The O6‐methylguanine‐DNA methyltransferase (MGMT) plays a major role in repairing DNA damage from alkylating agents. In several human neoplasms including low‐grade diffuse astrocytomas, promoter hypermethylation of MGMT has been shown to correlate with an increased frequency of p53 mutation. In the present study, we analyzed MGMT promoter methylation by the methylation‐specific PCR in 49 newly diagnosed WHO grade II astrocytomas and evaluated its clinical usefulness. MGMT promoter methylation was found in 21 (43%) of the 49 tumors. A tight correlation existed between MGMT methylation and p53 protein accumulation (P=0.0424). The presence of MGMT methylation was significantly associated with a shorter progression free survival (PFS) on both univariate analysis (P=0.0014) and multivariate analysis (P=0.0081). It was a more powerful determinant of the PFS than age, sex, performance status, proliferative activity, or p53 expression, and was independent of the extent of surgery. In terms of the overall survival, MGMT methylation demonstrated a prognostic utility in the univariate analysis but not in the multivariate analysis. The present findings indicate that aberrant methylation of the MGMT promoter independently augurs for an unfavorable clinical course in patients with low‐grade diffuse astrocytomas. Since the presence of MGMT methylation is expected to predict an increased sensitivity to alkylating chemotherapeutic agents, earlier chemotherapy could serve to improve an unfavorable natural history in tumors with MGMT methylation.
Pituitary adenomas are common benign intracranial neoplasms. However, their tumorigenesis is not yet clearly defined. Inactivation of genes involved in the negative cell-cycle regulatory p15(INK4b) - p16(INK4a) -cyclin D/CDK4-RB1-mediated pathway (RB1 pathway) is one of the most common and important mechanisms in the growth advantage of tumor cells. Recently, much attention has been focused on the importance of alternative mechanisms of gene inactivation, particularly promoter hypermethylation in the transcriptional silencing of such tumor-suppressor genes. Based on the rare occurrence of inactivation by gene mutations and deletions of the RB1 pathway in pituitary adenomas, we investigated the deregulation of the RB1 pathway in 42 sporadic human pituitary adenomas, especially focusing on the methylation status of this pathway as determined by a methylation-specific polymerase chain reaction assay. Homozygous deletion of the p15(INK4b) or p16(INK4a) gene was detected in one adenoma each. Amplification of the CDK4 gene was not apparent in any of the pituitary adenomas presently examined. Promoter hypermethylation of the p15(INK4b), p16(INK4a), and RB1 genes was detected in 15 (35.7%), 30 (71.4%), and 12 (28.6%) of the adenomas, respectively. Promoter hypermethylation of the p15(INK4b) gene coincided with p16(INK4a) alteration and/or RB1 methylation, whereas p16(INK4a) and RB1 methylations tended to be mutually exclusive (p = 0.019). Thus, the vast majority of the adenomas (38 of 42, 90.5%) displayed alterations of the RB1 pathway. None of the clinicopathologic features, including the proliferation cell index, was significantly correlated with any particular methylation status. Our results suggest that inactivation of the RB1 pathway may play a causal role in pituitary tumorigenesis, with hypermethylation of the p16(INK4a) gene being the most common deregulation, and further provide evidence that RB1 and p16(INK4a) methylations tend to be mutually exclusive but occasionally coincide with p15(INK4b) methylation.
Aberrant hypermethylation of CpG islands in the promoter region plays a causal role in the inactivation of various key genes involved in the cell cycle regulatory cascade, which could result in a loss of cell cycle control. The aim of the present study was to examine in more detail the prevalence and role of the promoter methylation of genes with a proven involvement in the cell cycle regulation of pituitary adenomas, since their tumorigenesis has not yet been clearly defined. We profiled the CpG island methylation status of a series of well-characterized cell cycle regulation genes: the RB1, p14(ARF), p15(INK4b), p16(INK4a), p21(Waf1/Cip1), p27(Kip1), and p73 genes, in 34 pituitary adenomas as determined by a methylation-specific polymerase chain reaction assay. Promoter hypermethylation of the RB1, p14(ARF), p15(INK4b), p16(INK4a), p21(Waf1/Cip1), p27(Kip1), and p73 genes was detected in 12 (35%), 2 (6%), 11 (32%), 20 (59%), 1 (3%), 0 (0%), and 4 (12%) of the adenomas, respectively. In total, 88% (30 of 34) of the adenomas displayed methylation of at least one of such cell cycle regulatory genes, especially methylation of the member genes of the RB1 pathway (29 of 34; 85%). Promoter hypermethylation of p15(INK4b) coincided with RB1 and/or p16(INK4a) methylation, whereas RB1 and p16(INK4a) methylations tended to be mutually exclusive (p = 0.0048). Furthermore, promoter hypermethylations of p14(ARF), p21(Waf1/Cip1), and p73 (not belonging to the member genes of the RB1 pathway) were also coincident with RB1 and/or p16(INK4a) methylation except in one p73 methylated case. In contrast, none of the clinicopathological features, including the cell proliferation index, was significantly correlated with any particular methylation status. Our results suggested that aberrant hypermethylation of the key cell cycle regulatory genes occurs at a relatively high frequency in pituitary adenomas, especially in RB1 pathway genes with promoter hypermethylation of the p16(INK4a) gene being the most common deregulation. We further obtained evidence to indicate that RB1 and p16(INK4a) methylations tended to be mutually exclusive, but did occasionally coincide with other cell cycle regulation gene methylations.
The precise mechanisms governing the direct effect of IFN-beta, including apoptosis induction, are not yet fully understood. To gain a better insight into these mechanisms, we investigated the signaling pathways focusing particularly on interferon regulatory factor 1 (IRF-1) and IRF-2 in glioblastoma cell lines. Furthermore, we attempted to determine whether or not IRF-1 and IRF-2 act as additional prognostic indicators in diffusely infiltrating astrocytomas (DIA). We first assessed the cytotoxic effects of IFN-beta based on a cell growth study and modified MTT assay, and then quantified the apoptosis using a sandwich enzyme immunoassay following IFN-beta treatment in the cell lines, U-87MG, T98G, and A-172. Subsequently, we carried out an analysis of apoptosis-related molecules as evaluated by densitometric analysis of Western blots, focusing on IRF-1 and IRF-2, and two major initiator caspases, caspase-8 and caspase-9. Furthermore, we assessed the expression of type I IFN receptor, IRF-1, and IRF-2 using immunohistochemical techniques in 63 DIA (15 of WHO grade II, 18 of grade III, and 30 of grade IV), and analyzed their impact on prognosis. An increase in apoptosis was apparent after 48 h of IFN-beta treatment (1 x 10(4) IU/ml) in T98G but not in U-87MG or A-172. IFN-beta treatment for 6 h significantly enhanced the expression of IRF-1 in all three cell lines. However, an enhanced expression of IRF-2 was observed only in the not-most-sensitive, non-apoptosis-induced U-87MG and A-172. While minimal processing of caspase-8 was noted in the three cell lines throughout the experiment, caspase-9 activation was observed in the apoptosis-detected T98G after 48 h of treatment, as indicated by a 1.33-fold increase (P=0.037). On the other hand, the IRF-1 LI and IRF-1/IRF-2 LI ratio were greater in low-grade DAI, and were negatively correlated with the histopathological grade in DIA (P=0.017 and P=0.001, respectively). Furthermore, the IRF-1/IRF-2 LI ratio was negatively correlated with the MIB-1 LI in DIA (P=0.004), and represented an independent and most powerful determinant of overall survival compared to other conventional prognostic factors (P=0.018). However, the relation was not statistically significant when only patients with high-grade DIA were assessed. Our findings suggest that up-regulation of IRF-1 and IRF-2 might be an important determinant of susceptibility to IFN-beta mediated cytotoxicity including apoptosis. Furthermore, the IRF-1/IRF-2 LI ratio may reflect the proliferative state of DIA and constitute an important prognostic marker in DIA. Thus, IRF-1 and IRF-2 could represent one of the therapeutic target sites for the regulation of cell growth in DIA.
Pleomorphic granular cell astrocytoma in the pineal region is exceedingly rare, and its clinicopathological features are distinctive. A 67-year-old woman was admitted with a staggering gait. Magnetic resonance imaging revealed a mass lesion at the pineal gland accompanied by obstructive hydrocephalus. Following surgery, pathological examinations demonstrated a pleomorphic granular cell astrocytoma. The patient has been free from recurrence for 24 months after surgery without adjuvant therapy. The specimen exhibited nuclear and cytoplasmic pleomorphism. The nuclei varied in size, shape and coarseness. Variability was also observed in the eosinophilic granular bodies, Rosenthal fibers and spindle-shaped tumor cells. GFAP, S-100 and vimentin were immunohistochemically positive. Reticulin network was absent between the tumor cells, and granular cells with ballooned cytoplasm showing positive staining for PAS. Pleomorphic granular cell astrocytoma is believed to be a form of astrocytoma originating from the pineal gland. Its clinicopathological features resemble those of pleomorphic xanthoastrocytoma. However, it can be differentiated from the latter by the absence of reticulin fibers, absence of basement membrane between adjacent cells, and presence of large numbers of mitochondria.
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