<b><i>Introduction:</i></b> The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) was proposed by the American Society of Cytopathology and the International Academy of Cytology to bring uniformity in the reporting system and the treatment protocol. A wide range of risk of malignancy for each category has been reported by various authors by applying the system. <b><i>Aim:</i></b> We intend to study the cytohistological concordance and the ROM for each of the diagnostic categories of the Milan system. <b><i>Materials and Methods:</i></b> The study included 292 cases of fine-needle aspiration cytology (FNAC) of salivary gland lesions over a period of 3 years. The diagnosis of these cases was reclassified into the 6 categories of the Milan system. The cytohistological concordance and ROM for each category of the Milan system were calculated based on the clinical and histopathological follow-up. <b><i>Results:</i></b> The patients’ age ranged from 3 to 81 years with the mean of 42.65 ± 16.3 years. The cases included 189 (64.7%) parotid, 82 (28.1%) submandibular, and 21 (7.2%) cases of minor salivary gland swellings. Follow-up histopathological diagnosis for 102 cases was available. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were calculated to be 64.28, 97.01, 90, 86.67, and 87.37%, respectively. After reclassification, the number of cases in each category was as follows: category I: 31 (10.62%), category II: 80 (27.4%), category III: 2 (0.68%), category IVA: 143 (48.97%), category IVB: 1 (0.34%), category V: 13 (4.45%), and category VI: 22 (7.53%). The calculated ROM was as follows: category I: 42.86%, category II: 26.67%, category III: 100% category IVA: 10.17%, category IVB: 0%, category V: 71.42%, category VI: 100%. <b><i>Conclusion:</i></b> FNAC is an excellent procedure to differentiate benign from malignant tumors, and MSRSGC is a useful system for risk assessment and deciding the further treatment protocol. Our findings also suggest that in addition to the surgical follow-up, inclusion of the clinical and radiological follow-up may be a better strategy for calculation of ROM, especially for categories I and II.

Mutations in H3.3-ATRX-DAXX chromatin remodeling pathway have been reported in pediatric GBMs. H3.3 (H3F3A) mutations may affect transcriptional regulation by altered global histone-methylation. Therefore, we analyzed yet partly understood global histone code (H3K-4/9/27/36) trimethylation pattern in H3F3A-ATRX mutants and wild-type. H3F3A, HIST1H3B, IDH1, ATRX, DAXX and Tp53 mutations were identified by sequencing/immunohistochemistry in 27 pediatric GBMs. Global histone-methylation H3K-4/9/27/36me3 and Polycomb-protein EZH2 expression were evaluated by immunohistochemistry. H3F3A-ATRX mutation was observed in 66.7 % (18/27) of pediatric GBMs. K27M and G34R-H3F3A mutations were found in 37 % (10/27) and 14.8 % (4/27) patients respectively. G34V-H3F3A, HIST1H3B and IDH1 mutations were absent. Notably, commonest global histone-methylation mark lost was H3K27me3 (17/25, 68 %) followed by H3K4me3 (45.5 %, 10/22) and H3K9me3 (18.2 %, 4/22). Global H3K36me3 showed no loss. Most significant observation was loss of one or more histone-trimethylation mark in 80 % (20/25) pediatric GBMs. Notably, simultaneous loss of H3K27me3 and H3K4me3 were present in 7/22 (31.8 %) of pediatric GBMs. Low expression of EZH2 was found in 12/24 (50 %) of cases. However no significant correlation of loss of histone-marks or EZH2 expression with H3F3A-ATRX mutants (loss of at least one histone-marks in 87.5 % (14/16) cases) versus wild-types (loss of at least one histone-marks in 75 % (6/8) cases) was seen. The present study highlights for the first time combinatorial loss of one or more histone-trimethylation marks associated with majority of pediatric GBMs and the finding suggests significant role of histone-code in the molecular biology that underlies pediatric GBMs. Hence therapies for patients with particular combinations of histone modifications present opportunity to design innovative patient-tailored treatment protocols.

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.

To evaluate age-related differences in histopathologic and molecular profile of glioblastomas (GBMs) at various age groups, with special reference to TP53 mutation, epidermal growth factor receptor (EGFR) amplification, EGFR vIII mutant, PTEN deletion, and IDH1 mutation. Agewise GBM incidence was calculated over a period of 5 years (2005 to 2009). Seventy-five GBMs were selected for molecular analysis. Majority of cases were in the age group of 41 to 60 years, and mean age was 43.6 years. Histology of all 75 cases selected for molecular profiling was identical. Primary adult GBMs showed EGFR amplification and PTEN deletion in majority (37.3% and 54.9%, respectively). TP53 and IDH1 mutations were rare (11.8% cases each). In secondary GBMs, TP53 (66.7%) and IDH1 mutations (44.4%) were most frequent. PTEN deletion was seen in 33.3% and none had EGFR amplification. Pediatric GBMs (<18 y) harbored frequent TP53 mutations (46.7%) and PTEN deletion in 40%. IDH1 mutations and EGFR amplification were absent. The molecular profile of primary GBMs in young adults (19 to 40 y) was distinctly different from that of adults older than 40 years. TP53 mutation was present in 20% cases. The frequency of EGFR amplification (13.3%) and PTEN deletion (33.3%) was significantly low (P=0.028 and 0.046, respectively). IDH1 mutation, which is rare in primary adult GBMs, was present in 40% of cases. Molecular heterogeneity exists within GBMs of different age cohorts. The molecular profile of GBMs in young adults is distinctly different. Thus, there is a strong need for further studies in various age groups to provide guidelines for therapeutic targeting.

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.

Molecular and clinical characteristics of pediatric meningiomas are poorly defined. Therefore, we analyzed clinical, morphological and molecular profiles of pediatric meningiomas. Forty pediatric meningiomas from January 2002 to June 2015 were studied. 1p36, 14q32 and 22q-deletion were assessed by fluorescent in situ hybridization and mutations of most relevant exons of AKT, SMO, KLF4, TRAF and pTERT using sequencing. Expression of GAB1, stathmin, progesterone receptor (PR), p53 along with MIB-1 LI was examined using immunohistochemistry. There were 36 sporadic and four NF2 associated meningiomas. Among sporadic meningiomas, the majority (72.2%) of cases harbored 22q-deletion. Difference in frequency of combined 1p/14q deletion in Grade-I versus Grade-II/III tumors was not significant (13.7% vs 28.5%, P = 0.57). PR immunoreactivity was seen in 65.5% of Grade-I and 14.2% of Grade-II/III tumors (P = 0.03). The majority (97.2%) of meningiomas were immunonegative for p53. Stathmin and GAB co-expression was observed in 58.3% of cases. Notably, AKT, SMO, KLF4, TRAF7 (exon 17) and pTERT mutations were seen in none of the cases analyzed. 1p/14q codeletion was frequent in skull base as compared to non-skull base meningiomas (23% vs 11.1%, P = 0.37). All NF2 meningiomas harbored 22q-deletion and showed GAB and stathmin co-expression while none showed 1p/14q loss. Pediatric meningiomas share certain phenotypic and cytogenetic characteristics with adult counterparts, but GAB and stathmin co-expression in the majority of cases and non-significant difference in frequency of 1p/14q co-deletion between low- and high-grade meningiomas indicate an inherently aggressive nature. Characteristic AKT/SMO, KLF4/TRAF7 and pTERT genetic alterations seen in adults are distinctly absent in pediatric meningiomas.

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.

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 Stupp's 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.