Temozolomide and radiotherapy in newly diagnosed glioblastoma patients:O^6-methylguanine-DNA methyltransferase (MGMT) promotor methylation status and Ki-67 as biomarkers for survival and response to treatment

Objective:This phase II study aimed at investigating the correlation between O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and protein expression,together with Ki-67 labeling index (LI),to response,time to progression (TTP),and overall survival (OS) in newly diagnosed glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ) concomitant with and adjuvant to radiotherapy (RT).Methods:From June 2005 to August 2008,34 adult patients (18-65 years),PS ≥70,with newly diagnosed GBM received TMZ 75 mg/m2 plus RT up to 60 Gy,followed by TMZ 175 mg/m2 5 days every 4 weeks for 12 doses.MGMT Methylation-specific PCR assay,MGMT protein expression,and Ki-67 expression using immunohistochemistry (IHC) were performed on the tissue blocks.The patients were followed by MRI while MR spectroscopy (MRS) was performed for the stable cases or to confirm progression and accordingly Bevacizumab 10 mg/kg every 2 weeks was added to 7 patients till further progression was proved.Results:31 cases were evaluable,12 (38.7%) had unmethylated MGMT,while 19 (61.3%) were methylated.Seventeen cases (55%) were MGMT immunonegative while 14 cases (45%) were immunopositive.The cut off value of Ki-67 LI in relation to survival was 17%,where 15 were < 17% (48.4%),and 16 were ≥ 17% (51.6%).Response evaluation started after the second dose of the adjuvant TMZ and was repeated every 2 months.The overall disease control rate (ODC) was 74.2%,where 2 patients had complete response (CR),14 had partial response (PR),and 7 had stable disease (SD),while 8 (25.8%) had progressive disease (PD).The ODC was significantly higher among methylated patients and in those with Ki-67 < 17% (P=0.0003).The median overall TTP was 12 months and the median OS was 20 months for all the patients including those who received Bevacizumab for some stable cases or as a salvage treatment in patients with good PS,the MGMT-methylated patients had a higher median TTP of 13 months (range 8 to 18 months,95% CI of 9.36 to 12.9),and OS of 24 months (range 12 to 31 months,95% CI of 16.1 to 21.32),while the unmethylated patients had a median TTP of 6.5 months and a median OS of 12 months,such correlations were highly significant (P=0.0001).MGMT immunoexpression failed to show significant correlation with MGMT promotor methylation or the outcome of the patients.Patients with Ki-67 < 17% had a median TTP of 16 months and median OS of 24 months compared to 7 and 12.5 months respectively for the patients with Ki-67 ≥17%.Significant correlation was found between the ODC,TTP,and OS with age < 52,near total excision,and TMZ doses received ≥ 10.The commonest grade 3 and 4 toxicities was neutropenia recorded in 3 patients (9.67%),thrombocytopenia in 4 patients (12.9%),and one patient with G3 nausea,vomiting,and constipations (3%),all were medically manageable.Conclusion:MGMT promotor methylation status and Ki-67 LI (but not the MGMT protein expression),could serve as prognostic markers for survival,also MGMT could identify the newly diagnosed GBM patients who will have better response to TMZ.

Expression of O⁶-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue

The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.

The interplay mechanism between IDH mutation, MGMT-promoter methylation, and PRMT5 activity in the progression of grade 4 astrocytoma: unraveling the complex triad theory

Background:The dysregulation of Isocitrate dehydrogenase(IDH)and the subsequent production of 2-Hydroxyglutrate(2HG)may alter the expression of epigenetic proteins in Grade 4 astrocytoma.The interplay mechanism between IDH,O-6-methylguanine-DNA methyltransferase(MGMT)-promoter methylation,and protein methyltransferase proteins-5(PRMT5)activity,with tumor progression has never been described.Methods:A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors.Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis.Inter-cohort statistical significance was evaluated.Results:Both IDH-mutant WHO grade 4 astrocytomas(n=22,64.7%)and IDH-wildtype glioblastomas(n=12,35.3%)had upregulated PRMT5 gene expression except in one case.Out of the 22 IDH-mutant tumors,10(45.5%)tumors showed MGMT-promoter methylation and 12(54.5%)tumors had unmethylated MGMT.All IDH-wildtype tumors had unmethylated MGMT.There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma(p-value=0.006).Statistically significant differences in progression-free survival(PFS)were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide(TMZ)or TMZ plus other chemotherapies,regardless of their IDH or MGMT-methylation status(p-value=0.0014).Specifically,IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation,who received only TMZ,have exhibited longer PFS.Conclusions:The relationship between PRMT5,MGMT-promoter,and IDH is not tridirectional.However,accumulation of D2-hydroxyglutarate(2-HG),which partially activates 2-OG-dependent deoxygenase,may not affect their activities.In IDH-wildtype glioblastomas,the 2HG-2OG pathway is typically inactive,leading to PRMT5 upregulation.TMZ alone,compared to TMZ-plus,can increase PFS in upregulated PRMT5 tumors.Thus,using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.

Study on the Relationship Between Depletion of O^6-Methylguanine-DNA Methyltransferase and Cancer Chemotherapy

Drug resistance is still one of the major problems in cancer therapy. However, during the past several years, many efforts have been focused on reversing or depleting the molecular basis of drug resistance along with the elucidating of the mechanisms involved.Nitrosourea compounds such as 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and 1, 3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) are an important class of useful antitumor drugs. They exert their antitumor activity by alkylating the O^6 position of guanine and leading to a DNA interstrand cross-link. Introcellular O^6-methylguanine-DNA methyltransferase (O^6-MT) can repair the mentioned DNA lesions and thus plays an important role in determining the sensitivity of

O^6-METHYLGUANINE-DNA METHYLTRANSFERASE AND HUMAN CANCER CHEMOTHERAPY

Two kinds of human tumor cell strains having different activity of O^6-methylguanine-DNA methyltransferase (O^6-MT) were transplanted into nude mice. Then the mice were inject-ed intraperitoneally with bifunctional alkylating agent 1--(4--amino--2-methyl--5--pyrimidinyl)methy1-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). The tumors with low O^6-MTactivity were quickly suppressed or cured. The result suggests that some tumors, if previ-sionally determined with low O^6-MT activity, might be efficiently cured by treatment withACNU. This probably opens a new way for human cancer chemotherapy.

Expression Silence of DNA Repair Gene hMGMT Induced by RNA Interference

Objective： MGMT protein expression has been associated with tumor resistance to alkylating agents. The objective of this paper is to construct the RNA interference vector which can specifically induce the expression silence of human DNA repair gene hMGMT. Methods： The hMGMT specific siRNA expression cassette was made by two steps PCR, linked with pUCI 9 to get pU6-MGMTi, co-transfected with pEGFP-CI into 16HBE and screened by G418. The MGMT mRNA and protein levels were detected by RT-PCR and Western Blot respectively. Results： hMGMT specific RNA interfere vector pU6-MGMTi was constructed successfully. In transfected 16HBE cells MGMT mRNA level could hardly be detected and the protein level was only 10% of control. Conclusion： MGMT specific RNAi expression cassette can effectively inhibit MGMT expression. MGMT silence cell line was built by co-transfection technology, which offered condition for studying the gene function of MGMT.

Molecular pathology and clinical implications of diffuse glioma

The prognosis for diffusely infiltrating gliomas at World Health Organization(WHO)grade 2-4 remains dismal due to their heterogeneity.The rapid development of genome-wide molecular-profiling-associated studies has greatly promoted the accuracy of glioma classification.Thus,the latest version of the WHO classification of the central nervous system tumors published in 2021 has incorporated more molecular biomarkers together with histological features for the diagnosis of gliomas.Advanced usage of molecular pathology in clinical diagnostic practice provides also new opportunities for the therapy of patients with glioma,including surgery,radiotherapy and chemotherapy,targeted therapy,immunotherapy,and more precision clinical trials.Herein,we highlight the updates in the classification of gliomas according to the latest WHO guidelines and summarize the clinically relevant molecular markers by focusing on their applications in clinical practice.We also review the advances in molecular features of gliomas,which can facilitate the development of glioma therapies,thereby discussing the challenges and future directions of molecular pathology toward precision medicine for patients with glioma.