|Year : 2022 | Volume
| Issue : 1 | Page : 9-13
An experience of Methylguanine-DNA Methylation Assay (MGMT) characterization in glial tumors
Amrit Kaur Kaler1, Renjan Thoma1, NG Suhas1, VK Prarthana1, Baby Dharman1, Gautam Balaram1, Mithua Ghosh1, R Veena2, Shekhar Patil3, PS Sridhar4, Shivakumar Swamy3, CT Satish3, S Bhattacharjee4, BS Ajaikumar4
1 Department of Molecular and Cancer Genomics, HCG Cancer Center, Bengaluru, Karnataka, India
2 Department of pathology, HCG Cancer Center, Bengaluru, Karnataka, India
3 Department of Medical Oncology, HCG Cancer Center, Bengaluru, Karnataka, India
4 Department of Radiation Oncology, HCG Cancer Center, Bengaluru, Karnataka, India
|Date of Submission||18-May-2021|
|Date of Decision||19-Nov-2021|
|Date of Acceptance||12-Mar-2022|
|Date of Web Publication||03-May-2022|
Dr. Amrit Kaur Kaler
Consultant Molecular Pathologist, Department of Laboratory Medicine, Kokilaben Dhirubhai Ambani Hospital, Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: MGMT (O6-methylguanine DNA methyltransferase) is a DNA repair enzyme, that rescues tumor cells from damage by alkylating agents like temozolomide (TMZ). Promotor methylation of MGMT leads to epigenetic silencing and potentially increased sensitivity to TMZ. MGMT methylation (mMGMT) is an independent favorable prognostic factor, and has confounded its role as a predictive biomarker in making therapeutic decisions for glial tumors, particularly glioblastoma multiforme (GBM).
Materials and Methods: This retrospective cross-sectional study was conducted for 5 years in high-grade tumors (HCG) Cancer Hospital between January 2016 and December 2020. Methylation-specific polymerase chain reaction of MGMT gene using bisulfite modification of tumor DNA was utilized to ascertain the methylation status of cases in the cohort.
Results: A total of 54 glial tumors comprising 35 males and 18 females between the age group 11 years to 76 years underwent mMGMT testing. About 64.8% (35 cases) of all glial tumors demonstrated mMGMT in the cohort; GBM accounting for a majority of the cases (80.0%; 28 cases). The percentage of mMGMT cases in males and females was found to be 60.0% and 73.0% of cases, respectively. The confluent necrosis commonly seen in GBM is present in 41.0% of methylated cases and minimal in 77.3% of unmethylated cases with a significant P < 0.05.
Conclusion: MMGMT is a valuable predictive biomarker and is essential in taking the therapeutic decision in newly diagnosed glial tumors. Necrosis can be used as an indicator of mMGMT status according to the present study.
Keywords: Epigenetics, female sex, gliomas, MGMT, necrosis, temozolomide
|How to cite this article:|
Kaler AK, Thoma R, Suhas N G, Prarthana V K, Dharman B, Balaram G, Ghosh M, Veena R, Patil S, Sridhar P S, Swamy S, Satish C T, Bhattacharjee S, Ajaikumar B S. An experience of Methylguanine-DNA Methylation Assay (MGMT) characterization in glial tumors. J Precis Oncol 2022;2:9-13
|How to cite this URL:|
Kaler AK, Thoma R, Suhas N G, Prarthana V K, Dharman B, Balaram G, Ghosh M, Veena R, Patil S, Sridhar P S, Swamy S, Satish C T, Bhattacharjee S, Ajaikumar B S. An experience of Methylguanine-DNA Methylation Assay (MGMT) characterization in glial tumors. J Precis Oncol [serial online] 2022 [cited 2022 May 22];2:9-13. Available from: https://www.jprecisiononcology.com//text.asp?2022/2/1/9/344538
| Introduction|| |
Gliomas are a group of molecularly heterogeneous brain tumors which accounts for 2% of total malignancies in the human body Glial tumors are categorized into low-grade gliomas (LGG) which includes WHO Grade I and II and high-grade tumors (HGG) categorized as WHO Grade III and IV tumors., The later are more common, of which Glioblastoma multiforme (GBM), Grade IV constitutes 77%–81% of total glial tumors and are clinically associated with poor prognosis. The clinical presentation, prognosis, and therapy vary according to tumor grade and histology, molecular profile, age, sex, and functional status of the patient. Molecular alterations such as IDH1 mutations, ATRX, p53 mutations, 1p/19q codeletions, telomerase reverse transcriptase (TERT), phosphatase and tensin homolog, loss of heterozygosity (LOH)10, epidermal growth factor receptor (EGFR) VIII have shown to assist diagnosis, determine the prognosis, and predict the response to therapy., One key molecular marker of importance in therapeutic decision-making in glial tumors is O6-methylguanine DNA methyltransferase (MGMT) which has demonstrated its role as an important prognostic marker in GBM and predicts response to the alkylating agent. Temozolamide (TMZ) acts by adding an alkyl group to the tumor cell which inhibits DNA replication resulting in cell death. Normally, MGMT negates the action of TMZ by removing the offending alkyl group and repairing the DNA. Methylation of MGMT (mMGMT) results in epigenetic silencing (CpG sites hypermethylation) allowing alkylation leading to enhanced anti-tumoral activity, resulting in better response and survival to TMZ.,
Glial tumors are seen in varied age groups from pediatric to the elderly and are more commonly seen in the male population. mMGMT is to be more frequently associated with the female population although different correlations on gene expression between the two sexes have been predicted to be associated with this gender parity. Morphologically, low-grade tumors show mild cytological atypia, while high-grade gliomas like GBM shows anaplasia, high mitotic activity, pseudo palisading of tumor cells around confluent necrotic areas, and microvascular proliferation, also known as glomeruloid bodies. However, mMGMT positivity has been recently observed in glioblastomas with minimal necrosis on morphology and is found to be an independent predictor of poor survival.
The aim of this study was to correlate mMGMT status in LGG and HGG tumors and to compare the clinicopathological parameters such as tumor subtype, tumor grade, tumor necrosis (pseudopalisading), age, and sex of the patient.
| Materials and Methods|| |
A retrospective cross-sectional study was conducted in the Department of Molecular Pathology and Genomics, HCG cancer Hospital for 5 years between January 2016 and December 2020. The study has been approved was taken by the Human Research Ethics Committee of the HCG Institute (EC Registration No: HIEC/50/21/09 A total of 54 patients biopsy-proven glial tumors on histopathology and immunohistochemistry referred for MGMT methylation (mMGMT) testing were included in the study. Before molecular evaluation, histopathology examination was done to ascertain the histopathological diagnosis and to measure the tumor burden. The viable tumor volume of >10% from formalin-fixed paraffin-embedded specimens (FFPE) was selected for processing. Samples below the tumor burden threshold were excluded as it decreases analytic specificity and requests for a repeat biopsy wherever possible. Specific gel-based polymerase chain reaction (PCR) is performed to detect MGMT gene promotor methylation in glial tumors.
DNA Extraction protocol
Genomic DNA was isolated from 10-μM sections of the selected FFPE blocks. Genomic DNA was extracted in accordance with the manufacturer's instruction using DNA extraction kit (QIAamp ® DNA Mini Kits, Qiagen TM) and quantified on Nanodrop Lite Spectrophotometer before PCR analysis.
Methylation-specific PCR is a useful tool for quantitative DNA analysis with multiple advantages, including the ease of design and execution, sensitivity in the ability to detect small quantities of methylated DNA. This assay requires a modification of the genomic DNA by sodium bisulfite and two independent primer sets for PCR amplification, one pair designed to recognize the methylated and the other unmethylated versions of the bisulfite modified sequence in glioma patients. This assay determines the methylation status of 6 CpG dinucleotides in a critical region of the MGMT promotor that contains Sp1 transcription factor binding sites. The results are considered positive or methylated if 1 or more CpG sites are methylated in this promotor region. The sensitivity and specificity of this assay are >1 and 80%, respectively.
A descriptive statistical analysis was done using software SPSS version 20.0 for Student's t-test and Chi-square was used to ascertain the significance of the findings. P < 0.05 was considered significant.
| Results|| |
A total of 54 glial tumors were referred and underwent MGMT testing, including 35 males and 16 females between the age group 11 years to 76 years with a median age of 49 years. [Figure 1] shows the distribution of mMGMT cases in the current study. Histo-morphological analysis of glial tumors [Table 1] revealed GBM as the most common tumor, constituting 81.6% (44 cases) of cases followed by LGG and anaplastic astrocytomas (AA) at 8.5%, while gliosarcomas accounted for 2.8% of the total cases. mMGMT was observed in 64.8% of the total glial tumors in the study cohort with GBM demonstrating a maximum mMGMT positivity rate of 80.0% (28 cases). Two cases (2.8%) of LGG i.e., pilocytic astrocytoma and gemistocytic astrocytoma were found to be positive of the total 4 cases referred for MGMT status. Three cases (10%) of AA are found to be positive and 1 case of gliosarcoma showed positivity (2.8%). In patients above 50 years, mMGMT was seen in 57.1% of cases of GBM while only 42.9% cases were positive below 50 years [Table 2]. All cases of mMGMT in LGG and AA are seen below 50 years. The percentage of methylated MGMT cases in males and females were found to be 60.0% and 73.0% cases, respectively [Table 3]. Necrosis was a predominant feature in GBM, but the confluent (sheets) necrosis commonly seen in GBM is present in 41% of methylated cases and absent in 77% of unmethylated cases [Table 4].
|Figure 1: Spectrum of glial tumors with methyltransferase methylation status|
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|Table 1: Different glial tumors with tumor grade and percentage of tumors showing methylation status|
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|Table 2: O6-methylguanine DNA methyltransferase methylation status and relation to age|
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|Table 3: Percentage of methylation status in glial tumors with sex ratio|
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| Discussion|| |
Gliomas are a group of molecular heterogeneous central nervous system (CNS) tumors originating from glial cells. In India, the incidence of CNS tumors ranges from 5 to 10/100,000 population and the number is increasing because of the increased diagnostic screening methods. They have been categorized by the WHO into LGG and HGG based on tumor histology and grade. LGG are WHO Grade I and II tumors which showed low-grade histology, slow growing with a peak incidence between 34 and 44 years and a median survival of 3.0–7 years. HGG is more common malignant CNS tumors; are WHO Grade IV tumors, Glioblastoma is most more common (60%–80%) and carries a poor prognosis with a median survival of 12–18 months., Kim et al. also reported that the most common malignant tumor in CNS is GBM which accounts for 12%–15% of all intracranial neoplasms and 60%–75% of glial tumors. The present study also showed maximum cases of glioblastomas, constituting 80% cases of glial tumors which is concordant with the prevalent literature.
WHO (2016) has recommended the evaluation of MGMT in all cases of Glioblastomas because of its role as a prognostic and predictive marker to TMZ. A total of 35 cases showed MGMT were found to be positive among 54 cases of glial tumors. In the present study, 63.0% of glioblastomas and Gliosarcomas (Grade IV) showed mMGMT. Wick et al. reported that mMGMT gene promoter has been observed in approximately 50% of Grade IV gliomas. Among the low-grade tumors, only 8.4% of total positive glial cases showed MGMT positivity. AA, a WHO Grade III tumor showed positivity in 8.5% of total cases but showed maximum positivity of the total AA cases. This finding could suggest increased mMGMT status in secondary GBM. However, the present study could not differentiate patients as between primary or secondary GBM due to lack of clinical details and information status on other markers in a tertiary center. The clinical impact of mMGMT in LGG is yet to be established as its prognostic value is well documented by other markers like IDH and 1p19q codeletion., Probably, further study on a larger number of LGG tumors on mMGMT needs to be carried out for better results.
Glial tumors have a wide range of presentations, but LGG is more common in the adolescent to middle-age population. GBM is a disease of the elderly with a median age of 64 at diagnosis. There have been very limited studies illustrating the correlation of mMGMT with age. In the present study, 57.1% of GBM cases showed MGMT positivity above 50 years, while 42.9% are below 50 years. While the clinical presentation and mMGMT positivity in LGG are below 50 years in limited cases of study. We conclude that mMGMT positivity is expressed in high-grade tumors with an age group above 50 years than below 50 years.
Glial tumors are more common in males while m mMGMT positivity is most commonly seen in females and this female predilection has gone unnoticed for quite some time and has aroused a special interest among the researchers. In the present study, a total of 33 males and 16 females were found while MGMT positivity was seen in 57.5% cases in men; and 62.5% of cases in females. Anja Smith et al., in their first cohort, described that the proportion of tumors with mMGMT is 59% in females than 39% in males. On their next cohort, they published 33% positivity in men while 56.6% in females. TCGA (2021) also confirmed the same findings. This protective effect is probably due to the sex hormones and is seen more commonly during menarche and menopause. The females with malignant tumors show higher mMGMT and better survival. This sex disparity in epigenetic profiling is vital to understanding gliomogenesis as it may lead to distinct prognosis in either sex. Maxwell et al. from TCGA reported GBM in females showed enrichment of transcription regulators in their genes which contributes toward methylation, the phenomenon could play a role in making therapeutic decisions. Interestingly, this overrepresentation of mMGMT in females is not a pan-cancer phenomenon and is more likely to be tumor-specific phenomenon.
The histopathological features of glioblastomas include high cellularity and pleomorphism with confluent necrotic areas surrounded by palisading tumor nuclei and proliferation of blood vessels forming a glomeruloid bodies. Necrosis in GBM signifies an independent prognostic factor that affects progression-free survival. The pathological evidence of necrosis was surprisingly observed in 39% of methylated cases and 70% of unmethylated cases in the present study. Arora et al. reported that mMGMT is associated with the absence of necrosis and can be used as an independent predictive factor. Eoli et al. in their study observed similar findings that necrosis was present in 67.5% of unmethylated cases and 32% of methylated cases. While Cankovic et al. reported no significant associations of MGMT status with histopathological features. There have been multiple studies on the necrosis in GBM which is caused by hypoxia leads to the secretion of tumor necrosis factor and might induce apoptosis further. This hypothesis explained the resistance to antiapoptotic drugs and its association with poor survival in GBM patients. The effect of necrosis on methylation or epigenetic modification or silencing has to be further studied at the genomic level to understand its effects on MGMT.
mMGMT is a prognostic test to indicate the response to TMZ, which is shown to affect patient survival. First, mMGMT gene promoter holds strong prognostic value, which might have confounded its role as a pure predictive factor of response to TMZ. TMZ an oral alkylating agent inhibits DNA replication by acting on O6 and N7 positions of guanine by methylation. The resultant O6-methylguanine adducts pair with thymidine and form DNA adducts resulting in genomic instability. In LGG, many studies are ongoing, to study the response of TMZ since the benefit has been seen differently in different patients. Hence, it is vital to know the incidence of mMGMT in both low-grade and high-grade glial tumors and to predict how the tumor type, sex status, and necrosis have an effect on methylation status. Further biomarkers studies on the tumor microenvironment around inflammatory cells and its possible association with necrosis might lead to better understanding.
| Conclusion|| |
mMGMT carries a favorable prognosis and is established as a good predictive marker to alkylating chemotherapy in high-grade gliomas. Necrosis can be used as an indicator of mMGMT status according to the present study. Its time to study the epigenetic modification of MGMT to various factors such as age, sex, and necrosis to understand the tumor biology and to establish the patient's overall survival. Currently, MGMT testing is a lynchpin of molecular testing in high-grade glioma with recent studies demonstrating its role in LGG. Further testing in larger cohorts is necessary to provide a true insight into the role of MGMT in glioma testing.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]