Stereotactic radiosurgery and radiotherapy for resected brain metastases: current pattern of care in the Radiosurgery and Stereotactic Radiotherapy Working Group of the German Association for Radiation Oncology (DEGRO).

PURPOSE: Preoperative stereotactic radiosurgery (SRS) of brain metastases may achieve similar local control and better leptomeningeal control rates than postoperative fractionated stereotactic radiotherapy (FSRT) in patients treated with elective metastasectomy. To plan a multicentre trial of preoperative SRS compared with postoperative FSRT, a survey of experts was conducted to determine current practice. METHODS: A survey with 15 questions was distributed to the DEGRO Radiosurgery and Stereotactic Radiotherapy Working Group. Participants were asked under what circumstances they offered SRS, FSRT, partial and/or whole brain radiotherapy before or after resection of a brain metastasis, as well as the feasibility of preoperative stereotactic radiosurgery and neurosurgical resection within 6 days. RESULTS: Of 25 participants from 24 centres, 22 completed 100% of the questions. 24 respondents were radiation oncologists and 1 was a neurosurgeon. All 24 centres have one or more dedicated radiosurgery platform and all offer postoperative FSRT. Preoperative SRS is offered by 4/24 (16.7%) centres, and 9/24 (37.5%) sometimes recommend single-fraction postoperative SRS. Partial brain irradiation is offered by 8/24 (33.3%) centres and 12/24 (50%) occasionally recommend whole-brain irradiation. Two centres are participating in clinical trials of preoperative SRS. SRS techniques and fractionation varied between centres. CONCLUSION: All responding centres currently offer postoperative FSRT after brain metastasectomy. Approximately one third offer single-fraction postoperative SRS and four already perform preoperative SRS. With regard to potential co-investigators, 18 were identified for the PREOP­2 multicentre trial, which will randomise between preoperative SRS and postoperative FSRT.

Factors of influence upon the SF-36-based health related quality of life of patients following surgery for petroclival and lateral posterior surface of pyramid meningiomas.

OBJECTIVE: To describe the patient's self assessed health related quality of life (saHRQoL) based upon the medical outcome study 36-item short form health survey (SF-36) as well as the factors of influence upon the saHRQoL following surgery for petroclival (PCM) and lateral posterior surface of the pyramid (LPPM) meningiomas. PATIENTS AND METHODS: In a series of 78 patients operated consecutively for PCM (n = 46) or LPPM (n = 32) the preoperative, intraoperative and postoperative data were collected retrospectively. The saHRQoL was obtained by mailing the SF-36 questionnaire to the patients. The SF-36 data of the whole patients group was compared with a healthy population. The SF-36 data of the PCM- and LPPM were compared to each other. The influence of pre-, intra- and postoperative findings upon the SF-36 was assessed by uni- and multifactorial analysis. RESULTS: 58 (69%) out of the 78 patients answered the SF-36 questionnaire at a median postoperative follow-up of 59 months. The patients, who answered the SF-36 questionnaire, had a significant lower perioperative complication rate than those who did not (46% vs. 75%, p = 0.019). The saHRQoL of the LPPM and PCM was reduced on several sub-scales, when compared to the German reference population. The outcome of PCM is, assessed by saHRQoL as well as by conventional neurosurgical grading scales, inferior to that of LPPM. The saHRQoL of LPPM correlated in the uni- and multivariate analysis with the early postoperative KPI on the sub-scales SF1 (physical functioning) and SF5 (vitality). Accordingly, the sub-scale SF2 (role-physical) of PCM correlated with the change of the KPI from preoperative to the last follow up. CONCLUSIONS: The saHRQoL of the evaluable patients was lower than that of the normal population. The saHRQoL score of PCM-patients was lower than that of LPPM-patients. For the future the saHRQol should be assessed routinely; It reflects the patients' perspective upon postoperative outcome and enables the comparison with other treatment modalities of these difficult to treat tumors.

hMYH and hMTH1 cooperate for survival in mismatch repair defective T-cell acute lymphoblastic leukemia.

hMTH1 is an 8-oxodGTPase that prevents mis-incorporation of free oxidized nucleotides into genomic DNA. Base excision and mismatch repair pathways also restrict the accumulation of oxidized lesions in DNA by removing the mis-inserted 8-oxo-7,8-dihydro-2'-deoxyguanosines (8-oxodGs). In this study, we aimed to investigate the interplay between hMYH DNA glycosylase and hMTH1 for cancer cell survival by using mismatch repair defective T-cell acute lymphoblastic leukemia (T-ALL) cells. To this end, MYH and MTH1 were silenced individually or simultaneously using small hairpin RNAs. Increased sub-G1 population and apoptotic cells were observed upon concurrent depletion of both enzymes. Elevated cell death was consistent with cleaved caspase 3 accumulation in double knockdown cells. Importantly, overexpression of the nuclear isoform of hMYH could remove the G1 arrest and partially rescue the toxicity observed in hMTH1-depleted cells. In addition, expression profiles of human DNA glycosylases were generated using quantitative reverse transcriptase-PCR in MTH1 and/or MYH knockdown cells. NEIL1 DNA glycosylase, involved in repair of oxidized nucleosides, was found to be significantly downregulated as a cellular response to MTH1-MYH co-suppression. Overall, the results suggest that hMYH and hMTH1 functionally cooperate for effective repair and survival in mismatch repair defective T-ALL Jurkat A3 cells.

Bevacizumab treatment in malignant meningioma with additional radiation necrosis. An MRI diffusion and perfusion case study.

BACKGROUND AND PURPOSE: Recently two retrospective cohort studies report efficacy of bevacizumab in patients with recurrent atypical and anaplastic meningioma. Another successful therapeutic option of bevacizumab seems to be treatment of cerebral radiation necrosis. However, the antiangiogenic effects in MRI diffusion and perfusion in meningiomas have not been previously described in detail. The objective of this research was to evaluate the clinical and MR imaging effects of bevacizumab in a malignant meningioma patient harboring additional cerebral radiation necrosis. CASE PRESENTATION: We report the case of an 80-year-old woman who underwent bevacizumab therapy (5 mg/kg every 2 weeks for 2 months) for treatment of a symptomatic radiation necrosis in malignant meningiomatosis of World Health Organization (WHO) grade III. The patient was closely monitored with MRI including diffusion and perfusion studies. Upon bevacizumab therapy, the clinical situation was well stabilized over a period of 4 months until the patient unfortunately died due to pneumonia/septicemia probably unrelated to bevacizumab therapy. Consecutive MRI demonstrated 4 important aspects: (1) considerable decrease of the contrast medium (CM)-enhanced radiation necrosis, (2) mixed response with respect to the meningiomatosis with stable and predominantly growing tumor lesions, (3) a new diffusion-weighted imaging (DWI) lesion in a CM-enhanced tumor as described in gliomas, which we did not interpret as a response to bevacizumab therapy, and (4) new thrombembolic infarcts, which are a known side-effect of bevacizumab treatment. CONCLUSION: Bevacizumab is effective in the treatment of radiation necrosis. We could not confirm the potential antitumor effect of bevacizumab in this patient. However, we could describe several new radiographic effects of bevacizumab therapy in malignant meningioma.

Ifosfamide, carboplatin and etoposide in recurrent malignant glioma.

After failure of temozolomide, there is no established standard salvage chemotherapy for patients with recurrent glioblastoma (GBM). Two phase II trials combining ifosfamide, carboplatin and etoposide chemotherapy (ICE) showed favorable results. We therefore applied the ICE protocol to 13 patients (10 GBM, 3 anaplastic astrocytomas). Partial or complete remissions were not observed. None of the 13 patients survived progression-free for 6 months. Our retrospective analysis suggests that the ICE regimen is not effective in patients with recurrent high-grade glioma if applied at second or third relapse.

Treatment results in patients with intracranial ependymomas.

OBJECTIVE: To evaluate the treatment outcomes of patients with intracranial ependymomas. METHODS: Between 1988-2007, 27 consecutive patients, with 9 patients (33%) aged under 16 years, were treated at our institution for an intracranial ependymoma. Pertinent clinical data were retrieved from the patients' charts. The histopathological findings in 25 cases were reviewed using the 2007 World Health Organization (WHO) classification system. Median follow-up was 84 months. RESULTS: Infratentorial tumors were diagnosed in 22, and supratentorial tumors in 5 patients. Histopathological findings were ependymoma WHO grade II (E II) in 14 patients, and anaplastic ependymoma WHO grade III (AE III) in 13 patients. A complete tumor resection was achieved with the first operation in 20 cases (74%). Primary adjuvant therapy consisted of chemotherapy alone in a 17-month-old child with an incompletely resected posterior fossa AE III, radiation therapy alone in 4 cases, and combined radiation therapy and chemotherapy in 7 cases. Tumor recurrence was seen in 10 cases (37%), including 5 patients with an E II and 5 patients with an AE III. The 5-year progression-free survival (PFS) was 74% and 67% for E II and AE III, respectively. The 5-year PFS was 80% following a complete resection, and 56% in patients with a residual tumor. CONCLUSIONS: Surgery alone, as the primary treatment, achieves a good outcome in most patients with E II. Good results can be achieved with surgery and adjuvant local radiotherapy in patients with AE III.

Simultaneous bedside assessment of global cerebral blood flow and effective cerebral perfusion pressure in patients with intracranial hypertension.

BACKGROUND: We examined a bedside technique transcerebral double-indicator dilution (TCID) for global cerebral blood flow (CBF) as well as the concept of effective cerebral perfusion pressure (CPP(eff)) during different treatment options for intracranial hypertension, and compared global CBF and CPP(eff) with simultaneously obtained conventional parameters. METHODS: Twenty-six patients developing intracranial hypertension in the course of traumatic brain injury or subarachnoid hemorrhage were prospectively analyzed using a combined assessment during elevated ventilation (n = 15) or osmotherapy (hypertonic saline or mannitol). For calculation of global CBF, injections of ice-cold indocyanine green boluses were performed and temperature and dye concentration changes were monitored in the thoracic aorta and the jugular bulb. CBF was then calculated according to the mean transit time principle. Estimation of CCP, the arterial pressure at which cerebral blood flow becomes zero, was performed by synchronized registration of corresponding values of blood flow velocity in the middle cerebral artery and arterial pressure and extrapolation to zero-flow velocity. CPP(eff) was calculated as mean arterial pressure minus critical closing pressure (CPP(eff) = MAP(c) - CCP). RESULTS: Elevated ventilation causes a decrease in both ICP (P < 0.001) and CBF (P < 0.001). While CPP(conv) increased (P < 0.001), CPP(eff) decreased during this observation (P = 0.002). Administration of osmotherapeutic agents resulted in a decrease of ICP (P < 0.001) and a temporary increase of CBF (P = 0.052). CPP(conv) and CPP(eff) showed no striking difference under osmotherapy. CONCLUSION: TCID allows repeated measurements of global CBF at the bedside. Elevated ventilation lowered and osmotherapy temporarily raised global CBF. In situations of increased vasotonus, CPP(eff) is a better indicator of blood flow changes than conventional CPP.

Interinstitutional variance of postoperative radiotherapy and follow up for meningiomas in Germany: impact of changes of the WHO classification.

OBJECTIVE: To document and critically analyse the impact of the revised WHO 2000 histological classification for meningiomas on postoperative radiotherapy/radiosurgery indications and MRI follow up protocols. METHODS: The current (2000) WHO classification was used to grade 57 meningiomas treated surgically at one institution. These had been reviewed previously in 1999. All German neurosurgical departments carrying out intracranial microsurgery were asked to detail their guidelines for radiation therapy and follow up for meningiomas of different WHO grades. RESULTS: Use of the current criteria downgraded seven of 15 atypical meningiomas (WHO grade II, MII) to grade I (MI), and four of six anaplastic tumours (WHO grade III, MIII) to grade II. Indications for radiotherapy/radiosurgery and MRI follow up protocols varied substantially with the histological grade and between institutions--for example, after an incomplete resection, radiotherapy/radiosurgery recommendations differed between MI and MII in 30 of 58 units (52%), and between MII and MIII in 34 of 56 units (61%). CONCLUSIONS: Correlative studies combining treatment and outcome data with a standardised histopathological analysis are warranted to define properly the indications for radiotherapy/radiosurgery and follow up protocols after surgery for meningiomas of different histological grades. The use of changing grading paradigms during recent years renders decision making based on local and published experience difficult. The relatively large number of meningiomas classified as atypical/WHO grade II in current practice would argue against an uncritically aggressive approach to these tumours.

Alterations of the tumor suppressor genes CDKN2A (p16(INK4a)), p14(ARF), CDKN2B (p15(INK4b)), and CDKN2C (p18(INK4c)) in atypical and anaplastic meningiomas.

We investigated 67 meningothelial tumors (20 benign meningiomas, 34 atypical meningiomas, and 13 anaplastic meningiomas) for losses of genetic information from chromosome arms 1p and 9p, as well as for deletion, mutation, and expression of the tumor suppressor genes CDKN2A (p16(INKa)/MTS1), p14(ARF), CDKN2B (p15(INK4b)/MTS2) (all located at 9p21) and CDKN2C (1p32). Comparative genomic hybridization and microsatellite analysis showed losses on 1p in 11 anaplastic meningiomas (85%), 23 atypical meningiomas (68%), and 5 benign meningiomas (25%). One atypical meningioma with loss of heterozygosity on 1p carried a somatic CDKN2C mutation (c.202C>T: R68X). Losses on 9p were found in five anaplastic meningiomas (38%), six atypical meningiomas (18%), and one benign meningioma (5%). Six anaplastic meningiomas (46%) and one atypical meningioma (3%) showed homozygous deletions of the CDKN2A, p14(ARF), and CDKN2B genes. Two anaplastic meningiomas carried somatic point mutations in CDKN2A (c.262G>T: E88X and c.262G>A: E88K) and p14(ARF) (c.305G>T: G102V and c.305G>A: G102E). One anaplastic meningioma, three atypical meningiomas, and one benign meningioma without a demonstrated homozygous deletion or mutation of CDKN2A, p14(ARF), or CDKN2B lacked detectable transcripts from at least one of these genes. Hypermethylation of CDKN2A, p14(ARF), and CDKN2B could be demonstrated in one of these cases. Taken together, our results indicate that CDKN2C is rarely altered in meningiomas. However, the majority of anaplastic meningiomas either show homozygous deletions of CDKN2A, p14(ARF), and CDKN2B, mutations in CDKN2A and p14(ARF), or lack of expression of one or more of these genes. Thus, inactivation of the G(1)/S-phase cell-cycle checkpoint is an important aberration in anaplastic meningiomas.

Analysis of human meningiomas for aberrations of the MADH2, MADH4, APM-1 and DCC tumor suppressor genes on the long arm of chromosome 18.

We have previously reported that losses of genomic material from the long arm of chromosome 18 are frequent in atypical and anaplastic meningiomas but rare in benign meningiomas. In the present study, we have investigated a series of 37 meningiomas for mutation and expression of 4 tumor suppressor genes (MADH2, MADH4, APM-1 and DCC) located at 18q21. Comparative genomic hybridization or loss of heterozygosity analysis showed losses on chromosome 18 that included sequences from 18q21 in 15 of 37 tumors. Mutation analysis of APM-1 revealed a missense mutation (c. 1819G>A: G607S) in 1 atypical meningioma. None of the tumors showed mutations of MADH2 and MADH4 or loss of detectable transcripts from MADH2, MADH4, APM-1 and DCC. In contrast to human brain tissue, normal leptomeninges and meningiomas showed preferential expression of a DCC splice variant lacking 60 base pairs from exon 17. Taken together, our data do not support a significant role for MADH2, MADH4, APM-1 and DCC alterations in the pathogenesis of meningiomas. The targeted gene that is inactivated in most meningiomas with 18q losses remains to be identified.

Reproducing the conformations of protein-bound ligands: a critical evaluation of several popular conformational searching tools.

Several programs (Catalyst, Confort, Flo99, MacroModel, and Omega) that are commonly used to generate conformational ensembles have been tested for their ability to reproduce bioactive conformations. The ligands from thirty-two different ligand-protein complexes determined by high-resolution (< 2.0 A) X-ray crystallography have been analyzed. The Low-Mode Conformational Search method (with AMBER* and the GB/SA hydration model), as implemented in MacroModel, was found to perform better than the other algorithms. The rule-based method Omega, which is orders of magnitude faster than the other methods, also gave reasonable results but were found to be dependent on the input structure. The methods supporting diverse sampling (Catalyst, Confort) performed least well. For the seven ligands in the set having eight or more rotatable bonds, none of the bioactive conformations were ever found, save for one exception (Flo99). These ligands do not bind in a local minimum conformation according to AMBER*\GB/SA. Taking these last two observations together, it is clear that geometrically similar structures should be collected in order to increase the probability of finding the bioactive conformation among the generated ensembles. Factors influencing bioactive conformational retrieval have been identified and are discussed.

Alterations of INK4a(p16-p14ARF)/INK4b(p15) expression and telomerase activation in meningioma progression.

Dysregulation of cell cycle progression and telomerase activation have been implicated in malignant tumor progression as well as in the evasion of senescence and immortalization. We have investigated expression of the cell cycle control and tumor suppressor genes INK4a(p16-p14ARF), INK4b(p15-p10) and RB, and their relation to telomerase activation during malignant meningioma progression. 7/26 (27%) benign, 3/12 (25%) atypical but 4/7 (57%) anaplastic tumors lacked both, p16 and p15 protein expression. 14/39 (36%) benign and atypical but 5/7 (71%) anaplastic meningiomas contained no p14ARF mRNA. 2/46 (4%) tumors failed to express pRB. We observed frequent differential loss of expression of the alternatively spliced INK4a tumor suppressors p16 and p14ARF. Exclusive expression of the alternative INK4b transcript p10 possibly at the expense of p15 and therefore resulting in loss of p15 tumor suppressor activity was noted in two meningiomas. We have previously described telomerase activity or expression of the telomerase catalytic subunit hTERT in this meningioma series. Telomerase activation was detected in 10/27 (37%) benign, but 18/19 (95%) non-benign meningiomas. We observed no significant overall correlation between loss of INK4a/INK4b expression and telomerase activation. In conclusion, our results suggest a greater role for losses of INK4a/INK4b gene products in meningioma formation and malignant progression than previously thought. Inactivation of p16/p15- and pl4ARF-dependent pathways possibly in conjunction with telomerase activation might be critical steps for a meningioma cell towards escape from senescence, that is, immortalization.

A pharmacophore model for dopamine D4 receptor antagonists.

A pharmacophore model for dopamine D4 antagonists has been developed on the basis of a previously reported dopamine D2 model. By using exhaustive conformational analyses (MM3* force field and the GB/SA hydration model) and least-squares molecular superimposition studies, a set of eighteen structurally diverse high affinity D4 antagonists have successfully been accommodated in the D4 pharmacophore model. Enantioselectivities may be rationalized by conformational energies required for the enantiomers to adopt their proposed bioactive conformations. The pharmacophore models for antagonists at the D4 and D2 receptor subtypes have been compared in order to get insight into molecular properties of importance for D2/D4 receptor selectivity. It is concluded that the bioactive conformations of antagonists at the two receptor subtypes are essentially identical. Receptor essential volumes previously identified for the D2 receptor are shown to be present also in the D4 receptor. In addition, a novel receptor essential volume in the D4 receptor, not present in the D2 receptor, has been identified. This feature may be exploited for the design of D4 selective antagonists. However, it is concluded that the major determinant for D2/D4 selectivity is the nature of the interactions between the receptor and aromatic ring systems. The effects of the electronic properties of these ring systems on the affinities for the two receptor subtypes differ substantially.

Multiple intracranial juvenile xanthogranulomas. Case report.

The authors report on an 11-year-old boy in whom proptosis of the eye caused by a benign intraosseous xanthofibroma of the left orbital wall became clinically apparent at the age of 4 years. Two years later he developed bilateral papilledema, at which time computerized tomography and magnetic resonance studies revealed multiple enhancing intracranial lesions. The largest mass was located in the left middle fossa; other lesions were located at the tentorium cerebelli, in both lateral ventricles, near the superior sagittal sinus, and extracranially near the left jugular vein. The mass in the left middle fossa was resected and diagnosed as juvenile xanthogranuloma (JXG). Thirty months later, the patient again became symptomatic, exhibiting behavioral abnormalities and a decrease in mental powers. At that time, the two remaining lesions in both lateral ventricles had grown enough to cause trapping of the temporal horns and raised intracranial pressure. These lesions were successively resected and histopathologically confirmed to be JXGs. However, resection of the second intraventricular lesion was complicated by postoperative bilateral amaurosis, presumably caused by postdecompression optic neuropathy. According to a review of the literature, fewer than 20 patients with JXG involving the central nervous system have been reported. The patient described in this report is the first in whom multiple intracranial JXGs developed in the absence of cutaneous manifestations. Although JXGs are biologically benign lesions, the treatment of patients with multifocal and/or progressive intracranial manifestations is problematic.

Allelic losses on chromosome arm 10q and mutation of the PTEN (MMAC1) tumour suppressor gene in primary and metastatic malignant melanomas.

Malignant melanomas frequently show loss of alleles on the long arm of chromosome 10. The PTEN (MMAC1) gene has been identified as a tumour suppressor gene at 10q23.3 that is mutated in various types of advanced human cancers. We have investigated a series of 40 sporadic melanomas from 37 patients (15 primary cutaneous melanomas and 25 melanoma metastases) for allelic losses on chromosome 10, as well as for deletion and mutation of the PTEN gene. Microsatellite analysis revealed loss of heterozygosity at loci located on 10q in tumours from 15 of 34 patients investigated (44%). Somatic PTEN mutations were identified in melanomas from 4 of 37 patients (11%), all of whom had metastatic disease. In two of these patients, the tumours had additionally lost one PTEN allele, indicating complete loss of wild-type PTEN in the tumour cells. Our findings corroborate that loss of heterozygosity on chromosome 10 is a frequent aberration in malignant melanomas and implicate PTEN as a tumour suppressor gene inactivated by somatic mutation in a fraction of these tumours.

Identification of two distinct deleted regions on the short arm of chromosome 1 and rare mutation of the CDKN2C gene from 1p32 in oligodendroglial tumors.

Oligodendroglial tumors frequently show allelic losses on the short arm of chromosome 1. To narrow down the putative tumor suppressor gene site(s) on 1p, we have investigated 35 oligodendrogliomas and 10 mixed gliomas (oligoastrocytomas) for loss of heterozygosity (LOH) at 21 highly polymorphic loci on chromosome 1 (19 loci on 1p and 2 loci on 1q). LOH at loci on 1p was found in 30 of the 45 tumors (67%). Two distinct regions of common allelic loss were identified: a distal region between D1S76 and D1S253 at 1p36.3, and a proximal region between D1S482 and D1S2743 at 1p34-p35. We also analyzed our tumor series for genetic alterations and expression of the cyclin dependent kinase inhibitor gene CDKN2C (p18INK4c) from 1p32. We found 1 recurrent anaplastic oligodendroglioma that carried a somatic CDKN2C mutation at codon 113 (GAA ==> TAA: Glu ==> Stop). The remaining 44 tumors of our series showed neither coding sequence mutations nor homozygous deletions of CDKN2C. Investigation of 35 tumors by differential reverse transcription-PCR revealed expression of CDKN2C transcripts in all instances. Our data thus provide evidence for more than a single oligodendroglioma-associated tumor suppressor gene on 1p and implicate CDKN2C as a candidate tumor suppressor gene altered in a low fraction of oligodendroglial tumors.

Prospective study of symptoms and gastro-oesophageal reflux 10 years after posterior partial fundoplication.

BACKGROUND: This was a prospective study of symptoms, and short-term and long-term reflux competence after partial fundoplication. METHODS: Some 101 patients were operated consecutively with posterior partial (270 degrees ) fundoplication. Indications for surgery were reflux disease without erosive oesophagitis in 25 patients, moderate oesophagitis in 43, severe oesophagitis in 25 and paraoesophageal hernia in eight. Symptom score, manometry and pH tests were performed before operation, 6 months after operation and after 6-14 years. RESULTS: All patients (n = 101) were free from heartburn and regurgitation at early follow-up. There was evidence of clinical recurrence at late follow-up (n = 87) in two of 22 patients without oesophagitis before operation, two of 39 with moderate oesophagitis before operation and three of 19 patients with severe oesophagitis before operation; 92 per cent had good reflux control at late follow-up. CONCLUSION: Posterior partial fundoplication shows excellent reflux control at early follow-up. Ten years later fewer than 10 per cent of patients have recurrence, which is more common in patients who had severe oesophagitis before operation.

Amplification and overexpression of the MDM4 (MDMX) gene from 1q32 in a subset of malignant gliomas without TP53 mutation or MDM2 amplification.

We have previously reported on the amplification and overexpression of the MDM2 proto-oncogene in a subset of malignant gliomas without TP53 mutation (G. Reifenberger et al, Cancer Res., 53: 2736-2739, 1993). Here, we show that the MDM4 (MDMX) gene located on 1q32 is a further target for amplification in malignant gliomas. MDM4 codes for a Mdm2-related protein that can bind to p53 and inhibits p53-mediated transcriptional transactivation. We investigated a series of 208 gliomas (106 glioblastomas, 46 anaplastic gliomas, and 56 low-grade gliomas) and identified 5 tumors (4 glioblastomas and 1 anaplastic oligodendroglioma) with MDM4 amplification and overexpression. Several other genes from 1q32 were found to be coamplified with MDM4, such as GAC1 in five tumors, REN in four tumors, and RBBP5 in three tumors. Additional analyses revealed that the malignant gliomas with MDM4 amplification and overexpression carried neither mutations in conserved regions of the TP53 gene nor amplification of the MDM2 gene. Taken together, our data indicate that amplification and overexpression of MDM4 is a novel molecular mechanism by which a small fraction of human malignant gliomas escapes p53-dependent growth control.

Conformational energy penalties of protein-bound ligands.

The conformational energies required for ligands to adopt their bioactive conformations were calculated for 33 ligand-protein complexes including 28 different ligands. In order to monitor the force field dependence of the results, two force fields, MM3* and AMBER*, were employed for the calculations. Conformational analyses were performed in vacuo and in aqueous solution by using the generalized Born/solvent accessible surface (GB/SA) solvation model. The protein-bound conformations were relaxed by using flat-bottomed Cartesian constraints. For about 70% of the ligand-protein complexes studied, the conformational energies of the bioactive conformations were calculated to be < or = 3 kcal/mol. It is demonstrated that the aqueous conformational ensemble for the unbound ligand must be used as a reference state in this type of calculations. The calculations for the ligand-protein complexes with conformational energy penalties of the ligand calculated to be larger than 3 kcal/mol suffer from uncertainties in the interpretation of the experimental data or limitations of the computational methods. For example, in the case of long-chain flexible ligands (e.g. fatty acids), it is demonstrated that several conformations may be found which are very similar to the conformation determined by X-ray crystallography and which display significantly lower conformational energy penalties for binding than obtained by using the experimental conformation. For strongly polar molecules, e.g. amino acids, the results indicate that further developments of the force fields and of the dielectric continuum solvation model are required for reliable calculations on the conformational properties of this type of compounds.