Prognostic Stratification of GBMs Using Combinatorial Assessment of IDH1 Mutation, MGMT Promoter Methylation, and TERT Mutation Status: Experience from a Tertiary Care Center in India

AbstractThis study aims to establish the best and simplified panel of molecular markers for prognostic stratification of glioblastomas (GBMs). One hundred fourteen cases of GBMs were studied for IDH1, TP53, and TERT mutation by Sanger sequencing; EGFR and PDGFRA amplification by fluorescence in situ hybridization; NF1expression by quantitative real time polymerase chain reaction (qRT-PCR); and MGMT promoter methylation by methylation-specific PCR. IDH1 mutant cases had significantly longer progression-free survival (PFS) and overall survival (OS) as compared to IDH1 wild-type cases. Combinatorial assessment of MGMT and TERT emerged as independent prognostic markers, especially in the IDH1 wild-type GBMs. Thus, within the IDH1 wild-type group, cases with only MGMT methylation (group 1) had the best outcome (median PFS: 83.3 weeks; OS: not reached), whereas GBMs with only TERT mutation (group 3) had the worst outcome (PFS: 19.7 weeks; OS: 32.8 weeks). Cases with both or none of these alterations (group 2) had intermediate prognosis (PFS: 47.6 weeks; OS: 89.2 weeks). Majority of the IDH1 mutant GBMs belonged to group 1 (75%), whereas only 18.7% and 6.2% showed group 2 and 3 signatures, respectively. Interestingly, none of the other genetic alterations were significantly associated with survival in IDH1 mutant or wild-type GBMs.Based on above findings, we recommend assessment of three markers, viz., IDH1, MGMT, and TERT, for GBM prognostication in routine practice. We show for the first time that IDH1 wild-type GBMs which constitute majority of the GBMs can be effectively stratified into three distinct prognostic subgroups based on MGMT and TERT status, irrespective of other genetic alterations

Limb girdle muscular dystrophy type 2A in India: A study based on semi-quantitative protein analysis, with clinical and histopathological correlation

Background : Limb girdle muscular dystrophy (LGMD) type 2A is caused by mutation in the gene encoding for calpain-3 resulting in total or partial loss of protein. Diagnosis of LGMD2A, the most prevalent form of LGMD, is established by analyzing calpain-3 protein deficiency or CAPN3 gene mutation. Since there is no data from India regarding the incidence of LGMD2A, this study was undertaken. Aims : To study the frequency of LGMD2A in Indian population on the basis of protein analysis by immunoblotting and to correlate pathological and clinical features with protein analysis. Settings and Design : One hundred and seventy-one muscle biopsies of clinically suspected LGMD, unclassified muscular dystrophy or myopathy were analyzed in a tertiary national referral centre for neurosciences. Materials and Methods : Histopathological, immunohistochemical and enzyme histochemical analysis of muscle biopsies was performed followed by protein analysis for calpain-3 and dysferlin by immunoblotting. Results : Immunoblot identified 75 patients (43.8%) with calpain-3 deficiency, of which 36 (45%) had complete loss and 39 (55%) had partial loss of calpain-3 protein. In patients with LGMD phenotype alone, the incidence of LGMD2A was 47%. The biopsies of these patients displayed variety of morphological changes ranging from dystrophic pattern with presence of active fibre necrosis, regeneration and lobulated fibres to end stage muscle disease. The mean age of presentation and disease onset was 24 and 18 years respectively. Conclusions : This series of 75 patients is probably the first confirmed cases of LGMD2A (calpainopathy) from India. Our study suggests that LGMD2A is the most frequent form of LGMD in India, similar to the Western data, thus, highlighting the importance of immunoblotting for an accurate diagnosis

Detection of allelic status of 1p and 19q by microsatellite-based PCR versus FISH: limitations and advantages in application to patient management

Combined loss of chromosome arms 1p and 19q in oligodendroglial tumors has become a powerful predictor of prognosis and treatment response, and hence clinical testing for their detection is widely used nowadays. Polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) analysis and fluorescence in-situ hybridization (FISH) are the 2 important relatively common clinical molecular diagnostic techniques used for this purpose and they have their unique advantages and limitations. The preference of methodology used depends on local expertise and existing laboratory capabilities. However, there is no consensus on which methodologic approach has a better potential. The objective of the study was to compare the accuracy, reliability, and feasibility of FISH and PCR in detecting the 1p and 19q LOH status. Using the PCR-based method, a LOH analysis was performed on 30 oligodendrogliomas and 10 glioblastomas using fresh-frozen tissue and peripheral blood as control. A FISH assay using paraffin blocks was performed on all the cases. Concordance for 1p and 19q was found in 92.5% (37 of 40) and 82.5% (33 of 40) cases, respectively. The relative advantages and limitations of both the techniques are analyzed and discussed. The main issue pertains to the use of the best technique in large clinical trials whose results are translated to patient care services. Unless the technique used is correct, the results of clinical trials and their correlations may be somewhat questionable

O6-methylguanine DNA methyltransferase gene promoter methylation status in gliomas and its correlation with other molecular alterations: first Indian report with review of challenges for use in customized treatment

Background: O<SUP>6</SUP>-methylguanine methyltransferase (MGMT) promoter methylation in adult glioblastomas (glioblastoma multiforme) is considered a promising molecular alteration, predictive of better response to temozolomide therapy and longer overall survival. Objective: To look at the frequency of MGMT methylation in glial tumors of all grades and types, and correlate this alteration with loss of heterozygosity 1p/19q, TP53 gene mutations, epidermal growth factor receptor (EGFR) amplification, and isocitrate dehydrogenase 1 (IDH1) mutations. Methods: One hundred two gliomas of various grades and subtypes were assessed by methylation-specific polymerase chain reaction for MGMT promoter methylation status. The results were correlated with 1p/19q status, EGFR amplification, TP53, and IDH1 mutations. Results: There was an inverse correlation of MGMT promoter methylation frequency with tumor grade, observed in 79.4%, 70.8%, and 56.8% of grade II, grade III, and grade IV gliomas, respectively. The difference was statistically significant in grade II vs IV tumors (P=.036). The majority of cases with 1p/19q loss of heterozygosity also showed MGMT methylation, although the association was not significant. There was no significant correlation of MGMT status with IDH1 mutation. In astrocytic tumors, there was no correlation of MGMT methylation with TP53 mutation or EGFR amplification. Conclusion: MGMT promoter methylation was observed in a considerable proportion of all grades and subtypes of gliomas, with no significant correlation with other known genetic alterations. On extensive literature review, in both low-and high-grade gliomas, wide variability of data on the frequency of MGMT methylation and its association with other molecular alterations from various centers was noted, mostly owing to technical causes. This raises questions regarding the capacity of this test for use as an objective and reproducible marker for customized treatment in individual cases

Loss of heterozygosity on chromosome 10q in glioblastomas, and its association with other genetic alterations and survival in Indian patients

Background: Glioblastoma multiforme (GBM) is the most common malignant central nervous system neoplasm. Loss of heterozygosity (LOH) on chromosome 10q in these tumors has been found to show variable association with prognosis. Aim: To evaluate LOH 10q status in cases of GBM, and to correlate these results with patient characteristics, other genetic alterations, and survival. Material and Methods: Fresh tumor tissue and blood samples were obtained for 25 cases of GBM diagnosed over a 2-year period. LOH 10q assay was performed on blood and tumor DNA by a PCR-based method using four microsatellite markers. TP53 mutation analysis and fluorescence in situ hybridization for epidermal growth factor receptor (EGFR) were performed. Histopathology was reviewed and clinical data were analyzed. Results: LOH 10q was identified in 17 of 25 cases (68%). Losses were frequent with markers D10S1765 (12/20 informative cases; 60%) and D10S587 (12/17 informative cases; 70.5%) in the regions of 10q23.3 and 10q26.1, respectively. D10S540 for 10q25.1 showed LOH in 4/12 informative cases (33.3%) and D10S1770 for 10q26-ter in none of the 25 cases. LOH with D10S1765 at the PTEN gene locus was found to correlate with overall LOH 10q status (P = 0.001). LOH 10q was more common in patients older than 40 years (16/19, 84.2%) than in those below (1/6, 16.7%) (P = 0.006). One of three pediatric patients included demonstrated LOH 10q. Survival rates for patients with LOH were lower than for patients with retained heterozygosity. Conclusion: LOH 10q is a frequent genetic abnormality in GBM in Indian patients, is seen more frequently in older adults, and its presence is associated with shorter survival. The single best marker to determine LOH 10q status is D10S1765 at the PTEN region

A clinicopathological and molecular analysis of glioblastoma multiforme with long-term survival

The median survival time of patients with glioblastoma multiforme (GBM) is 12 months, and only 3-5% of patients survive longer than 3 years. We performed histomorphological and detailed molecular analyses of seven long-term survivors of GBM to identify any prognostic factors that potentially contribute to survival. Morphology and immunohistochemistry for p53, phosphatase and tensin homologue (PTEN) and epidermal growth factor receptor (EGFR) protein expression were investigated. EGFR amplification and 1p/19q deletion were assessed by fluorescent in situ hybridization. The O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status was evaluated by performing methylation-specific polymerase chain reaction assays. All tumors were classical GBMs and no significant oligodendroglial differentiation was noted. The majority showed EGFR amplification (4/7), PTEN protein expression (6/7) and MGMT promoter methylation (5/6). Immunopositivity for p53 was noted in three of seven patients. Deletion of chromosome 1p/19q, either isolated or combined, was not identified in any of the se patients. All patients were treated by gross total resection followed by radiotherapy; six patients received additional temozolomide treatment. A relatively young age of onset (48 years), with a high MGMT promoter methylation and PTEN protein expression were favorable factors for long-term survival. The presence of EGFR amplification indicates that more than a single factor determines survival in GBM

Heterozygosity status of 1p and 19q and its correlation with p53 protein expression and EGFR amplification in patients with astrocytic tumors: novel series from India

There are few reports of loss of heterozygosity (LOH) of 1p and 19q in astrocytic tumors, especially glioblastoma multiforme (GBM). We evaluated 1p and 19q (either or both) heterozygosity status in 71 astrocytomas, including 6 pediatric cases: 20 diffuse astrocytomas (DA), 9 anaplastic astrocytomas (AA), and 42 GBM. In the GBMs, p53 protein expression was assessed by immunohistochemistry and epidermal growth factor receptor (EGFR) gene amplification by fluorescence in situ hybridization; TP53 sequencing was done in 15 of the GBMs. In adults, LOH of 1p or 19q was detected in 16% of DAs and 50% of GBMs; none of the AAs showed this alteration. In GBMs, LOH of 19q was most common (26%), followed by combined 1p and 19q LOH (13%) and 1p LOH (10%). Pediatric GBMs also harbored isolated 1p and 19q LOH (50%). Notably, LOH of 1p or 19q LOH was more frequent in p53 immunopositive secondary GBMs (61%) than in primary GBMs (17%). This suggests that LOH of 1p and 19q may be acquired during progression to secondary GBMs. Thus, 1p and 19q LOH can occur in astrocytic tumors, most commonly in secondary GBMs without morphological correlation with an oligodendroglial histology. The clinical significance of recognition of this subset of GBMs is based on several recent reports of association with better prognosis, although long-term follow-up studies are required