Search

Pilocytic astrocytomas occur rarely in adults and show aggressive tumor behavior. However, their underlying molecular-genetic events are largely uncharacterized. Hence, we studied 59 adult pilocytic astrocytoma (APA) cases of classical histology (MIB-1 LI:1-5%). Analysis of BRAF alterations using qRT-PCR, confirmed KIAA1549-BRAF fusion in 11(19%) and BRAF-gain in 2(3.4%) cases. BRAF-V600E mutation was noted in 1(1.7%) case by sequencing. FGFR1-mutation and FGFR-TKD duplication were seen in 7/59(11.9%) and 3/59(5%) cases respectively. Overall 36% of APAs harbored BRAF and/or FGFR genetic alterations. Notably, FGFR related genetic alterations were enriched in tumors of supratentorial region (8/25,32%) as compared to other locations (P=0.01). The difference in age of cases with FGFR1-mutation (Mean age±SD: 37.2±15) vs KIAA1549-BRAF fusion (Mean age±SD: 25.1±4.1) was statistically significant (P=0.03). Combined BRAF and FGFR alterations were identified in 3(5%) cases. Notably, the cases with more than one genetic alteration were in higher age group (Mean age±SD: 50±12) as compared to cases with single genetic alteration (Mean age±SD: 29±10)(P= 0.003). Immunopositivity of p-MAPK/p-MEK1 was found in all the cases examined. pS6-immunoreactivity, a marker of mTOR activation was observed in 34/39(87%) cases. Interestingly, cases with BRAF and/or FGFR related alteration showed significantly lower pS6-immunostatining (3/12;25%) as compared to those with wild-type BRAF and/or FGFR (16/27;59%)(P=0.04). Analysis of 7 IDH wild-type adult diffuse astrocytomas (DA) showed FGFR related genetic alterations in 43% cases. These and previous results suggest that APAs are genetically similar to IDH wild-type adult DAs. APAs harbor infrequent BRAF alterations but more frequent FGFR alterations as compared to pediatric cases. KIAA1549-BRAF fusion inversely correlates with increasing age whereas FGFR1-mutation associates with older age. Activation of MAPK/ERK/mTOR signaling appears to be an important oncogenic event in APAs and may be underlying event of aggressive tumor behavior. Our findings provide a rationale for potential therapeutic advantage of targeting MAPK/ERK/mTOR pathway in APAs. This article is protected by copyright. All rights reserved.

This 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.

This study aims (1) to evaluate ATRX expression in different grades and subtypes of gliomas and correlate with other hallmark genetic alterations, (2) to identify and characterize mosaic/heterogeneous staining in gliomas in terms of mutation status. One hundred seventy six cases of glioma were assessed for ATRX immunohistochemistry and subdivided into positive, negative and mosaic/heterogeneous staining patterns. Five cases with heterogeneous staining were further subjected to next generation sequencing. Higher frequency of ATRX immune-negativity was detected in grade II/III astrocytic, oligoastrocytic tumors and secondary glioblastomas (GBMs), while infrequent in primary GBMs and rare in oligodendrogliomas. Loss of expression was significantly associated with IDH1 and/or TP53 mutation, while mutually exclusive with 1p/19q codeletion. Mosaic/heterogeneous staining was detected exclusively in GBMs (21.2%). Two different types of mosaic staining were identified (1) Admixture of positive and negative nuclei or intermixed mosaic and (2) Separate fragments with positive and negative/intermixed mosaic staining. ATRX mutation was identified in 2/5 (40%) cases with mosaic staining while one case showed DAXX mutation. All these cases were characterized by distinctly separate immune-negative and positive/intermixed foci. Hence, it is suggested that cases with heterogeneous staining (especially those with distinctly negative fragments) should be subjected to mutation analysis.

INTRODUCTION: WHO 2016 classification recognizes four molecular-subgroups of medulloblastoma(MB) viz. WNT,SHH,Group3 and 4. This molecular classification is prognostically and therapeutically important and helps in better risk-stratification of patients. Translation of this molecular data to routine practice is a challenge. Immunohistochemistry(IHC), FISH and Nanostring are methods which can be done using FFPE tumor-tissue. OBJECTIVES: (a)To compare MB subgrouping by three-panel IHC and FISH vs Nanostring (b)To categorise non-WNT/non-SHH into Group3 and 4. METHODS: Forty-seven cases of MB with adequate tissue were retrieved. IHC was performed for beta-catenin,GAB1 and YAP1. MYC amplification was done using FISH. RNA was extracted from FFPE using Invitrogen-RecoverAll(TM) total nucleic-acid isolation, and then subjected to Nanostring-Assay. RESULTS: Among 47 cases, 39 were pediatric and 8 adult. IHC results revealed 5WNT, 8SHH, and 32non-WNT/non-SHH. MYC amplification was identified in 8 cases. Nanostring classified MBs into 6 WNT, 6 SHH, 7 Group3 and 28 Group4. Seven cases were unclassifiable; all of which had >8 year old blocks. Hence, successful subgroup assignment was seen in 40/47(85.1%)cases. Concordant subgroup assignment was noted in 35/40 cases(87.5%). Subgroup switching was identified in 5 cases. However, on correlation with follow-up, there was significant difference between MYC-amplified(worse) and non-amplified Non-WNT/SHH, and also between Group3(worse) and 4. CONCLUSIONS: Both IHC supplemented by FISH and Nanostring are robust methods for molecular subgrouping, albeit with few disadvantages. IHC cannot differentiate between group3 and 4, while Nanostring cannot classify older-archived tumors,and is not available at most centres. Thus, a neuropathologist can perform molecular subgrouping using IHC alone, IHC supplemented by FISH, or Nanostring, depending on availability of resources.

KLF2 (Kruppel-like factor 2) is a member of the zinc finger transcription factor family, which critically regulates embryonic lung development, function of endothelial cells and maintenance of quiescence in T-cells and monocytes. It is expressed in naive T-cells and monocytes, however its level of expression decreases during activation and differentiation. KLF2 also plays critical regulatory role in various inflammatory diseases and their pathogenesis. Nuclear factor-kappaB (NF-κB) is an important inducer of inflammation and the inflammation is mediated through the transcription of several proinflammatory cytokines, chemokines and adhesion molecules. So, both transcriptional factors KLF2 and NF-κB are being associated with the similar cellular functions and their maintenance. It was shown that KLF2 regulates most of the NF-κB-mediated activities. In this review, we focused on emphasizing the involvement of KLF2 in health and disease states and how they interact with transcriptional master regulator NF-κB.

Glioblastoma (GBM, WHO grade-IV) being the most malignant and aggressive form of glioma remains a major clinical challenge, with an overall 5-year survival rate of only 9.8%. Till recently, glioma diagnosis and grading were solely dependent on the phenotypic and histological features. However, with the advancement in the understanding of the molecular biology of glioma several molecules have been identified. The importance of these molecular/genotypic features of the tumor became evident by the inclusion of these molecular features by World Health Organization (WHO) in 2016 in glioma sub-grouping. Our lab is focused on studying the role of FAT1 gene (human ortholog of Drosophila tumor suppressor gene, fat) in glioma biology and aggressiveness. We observed FAT1 gene to have an oncogenic role in glioma where it has been found to upregulate migration/invasion, inflammatory microenvironment of the tumors, HIF1α expression/activity in the tumor-cells under severe hypoxia and in regulating EMT/stemness properties of GBM-cells under hypoxia. Here, we have characterized the molecular relationship between FAT1 related molecules and known- molecular markers of glioma with the hope of identifying glioma subgroup with a molecular signature of clinical significance by (i) analyzing the expression correlation of FAT1 and FAT1 regulated pro-inflammatroy molecules like COX2, IL1b and IL6 with the known- molecular markers of glioma like p53, IDH1, MGMT, EGFR, TERT in low-grade (grade-II) and high-grade (grade-III/IV) gliomas (n=50) by real-time PCR, sequencing, immunohistochemistry and in-silico analysis of TCGA-GBM-data (ii) Analyzed the regulatory role of FAT1 on the above known markers by siRNA mediated knockdown of FAT1 in in-vitro cell-culture system and (iii) further analyzed the identified molecular signature for their correlation with the patients prognosis/survival in the follow up patients. We observed a novel molecular signature with significant correlation with patients’ clinical outcome. Therapeutic targetting of FAT1 may benefit patients with high FAT1 expressing tumors.

Pilocytic astrocytomas occur rarely in adults and show aggressive tumor behavior. However, their underlying molecular‐genetic events are largely uncharacterized. Hence, 59 adult pilocytic astrocytoma (APA) cases of classical histology were studied (MIB‐1 LI: 1%–5%). Analysis of BRAF alterations using qRT‐PCR, confirmed KIAA1549‐BRAF fusion in 11 (19%) and BRAF‐gain in 2 (3.4%) cases. BRAF‐V600E mutation was noted in 1 (1.7%) case by sequencing. FGFR1‐mutation and FGFR‐TKD duplication were seen in 7/59 (11.9%) and 3/59 (5%) cases, respectively. Overall 36% of APAs harbored BRAF and/or FGFR genetic alterations. Notably, FGFR related genetic alterations were enriched in tumors of supratentorial region (8/25, 32%) as compared with other locations (P = 0.01). The difference in age of cases with FGFR1‐mutation (Mean age ± SD: 37.2 ± 15 years) vs. KIAA1549‐BRAF fusion (Mean age ± SD: 25.1 ± 4.1 years) was statistically significant (P = 0.03). Combined BRAF and FGFR alterations were identified in 3 (5%) cases. Notably, the cases with more than one genetic alteration were in higher age group (Mean age ± SD: 50 ± 12 years) as compared with cases with single genetic alteration (Mean age ± SD: 29 ± 10; P = 0.003). Immunopositivity of p‐MAPK/p‐MEK1 was found in all the cases examined. The pS6‐immunoreactivity, a marker of mTOR activation was observed in 34/39 (87%) cases. Interestingly, cases with BRAF and/or FGFR related alteration showed significantly lower pS6‐immunostatining (3/12; 25%) as compared with those with wild‐type BRAF and/or FGFR (16/27; 59%) (P = 0.04). Further, analysis of seven IDH wild‐type adult diffuse astrocytomas (DA) showed FGFR related genetic alterations in 43% cases. These and previous results suggest that APAs are genetically similar to IDH wild‐type adult DAs. APAs harbor infrequent BRAF alterations but more frequent FGFR alterations as compared with pediatric cases. KIAA1549‐BRAF fusion inversely correlates with increasing age whereas FGFR1‐mutation associates with older age. Activation of MAPK/ERK/mTOR signaling appears to be an important oncogenic event in APAs and may be underlying event of aggressive tumor behavior. The findings provided a rationale for potential therapeutic advantage of targeting MAPK/ERK/mTOR pathway in APAs.

Glioblastoma Multiforme (GBM) is a kind of brain tumor which arises from astrocytes present in the brain. It represents approximately 15% of all primary brain tumor and has been considered as one of the deadliest type of cancer with very poor prognosis. Thus, there is an urgent need to identify novel targets for GBM therapy. Recently, non-coding RNAs (ncRNAs) such as miRNAs and lncRNAs have emerged as one of the promising tools as diagnostic or prognostic biomarkers and as novel targets for therapy. There are recent evidences that small nucleolar RNAs (snoRNAs), a subtype of ncRNAs might also play a key role in various cancers. In this context, we aimed at studying the role of snoRNAs in GBM. For this, we did snoRNA profiling in GBM patients and found that a particular cluster of snoRNA called SNORD-X was highly downregulated in adult as well as pediatric GBM patients. We also found SNORD-X promoter to be methylated and shows maternal imprinting. We next did functional analyses of SNORD-X by generation of an overexpression construct. Our preliminary data suggests that overexpression of SNORD-X in GBM cell lines (U87MG and A172) increases cell proliferation in vitro. It also enhances the colony forming capability of GBM cells. Further, its overexpression causes decrease in apoptosis in a caspase-dependent manner. A target analysis of SNORD-X was done using RNAhybrid software. We found EGR1, DCUN1D3 and BRCA1 to be the direct targets of SNORD-X using a combination of qRT-PCR and wild-type/mutated 3’UTR luciferase analyses. Overall, our work shows for the first time correlation and functional analysis of SNORD-X cluster in GBM. Citation Format: Shikha Gupta, Prerana Jha, Chitra Sarkar, Ritu Kulshreshtha. SNORD-X in glioblastoma: regulation and functional analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3482. doi:10.1158/1538-7445.AM2017-3482

Pediatric high-grade gliomas (HGGs) are highly malignant tumors that remain incurable and relatively understudied. The crucial role of noncoding RNAs (ncRNAs) has been reported in various cancers. However, the study on miRNAs in pediatric HGGs is scant and there is no report till date on the status of other small ncRNAs. Genome-wide microarray analysis was performed to investigate small ncRNA expression in pediatric HGG (n = 14) and compared to adult glioblastoma (GBM) signature. The validation of miRNAs and small nucleolar RNAs (snoRNAs) was done by real-time polymerase chain reaction. TP53 and H3F3A mutation-specific miRNA and snoRNA profiles were generated and analyzed. Pediatric HGGs showed upregulation of miR-17/92 and its paralog clusters (miR106b/25 and miR-106a/363), whereas majority of downregulated miRNAs belonged to miR379/656 cluster (14q32). Unsupervised hierarchical clustering identified two distinct groups. Interestingly, Group 2 with downregulated 14q32 cluster showed better overall survival. The miRNAs unique to pediatric HGG as compared to adult GBM were predicted to affect PDGFR and SMAD2/3 pathways. Similarities were seen between pediatric HGG and TP53 mutant miRNA profiles as compared to wild types. Several of H3F3A mutation-regulated genes were found to be the targets of H3F3A mutant-specific miRNAs. Remarkably, a significant downregulation of HBII-52 snoRNA cluster was found in pediatric HGGs, and was specific to H3F3A nonmutants. This is the first genome-wide profiling study on miRNAs and snoRNAs in pediatric HGGs with respect to H3F3A and TP53 mutations. The comparison of miRNA profiles of pediatric HGGs and adult GBM reiterates the overlaps and differences as also seen with their gene expression and methylation signatures.

Background Hypoxia is a critical aspect of the glioma microenvironment and has been associated with poor prognosis and resistance to various therapies. However, the mechanisms responsible for hypoxic survival of glioma cells remain unclear. Recent studies strongly suggest that microRNAs act as critical mediators of the hypoxic response. We thus hypothesized their prominent role in hypoxia resistance in glioblastoma (GBM) and aimed to identify those. Results With this study, we present the first detailed analysis of small RNA transcriptome of cell line U87MG, a grade IV glioma cell line, and its alteration under hypoxic condition. Based on deep sequencing and microarray data, we identify a set of hypoxia regulated microRNAs, with the miR-210-3p and its isomiRs showing highest induction in GBM cell lines U87MG and U251MG. We show miR-210-3p, miR-1275, miR-376c-3p, miR-23b-3p, miR-193a-3p and miR-145-5p to be up-regulated, while miR-92b-3p, miR-20a-5p, miR-10b-5p, miR-181a-2-3p and miR-185-5p are down-regulated by hypoxia. Interestingly, certain hypoxia-induced miRNAs are also known to be over-expressed in GBM tumors, suggesting that hypoxia may be one of the factors involved in establishing the miRNA signature of GBM. Transcription factor binding sites for Hypoxia inducible factor 1 A (HIF1A) were identified in the promoter region (5 kb upstream) of 30 hypoxia-induced miRNAs. HIF-1A over-expression and silencing studies show regulation of specific miRNAs, including miR-210-3p, to be HIF1A dependent. On the other hand, miR-210-3p leads to an increase in transcriptional activity of HIF and its target genes vascular endothelial growth factor (VEGF) and carbonic anhydrase 9 (CA9). MiR-210-3p levels were found to be high in GBM patient samples and showed good correlation with the known hypoxia markers CA9 and VEGF. We show that miR-210-3p promotes hypoxic survival and chemoresistance in GBM cells and targets a negative regulator of hypoxic response, HIF3A. Additionally, a total of 139 novel miRNAs were discovered by the analysis of deep sequencing data and three of these were found to be differentially expressed under hypoxia. Conclusions Overall, our study reveals a novel miRNA signature of hypoxia in GBM and suggests miR-210-3p to be an oncogenic player and a novel potential intrinsic marker of hypoxia in glioblastoma. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-686) contains supplementary material, which is available to authorized users.

Background. Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH) 1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Methods. Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Results. Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine-phosphate-guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Conclusions. Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation.

Background MicroRNAs (miRNAs) are non-uniformly distributed in genomes and ~30% of the miRNAs in the human genome are clustered. In this study we have focused on the imprinted miRNA cluster miR-379/miR-656 on 14q32.31 (hereafter C14) to test their coordinated function. We have analyzed expression profile of >1000 human miRNAs in >1400 samples representing seven different human tissue types obtained from cancer patients along with matched and unmatched controls. Results We found 68% of the miRNAs in this cluster to be significantly downregulated in glioblastoma multiforme (GBM), 61% downregulated in kidney renal clear cell carcinoma (KIRC), 46% in breast invasive carcinoma (BRCA) and 14% in ovarian serous cystadenocarcinoma (OV). On a genome-wide scale C14 miRNAs accounted for 12-30% of the total downregulated miRNAs in different cancers. Pathway enrichment for the predicted targets of C14 miRNA was significant for cancer pathways, especially Glioma (p< 3.77x10-6, FDRMEF2, a crucial transcription factor for the cluster was observed which likely contribute to the observed downregulation of the entire miRNA cluster. Conclusion We provide compelling evidence that the entire C14 miRNA cluster is a tumor suppressor locus involved in multiple cancers, especially in GBM, and points toward a general mechanism of coordinated function for clustered miRNAs. Reviewers Reviewed by: Prof. Gregory J Goodall and Dr. Alexander Max Burroughs

The IDH1 gene on chromosome 2q33.3 encodes for isocitrate dehydrogenase 1 (IDH1), located in the cytoplasm and the peroxisomes. This enzyme catalyzes NADPH production via oxidative decarboxylation of isocitrate to alpha-ketoglutarate in the Krebs citric acid cycle.1 In 2008, for the first time, Parsons et al introduced to the medicine world the role of IDH1 in the pathogenesis of glioblastoma multiforme (GBM). In their genome-wide sequencing analysis, recurrent somatic mutations specifically involving the amino acid arginine at position 132 were detected in 12% of the GBM specimens.2 Subsequent studies have shown that IDH1 mutation is an early step in gliomagenesis and has been reported to occur in grades II and III astrocytomas, oligodendrogliomas (OG), oligoastrocytomas (OA), and secondary GBM.3–12 Hartmann et al, in their analysis of 1010 diffuse glioma tumors, demonstrated that most cases of diffuse astrocytomas (DA; 72.7%, 165/227), anaplastic astrocytomas (AA; 64.0%, 146/228), OG (82.0%, 105/128), anaplastic oligodendrogliomas (AOG; 69.5%, 121/174), OA (81.6%, 62/76), and anaplastic oligoastrocytomas (AOA; 66.1%, 117/177) had IDH1 mutations.13 Of importance, these mutations appear to be specific for these tumors as primary GBM, pilocytic astrocytoma World Health Organization (WHO) grade I and other central nervous system (CNS) and non-CNS neoplasms, with the exception of acute myeloid leukemia and cartilaginous neoplasms, harbor this genetic alteration much less frequently.3–11,14–19 Different types of mutations have been described, and the most frequent is G to A transitions at position 395 of the IDH1 transcript. This results in substitution of the amino acid arginine with histidine (R132H). Rarer ones include R132C, R132S, R132G, R132L, R132V and R132P.2,4,5,8–11,13,17,20,21 Mutations involving IDH2, a homologous gene, have also been detected in gliomas but at a much lower frequency ranging from 2% to 5%.8,10,13,17,21,22 Although these mutations are rare in the pediatric age group, in patients aged ≥18 years, they seem to be associated with younger age at presentation and have a favorable impact on the overall and progression-free survival associated with grade II-IV gliomas.2,6,7,10,12,13,22–28 IDH1 testing is being used as a standard diagnostic tool in many neuropathology laboratories. It is useful in differentiating gliomas from nonneoplastic CNS lesions,21,29–31 diffuse astrocytoma WHO grade II from pilocytic astrocytoma grade I,15,32 anaplastic astrocytomas WHO grade III from GBM,32 primary from secondary GBM,6,32 and astrocytomas from ependymomas.32 Most studies of IDH mutations are based on DNA sequencing, which is labor intensive, requiring trained personnel and sophisticated equipment, not available at every center. Moreover, false-negative results may be obtained in cases of inadequate tumor DNA availability because of small biopsy samples, extensive necrosis, or admixture with normal tissue elements. Alternate rapid methods, some based on routinely processed tissue specimens, have been recently suggested.17,21,26,29–36 Of these, a significant development was the introduction of mAb H0933 and IMab-134 antibodies, which are specific for the most common IDH1 mutation: R132H. In the present study, which is a continuation of our previous study on IDH1 mutation as assessed by direct DNA sequencing,11 we compared the results of immunohistochemistry (IHC) using mAb H09 for IDH1-R132 mutations with those of DNA sequencing in different types and grades of gliomas.

Several studies on molecular profiling of oligodendrogliomas (OGs) in adults have shown a distinctive genetic pattern characterized by combined deletions of chromosome arms 1 p and 19q, O6-methylguanine-methyltransferase (MGMT) methylation, and isocitrate dehydrogenase 1 (IDH1) mutation, which have potential diagnostic, prognostic, and even therapeutic relevance. OGs in pediatric and young adult patients are rare and have been poorly characterized on a molecular and biological basis, and it remains uncertain whether markers with prognostic significance in adults also have predictive value in these patients. Fourteen cases of OGs in young patients (age

Assessment of promoter methylation of the O 6 -methylguanine DNA methyltransferase (MGMT) gene has recently gained importance in molecular profiling of high-grade gliomas. It has emerged not only as an important prognostic marker but also as a predictive marker for response to temozolomide in patients with newly diagnosed glioblastoma. Further, recent studies indicate that MGMT promoter methylation has strong prognostic relevance even in anaplastic (grade III) gliomas, irrespective of therapy (chemotherapy or radiotherapy). This article provides an overview of its use as a predictive and prognostic biomarker, as well as the methods employed for its assessment and use in therapeutic decision making.

Pilocytic astrocytoma (PA), a WHO grade I tumor, occurs rarely in adults and shows aggressive tumor behavior. However, their underlying molecular-genetic events are largely uncharacterized. Hence, we studied 59 adult PA patients of classical histology with MIB-1 LI from 1-5% (median age at diagnosis 30 yrs, age-range 19-69 yrs). BRAF alterations examined in 59 cases confirmed KIAA1549-BRAF fusion in 11(19%), BRAF-gain in 2(3.4%) by qRT-PCR, and BRAF-V600E mutation in 1(1.7%) case using direct sequencing. FGFR1-mutation and FGFR-TKD duplication were seen in 7/59(11.9%) and 3/59(5%) cases respectively. Notably, FGFR related genetic alterations were enriched in supratentorial region (8/25, 32%) as compared to other tumor locations (P = 0.01). The association of age with respect to FGFR1-mutation (Mean age ± SD: 37.2 ± 15) compared to cases with KIAA1549-BRAF (Mean age ± SD: 25.1 ± 4.1) was significant (P = 0.03). Further, BRAF + FGFR duplicate genetic alterations were identified in 3(5%) cases (all in supratentorial tumors). Overall 36% of adult PAs harbored BRAF and/or FGFR related genetic alterations. Immunopositivity of p-MAPK/p-MEK1 corresponding to MAPK/ERK pathway activation was found in all the cases examined. In total, mTOR activator, pS6-immunoreactivity of any degree was observed in 34/39(87%) of cases. Interestingly, cases with BRAF and/or FGFR related alteration showed significantly lower pS6 immunostaining (3/12, 25%) as compared to BRAF and/or FGFR wild-type (16/27;59%) (P = 0.04). This study represents the largest reported adult PA patient cohort molecularly profiled to date. PAs with classical histology occur in adults but harbor infrequent BRAF alterations but enriched FGFR alterations in most instances compared to pediatric. The activation of constitutive BRAF-FGFR related MAPK/ERK signaling along with mTOR appears to be an important oncogenic event in adult PAs and may be constituting to the aggressive tumor behavior of adults. Therefore this study provides a rationale for potential therapeutic advantage of targeting MAPK/ERK and mTOR pathway in adult PAs.

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.

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.