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Background The prognosis of patients with diffusely infiltrating gliomas is dismal but varies greatly between individuals. While characterization of gliomas was primarily relied on typical histopathological features, specific molecular markers increasingly gained importance and play a key role in the recently published 5th edition of the World Health Organization (WHO) classification. Heme biosynthesis represents a crucial pathway due to its key role in oxygen transport, energy production or drug metabolism. Recently, we described a “heme biosynthesis mRNA expression signature” that correlates with histopathological glioma grades and patient survival. The aim of the current study was to correlate the heme biosynthesis mRNA expression signature with the most recent diagnostic molecular markers for glioma stratification. Material and Methods In this study, patient data were derived from the “The Cancer Genome Atlas” (TCGA) lower-grade glioma and glioblastoma cohorts. We identified diffusely infiltrating gliomas correlating molecular tumor diagnosis according to the most recent WHO classification with heme biosynthesis mRNA expression. The following molecular markers were analyzed: EGFR amplification, TERT promoter mutation, CDKN2A/B homozygous loss, concurrent chromosome 7 gain/10 loss, MGMT methylation, IDH mutation, ATRX loss, p53 mutation and 1p19q co-deletion. Subsequently, we calculated the heme biosynthesis mRNA expression signature and correlated this signature with distinct molecular glioma markers as well as the resulting molecular subgroups. Results A total of 649 patients with available data on up-to-date molecular markers and heme biosynthesis mRNA expression were included. According to analysis of individual molecular markers, we found a significantly higher heme biosynthesis mRNA expression signature in gliomas with IDH wildtype (p Conclusion Our data demonstrate a significant correlation between diagnostic molecular markers and heme biosynthesis regulation in diffusely infiltrating gliomas. Consequently, heme biosynthesis expression is a promising biomarker for glioma aggressiveness and might constitute a potential target for novel therapeutic approaches.

Background The prospects of a patient with suspected glioblastoma may rely heavily on the indication for surgical resection versus biopsy only. Biopsy percentages vary considerably across hospitals and guidelines for treatment of glioblastoma lack criteria for surgical decision-making. To identify patient and tumor characteristics associated with the decision to resect or biopsy a glioblastoma and to develop and validate a prediction model for decision support. Material and Methods Clinical data and pre-operative MRI scans were collected for adults who underwent first-time surgery for supratentorial glioblastoma from a registry-based cohort study of 12 hospitals from the Netherlands, Germany, France, Italy, and the United States between 1st of January 2007 and 31st of December 2011. The main outcome was the type of surgical procedure: surgical resection or biopsy only. Predictors were patient- and tumor-related characteristics. Radiological factors were extracted from MRI using an automated tumor segmentation method. A prediction model was constructed using multivariable logistic regression analysis. The model was cross-validated and externally validated with a leave-one-hospital-out approach. Results Out of 1053 patients treated for glioblastoma, 28% underwent biopsy only. Biopsy rates varied from 15-40% across hospitals. The prediction model showed excellent discrimination with an average area under the curve of 0.86. Of the patient-related characteristics, younger age was associated more with resection and Karnofsky Performance Score of 60 or less with biopsy. Of the tumor-related characteristics, a location in the right hemisphere, unifocality, no tumor midline crossing, and no involvement of the cortical spinal tract, were associated with resection, as well as a high expected resectability index, a location in the right occipital lobe, and a higher percentage of tumor in Schaefer’s dorsal or ventral attention, limbic, and default networks. External validation proved acceptable to outstanding discrimination with areas under the curve ranging between 0.79 and 0.92 for hospitals. Conclusion A prediction model is presented and validated to support the decision to resect or to biopsy a patient with a suspected supratentorial glioblastoma. In this prediction model, tumor-related characteristics were more informative than patient-related factors. This may support surgical decision-making for individual patients, or facilitate comparisons of patient cohorts between surgeons or institutions.

CLIN-NEURO-COGNITIVE NC-02. ASSESSING NEURO-COGNITIVE STATUS IN NEWLY PRESENTING PATIENTS WITH SUPRATENTORIAL INTRACRANIAL TUMOURS. Jennifer L. Scotland1, Ian R. Whittle1, and Ian J. Deary2; University of Edinburgh, Edinburgh, United Kingdom; Centre for Cognitive Ageing and Cognitive Epidemiology, Dept. of Psychology, University of Edinburgh, Edinburgh, United Kingdom INTRODUCTION: Neurocognitive endpoints are increasingly included in clinical trials in neuro-oncological settings (Meyers & Brown, 2006). There is a need to identify short, repeatable measures of cognition that are sensitive to impairment in this group. Previous authors have proposed such batteries. METHODS: Newly presenting patients with a radiological diagnosis of any intracranial tumour (n 1⁄4 118) were assessed, prior to any surgical intervention, on a number of standardised cognitive and other measures as part of a larger study (Scotland et al., 2012). Their performance was compared with that of patients admitted for elective spinal surgery (n 1⁄4 85) and a healthy control group (n 1⁄4 80). RESULTS: Analysis of covariance compared the performance of the three groups. Age, sex, and National Adult Reading Test score (used to assess premorbid function) were the covariates. Patients with intracranial tumours performed significantly worse on the majority of the tests. The intracranial tumour cohort had significantly lower scores than did the spinal surgery and healthy control groups on the following measures: immediate and delayed memory (Rey Auditory Verbal Learning Test, RAVLT; p , 0.001); Trail Making Test Part B (p , 0.001); digit-symbol coding (p , 0.001); and verbal fluency (Controlled Oral Word Association Test, COWAT; p 1⁄4 0.002 and p , 0.001, respectively). There was a moderate-to-large effect size in the participant group for all tests. DISCUSSION/CONCLUSIONS: The RAVLT, Trail Making Test Part B, and COWAT are sensitive measures of cognitive impairment in patients with brain tumours and support Meyers & Brown’s (2006) proposal for the inclusion of these tests in clinical trials that have neurocognitive endpoints. The verbal fluency test, in particular, has a number of potential advantages for use in neuro-oncological settings, given that it can be completed by patients with focal motor deficits and is available in alternate forms to reduce practice effects. NC-03. A STANDARD NEUROLINGUISTIC APPROACH TO

The 16th Annual Scientific Meeting of the Society for Neuro-Oncology in Conjunction with the AANS/CNS Section on Tumors, Orange County, California, USA, 17-20 November 2011. In Neuro-Oncology, 2011, v. 13 n. Suppl. 3, p. iii111, abstract no. ET-18

We analyzed the safety and efficacy of Mylotarg (gemtuzumab ozogamicin, an antibody-targeted chemotherapy consisting of a humanized anti-CD33 antibody linked to calicheamicin, a potent antitumor antibiotic) in the treatment of 101 patients > or =60 years of age with acute myeloid leukemia (AML) in untreated first relapse in three open-label trials. Mylotarg is administered as a 2-h intravenous infusion at 9 mg/m(2) for two doses with 14 days between doses. The overall remission rate was 28%, with complete remission (CR) in 13% of patients and complete remission with incomplete platelet recovery (CRp) in 15%. Median survival was 5.4 months for all patients and 14.5 months and 11.8 months for patients achieving CR and CRp, respectively. CD33 antigen is present on normal hematopoietic progenitor cells; thus, an expected high incidence of grade 3 or 4 neutropenia (99%) and thrombocytopenia (99%) was observed. The incidences of grade 3 or 4 elevations of bilirubin and hepatic transaminases were 24% and 15%, respectively. There was a low incidence of grade 3 or 4 mucositis (4%) and infections (27%) and no treatment-related cardiotoxicity, cerebellar toxicity, or alopecia. Mylotarg is an effective treatment for older patients with CD33-positive AML in first relapse and has acceptable toxicity.

The top-quark Yukawa infrared fixed-point solution of the renormalization group equations, with minimal supersymmetry and grand unification, defines a low-energy spectrum of supersymmetric particle masses in terms of a few GUT-scale parameters assuming universal boundary conditions. We give predictions in this model for the inclusive b\ensuremath{\rightarrow}s\ensuremath{\gamma} branching fraction and investigate the impact of nonuniversal scalar mass boundary conditions. We find our results do not depend significantly on the value of the GUT-scale trilinear coupling ${\mathit{A}}^{\mathit{G}}$. The small tan\ensuremath{\beta} region favors predictions for the inclusive b\ensuremath{\rightarrow}s\ensuremath{\gamma} branching fraction close to that of the standard model value. Nevertheless, forthcoming experimental results can eliminate some regions of the GUT parameter space.

We investigate the possibility of observing strong interactions of longitudinally polarized weak vector bosons in the process $\gamma \gamma \to ZZ$ at a photon linear collider. We make use of polarization of the photon beams and cuts on the decay products of the $Z$ bosons to enhance the signal relative to the background of transversely polarized $ZZ$ pairs. We find that the background overwhelms the signal unless there are strong resonant effects, as for instance from a technicolor analogue of the hadronic $f_2(1270)$ meson., Comment: 13 pages including 6 figures (uuencoded), some rewording to the text, LBL-35746, MAD/PH/837

Background: Bcl-2 family genes are frequently amplified in small cell lung cancer (SCLC). A phase I trial was conducted to evaluate the safety of obatoclax, a Bcl-2 family inhibitor, given in combination with standard chemotherapy. Methods: Eligible patients (3–6 per cohort) had extensive-stage SCLC, measurable disease, ⩽1 before therapy, Eastern Cooperative Oncology Group performance status 0 or 1, and adequate organ function. Patients were treated with escalating doses of obatoclax, either as a 3- or 24-h infusion, on days 1–3 of a 21-day cycle, in combination with carboplatin (area under the curve 5, day 1 only) and etoposide (100 mg m−2, days 1–3). The primary endpoint was to determine the maximum tolerated dose of obatoclax. Results: Twenty-five patients (56% male; median age 66 years) were enrolled in three dose cohorts for each schedule. Maximum tolerated dose was established with the 3-h infusion at 30 mg per day and was not reached with the 24-h infusion. Compared with the 24-h cohorts, the 3-h cohorts had higher incidence of central nervous system (CNS) adverse events (AEs); dose-limiting toxicities were somnolence, euphoria, and disorientation. These CNS AEs were transient, resolving shortly after the end of infusion, and without sequelae. The response rate was 81% in the 3-h and 44% in the 24-h infusion cohorts. Conclusion: Although associated with a higher incidence of transient CNS AEs than the 24-h infusion, 3-h obatoclax infusion combined with carboplatin–etoposide was generally well tolerated at doses of 30 mg per day. Though patient numbers were small, there was a suggestion of improved efficacy in the 3-h infusion group. Obatoclax 30 mg infused intravenously over 3 h on 3 consecutive days will be utilised in future SCLC studies.

Supersymmetry and Lorentz invariance are closely related as both are spacetime symmetries. Terms can be added to Lagrangians that explicitly break either supersymmetry or Lorentz invariance. It is possible to include terms which violate Lorentz invariance but maintain invariance under supersymmetric transformations. I illustrate this with some simple extensions of the original Wess-Zumino model., Comment: 8 pages, presented at Coral Gables Conference

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) confers resistance to methylating and chloroethylating agents in pediatric medulloblastoma- and glioma-derived cell lines and xenografts. Here, we assayed MGMT activity in 110 pediatric brain tumors to establish correlates with patient and tumor characteristics. We also assayed MGMT in histologically normal brain adjacent to 22 tumors to characterize changes in activity accompanying neurocarcinogenesis. MGMT activity was detected in 94% of tumors, ranging ca. 1,500-fold from 0.34 to 498 fmol/10(6) cells (approximately 205-300,000 molecules/cell). Mean activity was 25 +/- 66 fmol/10(6) cells, including six specimens with undetectable activity (Mer- phenotype;0.25 fmol/10(6) cells or 151 molecules/cell). MGMT content varied 10-fold among diagnostic groups and was associated with degree of malignancy, as evidenced by a 4-fold difference in activity between high- and low-grade tumors (P = 0.03). Tumor MGMT content was age dependent, being 5-fold higher in children 3-12 years old than in infants (P = 0.015) and adolescents (P = 0.015). Mean activity in tumors was 9-fold higher than in adjacent histologically normal brain (21 +/- 44 versus 2.4 +/- 4.0 fmol/10(6) cells; P = 0.05). By comparing tumor and adjacent normal tissue from the same patient, we found that 68% of cases exhibited an elevation of tumor activity that ranged from 2- to590-fold. Moreover, 67% of Mer- normal tissue was accompanied by Mer+ tumor. These observations indicate that MGMT activity is frequently elevated during pediatric neurocarcinogenesis. Significantly, enhanced MGMT activity may heighten resistance to alkylating agents, suggesting a potential role for MGMT inhibitors in therapy.

Temozolomide (TMZ) is a DNA-methylating agent that has recently been introduced into Phase II and III trials for the treatment of gliomas. TMZ produces O6-methylguanine in DNA, which mispairs with thymine during the next cycle of DNA replication. Subsequent futile cycles of DNA mismatch repair can lead to a p53-associated apoptotic cell death, although this mechanism has been described mostly in hematopoietic neoplasms. We studied the action of TMZ in gliomas and the role p53 might play by using U87 glioma cells that were either p53-wild-type or p53-deficient (by virtue of expression of the viral oncoprotein E6). LN-Z308 cells, in which p53 gene is deleted, were also used. p53-proficient U87 MG cells underwent a prolonged, p53- and p21(Waf1/Cip1)-associated G2-M arrest beginning 2 days after TMZ treatment. Although very few of these cells underwent apoptosis, most underwent senescence over a 10-day period. p53-deficient (E6-transfected U87 and LN-Z308) cells similarly underwent G2-M arrest in response to TMZ, but this arrest was accompanied by only minor changes in p53 or p21(Waf1/Cip1) and was reversed within 7 days of TMZ treatment in association with the appearance of cells with either 8n or subG1 DNA content. These results suggest that glioma cells respond to TMZ by undergoing G2-M arrest. p53 is not necessary for this G2-M arrest to occur but is important in the duration of G2-M arrest and in the ultimate fate of TMZ-treated cells. Therefore, the integrity of the G2-M cell cycle checkpoint may be important in the cytotoxicity of TMZ in glioma cells.

Glioblastoma multiforme (GBM) carries a dismal prognosis. However, a range of survival times exists, and parameters that define prognostic groups may help to optimize treatment. To identify such prognostic groups, we analyzed tumor tissue from 110 cases of newly diagnosed GBM from two clinical protocols. Similar to other studies, we found no association of epidermal growth factor receptor (EGFR) overexpression (as assessed by immunohistochemistry), p53 immunopositivity, or p53 mutation with survival in the entire sample. However, EGFR overexpression showed trends toward worse prognosis in patients younger than the median age, but better prognosis in patients older than the median age. This interaction of EGFR with age group was statistically significant and led us to focus our further analyses on the younger patients. In this group, a statistically significant association of EGFR overexpression with worse survival was identified in the p53-negative but not p53-positive tumors. We found a similar result after screening these cases for mutations in p53: EGFR overexpression was negatively associated with survival only in the p53 wild-type cases. To confirm this unexpected result, this finding was reproduced in a validation sample of an additional 42 tumors from younger patients on the same two clinical protocols. This complex relationship between EGFR and p53 in younger patients remained in a multivariate analysis that incorporated additional prognostic variables. The results suggest that analysis of prognostic markers in GBM is complex, and maximal information may require analysis of subgroups based on age and the status of specific markers such as p53. In addition, they suggest a specific group of patients on which to focus promising therapies targeting EGFR.

We study the impact of a set of horizontal symmetries on the requirements for producing the baryon asymmetry of the universe via leptogenesis. We find that Abelian horizontal symmetries lead to a simple description of the parameters describing leptogenesis in terms of the small expansion parameter that arises from spontaneous symmetry breaking. If the family symmetry is made discrete, then an enhancement in the amount of leptogenesis can result., Comment: 11 pages, 2 figures, version to appear in Physical Review D

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) contributes to the resistance of human brain tumor cell lines and xenografts to methylating and chloroethylating agents. We assayed MGMT in 174 newly diagnosed or recurrent gliomas to (a) quantitate changes in MGMT activity associated with alkylating agent-based chemotherapy; and (b) assess the contribution of MGMT to clinical outcome. Glioma MGMT activity ranged 300-fold, averaging 3,800+/-7,200 molecules/cell. Twenty-four percent of tumors lacked detectable activity [Methyl repair-deficient (Mer-) phenotype, defined here as151 molecules/cell or0.25 fmol/10(6) cells]. Tumors treated with surgery alone and tumors recurring after surgery and radiotherapy did not differ significantly in frequency of the Mer- phenotype (29% versus 24%). However, the frequency of the Mer- phenotype among tumors recurring after surgery, radiation, and alkylating agent-based chemotherapy was 7-fold lower than in tumors treated with surgery alone (4.3% versus 29%; Por = 0.02) and 6-fold lower than in tumors recurring after surgery and radiation (4.3% versus 24%; Por = 0.05). In contrast to gliomas, there was no relationship of alkylating agent-based therapy with the frequency of the Mer- phenotype in paired histologically normal brain. These data suggest that alkylating agents, either alone or synergistically with radiotherapy, selectively kill Mer- glioma cells in situ. Importantly, Mer- and Mer+ tumors did not differ in time to tumor progression following treatment with alkylating agents, indicating that although Mer- glioma cells may be differentially killed by alkylators, factors other than Mer phenotype were the principal determinants of time to clinical progression. Nonetheless, our results support the possibility that complete ablation of glioma MGMT with substrate analogue inhibitors could improve the efficacy of alkylating agent-based chemotherapy.

Malignant brain tumors pose diagnostic and therapeutic problems. Despite the advent of new brain imaging modalities, including magnetic resonance imaging (MRI) and [F-18]fluorodeoxyglucose (FDG) positron emission tomography (PET), determination of tumor viability and response to treatment is often difficult. Blood-brain barrier disruption can be caused by tumor or nonspecific reactions to treatment, making MRI interpretation ambiguous. The high metabolic background of the normal brain and its regional variability makes it difficult to identify small or less active tumors by FDG imaging of cellular energetics. We have investigated 2-[C-11]thymidine (dThd) and PET to image the rate of brain tumor cellular proliferation. A series of 13 patients underwent closely spaced dThd PET, FDG PET, and MRI procedures, and the image results were compared by standardized visual analysis. The resulting dThd scans were qualitatively different from the other two scans in approximately 50% of the cases, which suggests that dThd provided information distinct from FDG PET and MRI. In two cases, recurrent tumor was more apparent on the dThd study than on FDG; in two other patients, tumor dThd uptake was less than FDG uptake, and these patients had slower tumor progression than the three patients with both high dThd and FDG uptake. To better characterize tumor proliferation, kinetic modeling was applied to dynamic dThd PET uptake data and metabolite-analyzed blood data in a subset of patients. Kinetic analysis was able to remove the confounding influence of [C-11]CO2, the principal labeled metabolite of 2-[C-11]dThd, and to estimate the flux of dThd incorporation into DNA. Sequential, same-day [C-11]CO2 and [C-11]dThd imaging demonstrated the ability of kinetic analysis to model both dThd and CO2 simultaneously. Images of dThd flux obtained using the model along with the mixture analysis method for pixel-by-pixel parametric imaging significantly enhanced the contrast of tumor compared with normal brain. Comparison of model estimates of dThd transport versus dThd flux was able to discern increased dThd uptake simply on the basis of blood-brain barrier disruption retention on the basis of increased cellular proliferation. This preliminary study demonstrates the potential for imaging brain tumor cellular proliferation to provide unique information for guiding patient treatment.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) confers resistance to therapeutic methylating and chloroethylating agents in human brain tumor-derived cell lines. In this work, we assayed MGMT activity in 152 adult gliomas to establish correlates with patient and tumor characteristics. We also assayed MGMT in histologically normal brain adjacent to 87 tumors to characterize changes in activity accompanying neurocarcinogenesis. MGMT activity was detectable in 76% (115 of 152) of tumors, ranging approximately 300-fold from 0.30 to 89 fmol/10(6) cells (180-57,000 molecules/cell). Mean activity was 6.6 +/- 13 fmol/10(6) cells and varied 4-fold among diagnostic groups. The mean for oligodendrogliomas was 2-fold lower (P0.03), and for mixed oligodendroglioma-astrocytomas, the mean was 4-fold lower (P0.006) than for astroglial tumors. Twenty-five % of gliomas had no detectable MGMT activity (Mer- phenotype;0.25 fmol/10(6) cells or 150 molecules/cell). Glioma MGMT was inversely correlated with age (P0.01), consistent with the observed age dependence in the progenitor tissue of brain tumors (J. R. Silber et al., Proc. Natl. Acad. Sci. USA, 93: 6941-6946, 1996). Neither MGMT activity nor proportion of Mer- tumors differed by sex. Glioma MGMT was correlated with degree of aneuploidy (P0.006) but not with fraction of S-phase cells. Mean activity in tumors was 5-fold higher than in adjacent histologically normal brain (5.0 +/- 7.6 versus 1.1 +/- 1.9 fmol/10(6) cells; P0.001). Notably, elevation of tumor activity was observed in 62% of tissue pairs, ranging from 2-fold to105-fold. Moreover, 64% of Mer- normal tissue was accompanied by Mer+ tumor. These observations indicate that expression of MGMT activity is frequently activated and/or increased during human neurocarcinogenesis, and that the enhancement is not related to proliferation per se. Significantly, enhanced MGMT activity may heighten the resistance of brain tumors to therapeutic alkylating agents.

The basic helix-loop-helix (bHLH) class of transcription factors plays a pivotal role in tissue-specific determination and differentiation. Moreover, dysregulated expression or loss of function of these factors contributes to leukemogenesis and solid tumor development. Neurogenesis is regulated by genes of the NEUROD/atonal and ACHAETE SCUTE families. We analyzed expression of human NEUROD1, NEUROD2, NEUROD3, and ACHAETE SCUTE 1 (HASH1) in cerebellar and cerebral primitive neuroectodermal tumors (PNETs), gliomas, and cell lines derived from a variety of neuroectodermal tumors by Northern analysis and in situ hybridization. NEUROD1 was expressed in each of the 12 medulloblastoma specimens, whereas NEUROD2 and NEUROD3/neurogenin were expressed in partly overlapping subsets of medulloblastomas. All of the tumors that presented with distant metastases expressed NEUROD3. The only other NEUROD3-positive tumor progressed early in treatment. Human ACHAETE SCUTE homologue (HASH1) was not expressed in medulloblastomas (infratentorial PNETs) but was expressed in three of five supratentorial PNETs. Neuroectodermal tumor cell lines derived from other sites (e.g., neuroblastoma and retinoblastoma) expressed NeuroD and ACHAETE SCUTE family members. No NEUROD message was detected in glial tumors or cell lines. Neurogenic bHLH transcription factor expression patterns suggest that specific family members may contribute to or reflect biological differences that arise during malignant transformation.

The Papanicolaou (Pap) smear is a widely accepted tool for the detection and prevention of cervical cancer. Under the Bethesda Scoring System, the key internal control for adequacy of the smear is the presence of endocervical cells. Over the past several decades, much effort has been undertaken to maximize the adequacy of this acquisition. The present study determined if Pap smear sample adequacy is related to clinician experience.This study includes a retrospective analysis of 1,356 Pap smears acquired between January 1992 and January 1995. The procedures were performed by family practice residents and faculty physicians. Cervical smears were obtained with a combination of a cytobrush and an Ayre spatula and were placed on two slides. Adequacy was subsequently determined by a cytotechnologist. Chi-square analysis was used to determine if the adequacy rates for faculty and for PGY-1, PGY-2, and PGY-3 residents were significantly different.The adequacy rates for PGY-1, PGY-2, PGY-3 residents and faculty physicians were 80%, 89%, 89%, and 93%, respectively. The adequacy rates for the first-year residents were significantly different from that of all other studied groups.This study demonstrates the relationship between clinician experience and the ability to perform an adequate Pap smear. Considering the cost and potential medical risk of an inadequate Pap smear, family practice residency programs should increase the degree of PGY-1 education and experience in performing Pap smears.

DNA amplification is a common mechanism invoked by many human tumors to elicit overexpression of genes whose products are involved in drug resistance or cell proliferation. Although amplified regions in tumor DNA may exceed several megabases in size, segments of amplicons with a high probability of containing gene sequences may be amenable to detection by restriction landmark genomic scanning (RLGS), a high-resolution DNA analysis that separates labeled NotI fragments in two dimensions. Here, we tested this by applying RLGS to matched samples of glioma and normal brain DNA and found tumor-specific amplification of the gene encoding cyclin-dependent kinase 6 (CDK6), an observation not previously reported in human tumors. The CDK6 gene has been localized to chromosome 7q21-22, but in the gliomas studied here, it was not coamplified with either the syntenic MET (7q31) or epidermal growth factor receptor (7p11-p12) genes, suggesting that this may be part of a novel amplicon in gliomas. We then corroborated this finding by identifying both amplification-associated and amplification-independent increases in CDK6 protein levels in gliomas relative to matched normal brain samples. These data implicate the CDK6 gene in genomic amplification and illustrate the potential of RLGS for the more general identification and cloning of novel genes that are amplified in human cancer.

PURPOSE: To characterize transient intraspinal subdural enhancement (potentially mimicking the subarachnoid spread of tumor) seen on MR images in some children after suboccipital craniectomy for posterior fossa tumor resection. METHODS: Radiologic and medical records of 10 consecutive children who had MR imaging for spinal staging after resection of posterior fossa tumor during a 9-month period were reviewed retrospectively. In addition, one case with similar findings of intraspinal enhancement on spinal staging MR images obtained at another institution was included in the review. RESULTS: Intraspinal enhancement thought to be subdural was seen in four of 10 patients undergoing spinal staging MR imaging 6 to 12 days after surgery. In these four patients, MR studies 50 to 18 days later, without intervening treatment, showed resolution of the abnormal enhancement. A fifth patient (from another institution) with similar intraspinal enhancement underwent CT myelography 4 days later, which showed no subarachnoid lesions. No metastases have developed in any of these five patients during the 2.5- to 3.5-year follow-up period. conclusions: From analysis of the MR appearance and on the basis of prior myelographic experience, we suggest an extraarachnoid, probably subdural, location of this enhancement. Awareness of this phenomenon will reduce the rate of false-positive diagnoses of metastatic disease. Preoperative spinal staging should be considered for patients undergoing suboccipital craniectomy.

The product of the p16/CDKN2 locus, p16ink4, negatively regulates the cell cycle through binding and inactivation of cyclin-dependent kinases (CDKs) 4 and 6. This locus is frequently targeted for deletion in cell lines and primary tumor tissues. In gliomas, although up to 50% do not have detectable expression of p16/CDKN2 protein or mRNA, often the gene is wild type in sequence. Here, we tested the hypothesis that transcriptional repression of p16/CDKN2 in gliomas may be mediated by aberrant methylation of the CpG island, which is in the 5' region of the locus. Partial rather than complete p16/CDKN2 methylation was detected in 24% (10 of 42) of the gliomas, regardless of tumor grade, but was not observed in normal brain (0 of 10). We tested whether this partial methylation could inhibit expression in a human tumor cell line in which suppressed p16/CDKN2 expression was associated with both methylation and tightly compacted chromatin around the p16/CDKN2 promoter. Exposure of these cells to 5-aza-2-deoxycytidine resulted in a dramatic increase in promoter accessibility and induction of p16/CDKN2 expression, indicating that chromatin structure, CpG island methylation, and p16/CDKN2 expression are intimately associated. Taken together, these data suggest that methylation occurs in only a subset of cells within gliomas and that the methylation-associated inactivation of p16/CDKN2 expression observed in many common human cancers may mechanistically result from structural changes in the chromatin containing the p16/CDKN2 locus.

We have analyzed the sensitivity of 14 human medulloblastoma- and glioma-derived cell lines to the clinically used methylating agents temozolomide and streptozotocin. The cell lines responded similarly to these agents, displaying a 3-fold range in cytotoxicity, assessed as the 10% survival dose (LD10). The contribution of O6-methylguanine-DNA methyltransferase (MGMT) to resistance, measured as reduction in the LD10 by O6-benzylguanine (O6-BG), varied among the lines by 1 order of magnitude for both agents. However, in all MGMT-expressing lines, O6-BG eliminated a threshold dose that accounted for up to one-half of the LD10. The effect of O6-BG on the rate of killing varied 13-fold for temozolomide and 14-fold for streptozotocin. Some lines displayed two subpopulations with different rates of killing, with one subpopulation that comprised 20-60% of cells showing essentially no dependence of the rate of killing on MGMT. O6-BG increased the range of the LD10 for both agents. The persistent, heightened variability in cytotoxicity in the absence of MGMT, the lack of correlation between MGMT content of the lines and cytoxicity (LD10), and the lack of correlation between MGMT content and the contribution of MGMT to resistance (O6-BG-mediated reduction of the LD10) reflect the operation of resistance mechanisms other than MGMT. We also analyzed sensitivity to methyl methanesulfonate, observing little dependence of resistance on MGMT and persistent variability in cytotoxicity in the presence of O6-BG. We discuss the implications for clinical use of methylators and O6-BG.

P16INK4 is a cell cycle regulator that specifically binds to and inactivates cyclin-dependent kinase 4 (CDK4). Its encoding gene (p16/CDKN2) maps to chromosome 9p21, a region that undergoes frequent loss of heterozygosity in a variety of human tumors. We have analyzed the p16/CDKN2 gene and its expression in a series of primary glioma samples. Although homozygous deletion or mutation of the p16/CDKN2 gene was uncommon in this series and P16INK4 protein was detectable in all grade II tumors, it was present in only 50% of grade III and grade IV samples. Conversely, in some grade IV tumors that level of P16INK4 protein was elevated; in these cases, its target, CDK4, was amplified and overexpressed. These results suggest: (a) the involvement of P16INK4 in glioma progression; (b) that mechanisms other than mutation or deletion can down-regulate expression of the p16/CDKN2 gene; and (c) that the balance between CDK4 and its cognate inhibitor, P16INK4, may confer a cell growth advantage and facilitate tumor progression.

Perturbation of the DNA repair process appears to be responsible for the occurrence of a number of human diseases, which are usually associated with a propensity to develop internal malignancies and/or disorders of the central nervous system. We have been interested in the possibility that a subtle abnormality in DNA repair competency might be associated with the transformation of nonmalignant cells to the malignant state. To study this question, we assayed malignant and nonmalignant brain tissues from 19 individuals for mRNA expression levels of the human DNA repair genes ERCC1, ERCC2, and XPAC and for differential splicing of the ERCC1 transcript. We separately compared expression levels of these genes in the following situations: concordance of expression within malignant tissues; concordance of expression within nonmalignant tissues; concordance between malignant and nonmalignant tissues within individuals of the cohort; and concordance of gene expression between two nonmalignant tissue sites within a single individual. Linear regression analyses of mRNA values obtained suggested orderly concordance of these three DNA repair genes in nonmalignant tissues within the patient cohort and an excellent concordance of these genes between two separate biopsy sites from the same individual. In contrast, malignant tissues showed disruption of concordance between the full-length ERCC1 transcript and ERCC2, which have excision and helicase functions, respectively. Furthermore, within the same individuals, malignant tissues were discordant with nonmalignant tissues for ERCC1 and ERCC2, although concordance for XPAC was preserved. These data suggest that one molecular characteristic of human malignancy may be the disruption of the normal relationship between the excision and the helicase functions of the nucleotide excision repair pathway.

Glioblastomas were examined for abnormalities in fibroblast growth factor receptor (FGFR) expression by polymerase chain reaction and immunocytochemical analysis. Polymerase chain reaction analysis demonstrated that FGFR1 mRNA levels were significantly higher in glioblastomas than in normal brain adjacent to the tumor or in untransformed human brain. These results were consistent with immunocytochemical localization of FGFR1 protein in glioblastomas: glioblastoma cells exhibited intense FGFR1 immunoreactivity in frozen sections of tumor and low to undetectable FGFR1 immunoreactivity in adjacent normal brain or in normal white matter obtained from patients without neoplastic disease. Endothelial cells of capillaries and larger vessels within the tumor were devoid of FGFR1 immunoreactivity. All glioblastomas evaluated in the present study expressed FGFR1 mRNA and FGFR1 immunoreactivity. Examination of the FGFR1 gene by Southern blot analysis indicated that overexpression of FGFR1 mRNA in glioblastomas did not result from gene amplification. These results indicate that glioblastoma cells, in contrast to endothelial cells within the tumor, display increased levels of FGFR1. Therefore, FGFR1 signal transduction may be associated with increased autocrine growth activity of tumor cells and is probably not related to the increased endothelial cell proliferation associated with these tumors.

By altering the accessibility of DNA sequences for alkylation or platination, and/or for subsequent repair, topoisomerase II can potentially affect the level of DNA interstrand cross-links induced in cells by bifunctional agents. In this study, we investigated the extent to which inhibition of topoisomerase II activity in a human glioblastoma multiforme cell line alters the kinetics of both the formation and the repair of total genomic DNA interstrand cross-links, as well as the sensitivity of the tumor cells to cis-diamminedichloroplatinum II (cis-DDP) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Cells were incubated with and without 200 microM novobiocin, a known topoisomerase II inhibitor, for 24 h, followed by exposure to 50 microM BCNU and 25 microM cis-DDP. DNA interstrand cross-linking was determined at various time points over 72 h, using a modified ethidium bromide-DNA binding assay. Sensitivity of the cells to cis-DDP and BCNU was also determined with and without novobiocin pretreatment with 200 microM novobiocin. This concentration of novobiocin showed no significant direct cytotoxicity, although it inhibited topoisomerase II activity in tumor cell nuclear extracts by 73%. A significant decrease in the rate of repair of both cis-DDP and BCNU induced DNA interstrand cross-links, with a corresponding decrease in the clonogenic survival of the cells, was observed following novobiocin exposure. Although the peak cross-link indices of novobiocin-treated cells relative to controls were not significantly increased, residual DNA cross-linking in the cells after 72 h was increased by 1.4-fold for BCNU and 3-fold for cells treated with cis-DDP, thus, indicating a greater effect of topoisomerase II on cross-link repair than on cross-link formation. These data suggest that inhibition of topoisomerase II may provide a potentially effective clinical strategy for sensitizing human brain tumors, and possibly other tumors as well, to DNA cross-linking anticancer agents.

We make a numerical study of gauge and Yukawa unification in supersymmetric grand unified models and examine the quantitative implications of fermion mass ans\"{a}tze at the grand unified scale. Integrating the renormalization group equations with $\alpha _1(M_Z)$ and $\alpha _2(M_Z)$ as inputs, we find $\alpha _3(M_Z)\simeq 0.111 (0.122)$ for $M_{SUSY}^{}=m_t$ and $\alpha _3(M_Z)\simeq 0.106 (0.116)$ for $M_{SUSY}^{}=1$ TeV at one-loop (two-loop) order. Including $b$ and $\tau $ Yukawa couplings in the evolution, we find an upper limit $m_t\ltap 200$ GeV from Yukawa unification. For given $m_t\ltap 175$ GeV, there are two solutions for $\beta$, one with $\tan \beta > m_t/m_b$, and one with $\sin \beta \simeq 0.78(m_t/150\;{\rm GeV})$. Taking a popular ansatz for the mass matrices at the unified scale, we obtain a lower limit on the top quark mass of $m_t\gtap 150 (115)$ GeV for $\alpha _3(M_Z)=0.11 (0.12)$ and an upper limit on the supersymmetry parameter $\tan \beta \ltap 50$ if $\alpha _3(M_Z)=0.11$. The evolution of the quark mixing matrix elements is also evaluated., Comment: 46 pages + 11 figures not included (available on request), MAD/PH/711

In minimal SUSY-GUT models with $M_{SUSY}\ltap 1$ TeV, the renormalization group equations have a solution dominated by the infrared fixed point of the top Yukawa coupling. This fixed point predicts $m_t=(200\; {\rm GeV})\sin \beta $; combined with the LEP results it excludes $m_t\ltap 130$ GeV. For $m_t$ in the range 130-160 GeV, we discuss the sensitivity of the $m_t$ fixed point result to GUT threshold corrections and point out the implications for Higgs boson searches. The lightest scalar $h$ has mass 60-85 GeV and will be detectable at LEPII. At SSC/LHC, each of the five scalars $h$, $H$, $A$, $H^{\pm }$ may be detectable, but not all of them together; in one parameter region none will be detectable., Comment: 12 pages + 4 PS figures not included (available upon request), MAD/PH/755

By evolution of fermion mass matrices of the Fritzsch and the Georgi-Jarlskog forms from the supersymmetric grand unified scale, DHR obtained predictions for the quark masses and mixings. Using Monte Carlo methods we test these predictions against the latest determinations of the mixings, the CP-violating parameter epsilon_K and the B_d^0--Bbar_d^0 mixing parameter r_d. The acceptable solutions closely specify the quark masses and mixings, but lie at the edges of allowed regions at 90% confidence level., Comment: 11 pages, 1 figure (not included)