in part, by inhibition of Stat3 signaling through regulating the stability of the Socs3 protein.

TEMOZOLOMIDE AND BEVACIZUMAB THERAPY IN ADVANCED HEMANGIOPERICYTOMA/ SOLITARY FIBROUS TUMOR

Tumors comprising the spectrum of hemangiopericytoma/ malignant solitary fibrous tumor (HPC/SFT) are thought to arise from fibroblasts and represent a small subset of soft tissue sarcomas. Surgery is typically the treatment of choice for localized disease, with reported 10-year overall survival rates of 54-89% after complete surgical resection. However, for the approximately 20% of HPC/SFT patients who eventually develop local recurrences and/or distant metastases, options for effective treatment are limited and are poorly defined. Alternative therapeutic options are therefore needed for improved palliation and disease control. We hypothesize that HPC/SFT are a spectrum of soft tissue tumors with unique clinical, pathological, and molecular makeup and clinical behavior. HPC/SFT respond to unique therapeutic agents that specifically target aberrations specific to these tumors. We retrospectively reviewed the characteristics and the clinical outcomes for all HPC/SFT patients whose tumor specimens have been reviewed at the MD Anderson Cancer Center from January 1993 to June 2007 by a MD Anderson pathologist and were treated at the institution with available electronic medical records. We identified 128 patients, 79 with primary localized disease and 49 with recurrent and/or metastatic disease. For the 23 patients with advanced HPC/SFT who received adriamycin-based, gemcitabine based, or paclitaxel chemotherapy as first- or second-line therapy, the overall RECIST response rate was 0%. Most patients achieved a brief duration of disease stabilization on chemotherapy, with median progression-free survival (PFS) period of 4.6 months. For the 14 patients with advanced HPC/SFT who received temozolomide and bevacizumab systemic therapy, the overall RECIST response rate was 14%, with the overall Choi response rate of 79%. The median PFS for the cohort was 9.7 months with a median 6-month progression free rate of 78.6%. The most frequently observed toxic effect of temzolomide-bevacizumab therapy was myelosuppression. We have designed a phase II study to evaluate the safety and efficacy of temozolomide-bevaciumab in locally advanced, recurrent, and metastatic HPC/SFT in a prospective manner. Combination therapy with temozolomide and bevacizumab may be a potentially clinically beneficial regimen for advanced HPC/SFT patients.

Risk of Second Malignant Neoplasms Following Proton Arc Therapy and Volumetric Modulated Arc Therapy for Prostate Cancer

The risk of second malignant neoplasms (SMNs) following prostate radiotherapy is a concern due to the large population of survivors and decreasing age at diagnosis. It is known that parallel-opposed beam proton therapy carries a lower risk than photon IMRT. However, a comparison of SMN risk following proton and photon arc therapies has not previously been reported. The purpose of this study was to predict the ratio of excess relative risk (RRR) of SMN incidence following proton arc therapy to that after volumetric modulated arc therapy (VMAT). Additionally, we investigated the impact of margin size and the effect of risk-minimized proton beam weighting on predicted RRR. Physician-approved treatment plans were created for both modalities for three patients. Therapeutic dose was obtained with differential dose-volume histograms from the treatment planning system, and stray dose was estimated from the literature or calculated with Monte Carlo simulations. Then, various risk models were applied to the total dose. Additional treatment plans were also investigated with varying margin size and risk-minimized proton beam weighting. The mean RRR ranged from 0.74 to 0.99, depending on risk model. The additional treatment plans revealed that the RRR remained approximately constant with varying margin size, and that the predicted RRR was reduced by 12% using a risk-minimized proton arc therapy planning technique. In conclusion, proton arc therapy was found to provide an advantage over VMAT in regard to predicted risk of SMN following prostate radiotherapy. This advantage was independent of margin size and was amplified with risk-optimized proton beam weighting.

FoxM1B transcriptionally regulates vascular endothelial growth factor expression and promotes the angiogenesis and growth of glioma cells.

We previously found that FoxM1B is overexpressed in human glioblastomas and that forced FoxM1B expression in anaplastic astrocytoma cells leads to the formation of highly angiogenic glioblastoma in nude mice. However, the molecular mechanisms by which FoxM1B enhances glioma angiogenesis are currently unknown. In this study, we found that vascular endothelial growth factor (VEGF) is a direct transcriptional target of FoxM1B. FoxM1B overexpression increased VEGF expression, whereas blockade of FoxM1 expression suppressed VEGF expression in glioma cells. Transfection of FoxM1 into glioma cells directly activated the VEGF promoter, and inhibition of FoxM1 expression by FoxM1 siRNA suppressed VEGF promoter activation. We identified two FoxM1-binding sites in the VEGF promoter that specifically bound to the FoxM1 protein. Mutation of these FoxM1-binding sites significantly attenuated VEGF promoter activity. Furthermore, FoxM1 overexpression increased and inhibition of FoxM1 expression suppressed the angiogenic ability of glioma cells. Finally, an immunohistochemical analysis of 59 human glioblastoma specimens also showed a significant correlation between FoxM1 overexpression and elevated VEGF expression. Our findings provide both clinical and mechanistic evidence that FoxM1 contributes to glioma progression by enhancing VEGF gene transcription and thus tumor angiogenesis.

Robotic-assisted laparoscopic "salvage" rectopexy for recurrent ileoanal J-pouch prolapse.

Total restorative proctocolectomy with ileal pouch-anal anastomosis (RP/IPAA) has become the standard of care for the surgical treatment of ulcerative colitis. Despite its correlation with an excellent quality of life and favorable long-term outcomes, RP/IPAA has been associated with several complications. Prolapse of the ileoanal pouch is a rare and debilitating complication that should be considered in the differential diagnosis of pouch failure. Limited data exist regarding the prevalence and treatment of pouch prolapse. We present the case of a recurrent J-pouch prolapse treated with a novel minimally invasive "salvage" approach involving a robotic-assisted laparoscopic rectopexy with mesh.

Malignant solitary fibrous tumor involving the liver

Solitary fibrous tumors are predominantly benign and are most commonly found in the thoracic cavity and pleura; while reports exist in the literature of malignant solitary fibrous tumors and those located in extrathoracic organs, these cases are considered extremely rare. Herein, a case is reported of a malignant solitary fibrous tumor involving the liver that was diagnosed and treated in a 62-year-old woman. The patient presented with complaints of upper abdominal pain and unintentional weight loss. Computed tomography scan of the abdomen revealed a remarkably large mass, measuring 15 cm × 10 cm × 20 cm, which appeared to be unrelated to any particular organ. The intraoperative finding of a wide communication with the left liver suggested hepatic origin, and served as an indicator for tumor resection via left hemihepatectomy. The diagnosis of solitary fibrous tumor and its malignant nature was confirmed by histological and immunohistochemical examination of the resected tissues. Hepatic solitary fibrous tumor is very rare, and surgery remains the mainstay of treatment. Due to limited reports of such tumors in the literature, little can be said about the benefit of adjuvant therapy and prognosis for the rare cases with malignant histological findings.

Analysis of differential gene expression in nontumorigenic glioblastoma multiforme

Loss of chromosome 10 represents the most common cytogenetic abnormality in high grade gliomas (glioblastoma multiforme). To identify genes involved in the malignant progression of human gliomas, a subtractive hybridization was performed between a tumorigenic glioblastoma cell line (LG11) and a nontumorgenic hybrid cell (LG11.3) containing an introduced chromosome 10. LG11 mRNA was subtracted from LG11.3 cDNA to produce cDNA probes enriched for sequences whose expression differs quantitatively from the parental tumorigenic cells. Both known and novel sequences were identified as a result of the subtraction. Northern blot analysis was then used to confirm differential expression of several subtracted clones. One novel clone, clone 17, identified a 2.6 kb message that showed a consistent two to four fold increase in expression in the LG11.3 nontumorigenic cells. Clone 17 (340 bp) was used successfully to screen for a near full-length version, RIG (regulated in glioma), which was 2,569 bp in size. The RIG cDNA sequence showed homology to clone 17 and to an anonymous EST (IB666), but to no previously identified genes. This screening effort also identified several independent clones representing novel sequences, most of which failed to show increased expression in the nontumorigenic GBM cells. Tissue distribution studies of RIG indicated highest levels of expression in human brain with appreciably lower levels in heart and lung. In vitro transcription and translation experiments demonstrated the ability of RIG to direct the synthesis of a 13 kD protein product. However, open reading frame analysis revealed no identify with previously described motifs or any known proteins. Using a combination of somatic cell hybrid panels and in situ hybridization, the RIG gene was mapped to chromosome 11p14-11p15. Further study of RIG and related gene products may provide insight into the negative regulation of glial oncogenesis. ^

Expression, induction and regulation of the cytochrome P450 monooxygenase system in the rat glioma C6 cell line

The cytochrome P450 monooxygenase system consists of NADPH- cytochrome P450 reductase (P450 reductase) and cytochromes P450, which can catalyze the oxidation of a wide variety of endogenous and exogenous compounds, including steroid hormones, fatty acids, drugs, and pollutants. The functions of this system are as diverse as the substrates. P450 reductase transfers reducing equivalents from NADPH to P450, which in turn catalyzes metabolic reactions. This enzyme system has the highest level of activity in the liver. It is also present in other tissues, including brain. The functions of this enzyme system in brain seem to include: neurotransmission, neuroendocrinology, developmental and behavioral modulation, regulation of intracellular levels of cholesterol, and potential neurotoxicity.^ In this study, we have set up the rat glioma C6 cell line as an in vitro model system to examine the expression, induction, and tissue-specific regulation of P450s and P450 reductase. Rat glioma C6 cells were treated with P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). The presence of P450 reductase and of cytochrome P450 1A1, 1A2, 2A1, 2B1/2, 2C7, 2D1-5 and 2E1 was detected by reverse transcription followed by polymerase chain reaction (RT-PCR) and confirmed by restriction digestion. The induction of P450 1A1 and 2B1/2 and P450 reductase was quantified using competitive PCR. Ten- and five-fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected. Western blot analysis of microsomal preparations of glioma C6 cells demonstrated the presence of P450 1A, 2B and P450 reductase at the protein level. ELISAs showed that BA and PB induce P450 1A and 2B proteins 7.3- and 13.5-fold, respectively. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 CO difference spectra with absorption at or near 450 nm. Microsomes prepared from rat glioma C6 cells demonstrated much higher levels of ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activity, when treated with BA or PB, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active cytochrome P450 monooxygenase system which can be induced by P450 inducers. The mRNAs of P450 1A1 and 2B1/2 can not bind to the oligo(dT) column efficiently, indicating they have very short poly(A) tails. This finding leads us to study the tissue specific regulation of P450s at post-transcriptional level. The half lives of P450 1A1 and 2B1/2 mRNA in glioma C6 cells are only 1/10 and 1/3 of that in liver. This may partly contribute to the low expression level of P450s in glial cells. The induction of P450s by BA or PB did not change their mRNA half lives, indicating the induction may be due to transcriptional regulation. In summary of this study, we believe the presence of the cytochrome P450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors. ^

The equine DNAH3 gene: SNP discovery and exclusion of an involvement in recurrent airway obstruction (RAO) in European Warmblood horses

Recurrent airway obstruction is one of the most common airway diseases affecting mature horses. Increased bronchoalveolar mucus, neutrophil accumulation in airways, and airway obstruction are the main features of this disease. Mucociliary clearance is a key component of pulmonary defense mechanisms. Cilia are the motile part of this system and a complex linear array of dynein motors is responsible for their motility by moving along the microtubules in the axonemes of cilia and flagella. We previously detected a QTL for RAO on ECA 13 in a half-sib family of European Warmblood horses. The gene encoding DNAH3 is located in the peak of the detected QTL and encodes a dynein subunit. Therefore, we analysed this gene as a positional and functional candidate gene for RAO. In a mutation analysis of all 62 exons we detected 53 new polymorphisms including 7 non-synonymous variants. We performed an association study using 38 polymorphisms in a cohort of 422 animals. However, after correction for multiple testing we did not detect a significant association of any of these polymorphisms with RAO (P>0.05). Therefore, it seems unlikely that variants at the DNAH3 gene are responsible for the RAO QTL in European Warmblood horses.

Anatomic pathways of peripancreatic fluid draining to mediastinum in recurrent acute pancreatitis: visible human project and CT study

BACKGROUND In past reports, researchers have seldom attached importance to achievements in transforming digital anatomy to radiological diagnosis. However, investigators have been able to illustrate communication relationships in the retroperitoneal space by drawing potential routes in computerized tomography (CT) images or a virtual anatomical atlas. We established a new imaging anatomy research method for comparisons of the communication relationships of the retroperitoneal space in combination with the Visible Human Project and CT images. Specifically, the anatomic pathways of peripancreatic fluid extension to the mediastinum that may potentially transform into fistulas were studied. METHODS We explored potential pathways to the mediastinum based on American and Chinese Visible Human Project datasets. These drainage pathways to the mediastinum were confirmed or corrected in CT images of 51 patients with recurrent acute pancreatitis in 2011. We also investigated whether additional routes to the mediastinum were displayed in CT images that were not in Visible Human Project images. PRINCIPAL FINDINGS All hypothesized routes to the mediastinum displayed in Visible Human Project images, except for routes from the retromesenteric plane to the bilateral retrorenal plane across the bilateral fascial trifurcation and further to the retrocrural space via the aortic hiatus, were confirmed in CT images. In addition, route 13 via the narrow space between the left costal and crural diaphragm into the retrocrural space was demonstrated for the first time in CT images. CONCLUSION This type of exploration model related to imaging anatomy may be used to support research on the communication relationships of abdominal spaces, mediastinal spaces, cervical fascial spaces and other areas of the body.