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Purpose: To compare event-free survival (EFS) in children with high-risk medulloblastoma randomly assigned to receive either chemotherapy before radiation or chemotherapy after radiation., Patients and Methods: One hundred twelve patients were randomly assigned to each arm. Criteria used to categorize patients as high risk included M1-4 disease by modified Chang staging classification, T3b/T4 disease, or greater than 1.5 cm3 of residual tumor after surgery. Postoperatively, children with high-risk medulloblastoma were randomly assigned to two arms, either chemotherapy entailing three cycles of cisplatin and etoposide before radiation (chemotherapy first [CT1]) or the same chemotherapy regimen after radiation (radiation therapy first [RT1]). Both groups received consolidation chemotherapy consisting of vincristine and cyclophosphamide., Results: The median follow-up time was 6.4 years. Five-year EFS was 66.0% in the CT1 arm and 70.0% in the RT1 arm (P = .54), and 5-year overall survival in the two groups was 73.1% and 76.1%, respectively (P = .47). In the CT1 arm, 40 of the 62 patients with residual disease achieved either complete or partial remission., Conclusion: Five-year EFS did not differ significantly whether, after surgery, patients received chemotherapy before or after radiotherapy.

Background: Tumours are responsive to temozolomide (TMZ) if they are deficient in O(6)-methylguanine-DNA methyltransferase (MGMT), and mismatch repair (MMR) proficient., Methods: The effect of TMZ on medulloblastoma (MB) cell killing was analysed with clonogenic survival assays. Expression of DNA repair genes and enzymes was investigated using microarrays, western blot, and immunohistochemistry. DNA sequencing and promoter methylation analysis were employed to investigate the cause of loss of the expression of MMR gene MLH1., Results: Temozolomide exhibited potent cytotoxic activity in D425Med (MGMT deficient, MLH1 proficient; IC(50)=1.7 μM), moderate activity against D341Med (MGMT proficient, MLH1 deficient), and DAOY MB cells (MGMT proficient, MLH1 proficient). MGMT inhibitor O(6)-benzylguanine sensitised DAOY, but not D341Med cells to TMZ. Of 12 MB cell lines, D341Med, D283Med, and 1580WÜ cells exhibited MMR deficiency due to MLH1 promoter hypermethylation. DNA sequencing of these cells provided no evidence for somatic genetic alterations in MLH1. Expression analyses of MMR and MGMT in MB revealed that all patient specimens (n=74; expression array, n=61; immunostaining, n=13) are most likely MMR proficient, whereas some tumours had low MGMT expression levels (according to expression array) or were totally MGMT deficient (3 out of 13 according to immunohistochemistry)., Conclusion: A subset of MB may respond to TMZ as some patient specimens are MGMT deficient, and tumours appear to be MMR proficient.

Purpose: To determine the response, toxicity, and survival for children with progressive or recurrent medulloblastoma and germinoma using a single myeloablative course of chemotherapy supported by autologous hematopoietic stem cells., Patients and Methods: Subjects were in second remission or had minimal residual disease at the time of study entry. The conditioning regimen consisted of cyclophosphamide 6,000 mg/m(2) plus melphalan 180 mg/m(2)., Results: Twenty-nine evaluable pediatric patients were accrued. The most frequent major toxicities were myelosuppression, infections, and stomatitis, but no toxic deaths were recorded. Best responses were: CR = 6, CCR = 13, PR = 6, SD = 2, and PD = 2. There were 6 medulloblastoma and 3 germinoma survivors with a median follow-up of 7.5 years (range = 2.8-10). Two germinoma survivors received radiotherapy after autografting for presumptive progressive disease., Conclusion: Myeloablative chemotherapy consisting of cyclophosphamide and melphalan was tolerable in the relapsed brain tumor setting with 19/29 cases achieving CR or CCR status and 9/29 becoming long-term survivors., ((c) 2008 Wiley-Liss, Inc.)

The total expression profiles of two medulloblastoma cell lines resistant to the preactivated form of cyclophosphamide (4-hydroperoxycyclophosphamide, 4-HC) were examined using the Affymetrix GeneChip U133A array. Our primary objective was to look for possible genes, other than the well-studied aldehyde dehydrogenases (ALDH) that may be involved in cyclophosphamide (CP) resistance in medulloblastomas. We present here the lists of the most highly upregulated [30 for D341 MED (4-HCR); 20 for D283 MED (4-HCR)] and downregulated [19 for D341 MED (4-HCR); 15 for D283 MED (4-HCR)] genes which may be involved in conferring CP-resistance to the two medullobalstoma cell lines. The lists of genes from the two sublines almost had no overlap, suggesting different mechanisms of CP-resistance. One of the most noteworthy upregulated gene is TAP1 [90-fold increase in D341 MED (4-HCR) relative to D341 MED]. TAP1, a protein belonging to the ABC transporter family is normally involved in major histocompatibility class I (MHC I) antigen processing. This suggests the possible role of multidrug resistance (MDR), albeit atypical (which means it does not involve the usual MDR1 and MRP glycoproteins), in medulloblastoma's CP-resistance. Apart from TAP1, a number of other genes involved in MHC1 processing were upregulated in D341 MED (4HCR). D341 MED (4-HCR) also had a 20-fold increase in the expression of the aldo-keto reductase gene, AKR1B10, which may deactivate the reactive cyclophosphamide metabolite, aldophosphamide. For D283 MED (4-HCR), the most notable increase in expression is that of ALDH1B1, a member of the aldehyde dehydrogenase (ALDH) family of proteins.

Purpose: An inverse correlation has been established between tumor levels of the DNA repair protein alkylguanine-DNA alkyltransferase (AGT) and a positive outcome after alkylator chemotherapy. Quantitative imaging of AGT could provide important information for patient-specific cancer treatment. Several radiolabeled analogues of O6-benzylguanine (BG), a potent AGT inactivator, have been developed and shown to be capable of labeling pure AGT protein. Herein, two of these analogues--O6-3-[*I]iodobenzylguanine ([*I]IBG) and O6-3-[*I]iodobenzyl-2'-deoxyguanosine ([*I]IBdG)--were further evaluated in two murine xenograft models. (AcO)2-[131I]IBdG, a peracetylated derivative of IBdG, also was investigated as an alternative agent., Methods: Several biodistribution studies of radioiodinated IBG and IBdG were performed in TE-671 human rhabdomyosarcoma and DAOY human medulloblastoma murine xenograft models. Mice were treated with BG or its nucleoside analogue dBG to deplete the tumor AGT content. The effect of unlabeled IBG and that of 7,8-benzoflavone (BF), an inhibitor of the cytochrome P-450 isozyme CYP1A2, on the tumor uptake of the tracers was determined. The uptake of (AcO)2-[131I]IBdG along with that of [125I]IBdG in DAOY cells in vitro was determined in the presence and absence of a nucleoside transporter inhibitor, dipyridamole., Results: Pretreatment of mice either with BG or dBG failed to reduce tumor levels of [*I]IBG or [*I]IBdG even though such treatments completely depleted tumor AGT content. Treatment of mice with BF increased tumor uptake of [125I]IBG by 56%; however, differentiation of tumors with and without AGT still was not possible. (AcO)2-[131I]IBdG, a peracetylated derivative of IBdG, had a higher uptake in vitro in DAOY tumor cells. However, its uptake, like that of [125I]IBdG, was blocked by dipyridamole., Conclusions: Taken together, these results suggest that labeled agents that are more specific for cellular AGT and that are more metabolically stable are needed.

495 medulloblastomas (MBs) from 6 Pediatric Oncology Group (POG) protocols were reviewed to assess the incidence and prognostic significance of "large cell" and "anaplastic" variants. "Large cell" medulloblastomas (LC MBs) were those with focal or diffuse, large, round neoplastic cells with prominent nucleoli. "Anaplastic" MBs (A MBs) were those with nuclei that were also large but markedly atypical with coarse chromatin and irregular shapes. Twenty-one cases were identified in the combined LC/A MB group, comprising about 4% of all MBs. Survival curves and Kaplan-Meier estimates of survival probabilities were examined separately for the LC/A MB and control groups. The logrank test for detecting poorer survival in the 21 cases was significant (p < 0.0001). Fluorescence in situ hybridization for c-myc showed amplification in 4 of 11 cases of the LC/A phenotype and 1 additional case of high level gain at 8q24 was disclosed by comparative genomic hybridization. Comparative genomic hybridization confirmed c-myc amplification and found evidence for isochromosome 17q in 3 of 4 LC/A cases studied successfully. One additional tumor showed high level gain restricted to 2p13 consistent with n-myc amplification. Monosomy 22, common in atypical teratoid/rhabdoid tumors, was not found. These results suggest that LC/A MB phenotype could be, at least in part, a correlate of c-myc, and possibly n-myc, amplification. The study thus confirms original observations about the LC MB in regard to histological features, immunohistochemical findings, c-myc amplification, cytogenetic findings, and poor prognosis.

Purpose: To evaluate prospectively the effects on survival, relapse-free survival, and patterns of relapse of reduced-dose (23.4 Gy in 13 fractions) compared with standard-dose (36 Gy in 20 fractions) neuraxis irradiation in patients 3 to 21 years of age with low-stage medulloblastoma, minimal postoperative residual disease, and no evidence of neuraxis disease., Patients and Methods: The Pediatric Oncology Group and Children's Cancer Group randomized 126 patients to the study. All patients received posterior fossa irradiation to a total dose of 54 Gy in addition to the neuraxis treatment. Patients were staged postoperatively with contrast-enhanced cranial computed tomography, myelography, and CSF cytology. Of the registered patients, 38 were ineligible., Results: The planned interim analysis that resulted in closure of the protocol showed that patients randomized to the reduced neuraxis treatment had increased frequency of relapse. In the final analysis, eligible patients receiving standard-dose neuraxis irradiation had 67% event-free survival (EFS) at 5 years (SE = 7.4%), whereas eligible patients receiving reduced-dose neuraxis irradiation had 52% event-free survival at 5 years (SE = 7.7%) (P =.080). At 8 years, the respective EFS proportions were also 67% (SE = 8.8%) and 52% (SE = 11%) (P =.141). These data confirm the original one-sided conclusions but suggest that differences are less marked with time., Conclusion: Reduced-dose neuraxis irradiation (23.4 Gy) is associated with increased risk of early relapse, early isolated neuraxis relapse, and lower 5-year EFS and overall survival than standard irradiation (36 Gy). The 5-year EFS for patients receiving standard-dose irradiation is suboptimal, and improved techniques and/or therapies are needed to improve ultimate outcome. Chemotherapy may contribute to this improvement.

Loss of portions of chromosome 17p, usually through the formation of i(17qp) is a well-known finding in medulloblastomas. Loss of heterozygosity (LOH) studies, however, occasionally demonstrate loss of the more distal portions of 17p, a pattern which is more consistent with a terminal deletion. Here we use a combination of routine karyotyping, fluorescence in situ hybridization (FISH) and LOH studies on four medulloblastoma cell lines and one xenograft to demonstrate the spectrum of chromosome 17 abnormalities which occur in these tumors. Cell line D-556 Med showed a typical dicentric i(17q) and cell line D-721 Med showed two normal copies of chromosome 17 by all methods. Cell line D-425 Med showed loss of terminal 17p by LOH, while the karyotype showed what appeared to be an i(17q). FISH and chromosome 17 painting, however, demonstrated that the abnormal chromosome 17 was actually formed through an unbalanced translocation involving two copies of chromosome 17, with breakpoints at p12 and q11-1, an explanation which reconciled the cytogenetic and LOH findings. Cell line D 581 Med had a terminal deletion at 17p11.2. The finding of two cells with i(17q) in this case by interphase FISH suggests that the terminal deletion arose from breakage of an i(17q). Finally, xenograft D 690 Med showed LOH for regions distal to 17p12, whereas karyotyping, FISH using probes on 17p, and chromosome 17 painting showed two intact copies of chromosome 17. This pattern can be explained by homologous recombination. These data support the concept that the critical deletion of 17p can occur through a variety of mechanisms in the medulloblastoma. The losses may occur through typical i(17q), as well as other mechanisms such as terminal deletions, possibly through breakage of i(17q), unbalanced translocations and homologous recombination.

Aims: We have reviewed immunohistochemically 17 paediatric medulloblastomas in order to determine if correlations exist that might be useful in subclassifying these tumours., Methods and Results: The patient group included 11 children who had died (mean survival 13 months) and six still alive (followed for up to 10 years). Ten tumours were diffuse and six were nodular (one biopsy had only perivascular tumour). Of the 10 diffuse tumours, three were desmoplastic: of the six nodular tumours, all six were desmoplastic. All 17 tumours were synaptophysin-reactive: three nodular tumours were glial fibrillary acidic protein (GFAP)-reactive in the nodules (two of three S 100-reactive tumours were also GFAP-reactive). MIB-1 labelling indices (LI) ranged from 5 to 80%. Six tumours exhibited at least 1% LI against Tp53 (Mab D07 and/or Mab 1801). Eight cases were 100% bcl2-reactive with nine cases having an LI <80% ('low labelling'). All nine 'low labelling' bcl2 cases were TP53 non-reactive; all six Tp53-reactive cases were bcl2 100% reactive. Six of 10 patients with diffuse medulloblastomas survived 18 months or less while four of 10 are alive up to 10 years. In contrast, five of six patients with nodular neoplasms died within 48 months of diagnosis with one patient followed up for less than 1 year., Conclusions: Immunohistochemistry is a useful adjunct in characterizing subsets of paediatric medulloblastomas and confirms that larger co-operative studies may be fruitful in identifying a prognostic utility of a combined histochemical/immunohistochemical analysis on these tumours.

Purpose: We have previously reported preferential repair of DNA interstrand crosslinks in the 4-hydroperoxycyclophosphamide-resistant human medulloblastoma cell line D-283 Med (4-HCR). We now report further studies that explored the potential mechanisms underlying this repair., Methods: Limiting dilution assays and Western, Southern, and Northern blots were used to compare specific differences between D-283 Med (4-HCR) and its parental line D-283 Med., Results: D-283 Med (4-HCR) was cross-resistant to melphalan and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with O6-alkylguanine-DNA alkyltransferase (AGT) levels of 466+/-164 fmol/mg protein; AGT levels in the parental line, D-283 Med, were 76+/-96 fmol/mg. The increase in AGT activity was not a result of gene amplification. Depleting AGT with O6-benzylguanine partially restored sensitivity to BCNU. Both cell lines were deficient in the human mismatch protein MutLalpha. ERCC4 mRNA and poly(ADP-ribose) polymerase levels were similar in both cell lines, and ERCC1 mRNA levels were 2- to 2.5-fold lower in D-283 Med (4-HCR). Topoisomerase I levels were 2- to 2.5-fold higher in D-283 Med compared with D-283 Med (4-HCR)., Conclusion: These results, while illustrating the multiple differences between D-283 Med and D-283 Med (4-HCR), do not explain the enhanced DNA interstrand crosslink repair seen in D-283 Med (4-HCR).

During replication, the primary function of the eukaryotic DNA mismatch repair (MMR) system is to recognize and correct mismatched base pairs within the DNA helix. Deficiencies in MMR have been reported previously in cases of hereditary nonpolyposis colorectal cancer and sporadic tumors occurring in a variety of tissues including gliomas. Furthermore, recent evidence indicates that the MMR system may be involved in mediating therapeutic sensitivity to alkylating agents. In this study, 22 neoplastic tissue samples from 22 patients who underwent surgical resection for medulloblastoma, a common cerebellar tumor of childhood, were assayed for the presence or absence of MMR polypeptides using Western blot and immunohistochemical techniques. Results from these experiments indicate that the MMR system is not commonly deficient in medulloblastoma.

Purpose: The purpose of this study was to test the hypothesis that survivors of medulloblastoma who were younger at diagnosis and those who received standard-dose cranial irradiation (SRT) of 36 Gy would have a lower performance on standardized tests of cognitive function and achievement than children who were older and those treated with reduced-dose cranial irradiation (RRT) of 23.4 Gy., Patients and Methods: Eligible patients had been treated on Pediatric Oncology Group (POG) study 8631 for low-risk medulloblastoma that randomized patients to receive RRT or SRT after surgical resection. Those who were alive and free of progressive disease 6.1 to 9.9 years from completion of treatment were eligible for this study. Of the 35 eligible patients, 22 patients (13 SRT, nine RRT) participated in a battery of tests that included intellectual and academic development as well as ratings of health-related quality of life., Results: Patients were stratified by treatment group (SRT v RRT) and into younger (Y) and older (O) groups by the median age at diagnosis (8.85 years), which resulted in four groups that we hypothesized would show neuropsychologic test scores in the following order: Y/SRT less than Y/RRT less than O/SRT less than O/RRT. Evidence to support the hypothesized ordering of groups in terms of neuropsychologic toxicity was obtained with regard to Performance Intelligence Quotient (IQ), Full Scale IQ, Attention, Reading, and Arithmetic., Conclusion: Children treated for medulloblastoma experienced less severe neuropsychologic toxicity when treated with 23.4 Gy instead of 36 Gy cranial irradiation. Older children experienced less toxicity than children who were younger at the time of irradiation.

In the present cytogenetic analysis of 116 pediatric brain tumors, chromosomal abnormalities were demonstrated in 44 cases, 48 cases revealed only 46,XX or 46,XY cells, and 24 cases were nonproductive. In contrast to studies of adult brain tumors in which isolated loss of one X or the Y chromosome is often encountered, 45,X,-X and 45,X-Y stemlines or sidelines were not observed in this series of childhood tumors. Among the 17 medulloblastomas with cytogenetic abnormalities, chromosome 1 was most frequently affected by structural deviations; the most prevalent specific alteration (7 of 17 tumors) was loss of 17p, through i(17)(q10) or unbalanced translocation. The majority of low grade astrocytomas had normal stemlines, although one pilocytic astrocytoma and one cerebellar astrocytoma had frequent telomeric associations and a second pilocytic astrocytoma had a clone with trisomy 11. Thirteen of 19 high-grade and recurrent astrocytic tumors had abnormal stemlines that were approximately equally divided among cases with chromosomal counts in the near-diploid, hyperdiploid, and near-triploid-tetraploid ranges. Although no consistent abnormalities were observed, subsets of cases had structural abnormalities of chromosome 3, 7q, 9q, or 17p. The cases of childhood brain tumors described here demonstrate that 45,X,-X, and 45,X,-Y stemlines or sidelines are rare in these tumors and confirm frequent loss of 17p in medulloblastomas. High-grade astrocytic tumors in children frequently have abnormal stemlines, often in the hyperdiploid and polyploid ranges, and they differ from high-grade gliomas in the adult by lacking consistent numerical and structural deviations.

In recent years, major advances in the diagnosis and treatment of patients with brain tumors have been seen. Today, evaluation of the central nervous system almost always includes magnetic resonance imaging (MRI). The appearance of a new lesion on the MRI scan of a patient previously treated for a central nervous system (CNS) tumor raises concern for recurrent disease with the need for selection of new, potentially toxic therapy. However, the sensitivity of MRI may allow demonstration of new lesions which are not due to tumor. We now report three patients with medulloblastoma who demonstrated new enhancing lesions on MRI following treatment of their tumors with surgery (3 patients), chemotherapy (2 patients), and radiotherapy (2 patients). Two patients underwent resection of the lesion revealing gliosis. One patient had serial imaging that showed disappearance of the lesions. This suggests that not all new enhancing lesions in previously treated brain tumor patients represent tumor. Histologic proof of a suspicious lesion should be demonstrated prior to initiation of new therapy.

Background: Does a "delay in diagnosis" lead to a child being diagnosed with advanced stage as opposed to early stage medulloblastoma? Correlation between the duration of a patient's presenting symptoms and stage at diagnosis was examined., Methods: The population consisted of 72 consecutive patients with histologically proven medulloblastoma diagnosed between July 1, 1983 and July 31, 1995. A standard history and physical examination format was used to record the nature and duration of presenting symptoms. Patients were staged by use of the operative findings, pre- and postoperative cranial computed tomography (CT) scans and, later in the series, cranial magnetic resonance imaging (MRI) studies and, for determination of the M stage, myelography, spinal MRI, and postoperative cerebrospinal fluid cytology., Results: There were 40 males (56%) and 32 females (44%) with a mean age of 11.8 years. The most common presenting symptoms were vomiting (67%), headache (60%), ataxia (40%), and nausea (39%). By the Chang-Harisiadis (CH) system, 39 patients (54%) were found to have high stage medulloblastoma (T3b-4M0 or any TM1-4), 27 (38%) had low stage disease (T1-3aM0), and in 6 (8%) the stage could not be fully determined. By the Langston modification of the Change-Harisiadis system (LCH) 38 patients (54%) had high stage, 24 (32%) had low stage, and in 10 (14%) the stage could not be fully determined. Fifty of the 66 patients for whom the duration of symptoms was known (76%) had < or = 3 months of symptoms prior to stage. High CH stage patients had a mean duration of symptoms of 7.4 +/- 6.9 weeks versus 19.5 +/- 22.5 weeks for low stage patients. (P < 0.001). High LCH stage patients had a mean duration of symptoms of 7 +/- 6.6 weeks versus 15.4 +/- 16.4 weeks for low stage patients (P < 0.01). Patients ultimately found to have MO disease were diagnosed more slowly (16.1 +/- 20 weeks) than those with M1 (7.3 +/- 5.3 weeks), M2 (6 +/- 5.3 weeks), or M3 disease (6.8 +/- 5.9 weeks) M0 vs. M1-3, P < 0.02). No patients had M4 disease. Using an alternative definition of high versus low stage (T4M0 or any TM1-4 vs. T1-3bM0) currently under consideration by pediatric oncologists, the duration of symptoms remained significantly longer for low stage disease in the CH system (high vs. low, 7.2 +/- 5.8 weeks vs. 17.5 +/- 19.1 weeks, P < 0.01) but not in the LH system (high vs. low, 10.6 +/- 16.1 weeks vs. 13.9 +/- 15.9 weeks, P not significant)., Conclusions: A short duration of symptoms is associated with the diagnosis of more advanced medulloblastoma. This finding has significant potential implications for the identification of prognostic groups in medulloblastoma as well as medical-legal claims of "delay in diagnosis" and capitated health care issues.

Purpose: The outcome for patients with recurrent medulloblastoma has historically been poor, with most patients dying of disseminated disease. Here, we report on seven patients with recurrent medulloblastoma, most heavily pretreated with a variety of chemotherapeutic agents, including parenteral etoposide (VP-16), who showed responses to the administration of repeated courses of low-dose oral VP-16., Patients and Methods: Seven patients age 4 to 16 years were treated with VP-16 after neuroradiographic and clinical evidence of tumor progression. Six had received prior irradiation. All seven had been pretreated with a variety of chemotherapeutic agents and schedules, including parenteral VP-16. VP-16 was administered orally as repeated 21-day courses at 50 mg/m2/d with a 7-day interval between courses. Evaluation consisted of neuroradiographic and clinical examination after completion of every two courses of therapy. Complete blood cell counts were performed weekly., Results: The major toxicity of oral VP-16 was hematologic, with two patients requiring platelet transfusions due to thrombocytopenia and two requiring RBC transfusions. All seven patients developed treatment-related neutropenia. Two patients were supported with granulocyte colony-stimulating factor (G-CSF) between courses. One patient developed infectious epididymitis after course 2 and required intravenous antibiotics; this illness was complicated by Clostridium difficile colitis. There was one episode of fever associated with neutropenia. There were no treatment-related deaths. Of seven patients assessed, six have demonstrated partial responses (PRs) and the remaining patient had stable disease (SD)., Conclusion: This report demonstrates the activity of oral VP-16 in the treatment of a small cohort of pretreated patients with recurrent medulloblastoma. This form of administration of oral VP-16 was well tolerated and produced modest toxicity.

Cyclophosphamide is one of the most active agents in the treatment of medulloblastoma. However, development of resistance to this alkylator frequently occurs and is the harbinger of tumor progression and death. In order to understand the biochemical basis of this resistance, we generated a panel of medulloblastoma cell lines in our laboratory that were resistant to 4-hydroperoxycyclophosphamide (4-HC). Previously, we have shown that elevated levels of aldehyde dehydrogenase and glutathione mediate cellular resistance to 4-HC. The present study was conducted to identify the third unknown mechanism mediating the resistance of cell line D283 Med (4-HCR) to 4-HC, testing the hypothesis that this resistance is mediated by an increased repair of DNA interstrand crosslinks (ICLs). The doses of 4-HC that produced a one- and two-log cell kill of D283 Med cells were 25 and 50 microM, respectively, compared with values of 125 and 165 microM in D283 Med (4-HCR), the resistant cell line. The formation and disappearance of 4-HC-induced DNA ICLs at the c-myc gene were subsequently studied by DNA denaturing/renaturing gel electrophoresis and Southern blot analysis. 4-HC-induced DNA ICLs in the c-myc gene exhibited a dose-dependent relationship. The percentage of the c-myc gene that was crosslinked was approximately 1-3% at a dose of 100 microM. More than 50% of the DNA crosslinking in D283 Med (4-HCR) cells was removed by 6 h after drug treatment, whereas, in D283 Med cells, more than 90% of the DNA crosslinking was still present at 6 h. These findings suggest that the increased repair of DNA ICLs in D283 Med (4-HCR) may contribute significantly to its resistance to 4-HC.

Purpose/objective: Some malignant brain tumors shed cells into the cerebrospinal fluid (CSF). These tumors may implant throughout the neuroaxis via the CSF. With the placement of a ventriculoperitoneal (VP) or ventriculoatrial (VA) shunt, tumor cells free-floating in the CSF may be carried through the shunt to the remainder of the body. Mechanical filtration devices to prevent this are not reliable. We report the development of a new device capable of reducing the incidence of shunt promoted metastasis., Materials & Methods: The device exposes the draining CSF, as it passes through a baffle system, to a localized high-intensity radiation field adequately shielded from surrounding normal tissue. The prototype consists of geometrically fixed iodine-125 (125I) sources. The device accommodates the maximum CSF flow rate of 500 ml/24 hours. Radiation exposure to clonogenic cells occurs as they transit through the baffle system. Since the volume of the prototype device is 14 ml, a tumor cell floating through the device will be exposed to radiation for 40 minutes. Utilizing the human medulloblastoma cell line D425 MED, a limiting dilution clonogenic assay was performed. Suspensions of tumor cells in liquid medium were pumped through the device at the maximum anticipated CSF production rate of 0.35 ml/min. After the cells, with their tissue culture medium, were received from the device, a series of nine 5-fold dilutions were prepared from the suspensions which initially contained 10(6) tumor cell/ml. Plates were then incubated and growth was demonstrated by visual scoring of colonies of more than 20 cells. Limiting dilution data analysis was performed. Radiation surveys of the fully loaded (approximately 1.8 Ci) 125I prototype were conducted. A well calibrator was used to measure the activity of the fully loaded device., Results: When the device was loaded with 125I seeds providing a dose of 364-479 cGy the probability of clonogen survival was 0.033. Radiation exposure levels at the exterior surface of the shielded device were in the range of 2-5 mR/hr and thus fell within guidelines for acceptable normal tissue exposure. Attenuation of radiation by the shielding case for the fully loaded device was 10(-5)., Conclusion: The device kills medulloblastoma cells as they are pumped through it. If the risk of metastasis is linearly related to the number of clonogenic cells, then the device would, we infer, reduce the risk of shunt-born metastasis by a factor of 0.033 and merits further investigation.

DNA derived from medulloblastoma biopsies was analyzed to determine if deletions of the 17p region, mutations of the TP53 gene, or amplification of the c-myc, N-myc, EGFR (epidermal growth factor receptor), or MDM2 (murine double-minute-2) genes was indicative of a poor prognosis. Loss of heterozygosity for 17p, observed in 8/28 (29%) paired samples, was associated with a shortened survival period (p = 0.045 by the logrank test). TP53 mutations occurred in 2/46 (4.3%) tumor samples. c-myc Amplification was seen in 3/43 (6.9%) cases, while none of the tumors contained amplified N-myc, EGFR, or MDM2 genes. These results demonstrate that, while only rare medulloblastomas contain TP53 gene mutations or amplification of the c-myc gene, loss of heterozygosity on chromosome 17p is indicative of a significantly worse prognosis among patients with these tumors. Further, these results provide a strong impetus for a prospective analysis of loss of heterozygosity in a cooperative group setting, which would include tumor staging, a selection of treatment modalities, and multivariate analyses.

Medulloblastoma, the most common malignancy of childhood, was originally shown to be sensitive to cyclophosphamide in 1981. We have used combined laboratory and clinical investigations to demonstrate the synergy of cyclophosphamide and vincristine in the treatment of this tumor, the therapeutic gain associated with escalation of the dosage of cyclophosphamide, the consequence of and mechanisms underlying resistance of medulloblastoma to cyclophosphamide, the emerging importance of the neuraxis as a site of relapse of medulloblastoma, and newer approaches, including intrathecal 4-hydroperoxycyclophosphamide and busulfan, to treat neuraxis disease. These studies serve as a paradigm for laboratory-clinical translational research.

Conventional treatment for medulloblastoma includes surgical resection followed by craniospinal irradiation, with the potential risk of subsequent radiotherapy-induced secondary neoplasms. We report a 23-year-old woman previously irradiated nine years earlier for a cerebellar medulloblastoma who developed a new enhancing lesion in the primary radiation field four weeks following completion of high dose chemotherapy with bone marrow transplantation for recurrent disease. Resection of this lesion revealed a low grade glioma with no evidence of recurrent medulloblastoma.

We evaluated the antitumor activity of busulfan against a panel of tumor cell lines and xenografts in athymic nude mice derived from childhood high-grade glioma, adult high-grade glioma, ependymoma, and medulloblastoma. Busulfan displayed similar activity against a panel of four medulloblastoma cell lines (D283 Med, Daoy, D341 Med, and D425 Med) and four corresponding sublines with laboratory-generated or clinically acquired resistance to 4-hydroperoxycyclophosphamide [D283 Med (4-HCR), Daoy (4-HCR), D341 Med (4-HCR), and D458 Med] and cross-resistance to melphalan. This is consistent with a nearly total lack of cross-resistance of busulfan to 4-hydroperoxycyclophosphamide. Busulfan was active in the therapy of all but one of the subcutaneous xenografts tested, with growth delays ranging from 14.3 days in D612 EP to 58.4 days in D528 EP. Busulfan produced statistically significant increases in the median survival of mice bearing intracranial D456 MG (66%-90%), D612 EP (18%-33%), and D528 EP (89%) xenografts. These studies suggest that busulfan may be active against medulloblastomas, high-grade gliomas, and ependymomas as well as against cyclophosphamide-resistant neoplasms.

Magnetic resonance imaging (MRI) is becoming the method of choice for evaluation of central nervous system tumors. However, the sensitivity of this modality raises concern that new lesions in patients previously diagnosed with a brain tumor may not necessarily represent recurrent disease. We report a patient previously treated with surgery, radiotherapy, and chemotherapy for a medulloblastoma who developed a new lesion in the floor of the fourth ventricle. Histologic review following excision revealed an arteriovenous malformation.

Background: Among patients with malignant brain tumors, infants and very young children have the worst prognosis and the most severe treatment-related neurotoxic effects. Therefore, in 1986, the Pediatric Oncology Group began a study in which postoperative chemotherapy was given in order to permit a delay in the delivery of radiation to the developing brain., Methods: Children under 36 months of age with biopsy-proved malignant brain tumors were treated postoperatively with two 28-day cycles of cyclophosphamide plus vincristine, followed by one 28-day cycle of cisplatin plus etoposide. This sequence was repeated until the disease progressed or for two years in 132 children 24 months of age at diagnosis and for one year in 66 children 24 to 36 months of age at diagnosis. After this, the patients received radiation therapy. The response to the first two cycles of chemotherapy was measured in 102 patients with residual postoperative disease., Results: The first two cycles of cyclophosphamide and vincristine produced complete or partial responses in 39 percent of the 102 patients who could be evaluated. The response rates were highest among patients with medulloblastomas, malignant gliomas, or ependymomas. Patients with brain-stem gliomas or embryonal tumors (primitive neuroectodermal tumors) had little or no response. The progression-free survival rate was 41 percent at one year for children who were 24 to 36 months old at diagnosis and 39 percent at two years for those under 24 months of age at diagnosis. Multivariate analysis identified embryonal tumors as a significant adverse prognostic feature (relative risk, 2.2; 95 percent confidence interval, 1.4 to 3.4) and complete resection as a favorable feature (relative risk, 0.33; 95 percent confidence interval, 0.20 to 0.54). Complete responses to chemotherapy were associated with a progression-free survival rate approaching that achieved with gross total resection. A comparison of cognitive evaluations obtained at base line and after one year of chemotherapy revealed no evidence of deterioration in cognitive function., Conclusions: Chemotherapy appears to be an effective primary postoperative treatment for many malignant brain tumors in young children. Disease control for one or two years in a large minority of patients permitted a delay in the delivery of radiation and, on the basis of preliminary results, a reduction in neurotoxicity. For patients who had undergone total surgical resection or who had a complete response to chemotherapy, the results are sufficiently encouraging to suggest that radiation therapy may not be needed in this subgroup of children after at least one year of chemotherapy.

O6-Alkylguanine-DNA alkyltransferase (AT) is a cellular protein that protects cells from the cytotoxic effects of nitrosoureas by repairing alkyl lesions at the O6 position of guanine. We have studied the ability of O6-benzylguanine to deplete AT activity in brain tumor xenografts and thereby increase the sensitivity of these tumors to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In toxicity studies, pretreatment of athymic mice with O6-benzylguanine increased the toxicity of BCNU significantly. After i.p. injection of O6-benzylguanine into athymic mice carrying subcutaneous (s.c.) D341MED, a human medulloblastoma xenograft with a high AT activity, the AT activity of the tumors became undetectable within 1 h and remained depleted until 36 h. In s.c. xenografts to D341MED, treatment with O6-benzylguanine followed 1 h later by BCNU produced a significantly greater growth delay (14.8 days) than was seen with BCNU alone (2.3 days). A lower pretreatment dose of O6-benzylguanine produced a significantly smaller therapeutic effect. Delaying the administration of BCNU until 36 h after O6-benzylguanine resulted in a growth delay (1.2 days) that was not significantly different from that produced by the control or BCNU alone. In athymic mice with intracranial (i.c.) xenografts of D341MED, pretreatment with O6-benzylguanine followed 1 h later by BCNU produced a significantly increased survival as compared with that of the control, BCNU alone, O6-benzylguanine alone, and O6-benzylguanine followed 36 h later by BCNU. In experiments with s.c. xenografts of D245MG, a human glioma xenograft with undetectable AT activity, pretreatment with O6-benzylguanine 1 h prior to BCNU produced a significantly greater effect than was seen with BCNU treatment alone. The combination regimen, however, was not as effective as an equitoxic dose of BCNU alone. These studies suggest that O6-benzylguanine may be a useful adjuvant to nitrosourea therapy in human malignancies that exhibit a range of AT activities and that dose and timing are important variables in achieving therapeutic success. These data also indicate that therapeutic potentiation of BCNU by O6-benzylguanine can be achieved in i.c. tumors. As a result, this approach may be useful in the treatment of neoplasms of the central nervous system.

Background: Although chemotherapy offers promise of increased survival for children with medulloblastoma and glioblastoma multiforme, drug resistance occurs frequently, resulting in tumor progression and death. Resistance to nitrosoureas and methylating agents, which damage DNA, can be mediated by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT). Depletion of this protein with alkylguanines or methylating agents, however, restores tumor cell sensitivity to the cytotoxicity of chloroethylnitrosoureas (e.g., carmustine [BCNU])., Purpose: This study was designed to determine whether resistance to the activity of nitrosourea (the drug BCNU) in BCNU-resistant human medulloblastoma (D341 Med) and human glioblastoma multiforme (D-456 MG) can be reversed by the methylating agent streptozocin and the O6-substituted guanines O6-methylguanine and O6-benzylguanine., Methods: Xenografts were grown subcutaneously in athymic BALB/c mice. BCNU was administered as a single intraperitoneal injection at doses of 100 mg/m2, 75 mg/m2, or 38 mg/m2--i.e., 1.0, 0.75, or 0.38, respectively, of the dose lethal to 10% of treated animals (LD10). Mice were treated intraperitoneally with a single dose of O6-benzylguanine or O6-methylguanine (240 mg/m2) or with streptozocin (600 mg/m2) daily for 4 days. Response was assessed by tumor growth delay and tumor regression. AGAT activity in the xenografts was measured at 1 and 6 hours after pretreatment, at the time tumors were excised., Results: Pretreatment with O6-benzylguanine, O6-methylguanine, or streptozocin reduced AGAT activity to 4%, 25%, and 95% of control values, respectively, in D341 Med and 0%, 0%, and 25% of control values, respectively, in D-456 MG 1 hour after injection. After 6 hours, levels changed to 7%, 61%, and 116% of control values in D341 Med and 0%, 79%, and 21% of control values in D-456 MG, respectively. Both D341 Med and D-456 MG xenografts were completely resistant to BCNU at its LD10. Pretreatment with O6-benzylguanine increased BCNU sensitivity in both types of xenograft. In contrast, treatment with BCNU plus O6-methylguanine or streptozocin did not produce growth delays substantially different from those produced by BCNU alone, reflecting the more efficient depletion of AGAT by O6-benzylguanine. Following therapy with BCNU plus O6-benzylguanine at 0.38 LD10, tumor regressions were seen in eight of 10 D341 Med and in all 10 D-456 MG xenografts., Conclusion: We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials.

Mechanisms of tumor resistance to 4-hydroperoxycyclophosphamide (4-HC) were studied by using a panel of human medulloblastoma cell lines either passaged in the laboratory for resistance to 4-HC or established from tumors showing clinical resistance to cyclophosphamide. Multiple distinct mechanisms of resistance were demonstrated. Daoy (4-HCR), a line that was 6-fold more resistant than Daoy, contained elevated levels of aldehyde dehydrogenase (ALDH). Most of the difference in sensitivity between the Daoy (4-HCR) and Daoy cell lines was abolished when 4-HC was replaced with phenylketocyclophosphamide, a 4-HC analogue that cannot be detoxified by ALDH. Thus, elevated levels of ALDH appear to play a role in the resistance of Daoy (4-HCR). Several of the cell lines [D283 Med (4-HCR), D341 Med (4-HCR), Daoy (4-HCR), D458 Med] contained elevated levels of glutathione (GSH). No changes in glutathione-S-transferase activity or isozyme pattern were observed, but in two of these three lines, the elevation in GSH was accompanied by elevated levels of gamma-glutamyl transpeptidase. To confirm the role of elevated GSH content in 4-HC resistance, the sensitivity of the cell lines to 4-HC was repeated after depletion of GSH by treatment with L-buthionine-S,R-sulfoximine. In medulloblastoma cell lines without other mechanisms of resistance, a linear relationship was seen between GSH content and resistance to 4-HC. Moreover, cells with GSH content greater than 5 nmol/mg protein and no other overriding mechanism of resistance could be sensitized to 4-HC treatment with L-buthionine-S,R-sulfoximine. Finally, D283 Med (4-HCR) cells had mild elevations in both ALDH and GSH content, but were resistant to phenylketocyclophosphamide and were not significantly sensitized by L-buthionine-S,R-sulfoximine. This cell line appears to demonstrate a third mechanism of resistance to 4-HC. These results suggest that 4-HC resistance in medulloblastoma can be multifactorial.

Six well characterized human medulloblastoma cell lines (D283 Med, Daoy, D341 Med, D384 Med, D425 Med, and D458 Med) were examined for the expression of O6-methylguanine-DNA methyltransferase (MGMT) by activity and Western and Northern blot analysis. High levels of MGMT activity were present in D283 Med, Daoy, D341 Med, and D384 Med (1.36, 0.80, 1.68, and 1.62 pmol/mg of protein, respectively), but negligible MGMT activity was detected in D425 Med and D458 Med (0.06 and 0.05 pmol/mg of protein, respectively), which were derived separately at different times from the same patient. The presence of MGMT protein and its transcript was demonstrated in D283 Med, Daoy, D341 Med, and D384 Med, but both the protein and the mRNA were undetectable in D425 Med and D458 Med. Nevertheless, all six cell lines contained an apparently unaltered MGMT gene, as determined by Southern blot analysis. The absence of MGMT activity in D425 Med and D458 Med is likely due to the absence of the protein, resulting from a lack of transcription of the MGMT gene. The varying levels of expression of MGMT in medulloblastoma cells found in this study should provide a molecular basis for drug design and selection in chemotherapy of this tumor.

Cytogenetic and molecular studies of medulloblastomas have demonstrated frequent loss of sequences from the short arm of chromosome 17, possibly implicating loss or inactivation of the p53 tumor suppressor gene. We amplified exons 5 through 8 of the p53 gene by the polymerase chain reaction technique. These segments, which encompass the regions usually mutated in human tumors, were sequenced to search for p53 mutations in 12 medulloblastoma tumors, 8 xenografts, and 3 permanent cell lines. Mutation of the p53 gene was found in only 1 of 3 cell lines tested and in none of the xenografts or primary tumors studied. Our results suggest that p53 is mutated in an unusual way or that a second tumor suppressor gene on the short arm of chromosome 17 is involved in the pathogenesis of medulloblastoma.

Medulloblastoma is the most common central nervous system malignancy of childhood, with approximately 350 new cases seen in the United States each year. Although the low incidence of this tumor, compared with adult neoplasms such as breast or colon carcinoma, has limited laboratory and clinical research, recent studies have greatly enhanced our understanding of the biology, phenotype, genotype, and therapy of medulloblastoma. Further efforts to integrate laboratory and clinical studies of this tumor may allow design of novel therapeutic approaches resulting in enhanced disease-free survival.

Three new human medulloblastoma (MB) cell lines (D384 Med, D425 Med, and D458 Med) and their transplantable xenografts were examined for antigenic expression with antibodies against neuroectodermal antigens, cytoskeletal proteins, neuroendocrine markers, glioma-associated antigens, tenascin, human lymphocyte antigen molecules, epidermal growth factor receptor, and T-cell antigen by indirect immunofluorescence, avidin-biotin complex peroxidase immunohistochemistry, and immunoblot methods. We found that each of the three cell lines expressed vimentin; low-, middle-, and high-molecular-weight neurofilament proteins; and the synaptic vesicle membrane glycoprotein synaptophysin. Each of the cell lines also reacted with antibodies against neural cell adhesion molecules, but none of them were positive for antibodies against glial fibrillary acidic protein, keratin, microtubule-associated protein tau and microtubule-associated protein 2, human lymphocyte antigen-DR, epidermal growth factor receptor, and T-cell antigen. Immunoreactivities with anti-tenascin and anti-glioma-associated antibodies were variable in these cell lines. Anti-human lymphocyte antigen-A,B and anti-beta 2-microglobulin antibodies reacted with xenografts of D384 Med and D425 Med and were weakly positive for a small population of D384 Med cultured cells. In summary, the detection of neurofilament proteins and synaptophysin and the absence of glial fibrillary acidic protein provide strong evidence for a neuronal phenotype of D384 Med, D425 Med, and D458 Med.

Virtually all brain tumor models studied to date have demonstrated the activity of the nitrosoureas, an observation confirmed in a cohort of adult patients with high-grade gliomas. However, studies with animal brain tumor models have not led to the selection of other clinically active agents (with the possible exception of AZQ), leading investigators to conduct experiments with human CNS tumor-derived cell lines and xenografts. These studies have identified agents subsequently shown to be active in phase II clinical trials against high-grade glioma and medulloblastoma. Clinical phase III trials in progress will demonstrate if these agents result in increased disease-free survival, which is the ultimate goal of all preclinical therapeutic studies. Future research with these laboratory models may ultimately allow the definition of the mechanisms of drug resistance and the identification of modulations effective in bypassing or reversing this resistance.

Medulloblastoma biopsies are heterogenous and might contain normal brain tissue, which limits the usefulness of such tumor material for biochemical analyses. We have, therefore, examined the gangliosides and their metabolism using the medulloblastoma cell lines. Daoy and D341 Med, cultured both in vitro and as xenografts in nude mice. The ganglioside patterns in the Daoy showed a switch from a high GM2, 70% (mol% of total ganglioside sialic acid) and low lactoseries gangliosides (2%) content in monolayer cultures, to a high proportion of lactoseries gangliosides (50%) and virtually no GM2 (1%) in xenografts, but an increased proportion of other a-series gangliosides. The D341 Med showed a similar change regarding the lacto-series gangliosides from 1% in suspension culture to 10% in xenografts. The activity of five glycosyltransferases, GM3, GD3, GM2, GM1 and LA2 synthases, did not parallel the ganglioside patterns and could not account for the noted variations therein. In the Daoy cell line the LA2 synthase as well as the GM2 synthase activity was relatively high in both culture systems, despite the marked difference in the expression of GM2 and the lactoseries gangliosides. These results suggest that environmental factors play a crucial role for the in vivo activity of the glycosyltransferases.

Medulloblastoma, a highly malignant pediatric tumor of the posterior fossa, demonstrates a marked propensity for leptomeningeal dissemination. Although the predominant site of relapse is the posterior fossa, the prevention of subarachnoid spread would be of significant therapeutic value. The established medulloblastoma cell lines D283 Med, D341 Med, D384 Med, D425 Med, D458 Med and Daoy have been investigated in in vitro adhesion assays for their capacity to bind to the predominant components of the leptomeningeal extracellular matrix: fibronectin, laminin and collagen IV. Growth on the reconstituted basement membrane matrix, Matrigel, was also assayed. Of the five neuronal phenotype DMed lines, all of which grow spontaneously as macrospheroids in standard fetal calf serum-containing tissue culture medium, only D425 Med and its sibline, D458 Med, derived from a subsequent sample from the same patient, displayed adherence to any of the substrata: approximately 20% of input D425 Med cells attached and exhibited cell spread and some extension (adhesion) on fibronectin. All other DMed lines failed to attach to these substrates. The glial phenotype cell line Daoy, which grows as an adherent monolayer under normal culture conditions, exhibited attachment, extension and growth on all substrata as did the glioma cell line U-251 MG and the neuroblastoma cell line SK-N-SH. The lack of attachment, and thus spread on components of the leptomeningeal extracellular matrix under in vitro assay conditions by 5/6 of the medulloblastoma cell lines investigated, is characteristic of neuronally differentiated cells, thus reinforcing the previously described neuronal phenotype of these lines. The readily demonstrated expression of N-CAM and L1 by all of the medulloblastoma cell lines suggests that the primary mode of leptomeningeal extension in vivo may be dependent on such other cell-cell and cell-substrate binding mechanisms.

Despite a considerable amount of information concerning chromosomal and molecular abnormalities found in gliomas in adults, relatively little is known regarding these abnormalities in pediatric brain tumors. We have analyzed DNA from 37 primary brain tumors and 4 tumor-derived cell lines for oncogene amplification. Probes utilized represent 11 known oncogenes (erbB1, gli, neu, myc, L-myc, N-myc, H-ras, K-ras, N-ras, sis, and src). Of 20 primary medulloblastomas studied, only one tumor was found to have erbB1 amplification. In contrast, of the 4 medulloblastoma cell lines studied, 1 had c-myc amplification, 1 had erbB1 amplification, and 1 had amplification of N-myc. Twelve glial brain tumors were analyzed, and only 1 case with amplification of the erbB1 oncogene was found. Other tumors studied include 1 meningioma, 2 ependymomas, 1 anaplastic ependymoma, and 1 cerebral primitive neuroectodermal tumor, none of which had oncogene amplification. These results suggest that oncogene amplification is relatively uncommon in primary medulloblastomas, but the frequency and diversity of oncogene amplification is greater in tumors that can be established as cell lines. The lower frequency of erbB1 amplification in glial brain tumors in children compared to adults is consistent with the generally lower grade of glial tumor histology seen in pediatric patients. However, the case with amplification of the erbB1 oncogene represented 1 of 2 cases of glioblastoma multiforme we studied, which suggests that pediatric glioblastoma multiforme may have a similar frequency of erbB1 oncogene amplification to glioblastomas seen in adults. Our results suggest that oncogene amplification is a relatively uncommon mechanism of oncogene activation in pediatric brain tumors, and they provide molecular evidence for heterogeneity in tumors classified as medulloblastomas.

The development of the Cavitron ultrasonic surgical aspirator (CUSA) has facilitated neurosurgical intervention for removal of central or peripheral nervous system tumors adjacent to or within vital structures. However, laboratory studies defining the phenotypic and genotypic properties of these tumors, both in cell culture and as xenografts in immunoincompetent animals, require viable tumor fragments free of microbial or red blood cell contamination. This report describes the use of a readily available sterile trap with the CUSA which, in conjunction with centrifugation and ammonium chloride lysis of the bloody aspirate, allowed collection of concentrated viable human medulloblastoma tumor cells. These cells were successfully established in cell culture and as transplantable xenografts in athymic mice.

Cultured cell lines and xenografts derived from 7 human medulloblastomas were evaluated for amplification of the c-myc, N-myc, epidermal growth factor receptor, and gli genes by Southern blot analysis. Karyotypes of the original biopsies and early passaged cells demonstrated double minute chromosomes in 4 of the 7 cases. All 7 samples (3 cell lines and 4 xenografts) from the 4 tumors with double minute chromosomes contained amplification of the c-myc gene. Cell lines and xenografts derived from the 3 biopsies without double minute chromosomes failed to demonstrate amplification of the 4 genes which were tested, but a rearrangement of the c-myc gene occurred in 1 of the 3 tumors. These observations demonstrate that the c-myc gene is often amplified and/or rearranged in human medulloblastomas and suggest that amplification of this gene provides a growth advantage for medulloblastoma cells in vitro and in vivo.

Three children with known primary brain neoplasms and leptomeningeal disease were evaluated with MR imaging. Two of the patients had medulloblastoma and one had pineoblastoma. The presence of leptomeningeal tumor spread was established by positive CSF cytopathology in conjunction with compatible contrast-enhanced CT findings. Contrast-enhanced CT, nonenhanced MR, and Gd-DTPA-enhanced MR studies were then compared. In two cases, leptomeningeal lesions were seen better with Gd-DTPA-enhanced MR than with contrast-enhanced CT. In all three cases, Gd-DTPA MR imaging revealed lesions that were not identified on noncontrast MR. Gd-DTPA-enhanced MR imaging is useful when searching for intracranial leptomeningeal tumor deposits in pediatric patients at risk for this condition.

D341 Med is a new continuous cell line and transplantable xenograft derived from a cerebellar medulloblastoma. This line grew in vitro in suspension culture with spontaneous macroscopic spheroid formation and demonstrated 20-fold amplification of c-myc. Cultured D341 Med cells injected subcutaneously into athymic mice grew as markedly cellular, highly invasive undifferentiated neoplasms. Intracranial tumors grew as markedly cellular mitotically active neoplasms largely located within the subarachnoid space or lining the ventricular system. Immunocytochemical analysis of the cell line and SQ tumors revealed the high (NFP-H) and middle (NFP-M) molecular weight (Mr) neurofilament proteins (NFPs). Immunoblots demonstrated the presence of molecular species that co-migrated with authentic human NFP-H and NFP-M. This cell line and transplantable xenograft may allow, in conjunction with the authors' other models of human medulloblastoma, analysis of the heterogeneous biologic properties and therapeutic sensitivity of this tumor.

An extensive panel of monoclonal antibodies (MAb) and monospecific antisera reactive against neuroectodermal-, neuronal-, glial-, and lymphoid-associated antigens, extracellular matrix, HLA, and cell-surface receptors was used to characterize the phenotype of four continuous, karyotypically distinct medulloblastoma cell lines and transplantable xenografts. All four cell lines demonstrated significant reactivity with anti-neuroectodermal-associated MAb. No apparent pattern of reactivity with anti-lymphoid MAb was seen; notably, there was a uniform absence of detectable Thy-1. Review of the complete antibody reactivity profile revealed a dichotomy between lines TE-671 and Daoy and lines D283 Med and D341 Med, which have been previously shown to express neurofilament protein in culture and xenografts, and to exhibit neuroblastic morphological features in biopsy and xenograft tissue sections. TE-671 and Daoy reacted with the MAb directed against tenascin, epidermal growth factor (EGF) receptor, HLA-A,B epitopes, beta 2-microglobulin and 5/8 of the glioma-associated antigens, but did not react with the anti-neurofilament protein (NFP) MAb. D283 Med and D341 Med expressed NFP but did not react with MAb against tenascin, EGF receptor, HLA-A,B epitopes, beta 2-microglobulin or 6/8 and 7/8 (respectively) of the glioma-associated antigens. The observed phenotypic differences provide a conceptual framework for investigating basic differences in the biological behavior of medulloblastoma. Moreover, the subdivisions can be evaluated for prospective value in tissue diagnosis, cerebrospinal fluid cytology and antibody-mediated imaging and therapy.

The human medulloblastoma cell line TE-671 was grown s.c. and intracranially in athymic nude mice. Tumor-bearing animals treated with chemotherapeutic agents or radiation were compared to untreated tumor-bearing controls. Tumors growing s.c. were sensitive to cyclophosphamide and vincristine with growth delays in duplicate trials of 15.8/16.5 and 12.9/15.0 days, respectively. These tumors were minimally responsive to the 2,5-bis(1-aziridinyl-3,6-dioxodiethyl ester of 1,4-cyclohexadiene-1,4-dicarbamic acid (NSC 182986) and cis-diamminedichloroplatinum II and unresponsive to methotrexate, 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido(2,3-d)pyrimidine (NSC 351521), 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC 409962), and procarbazine. Radiation therapy with 2500 or 1500 rads as a single fraction produced a marked response, with growth delays of 39.5 and 21.1 days, respectively. Cyclophosphamide produced a significant (p less than 0.0005) increase in the median survival of mice with intracranial tumors. Vincristine produced a minimal increase in the median survival while no response was seen to the 2,5-bis(1-aziridinyl-3,6-dioxodiethyl ester of (1,4-cyclohexadiene-1,4-dicarbamic acid at the dose level and schedule tested. This model system will allow further analysis of the therapeutic sensitivity of human medulloblastoma to other agents or combined-modality regimens.

Seven classical alkylators were tested for activity against the continuous human medulloblastoma cell line TE-671 grown in vitro and as s.c. and intracranial xenografts in athymic mice. Drugs tested included melphalan, cyclophosphamide (4-hydroperoxycyclophosphamide in vitro), iphosphamide (4-hydroperoxyiphosphamide in vitro), phenylketocyclo-phosphamide, phenylketoiphosphamide, Asta Z 7557, and thiotriethyl-enephosphoramide. All agents were active, with melphalan demonstrating the most activity in vitro and in vivo. Comparative studies of cyclophosphamide and phenylketocyclophosphamide revealed partition coefficients (log P) of 0.73 and greater than 1.69, respectively, and cyclophosphamide exhibited greater cytotoxic activity in post- (equitoxic) drug administration murine plasma. Hematological toxicity was limited to leukopenia/neutropenia for both of these agents. These studies suggest that the classical alkylators may have a role in the treatment of medulloblastoma and provide a means to further analyze their therapeutic potential.

A model was developed for the in vitro and in vivo study of the continuous cell line, TE-671, derived from a human medulloblastoma. TE-671 grew in vitro as sheets of uniform triangular- to spindle-shaped cells with round to oval nuclei and sparse cytoplasm. The in vitro population doubling time was 31.48 hours (h). Mean colony forming efficiency in an agarose medium was 9.0 +/- 3.0%. TE-671 grew subcutaneously in athymic mice as lobulated masses composed of sheets of small uniform cells with round hyperchromatic nuclei and scanty cytoplasm. Perivascular pseudorosettes were commonly seen in early in vivo passage. The in vivo doubling time of subcutaneous tumors was 3.17 +/- 1.02 days, with a latency to 500 mm3 of 20.66 +/- 1.48 days. The cell cycle time (Tc) was 24 h, S phase was 14 h, G1 phase was seven h, and G2 phase was three h. Intracerebral tumors grew as discrete masses composed of diffuse sheets of small anaplastic cells with frequent mitoses and perivascular pseudorosettes. Both in vitro and in vivo chromosome studies revealed modal chromosome counts in the near tetraploid range with the same marker chromosomes as described in the original report of this cell line. The in vitro and in vivo growth of TE-671 provides the best available experimental model for the analysis of the biological properties and chemosensitivity of human medulloblastoma.

The human glioma-derived cell line D-54 MG and the human medulloblastoma-derived cell line TE-671 have been shown to be sensitive in culture to the pharmacological interference with glutamine metabolism by acivicin, 6-diazo-5-oxo-L-norleucine, and methionine sulfoximine. Using as a guide the multiple contributions of glutamine to the biosynthesis of proteins, purines, and pyrimidines, we now have identified six additional antimetabolites active against these lines in vitro at clinically relevant concentrations. The 50% growth-inhibitory levels of the drugs against D-54 MG in 6-day continuous exposure experiments were: L-asparaginase, 0.057 IU/ml; 5-fluorouracil, 0.5 micrograms/ml; 6-mercaptopurine, 0.8 micrograms/ml; actinomycin D, 0.0007 micrograms/ml; N-phosphonacetyl-L-aspartic acid, 2.3 micrograms/ml; and 5-azacytidine, 0.2 micrograms/ml (3-day exposure. The corresponding 50% growth-inhibitory values in TE-671 were: L-asparaginase, 0.54 IU/ml; 5-fluorouracil, 1.5 micrograms/ml; 6-mercaptopurine, 4.7 micrograms/ml; actinomycin D, 0.00044 micrograms/ml; N-phosphonacetyl-L-aspartic acid, 4.5 micrograms/ml; and 5-azacytidine, 0.49 micrograms/ml. Dipyridamole up to 10 micrograms/ml was inactive against both lines. The isobologram method was used to evaluate the effectiveness of several two-drug combinations which were biochemically designed. The sums of the optimal fractional inhibitory concentrations for the pairs were: acivicin plus L-asparaginase, 0.14; acivicin plus methionine sulfoximine, 0.40; 6-diazo-5-oxo-L-norleucine plus methionine sulfoximine, 0.60; acivicin plus 6-mercaptopurine, 1.0, all in TE-671; and acivicin plus 5-fluorouracil, 0.79, in D-54 MG. Our findings suggest that an antimetabolite regimen exploiting glutamine sensitivity might improve the chemotherapy of some human gliomas and medulloblastomas.

An in vitro clonogenic assay was used to test the chemosensitivity of the human medulloblastoma cell line TE-671. Dose-response relationships for reduction in colony formation were generated for cyclophosphamide, vincristine, Adriamycin, 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC 409962), and 1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-diethylester (NSC 182986); and the in vitro drug dose at which there is a 75% reduction in the number of colonies in comparison to controls (ID75S) were derived from these data. Methotrexate produced no colony reduction at any dose tested up to 1000 micrograms/ml. The in vitro results were compared to growth delays in s.c. TE-671 xenografts in athymic mice treated with the same agents. Agents with an ID75 less than assumed in vivo plasma drug concentrations were all active in vivo, whereas two of the three agents with an ID75 greater than assumed in vivo plasma drug concentrations demonstrated no in vivo activity. These results suggest that for these agents, the relationship between the ID75 of the drug and its in vivo concentration allows in vitro clonogenic assay results to agree with in vivo growth delay responses.

We have evaluated the efficacy of intravenous 2,5-diaziridinyl-3,6-biscarboethoxyamino-1,4-benzoquinone (diaziquone or AZQ, NSC-182986) in the treatment of recurrent primary anaplastic brain tumors. Three of 16 evaluable patients (18.8%) showed clinical and radiographic improvement that permitted discontinuation of corticosteroids, 4 patients (25%) showed either clinical or radiographic improvement and were considered partial responders, and 9 patients (56.2%) showed no effects after two courses of AZQ. The treatment was well tolerated, and hematologic toxicity was mild. Pharmacokinetic studies indicated rapid decay of the parent compound from plasma using two different infusion schedules. These results compare favorably with those obtained using the nitrosoureas or procarbazine as single agents.

The level of O6-alkylguanine-deoxyribonucleic acid (DNA) alkyltransferase (AT) was determined in 15 human brain-tumor xenografts in athymic mice. This enzyme is a primary intracellular repair mechanism for lesions produced at the O6 position of guanine by a wide range of alkylating agents, including nitrosoureas and procarbazine. Its activity ranged from undetectable in five tumor lines to 2338 fmol/mg protein in N-1941, a human glioblastoma xenograft. The sensitivity of 10 of these xenografts to procarbazine was determined and it was found that four of the five tumor lines with AT levels of more than 100 fmol/mg protein had growth delays after procarbazine treatment of less than 20 days, whereas all five lines with undetectable AT levels had growth delays of over 30 days. The primary cytotoxic DNA adduct produced by procarbazine (namely, O6-methylguanine) was found to be significantly higher in two sensitive lines with low AT levels than in a highly resistant line with a high AT level. These data suggest that the AT levels of individual brain tumors can be used as predictive indicators of their susceptibility to drugs that exert their antineoplastic effect primarily by O6-alkylation of guanine in nuclear DNA.