Safety evaluation of substituting clopidogrel for ticlopidine in Japanese patients with ischemic stroke--Hiroshima ticlopidine, clopidogrel safe exchange trial.

BACKGROUND: Clopidogrel is sometimes substituted for ticlopidine when cerebrovascular or cardiovascular patients develop hematologic abnormalities after ticlopidine treatment. However, the adverse event rate after the substitution to clopidogrel remains undetermined. Therefore, in this study, we aimed to define the risk of adverse events after substituting clopidogrel for ticlopidine without a washout period. METHODS: We prospectively enrolled patients older than 20 years who had a history of noncardioembolic strokes, including transient ischemic attacks, were treated with ticlopidine for at least 6 months. This study was conducted from August 26, 2008, when the first patient was enrolled, to January 16, 2012, the date of the last patient examination, at 8 active stroke centers in Hiroshima, Japan. We excluded patients who had severe disabilities, evidence of cardioembolic stroke, or history of a bleeding event. Each patient received clopidogrel (either 50 mg or 75 mg) once a day in place of ticlopidine without a washout period. Follow-up exams were scheduled within 12 months after the medication substitution. The primary end point of this study was adverse events of interest, including clinically significant reduced blood cell counts, hepatic dysfunction, bleeding, and other serious side effects. RESULTS: In this study, 110 patients were enrolled and analyzed in an intent-to-treat manner (modified intent to treat). Within the scheduled follow-up periods, 9 primary end point events were observed in separate patients. The primary end point events were observed at a rate of 8.4% per year (Kaplan-Meier method). At the time of enrolment, 16 patients met the exclusion criteria, of which 8 recovered from their abnormal hematologic results to the institutional normal limit after the substitution of ticlopidine for clopidogrel (57.4% per year). CONCLUSIONS: The adverse event rates after the substitution of ticlopidine for clopidogrel is similar to the adverse event rates of patients who were initially treated with clopidogrel. The substitution of clopidogrel for ticlopidine should be considered for patients who develop hematologic abnormalities from ticlopidine treatment.

Pleomorphic granular cell astrocytoma in the pineal gland: case report.

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.

Promoter hypermethylation profile of cell cycle regulator genes in pituitary adenomas.

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.

Therapeutic implications of interferon regulatory factor (IRF)-1 and IRF-2 in diffusely infiltrating astrocytomas (DIA): response to interferon (IFN)-beta in glioblastoma cells and prognostic value for DIA.

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.

Aberrant TP53 protein accumulation in the neuronal component of ganglioglioma.

Gangliogliomas are characterized by their different phenotypic composition of ganglion cells and glial cells. In contrast to the glial cells that are capable of mitotic activity, the ganglion cells are generally considered to lack a neoplastic nature. The authors report here the first unequivocal case of a ganglioglioma harboring aberrant TP53 product that was expressed predominantly in the neuronal component. GeneChip TP53 assay revealed a point mutation resulting in an exchange of amino acid. This case suggests that ganglion cells can participate in the neoplastic process of gangliogliomas.

The p15(INK4b)/p16(INK4a)/RB1 pathway is frequently deregulated in human pituitary adenomas.

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.

Metastatic pituitary tumor from renal cell carcinoma treated by fractionated stereotactic radiotherapy--case report.

A 63-year-old man presented with rapidly progressive visual field deficit and hypopituitarism including diabetes insipidus, 8 years after treatment for a renal cell carcinoma. Neuroimaging studies revealed a dumbbell-shaped pituitary mass that had destroyed the sellar floor and abutted against the optic apparatus. Fractionated stereotactic radiotherapy (SRT), employing computer-image integration techniques and a frame that could be relocated to facilitate a fractionated dosing scheme, was carried out under a plan for reducing the treatment risk to the optic apparatus. Three months later, the patient exhibited marked improvement in the visual field deficit and visual acuity concomitant with a reduction in tumor volume. Magnetic resonance imaging of the sellar region confirmed striking shrinkage of the metastasis. His neurological status remained stable at 12 months after the SRT with no complications. Fractionated SRT appears to be effective for preserving or improving the residual vision in patients with visual loss secondary to metastatic tumor of the pituitary gland, and may result in a longer and better quality of life.

Deregulation of the TP53/p14ARF tumor suppressor pathway in low-grade diffuse astrocytomas and its influence on clinical course.

PURPOSE: The chromosome 9p21 region harbors three tumor suppressor genes, p14(ARF), p15(INK4b), and p16(INK4a), all of which can be targets for hypermethylation-associated inactivation in low-grade gliomas. p16(INK4a) and p15(INK4b) are critically involved in the RB1 pathway, whereas p14(ARF) acts as an upstream regulator of the TP53 pathway. The role of each tumor suppressor pathway in low-grade diffuse astrocytomas and their relationships with clinical behavior remain to be elucidated. EXPERIMENTAL DESIGN: We assessed the alterations of the RB1/CDK4/p16(INK4a)/p15(INK4b) and the TP53/MDM2/p14(ARF) pathways in 46 WHO grade II astrocytomas and analyzed their impact on prognosis. RESULTS: The TP53/MDM2/p14(ARF) pathway was altered in 32 of 46 cases (70%) by either TP53 mutation (25 cases) or p14(ARF) methylation (9 cases). The RB1/CDK4/p16(INK4a)/p15(INK4b) pathway was disrupted in 6 of 46 cases (13%) by either RB1 methylation (1 case), p16(INK4a) methylation (3 cases), or p15(INK4b) methylation or homozygous deletion (3 cases). Generally speaking, individual tumors thus tended to display alteration of only one component from both pathways. Any independently analyzed genetic alteration failed to provide statistically prognostic information. The alternate or simultaneous presence of TP53 mutation and p14(ARF) methylation emerged as a univariate predictor of a shorter progression-free survival (P = 0.0456) but was not statistically significant when age and extent of surgery were included in the analysis. CONCLUSIONS: Alternative disruption of the TP53/p14(ARF) pathway represents a frequent event in low-grade diffuse astrocytomas and correlates with an unfavorable clinical course. However, its value is unlikely to include prognostic utility that is independent of other conventional prognostic factors.

[Therapeutic efficacy and prognostic factors in diffuse astrocytomas].

Diffuse astrocytomas are slowly growing tumors with a relatively long overall survival. Considerable controversy exists as to the best therapeutic management for patients with such tumors. In the present study, we retrospectively analyzed a series of 64 patients with WHO grade II astrocytomas of the cerebral hemispheres. Gross total resection and interferon-beta therapy were significantly associated with both longer progression free survival (PFS) and overall survival (OS). Immediate postoperative radiation therapy did not prolong either the PFS or OS. The presence of promoter hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene was an independent predictor of a shorter PFS. Our data suggest that radical surgery plus interferon-beta therapy may offer the best chance for long survival. Since the presence of MGMT methylation is a probable indication of an increased sensitivity to alkylating chemotherapeutic agents, determining the methylation status of MGMT could provide a potential basis for logical therapeutic intervention in identifying a subgroup of patients who could be candidates for early chemotherapy.

Long-term remission of primary central nervous system lymphoma by intensified methotrexate chemotherapy.

High-dose (1-3.5 g/m2) methotrexate (MTX) followed by whole-brain radiation therapy (WBRT) has consistently improved length of survival in primary central nervous system lymphoma (PCNSL), but the prognosis remains dismal. To optimize and enhance the dose intensity of MTX, we applied MTX at 8 g/m2 to 20 patients with PCNSL. In an effort to lower the risk of neurotoxic treatment sequelae, the WBRT dose was reduced to 30 Gy in cases of complete remission after MTX therapy. Further, omission of WBRT and administration of stereotactic radiotherapy (SRT) were undertaken in 3 older patients. The overall response rate to the MTX therapy was 83%. The median progression free survival (PFS) was 54 months with a median overall survival (OS) of 57 months. Achieving a complete response after MTX therapy was significantly associated with a longer PFS. Late neurotoxicity was encountered in 4 (50%) of 8 patients who were aged 60 years or older and received WBRT, but in none of 12 patients who were aged less than 60 years or avoided WBRT. All older patients who underwent SRT sustained complete remission without a dementing disease. Intensifying the MTX dosage to 8 g/m2 appears more promising in comparison to results reported with MTX doses of 1-3.5 g/m2. In younger patients, the establishment of complete remission by intensified MTX therapy and subsequent WBRT with a relatively lower dose could promise durable tumor remission with an acceptable neurotoxicity. In older patients, WBRT should be avoided to sustain a meaningful survival, and SRT may provide a valid strategy in terms of enhancing local disease control without undue risk.

Telomerase activity in pituitary adenomas: significance of telomerase expression in predicting pituitary adenoma recurrence.

Clinical and histopathological evaluations are inadequate for assessing biological aggressiveness and regrowth potential in benign pituitary adenomas. To develop reliable and prognostically informative means of predicting behavior remains an intractable problem. Telomerase, a reverse transcriptase that extends telomere length, may facilitate tumorigenesis and tumor immortality. In the present study, we investigated the telomerase activity of pituitary adenomas, and attempted to assess the value of telomerase expression for predicting their clinical course. In total, 31 (30 patients) benign pituitary adenoma samples including 8 recurrent adenomas were studied. Telomerase expression was evaluated by polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay and telomerase activity levels were quantitated by improved PCR enzyme-linked immunosorbent assay (ELISA). The data were analyzed in relation to clinical course which was reviewed at 4-5.5 years (median follow-up time, 52.5 months) after surgery. The relative values of the telomerase expression for predicting the clinical course were compared with the MIB-1 antigen-based proliferative cell index (PCI) and p53 immunoreactivity which have recently been suggested to correlate with aggressive behavior in pituitary adenomas. Overall, telomerase expression was detected in 13% of the adenomas (4 tumor tissues, 3 patients). These adenomas comprised large, invasive, and functioning adenomas. The number of telomerase-positive adenomas was small; however, the PCI was higher in cases with telomerase expression (4 tumor tissues; mean, 4.2 +/- 2.4%) than in those without it (27 tumor tissues; 1.4 +/- 1.3%) (p = 0.01). One tumor with detectable telomerase expression, which did not undergo additional pharmacological or radiotherapeutic intervention after first surgery, recurred rapidly despite gross total surgical resection, although the PCI of both the primary and recurrent adenomas was not high. Detection of telomerase expression may represent an additional useful means of identifying aggressive behavior, complementing the histopathological evaluation of benign-appearing pituitary adenomas.

Promoter hypermethylation of the DNA repair gene O6-methylguanine-DNA methyltransferase is an independent predictor of shortened progression free survival in patients with low-grade diffuse astrocytomas.

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.

Treatment of low-grade diffuse astrocytomas by surgery and human fibroblast interferon without radiation therapy.

Low-grade diffuse astrocytomas are slowly growing tumors with a relatively long overall survival. However, a substantial proportion undergoes dedifferentiation to a more malignant phenotype. Considerable controversy exists as to the best therapeutic management for patients with such tumors. Over the past decade, we have applied human fibroblast interferon (HFIF) therapy without radiation therapy to low-grade astrocytomas. We investigated 28 patients with WHO grade II astrocytomas of the cerebral hemispheres treated by surgery plus HFIF therapy. The overall response rate to the HFIF therapy was 36%. All side-effects of HFIF were transient, tolerable and manageable. The 5-year progression free survival and overall survival probabilities were 65% and 96%, respectively. Although our data from small cohort of patients may have modest value, our results suggest that HFIF may be useful in treating low-grade diffuse astrocytomas.