Suvendu Purkait

CDKN2A deletion in pediatric versus adult glioblastomas and predictive value of p16 immunohistochemistry

Cell cycle regulator genes are major target of mutation in many human malignancies including glioblastomas (GBMs). CDKN2A is one such tumor suppressor gene which encodes p16INK4a protein and serves as an inhibitor of cell cycle progression. Very few studies are available regarding the association of CDKN2A deletion with p16 protein expression in GBMs. There is limited data on the frequency of CDKN2A deletion in different age groups. The aim of the present study was to analyze the frequency of CDKN2A gene deletions in GBM and correlate CDKN2A deletional status with (i) age of the patient (ii) p16 protein expression and (iii) other genetic alterations, namely EGFR amplification and TP53 mutation. A combined retrospective and prospective study was conducted. Sixty seven cases were included. The patients were grouped into pediatric (≤18 years), young adults (19‐40 years) and older adults (>40 years). CDKN2A and EGFR status were assessed by Fluorescence in situ Hybridization.TP53 mutation was analyzed by PCR based method. p16 expression was assessed using immunohistochemistry. CDKN2A deletion was noted in 40.3% cases of GBM with majority being homozygous deletion (74%). It was commoner in primary GBMs (65.8%) and cases with EGFR amplification (50%). A variable frequency of CDKN2A was observed in older adults (42.3%), young adults (44%), and pediatric patients (31.25%). The difference however was not statistically significant. There was statistically significant association between CDKN2A deletion and p16 immunonegativity with a high negative predictive value of immunohistochemistry.

Genome-wide small noncoding RNA profiling of pediatric high-grade gliomas reveals deregulation of several miRNAs, identifies downregulation of snoRNA cluster HBII-52 and delineates H3F3A and TP53 mutant-specific miRNAs and snoRNAs.

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.

A study of clinico‐pathological parameters and O6 – methylguanine DNA methyltransferase (MGMT) promoter methylation status in the prognostication of gliosarcoma

Gliosarcoma is a rare variant of glioblastoma multiforme (GBM) with similar clinical presentation and prognosis but a distinct genetic profile. The clinicopathological features of 22 cases of gliosarcoma were analyzed with respect to age, sex, KPS score, operative diagnosis, extent of resection and histopathological subtype (predominantly sarcomatous [PS], predominantly gliomatous [PG] or mixed). Twelve cases were PS, six were PG and four were mixed. The histological subtype did not correlate with the operative diagnosis; however, it did significantly correlate with the extent of resection (P = 0.014). In 14 cases with available survival data it was found that none of the clinicopathological parameters significantly correlated with survival (P > 0.05). Methyl guanine DNA methyl transferase promoter methylation studies were performed using methylation‐specific PCR in 16 cases which showed a methylation rate of 31.25% (5/16). The promoter methylation status did not correlate with the histological subtype and did not significantly affect survival (P > 0.05). Although gliosarcomas continue to be treated in the same way as GBM, the role of chemotherapy with temozolomide is not clear. This cohort is the largest to date to uniformly receive the Stupps protocol which is currently “standard of care” for GBM. A median overall survival of 18.5 months is substantially higher than previous studies, suggesting that temozolomide should be included in gliosarcoma therapy.

Oncogenic KIAA1549-BRAF fusion with activation of the MAPK/ERK pathway in pediatric oligodendrogliomas

Pediatric oligodendrogliomas (pODGs) are rare central nervous system tumors, and comparatively little is known about their molecular pathogenesis. Co-deletion of 1p/19q; and IDH1, CIC, and FUBP1 mutations, which are molecular signatures of adult oligodendrogliomas, are extremely rare in pODGs. In this report, two pODGs, one each of grade II and grade III, were evaluated using clinical, radiological, histopathologic, and follow-up methods. IDH1, TP53, CIC, H3F3A, and BRAF-V600 E mutations were analyzed by Sanger sequencing and immunohistochemical methods, and 1p/19q co-deletion was analyzed by fluorescence in situ hybridization. PDGFRA amplification, BRAF gain, intragenic duplication of FGFR-TKD, and KIAA1549-BRAF fusion (validated by Sanger sequencing) were analyzed by real-time reverse transcription PCR. Notably, both cases showed the oncogenic KIAA1549_Ex15-BRAF_Ex9 fusion transcript. Further, immunohistochemical analysis showed activation of the MAPK/ERK pathway in both of these cases. However, neither 1p/19q co-deletion; IDH1, TP53, CIC, H3F3A, nor BRAF-V600 E mutation; PDGFRA amplification; BRAF gain; nor duplication of FGFR-TKD was identified. Overall, this study highlights that pODGs can harbor the KIAA1549-BRAF fusion with aberrant MAPK/ERK signaling, and there exists an option of targeting these pathways in such patients. These results indicate that pODGs with the KIAA1549-BRAF fusion may represent a subset of this rare tumor that shares molecular and genetic features of pilocytic astrocytomas. These findings will increase our understanding of pODGs and may have clinical implications.

Expression of DNA methyltransferases 1 and 3B correlates with EZH2 and this 3-marker epigenetic signature predicts outcome in glioblastomas.

This study aims to analyze expression of EZH2 and DNA-methyltransferases (DNMT1, 3A and 3B) in astrocytic tumors and investigate their link as well as their correlation with survival, especially in GBMs. Expression of EZH2 and DNMTs (DNMT1, DNMT3A and DNMT3B) in different grades of astrocytomas (n=93) was assessed by qRT-PCR and immunohistochemistry. GBM-U87MG cell line was used for functional studies. Strong immunopositivity (LI≥25%) for EZH2, DNMT1 and DNMT3B was detected in 52%, 56% and 64% cases of GBMs respectively, which was significantly higher as compared to Grade II/III cases. Similarly, their median fold change of mRNA expression was also significantly higher in GBMs. There was also a significant positive correlation between DNMT1/DNMT3B and EZH2 mRNA and protein expression, which was in concordance with TCGA data set. Inhibition of DNMTs in cell line by Azacytidine resulted in down-regulation of EZH2, while knock-down of EZH2 by siRNA was not associated with any significant alteration of DNMTs, indicating that EZH2 expression in GBMs is possibly regulated by DNMTs, but not the reverse. Strong immunopositivity for EZH2, DNMT1 and DNMT3B were individually associated with significantly shorter survival and showed no correlation with IDH1 mutation status. In addition, the combination of these 3 markers represented an independent prognostic signature with cases having weak/negative expression of all 3 markers being associated with best prognosis. For the first time, the present study describes an epigenetic prognostic signature in GBMs based on immunohistochemical expression of EZH2, DNMT1 and 3B which can be used easily in routine neuropathology practice.

Genome-wide ChIP-seq analysis of EZH2-mediated H3K27me3 target gene profile highlights differences between low- and high-grade astrocytic tumors

Enhancer of zeste homolog-2(EZH2) is a key epigenetic regulator that functions as oncogene and also known for inducing altered trimethylation of histone at lysine-27 (H3K27me3) mark in various tumors. However, H3K27me3 targets and their precise relationship with gene expression are largely unknown in astrocytic tumors. In this study, we checked EZH2 messenger RNA and protein expression in 90 astrocytic tumors of different grades using quantitative PCR and immunohistochemistry, respectively. Further, genome-wide ChIP-seq analysis for H3K27me3 modification was also performed on 11 glioblastomas (GBMs) and 2 diffuse astrocytoma (DA) samples. Our results showed EZH2 to be highly overexpressed in astrocytic tumors with a significant positive correlation with grade. Interestingly, ChIP-seq mapping revealed distinct differences in genes and pathways targeted by these H3K27me3 modifications between GBM versus DA. Neuroactive ligand receptor pathway was found most enriched in GBM (P = 9.4 × 10-25), whereas DA were found to be enriched in metabolic pathways. Also, GBM showed a higher enrichment of H3K27me3 targets reported in embryonic stem cells and glioma stem cells as compared with DAs. Our results show majority of these H3K27me3 target genes were downregulated, not only due to H3K27me3 modification but also due to concomitant DNA methylation. Further, H3K27me3 modification-associated gene silencing was not restricted to promoter but also present in gene body and transcription start site regions. To the best of our knowledge, this is the first high-resolution genome-wide mapping of H3K27me3 modification in adult astrocytic primary tissue samples of human, highlighting the differences between grades. Interestingly, we identified SLC25A23 as important target of H3K27me3 modification, which was downregulated in GBM and its low expression was associated with poor prognosis in GBMs.

Genetic alterations related to BRAF‐FGFR genes and dysregulated MAPK/ERK/mTOR signaling in adult pilocytic astrocytoma

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.

Analysis of EZH2: micro-RNA network in low and high grade astrocytic tumors

Enhancer of Zeste homologue2 (EZH2) is an epigenetic regulator that functions as oncogene in astrocytic tumors, however, EZH2 regulation remains little studied. In this study, we measured EZH2 levels in low (Gr-II,DA) and high grade (Gr-IV,GBM) astrocytic tumors and found significant increased EZH2 transcript level with grade(median DA-8.5, GBM-28.9).However, a different trend was reflected in protein levels, with GBMs showing high EZH2 LI(median-26.5) compared to DA (median 0.3). This difference in correlation of EZH2 protein and RNA levels suggested post-transcriptional regulation of EZH2, likely mediated by miRNAs. We selected eleven miRNAs that strongly predicted to target EZH2 and measured their expression. Three miRNAs (miR-26a-5p,miR27a-3p and miR-498) showed significant correlation with EZH2 protein, suggesting them as regulators of EZH2, however miR-26a-5p levels decreased with grade. ChIP analyses revealed H3K27me3 modifications in miR-26a promoter suggesting feedback loop between EZH2 and miR26a. We further measured six downstream miRNA targets of EZH2 and found significant downregulation of four (miR-181a/b and 200b/c) in GBM. Interestingly, EZH2 associated miRNAs were predicted to target 25 genes in glioma-pathway, suggesting their role in tumor formation or progression. Collectively, our work suggests EZH2 and its miRNA interactors may serve as promising biomarkers for progression of astrocytic tumors and may offer novel therapeutic strategies.

EZH2 expression in gliomas: Correlation with CDKN2A gene deletion/ p16 loss and MIB‐1 proliferation index

Enhancer of zeste homolog 2 (EZH2) mediated down‐regulation of CDKN2A/p16 has been observed in cell lines as well as in a few carcinomas. However, there is no study correlating EZH2 expression with CDKN2A/p16 status in gliomas. Hence, the present study was conducted to evaluate EZH2 expression in astrocytic and oligodendroglial tumors and correlate with CDKN2A/p16 status as well as MIB‐1 labeling index (LI). Gliomas of all grades (n = 118) were studied using immunohistochemistry to assess EZH2, p16 and MIB‐1 LI and fluorescence in situ hybrization to evaluate CDKN2A gene status. EZH2 expression and CDKN2A homozygous deletion (HD) were both significantly more frequent in high‐grade gliomas (HGG). Further, strong EZH2 expression (LI ≥ 25%) was significantly more common in HGGs without CDKN2A HD (48.7%; 19/39) as compared to cases with deletion (15.8%; 3/19). Loss of p16 expression was noted in 100% and 51.3% of CDKN2A deleted and non‐deleted tumors, respectively. Notably, 80% (16/20) of the CDKN2A non‐deleted HGGs with p16 loss had strong EZH2 expression, in contrast to only 15.8% (3/19) in the deleted group. Loss of p16 expression significantly correlated with MIB‐1 LI, irrespective of EZH2 status. Thus, this study shows that EZH2 expression correlates with tumor grade in both astrocytic and oligodendroglial tumors and hence can be used as a diagnostic marker to differentiate between low and HGGs. Further, this is the first report demonstrating an inverse correlation of strong EZH2 expression with CDKN2A HD in HGGs. Loss of p16 protein expression is mostly attributable to CDKN2A HD and correlates significantly with MIB‐1 LI. Notably, our study for the first time suggests a possible epigenetic mechanism of p16 loss in CDKN2A non‐deleted HGGs mediated by strong EZH2 expression. A hypothetical model for control of proliferative activity in low versus HGGs is therefore proposed.

ATRX in diffuse gliomas with its mosaic/heterogeneous expression in a subset.

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.