Chiaki Komine

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.

O6‐methylguanine‐DNA methyltransferase methylation and TP53 mutation in malignant astrocytomas and their relationships with clinical course

Epigenetic silencing of O6‐methylguanine‐DNA methyltransferase (MGMT) by promoter methylation can confer cancer cells with an increased sensitivity to alkylating chemotherapeutic agents and a higher susceptibility to TP53 transition mutations. The aim of our study was to assess the correlation of promoter methylation of the MGMT gene with TP53 mutations and the clinical characteristics of malignant astrocytomas. We analyzed the MGMT promoter methylation and TP53 mutations in 45 malignant astrocytomas (16 anaplastic astrocytomas and 29 glioblastomas multiforme) treated prospectively with 1‐(4‐amino‐2‐methyl‐5‐pyrimidinyl)methyl‐3‐2(2‐chloroethyl)‐3‐nitrosourea, interferon‐β and radiation therapy, and evaluated their clinical usefulness. MGMT promoter methylation was found in 17 (38%) of the 45 newly diagnosed malignant astrocytomas. A clear trend existed between MGMT methylation and G:C to A:T transition mutations of TP53 (p = 0.0596). Patients with MGMT‐methylated tumors displayed a greater chance of responding to adjuvant therapy as compared with those with MGMT‐unmethylated tumors (p = 0.0393). TP53 mutation was not significantly associated with the clinical response (p = 0.1310). While neither MGMT methylation nor TP53 mutation had a significant effect on prognosis of the whole population, the presence of MGMT methylation emerged as a significant predictor of a longer survival when exclusively analyzing 29 patients with glioblastomas multiforme. These findings highlight the importance of MGMT methylation as a specific predictive factor for responsiveness to nitrosourea chemotherapy.

Aberrant hypermethylation of p14ARF and O6-methylguanine-DNA methyltransferase genes in astrocytoma progression.

The aim of the present study was to elucidate genetic alterations that are critically involved in astrocytoma progression. We characterized 27 World Health Organization grade II fibrillary astrocytomas which later underwent recurrence or progression, paying specific attention to the CpG island methylation status of critical growth regulatory genes. p14ARF and O6‐methylguanine‐DNA methyltransferase (MGMT) hypermethylation represented frequent events (26% and 63%, respectively), which were mutually exclusive except in one case, with alternate or simultaneous methylation of these two genes occurring in 85% of our tumor series. Seventeen tumors (63%) contained TP53 mutations, which were closely related to the presence of MGMT methylation. Methylation of the p21Waf1/Cip1, p27Kip1 and p73 genes and homozygous deletion of the p16INK4a, p15INK4b and p14ARF genes were not detected in any of the primary low‐grade tumors. The presence of p14ARF methylation at first biopsy was associated with shorter patient survival, whereas the presence of MGMT methylation carried a better clinical outcome after salvage therapy. Examination of 20 cases whose histological data for recurrent tumors were available revealed that malignant progression occurred in all of the tumors with p14ARF methylation but less frequently (50%) in the lesions with MGMT methylation. On analysis of their respective recurrent tumors, five of six patients whose primary low‐grade tumors carried p14ARF methylation exhibited homozygous co‐deletions of the p14ARF, p15INK4b and p16INK4a genes, which were restricted to glioblastoma as the most malignant end point. Our findings suggest that p14ARF hypermethylation and MGMT hypermethylation constitute distinct molecular pathways of astrocytoma progression, which could differ in biological behavior and clinical outcome.

A small amount of singlet oxygen generated via excited methylene blue by photodynamic therapy induces the sterilization of Enterococcus faecalis.

INTRODUCTION The present study aimed to clarify the relationship between the amount of singlet oxygen ((1)O(2)) generated from excited methylene blue (MB) and the bactericidal effects on Enterococcus faecalis. METHODS A diode laser was used as the laser irradiation source (λ = 660 nm, 200 mW). The laser irradiation time periods were 300, 600, and 900 seconds. In experiment 1, the amount of (1)O(2) generated from each concentration (0.0001%-1.0%) of excited MB was examined by using electron spin resonance to determine the optimal concentration of MB. In experiment 2, the bactericidal effects of (1)O(2) on E. faecalis were examined. Experimental groups were with laser irradiation, L(+); without laser irradiation, L(-); including MB, M(+); and not including MB, M(-). These were combined to form 4 groups: L(+)M(+), L(+)M(-), L(-)M(+), and L(-)M(-). After treatment, E. faecalis was incubated for 48 hours at 37°C, and the bactericidal effects of (1)O(2) on E. faecalis were determined on the basis of the number of colony-forming units per milliliter. RESULTS The largest amount of (1)O(2) was generated from 0.01% excited MB. After 300, 600, and 900 seconds of irradiation, 35.2, 87.2, and 117.1 μmol/L (1)O(2) were detected, respectively. In group L(+)M(+),colony-forming units per milliliter of E. faecalis dramatically decreased depending on the amount of (1)O(2) generated. No other groups showed any bactericidal effects. CONCLUSIONS Our findings suggest that 0.001%-0.01% of MB is the most effective range for generating (1)O(2) during the application of antimicrobial photodynamic therapy. At least 35.2 μmol/L generated (1)O(2) was necessary to achieve the sterilization of E. faecalis.

Promoter Hypermethylation of Mismatch Repair Gene hMLH1 Predicts the Clinical Response of Malignant Astrocytomas to Nitrosourea

Purpose: In certain types of human cancers, transcriptional inactivation of hMLH1 by promoter hypermethylation plays a causal role in the loss of mismatch repair functions that modulate cytotoxic pathways in response to DNA-damaging agents. The aim of the present study was to investigate the role of promoter methylation of the hMLH1 gene in malignant astrocytomas. Experimental Design: We examined the hMLH1 promoter methylation in a homogeneous cohort of patients with 41 malignant astrocytomas treated by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-2(2-chloroethyl)-3-nitrosourea chemotherapy in combination with radiation and interferon therapy, and assessed the correlation of such methylation with clinical outcome. Results: hMLH1 promoter methylation was found in 6 (15%) of the 41 newly diagnosed malignant astrocytomas. Hypermethylation of the hMLH1 promoter corresponded closely with a loss of immunohistochemical staining for hMLH1 protein (P = 0.0013). Patients with hMLH1-methylated tumors displayed a greater chance of responding to adjuvant therapy as compared with those with hMLH1-unmethylated tumors (P = 0.0150). The presence of hMLH1 hypermethylation was significantly associated with a longer progression-free survival on both univariate analysis (P = 0.0340) and multivariate analysis (P = 0.0161). Conclusions: The present study identified hMLH1 methylation status as a predictor of the clinical response of malignant astrocytomas to chloroethylnitrosourea-based adjuvant therapy. The findings obtained suggest that determination of the methylation status of hMLH1 could provide a potential basis for designing rational chemotherapeutic strategies, as well as for predicting prognosis.

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.

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.

Apoplexy accompanying pituitary adenoma as a complication of preoperative anterior pituitary function tests

SummaryPituitary apoplexy occurs as a very rare complication of the pituitary function test. We have experienced two cases of pituitary apoplexy following anterior pituitary function tests for preoperative assessment: a triple bolus test and a TRH test. To elucidate such a rare complication, we outline our two cases and review 28 cases from the literature. The clinical characteristics, etiology, pathophysiology, and diagnostic and therapeutic implications are also discussed. The combined data suggest that pituitary function tests have the potential to precipitate pituitary apoplexy, and its manifestations range from a clinically benign event to a catastrophic presentation with permanent neurological deficits or even death, although most patients may fortunately have a good outcome. We suggest that the pituitary function test should not be done as a routine test, and when such a test is planned, the patient should be observed with caution for any symptomatic changes for at least 2 hours following the test for appropriate treatment. Further, MRI, especially enhanced studies, may provide an earlier diagnosis of the pituitary apoplexy since CT scan images often fail to demonstrate either density changes or obvious enlargement of the pituitary adenoma at the acute stage.

Therapeutic Implications of Interferon Regulatory Factor (IRF)-1 and IRF-2 in Diffusely Infiltrating Astrocytomas (DIA): Response to Interferon (IFN)-β in Glioblastoma Cells and Prognostic Value for DIA

SummaryThe precise mechanisms governing the direct effect of IFN-β, 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-β based on a cell growth study and modified MTT assay, and then quantified the apoptosis using a sandwich enzyme immunoassay following IFN-β 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-β treatment (1 × 104 IU/ml) in T98G but not in U-87MG or A-172. IFN-β 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-β 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.

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.