Allan J. Yates

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Alterations of chromosome arms 1p and 19q as predictors of survival in oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas

PURPOSE A recent report suggests that alterations of chromosome arms 1p and 19q are associated with chemotherapeutic response and overall survival in anaplastic oligodendroglioma patients treated with procarbazine, lomustine, and vincristine chemotherapy. We set out to further clarify the diagnostic and prognostic implications of these alterations in a broader set of diffuse gliomas, including astrocytic neoplasms and low-grade oligodendrogliomas. PATIENTS AND METHODS Fluorescence in situ hybridization (FISH) signals from DNA probes mapping to 1p and 19q common deletion regions were enumerated in 162 diffuse gliomas (79 astrocytomas, 52 oligodendrogliomas, and 31 mixed oligoastrocytomas), collected as part of an ongoing prospective investigation of CNS tumors. RESULTS The oligodendroglial phenotype was highly associated with loss of 1p (P =.0002), loss of 19q (P <.0001), and combined loss of 1p and 19q (P <.0001). Combined loss of 1p and 19q was identified as a univariate predictor of prolonged overall survival among patients with pure oligodendroglioma (log-rank, P =.03) and remained a significant predictor after adjusting for the effects of patient age and tumor grade (P <.01). This favorable association was not evident in patients with astrocytoma or mixed oligoastrocytoma. CONCLUSION Combined loss of 1p and 19q is a statistically significant predictor of prolonged survival in patients with pure oligodendroglioma, independent of tumor grade. Given the lack of this association in patients with astrocytic neoplasms and the previously demonstrated chemosensitivity of oligodendrogliomas, a combined approach of histologic and genotypic assessment could potentially improve existing strategies for patient stratification and management.

Improving diagnostic accuracy and interobserver concordance in the classification and grading of primary gliomas

Accurate histologic diagnosis of gliomas is fundamental to proper patient management and to the interpretation of basic and clinical investigations. Diagnostic accuracy and reproducibility are compromised by the subjective histologic criteria currently used to classify and grade gliomas.

Localization of common deletion regions on 1p and 19q in human gliomas and their association with histological subtype

Allelic alterations of chromosomes 1 and 19 are frequent events in human diffuse gliomas and have recently proven to be strong predictors of chemotherapeutic response and prolonged survival in oligodendrogliomas (Cairncross et al., 1998; Smith et al., submitted). Using 115 human diffuse gliomas, we localized regions of common allelic loss on chromosomes 1 and 19 and assessed the association of these deletion intervals with glioma histological subtypes. Further, we evaluated the capacity of multiple modalities to detect these alterations, including loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH). The correlation coefficients for detection of 1p and 19q alterations, respectively, between modalities were: 0.98 and 0.87 for LOH and FISH, 0.79 and 0.60 for LOH and CGH, and 0.79 and 0.53 for FISH and CGH. Minimal deletion regions were defined on 19q13.3 (D19S412-D19S596) and 1p (D1S468-D1S1612). Loss of the 1p36 region was found in 18% of astrocytomas (10/55) and in 73% (24/33) of oligodendrogliomas (P<0.0001), and loss of the 19q13.3 region was found in 38% (21/55) of astrocytomas and 73% (24/33) of oligodendrogliomas (P=0.0017). Loss of both regions was found in 11% (6/55) of astrocytomas and in 64% (21/33) of oligodendrogliomas (P<0.0001). All gliomas with LOH on either 1p or 19q demonstrated loss of the corresponding FISH probe, 1p36 or 19q13.3, suggesting not only locations of putative tumor suppressor genes, but also a simple assay for assessment of 1p and 19q alterations as diagnostic and prognostic markers.

Randomized phase III trial in childhood high-grade astrocytoma comparing vincristine, lomustine, and prednisone with the eight-drugs-in-1-day regimen. Childrens Cancer Group.

PURPOSE In a previous randomized trial, the addition of adjuvant chemotherapy to postoperative radiotherapy proved beneficial in the treatment of childhood high-grade astrocytomas. The present study tests the hypothesis that an eight-drug adjuvant chemotherapy regimen would improve survival in such children compared with the three-drug regimen of the prior study. PATIENTS AND METHODS Between April 1985 and May 1990, patients between the ages of 18 months and 21 years with newly diagnosed high-grade astrocytomas were eligible for this study, as determined by the treating institutions histopathologic diagnosis. Treatment consisted of postoperative local-field radiotherapy and adjuvant chemotherapy, either lomustine (CCNU), vincristine, and prednisone (control regimen) or eight-drugs-in-1-day chemotherapy (experimental regimen). Two cycles of postoperative preirradiation chemotherapy were administered in the experimental regimen. Patients were evaluated radiographically every 3 months after irradiation. RESULTS Eighty-five eligible patients were randomized to the control regimen and 87 to the experimental regimen. The progression-free survival (PFS) and overall survival (OS) at 5 years were 33% (SE = 5%) and 36% (SE = 6%), respectively. There was no statistical difference in outcome between the two chemotherapy regimens. In patients with confirmed diagnoses of anaplastic astrocytoma (AA) or glioblastoma multiforme (GBM), anaplastic astrocytoma, greater than 90% resection, and nonmidline tumor location were characteristics predictive of an improved PFS. There was a difference in toxicity between the two chemotherapeutic regimens, with greater myelosuppression and hearing loss in the experimental regimen. Tumor recurrence occurred primarily within the primary tumor site. CONCLUSIONS There is no benefit to the treatment of high-grade astrocytomas in children with eight-drugs-in-1-day chemotherapy compared with CCNU, vincristine, and prednisone. Extent of tumor resection and histopathologic diagnosis are significant prognostic variables. The overall outcome for children with high-grade astrocytomas remains poor.

Pilot study of high-dose thiotepa and etoposide with autologous bone marrow rescue in children and young adults with recurrent CNS tumors. The Children's Cancer Group.

PURPOSE This study was designed to determine the toxicity, radiographic response rate, and outcome following high-dose thiotepa, etoposide, and autologous bone marrow rescue (ABMR) for young patients with recurrent malignant brain tumors. METHODS Eligibility criteria required adequate renal, hepatic, and pulmonary function, and no bone marrow infiltration. Thiotepa 300 mg/m2 and etoposide 500 mg/ m2 were infused on 3 consecutive days, and autologous bone marrow was infused 72 hours following chemotherapy. RESULTS Forty-five patients with recurrent high-grade brain tumors, aged 8 months to 36 years (median, 8 years), were treated. Seven patients (16%) died of treatment-related toxicities within 56 days of marrow reinfusion. Delayed platelet engraftment occurred in 44% of patients who survived beyond day 56. Of 35 patients with radiographically measurable disease who survived at least 28 days following ABMR, there were two complete responses (CRs) and six partial responses (PRs), for an overall response (CRs plus PRs) rate of 23% (SE = 7%). Objective responses were observed in four of 14 assessable patients with high-grade glioma and in two of six with primitive neuroectodermal tumors (PNETs)/ medulloblastoma. Survival was significantly improved in patients treated with minimal residual disease (P < .0005). Five of 18 patients (28%) with high-grade gliomas remain free of disease at 39+, 44+, 49+, 52+, and 59+ months post-ABMR. CONCLUSION The combination of high-dose thiotepa and etoposide has activity against a variety of recurrent childhood brain tumors. These results merit further evaluation in children and young adults with both recurrent and newly diagnosed high-grade brain tumors.

High-dose carboplatin, thiotepa, and etoposide with autologous stem-cell rescue for patients with recurrent medulloblastoma. Children's Cancer Group.

PURPOSE Medulloblastoma is a highly lethal disease when it recurs. Very few patients survive with conventional treatment. This study evaluated the use of high-dose carboplatin, thiotepa, and etoposide with autologous stem-cell rescue (ASCR) in patients with recurrent medulloblastoma. METHODS Chemotherapy consisted of carboplatin 500 mg/m2 (or area under the curve = 7 mg/mL x min via Calvert formula) on days -8, -7, and -6; and thiotepa 300 mg/m2 and etoposide 250 mg/m2 on days -5, -4, and -3; followed by ASCR on day 0. In addition to the study-prescribed therapy, 21 patients received other treatment: neurosurgical resection in seven, conventional chemotherapy in 17, and external-beam irradiation in 11 cases. RESULTS Twenty-three patients with recurrent medulloblastoma, aged two to 44 years (median, 13 years) at ASCR, were treated. Three patients died of treatment-related toxicities within 21 days of ASCR; multiorgan system failure in two, and Aspergillus infection with venoocclusive disease in one. Seven of 23 patients (30%) are event-free survivors at a median of 54 months post-ASCR (range, 24 to 78 months). Kaplan-Meier estimates of event-free (EFS) and overall survival are 34% +/- 10% and 46% +/- 11%, respectively, at 36 months post-ASCR. CONCLUSION This strategy may provide long-term survival for some patients with recurrent medulloblastoma.

O6-Methylguanine-DNA Methyltransferase Expression Strongly Correlates With Outcome in Childhood Malignant Gliomas: Results From the CCG-945 Cohort

PURPOSE O6-methylguanine-DNA methyltransferase (MGMT) functions to counteract the cytotoxic effects of alkylating agents, such as nitrosoureas, which play a central role in the treatment of childhood malignant gliomas. Epigenetic silencing of MGMT has been associated with prolonged survival in adults with malignant gliomas, although the association between MGMT expression status and outcome in pediatric malignant gliomas has not been defined. METHODS We examined the association between MGMT expression and survival duration using tumor samples from the Childrens Cancer Group 945 study, the largest randomized trial for childhood malignant gliomas completed to date. All patients received alkylator-based chemotherapy as a component of adjuvant therapy. Archival histopathologic material yielded tissue of sufficient quality for immunohistochemical assessment of MGMT expression status in 109 specimens. RESULTS Twelve of the 109 samples demonstrated overexpression of MGMT compared with normal brain. Five-year progression-free survival was 42.1% +/- 5% in the 97 patients whose tumors had low levels of MGMT expression versus 8.3% +/- 8% in the 12 patients whose tumors overexpressed MGMT (P = .017, exact log-rank test). The association between MGMT overexpression and adverse outcome remained significant after stratifying for institutional histologic diagnosis (eg, anaplastic astrocytoma or glioblastoma multiforme), as well as age, amount of residual tumor, and tumor location. CONCLUSION Overexpression of MGMT in childhood malignant gliomas is strongly associated with an adverse outcome in children treated with alkylator-based chemotherapy, independently of a variety of clinical prognostic factors.

'spindle cell oncocytoma' of the adenohypophysis: A tumor of folliculostellate cells?

We describe five primary tumors of the adenohypophysis featuring mitochondrion-rich spindle cells. The patient ages ranged from 53 to 71 years (mean 61.6 years); two were female. All presented with panhypopituitarism. Two also had visual field defect. On neuroimaging all tumors showed suprasellar extension and were indistinguishable from pituitary adenoma. None showed imaging or operative evidence of dural involvement. All were gross totally removed: four by transsphenoidal surgery and one by frontal craniotomy. Follow-up ranged from 2 to 68 months (mean 35.4 months). No recurrences were noted. The clinical workup was noncontributory in all but two patients: one (case no. 4) with an oncocytic thyroid adenoma and another (case no. 5) with squamous carcinoma of both the uterine cervix and of vocal cord. Histologically, the five tumors were composed mainly of fascicles of spindle cells with eosinophilic, granular cytoplasm. Mitoses were rare and necrosis was absent. Neoplastic cells were immunoreactive for vimentin, epithelial membrane antigen, S-100 protein, and galectin-3. Stains for pituitary hormones, synaptophysin, chromogranin, glial fibrillary acidic protein, cytokeratin CAM5.2, smooth muscle actin, CD34, and CD68 were negative. No thyroglobulin immunoreactivity was noted in the tumor of case no. 4. Ultrastructurally, the neoplastic cells contained numerous mitochondria with lamellar cristae. The neoplastic cells were linked by intermediate junctions and desmosomes. No secretory granules were noted. The histologic, immunohistochemical, and fine structural features of these tumors were unlike those of pituitary adenoma or any other primary sellar tumor. A derivation from adenohypophyseal folliculostellate cells is suggested.

A Review and Predictive Models of Gang ioside Uptake by Biological Membranes

Gangliosides are sialic acid-containing glycolipids that are present in the plasma membrane of all mammalian cells (Ledeen and Yu, 1982; Stults et al., 1989). They are particularly abundant in neurons, comprising 10% of the total lipids (Wiegandt, 1971; Ledeen, 1978). Gangliosides are located asymmetrically in the plasmalemma with their hydrophilic, oligosaccharide chains protruding from the extracellular surface and their nonpolar portion (ceramide) inserted into the hydrophobic regions of the membrane (Wiegandt, 197 1, 1982; Bretscher, 1973; Fishman and Brady, 1976; Hansson et al., 1977). Gangliosides added exogenously to culture media are taken up by a wide range of cells in vitro (Leon et al., 1982, 1988; Ghidoni et al., 1989; Riboni et al., 1990), including astrocytes (Masco et al., 1989), glioma (Fishman et al., 1980), and neuroblastoma (Leskawa et al., 1989) cells, fibroblasts (Keenan et al., 1974; Fishman et al., 1977; Giglioni et al., 1990), both red and white human blood cells (Ackerman et al., 1980), and leukemic and nonleukemic lymphocytes (Krishnaraj et al., 1980). Gangliosides delivered to cells this way have a variety of biological effects depending upon the cell type. One such effect that has been studied extensively is neurite formation (neuritogenesis) by neuronal cells (Morgan and Seifert, 1979; Byrne et al., 1983; Ferrari et al., 1983; Facci et al., 1984; Schengrund, 1990). Exogenous gangliosides are incorporated into the plasma membrane (Cuatrecasas, 1973; Keenan et al., 1974, 1975; Gill and King, 1975; Holmgren et al., 1975; Naiki and Marcus, 1976; Sharom and Grant, 1978; Yamakawa and Nagai, 1978; Kanda et al., 1982~ ; Facci et al., 1984; Riboni and Tettamanti, 199 1) by a timeand concentration-dependent process (Moss et al., 1976; Morgan and Seifert, 1979; Skaper et al., 1988) without a change in their chemical nature, so that, following incorporation, they are functionally and metabolically active (Moss et al., 1976). The mechanisms through which gangliosides exert their effects are not clear, but several have been proposed (Fass and Ramirez, 1984; Karpiak and Mahadik, 1984; Leon et al., 1984; Schonfeld et al., 1984; Toffano et al., 1984; Vaswani et al., 1990; Chu and Sharom, 199 1). These include alterations of several enzyme activities (Partington and Daly, 1979; Leon et al., 1981; Yates et al., 1988; Chan, 1989), protein phosphorylation (Bremer et al., 1984; Chan, 1988; Tsuji et al., 1988; Kai-Foon, 1989; Rafi et al., 1990; Bassi et al., 199 1 ; Weis and Davis, 199 l), some second messenger systems (Leon et al., 1982), and membrane permeability (Sarti et al., 1990). Although the addition of exogenous gangliosides is a widely used approach to studying their effects on cellular systems in vitro, the conditions under which they are added have not yet been standardized, making specific comparisons among different experiments difficult. In this article, we review many of the variables that are involved in the uptake and metabolism of exogenous gangliosides by cells and biological membranes. Under Predictive Models of Ganglioside Uptake, we present equations we have developed that relate the amounts of incorporated ganglioside to the major variables affecting ganglioside uptake. Under Factors that Affect Net Ganglioside Incorporation, we discuss several of the variables that are either qualitative or quantitative, but for which currently available data are insufficient to treat in a quantitative fashion.