MYCN amplification in spinal ependymoma: A five-year retrospective study.

Spinal ependymoma with MYCN amplification is a newly recognized type of spinal ependymoma that is known to be associated with poor prognosis. Available studies on this relatively rare tumor type have observed that these tumors tend to disseminate along the spinal cord and behave aggressively with worse overall and progression-free survival compared to the other types of ependymoma. In this study, we describe the clinical and histopathological features of spinal ependymomas in a single institution cohort with emphasis on those with MYCN amplification.

IRX1 is a novel gene, overexpressed in high-grade IDH-mutant astrocytomas.

BACKGROUND: IDH-mutant astrocytomas include CNS WHO grade 2 (A2), grade 3 (A3) and grade 4 (A4), of which A3 and A4 are high-grade. A3 has a heterogenous clinical outcome that cannot be explained entirely by the existing molecular biomarkers. We comprehensively studied the transcriptome profile of A3 to determine clinical significance. METHODS: TCGA mRNA-sequencing data of A3 was analyzed to derive differentially expressed genes (DEG), which were short-listed using various approaches. mRNA expression of the short-listed genes was validated using NanoString platform on a uniformly treated and molecularly characterized A3 cohort. Protein expression of one prognostically significant gene, Iroquois-class homeodomain (IRX1) was assessed by immunohistochemistry and correlated with patient survival and tumor recurrence. IRX1 expression was also studied in different grades of astrocytoma. Since DNA methyltransferase 3 alpha (DNMT3A) influences IRX1 expression, its mutations were evaluated in a subset of tumors. RESULTS: TCGA analysis identified 96 DEG in A3 tumours. 57 genes were short-listed and finally narrowed down to 14 genes. mRNA values of 12/14 genes validated in our cohort. On multiple-variable analysis, IRX1 was the most prognostically relevant gene, with respect to progression free survival of patients. Further, IRX1 immunoexpression was significantly higher in A3 and A4 when compared to A2 and glioblastoma. Higher IRX1 immunoexpression correlated with poor prognosis in patients with A3 tumours. Also, a higher IRX1 expression was associated with DNMT3A mutation. CONCLUSION: Our study identifies IRX1 as a novel biomarker overexpressed in high-grade IDH-mutant astrocytomas with prognostic significance in A3. DNMT3A mutation probably modulates IRX1 expression.

Chitinase 3-Like 2.

OBJECTIVES: We aimed to evaluate the expression pattern of chitinase 3-like 2 (CHI3L2) in the tumor core and peritumoral brain zone (PBZ) of newly diagnosed glioblastoma (GBM) in recurrent tumors and its association with patient prognosis. METHODS: The study was conducted on three sample sets derived from different patient cohorts. Messenger RNA (mRNA) expression of CHI3L2 in the tumor core and PBZ (n = 34) compared with control (n = 20) tissues was studied by quantitative polymerase chain reaction in sample set 1. Sample set 2 included 19 paired, primary-recurrent GBM tissues. Sample set 3 comprised 82 GBM tissues of patients with treatment and follow-up information. Immunohistochemistry (IHC) was performed on all three sample sets. RESULTS: mRNA expression of CHI3L2 was significantly higher in the tumor core and PBZ compared with control (P < .0001). By IHC, CHI3L2 showed strong cytoplasmic staining in tumor cells. Recurrent tumors had a higher expression of CHI3L2 compared with primary tumors (P = .007). Survival analysis showed CHI3L2 expression was associated with shorter overall survival (P = .034) and progression-free survival (P = .010), which was in line with The Cancer Genome Atlas cohort (P = .043). CONCLUSIONS: High expression of CHI3L2 in the tumor core and PBZ, as well as its association with tumor recurrence and poor patient prognosis, suggests it might be contributing to tumor spread and recurrence.

Spinal astroblastoma: a rare tumour in an unusual location.

Astroblastomas are central nervous system tumours with unknown cell of origin and clinical behaviour. These tumours occur most commonly in cerebral hemispheres with spinal astroblastomas being very rare. We report a case of spinal astroblastoma which harboured MN1 alteration.

Differential gene expression in peritumoral brain zone of glioblastoma: role of SERPINA3 in promoting invasion, stemness and radioresistance of glioma cells and association with poor patient prognosis and recurrence.

PURPOSE: Glioblastoma (GBM) is a highly invasive tumor. Despite advances in treatment modalities, tumor recurrence is common, seen mainly in the peritumoral brain zone (PBZ). We aimed to molecularly characterize PBZ, to understand the pathobiology of tumor recurrence. METHODS/PATIENTS: We selected eight differentially regulated genes from our previous transcriptome profiling study on tumor core and PBZ. Expression of selected genes were validated in GBM (tumor core and PBZ, n = 37) and control (n = 22) samples by real time quantitative polymerase chain reaction (qPCR). Serine protease inhibitor clade A, member 3 (SERPINA3) was selected for further functional characterization in vitro by gene knockdown approach in glioma cells. Its protein expression by immunohistochemistry (IHC) was correlated with other clinically relevant GBM markers, patient prognosis and tumor recurrence. RESULTS: The mRNA expression of selected genes from the microarray data validated in tumor core and PBZ and was similar to publicly available databases. SERPINA3 knock down in vitro showed decreased tumor cell proliferation, invasion, migration, transition to mesenchymal phenotype, stemness and radioresistance. SERPINA3 protein expression was higher in PBZ compared to tumor core and also was higher in older patients, IDH wild type and recurrent tumors. Finally, its expression showed positive correlation with poor patient prognosis. CONCLUSIONS: SERPINA3 expression contributes to aggressive GBM phenotype by regulating pro-tumorigenic actions in vitro and is associated with adverse clinical outcome.

Central nervous system high grade neuroepithelial tumor with BCOR immunopositivity: Is there a molecular heterogeneity?

Central nervous system high grade neuroepithelial tumor - BCOR altered is a newly defined entity which is characterised by internal tandem duplication (ITD) in exon 15 of BCOR. These tumors resemble high grade glioma histologically and exhibit BCOR immunopositivity. However, recently fusions of BCOR are also described in CNS lower grade gliomas, thus questioning the sensitivity and specificity of BCOR immunohistochemistry for identification of BCOR-ITD. We describe four cases of high grade neuroepithelial tumor with BCOR immunopositivity which were diagnosed over a period of one year at our institute. Amongst these, only one tumor revealed BCOR-ITD on sequencing. SATB2 immunopositivity which is a sensitive marker of BCOR-ITD, BCOR fusions and YWHAE fusions was noted in three out of four cases. Our study suggests that BCOR immunopositive CNS high grade tumors are molecularly heterogeneous and could harbour genetic alterations other than BCOR-ITD.

Low mitochondrial DNA copy number is associated with poor prognosis and treatment resistance in glioblastoma.

INTRODUCTION: Mitochondrial DNA (mtDNA) content in several solid tumors was found to be lower than in their normal counterparts. However, there is paucity of literature on the clinical significance of mtDNA content in glioblastoma and its effect on treatment response. Hence, we studied the prognostic significance of mtDNA content in glioblastoma tumor tissue and the effect of mtDNA depletion in glioblastoma cells on response to treatment. MATERIALS AND METHODS: 130 newly diagnosed glioblastomas, 32 paired newly diagnosed and recurrent glioblastomas and 35 non-neoplastic brain tissues were utilized for the study. mtDNA content in the patient tumor tissue was assessed and compared with known biomarkers and patient survival. mtDNA was chemically depleted in malignant glioma cell lines, U87, LN229. The biology and treatment response of parent and depleted cells were compared. RESULTS: Lower range of mtDNA copy number in glioblastoma was associated with poor overall survival (p = 0.01), progression free survival (p = 0.04) and also with wild type IDH (p = 0.02). In recurrent glioblastoma, mtDNA copy number was higher than newly diagnosed glioblastoma in the patients who received RT (p = 0.01). mtDNA depleted U87 and LN229 cells showed higher survival fraction post radiation exposure when compared to parent lines. The IC50 of TMZ was also higher for mtDNA depleted U87 and LN229 cells. The depleted cells formed more neurospheres than their parent counterparts, thus showing increased stemness of mtDNA depleted cells. CONCLUSION: Low mtDNA copy number in glioblastoma is associated with poor patient survival and treatment resistance in cell lines possibly by impacting stemness of the glioblastoma cells.

Alpha Internexin: A Surrogate Marker for 1p/19q Codeletion and Prognostic Marker in Anaplastic (WHO grade III) Gliomas.

BACKGROUND: The WHO 2016 classification of diffuse gliomas has incorporated molecular markers isocitrate dehydrogenase (IDH) gene mutations (IDHmut) and codeletion of chromosomal arms 1p and 19q (1p/19q codeletion) as tumor defining entities. The diagnosis of diffuse oligodendrogliomas (ODG) and anaplastic oligodendroglioma (AO) mandatorily requires the demonstration of IDH1 and/or IDH2 mutations along with 1p/19q codeletion, whereas the 1p/19q noncodeleted diffuse gliomas are labeled as astrocytomas. The current methodologies for assessing 1p/19q codeletion status are expensive and not widely available. Studies have proposed alpha internexin (INA) expression on immunohistochemistry (IHC) as a surrogate marker for 1p/19q codeletion and a good prognostic marker in gliomas. MATERIALS AND METHODS: In this study, we performed IHC for INA expression on the retrospective cohort of anaplastic gliomas (AGs) from our previously published study. RESULTS: INA positivity on IHC showed a significant positive correlation with 1p/19q codeletion (P < 0.001) with a Spearman's rank correlation coefficient (Rho) of 0.804, sensitivity of 87.5%, specificity of 93.0%, and a diagnostic odds ratio of 93:1 in AGs. Similar to the 1p/19q codeletion status, INA positivity showed a positive correlation with IDHmut (P = 0.002) and a negative correlation with α-thalassemia mental retardation X-linked protein (ATRX) loss of expression (P < 0.001). On univariate survival analysis, INA positivity was associated with significantly prolonged overall survival (OS) and recurrent free survival (RFS) in AGs (P < 0.001). Furthermore, within AO, INA positivity significantly improved RFS (P = 0.022) with OS trending towards significance (P = 0.094). CONCLUSIONS: INA expression on IHC could serve as a potential surrogate marker for 1p/19q, and highlights its prognostic value in AO and AGs.

Proteomic profiling of medulloblastoma reveals novel proteins differentially expressed within each molecular subgroup.

OBJECTIVES: The objective of the study was to identify novel medulloblastoma (MB) biomarkers through proteomic profiling, correlate it with the molecular subgroups of MB and assess the clinical significance. METHODS: Archived paraffin embedded tumor tissue blocks from 118 MB patients, operated at our institute were retrieved. Clinical information was documented from the hospital database. Tumours were stratified into molecular subgroups using the IHC markers- ß Catenin, GAB-1, YAP-1 and p53. Six fresh MB tumour tissues and two control cerebellar tissues were subjected to proteomic profiling to study differential protein expression in molecular subgroups using high resolution mass spectrometry. Prominent signalling pathways activated in each subgroup were identified using the Panther pathway software. RESULTS: Non WNT/SHH group was the most common (61.1 %), followed by SHH and WNT. p53 immunopositivity did not correlate with prognosis in any subgroup. Proteomic profiling revealed several novel proteins differentially expressed between MB molecular subgroups. Signalling pathways exclusively enriched in each molecular subgroup were also identified. The top upregulated proteins were PMEL and FBN2 in the WNT subgroup, SYNGR2 in the SHH subgroup and GFAP, IMPG2 and MAGEA10 in the Non WNT/Non SHH group. We validated GFAP by immunohistochemistry on the archived samples (n = 118) and noted two types of staining pattern in MBs - reactive (stellate) astrocytes and tumour cell staining. GFAP immunopositivity in tumor cells of SHH subgroup correlated with a better prognosis. CONCLUSIONS: Proteomic profile identified several novel proteins differentially regulated within the molecular subgroups that could serve as potential diagnostic /prognostic biomarkers. Notably, GFAP, which was derived from proteomics data, when validated by IHC, revealed a variable staining pattern in MB tumours. The prognostic significance of GFAP in SHH tumor patients further points at the heterogeneity of this subgroup. The study also throws light on the signaling pathways activated in MB and in turn its plausible role in the tumorigenesis.

Medulloblastoma: Distinctive Histo-Molecular Correlation with Clinical Profile, Radiologic Characteristics, and Surgical Outcome.

OBJECTIVE: Medulloblastoma (MB) is a heterogenous tumor, and the prognosis is influenced by various clinical, histological, and molecular factors. The aim of the study is to determine the clinical profile and radiologic characteristics among the histo-molecular subgroups, the predictors of surgical outcome, and the pattern of relapse in pediatric and adult MB. METHOD: An analysis of 118 patients of MB who underwent surgical treatment at National Institute of Mental Health and Neurosciences, India, over a 7-year period (2005-2011) is presented. The clinical profile, radiologic characteristics, surgical nuances, and survival patterns are discussed. The relevant statistical analysis was done using SPSS software, version 22.0. RESULTS: The mean age of the cohort was 12 years (12.3 ± 8.7). The primary manifestation was raised intracranial tension headache in 53 patients (44.9%), which was the predominant symptom in large cell/anaplastic (LCA)- and WNT-activated subgroups. The median preoperative Karnofsky performance score was 60 (60.6 ± 12.9). Vermian and hemispheric location of tumor was most commonly observed in non-WNT/non-SHH (groups 3 and 4; 91.7%) and SHH-activated (42.9%) subgroups, respectively. Ninety-two patients (78%) underwent preoperative ventriculoperitoneal shunts (VPS) for obstructive hydrocephalus (HCP) and 14 patients (11.8%) underwent VPS in the postoperative period. The median overall survival (OS) for the whole group was 82.1 ± 5.7 months and the median recurrence-free survival was 51.0 ± 4.8 months. While radiotherapy had a significant influence on OS, progression-free survival was influenced by radiotherapy as well as chemotherapy in both pediatric and adult cohort. Desmoplastic/nodular subtype and WNT-activated subgroup had the best prognosis; LCA and non-WNT/non-SHH had the worst prognosis. CONCLUSIONS: Majority of the patients were pediatric in the study. Age, hemispheric location of tumor, extent of resection, and adjuvant treatment status were the important clinical prognostic factors for survival. Surgery for MB is formidable, and VPS can be considered in persistent symptomatic and progressive HCP. Our study on pediatric and adult MB validates the prognostic significance of various clinical, radiologic, and histo-molecular parameters of MB.

Expression pattern and prognostic significance of myosin light chain 9 (MYL9): a novel biomarker in glioblastoma.

AIMS: Tumour recurrence is inevitable in glioblastoma (GBM) and mostly noted in the peritumoural brain zone (PT). In our previous microarray-based study, we identified Myosin Light Chain 9 (MYL9) as a highly expressed gene in the PT of GBM. Therefore, we aimed to study the expression pattern and clinical significance of MYL9 in GBM. METHODS: Patient samples included three retrospective cohorts: 25 GBM cases with differential biopsies of tumour core and PT, 62 retrospective cases of newly diagnosed GBM with survival information and 20 paired samples (newly diagnosed and recurrent GBM). All tumour tissues, archived as formalin fixed paraffin embedded blocks were retrieved and immunohistochemistry for MYL9 and IDH1 R132H was performed. MYL9 expression was correlated with patient prognosis in our cohort and in The Cancer Genome Atlas (TCGA) and Rembrandt cohorts. It was further evaluated in the 20 paired samples of GBM. RESULTS: MYL9 showed a cytoplasmic membranous staining of tumour cells. The staining pattern was variable and patchy within tumours. Higher MYL9 expression was associated with poor overall and progression-free survival in our and in TCGA and Rembrandt cohorts. The expression of MYL9 was higher in IDH1 R132H immunonegative cases. CONCLUSIONS: We show MYL9 as a novel biomarker, variably expressed in GBM. The association of high MYL9 expression with poor prognosis in newly diagnosed GBM patients and increased expression in recurrent GBM is indicative of its role in conferring tumour aggressiveness.

H3K27M, IDH1, and ATRX expression in pediatric GBM and their clinical and prognostic significance.

PURPOSE: Pediatric glioblastoma (pGBM) tumors have been identified as an entity distinct and different from the adult variety of GBM not only with respect to pathogenesis, genetics, and molecular alterations but also in clinical outcomes and overall survival. This study aims to evaluate the immunohistochemical profile of molecular markers in pediatric GBM and correlate them with clinical features and prognosis. MATERIALS AND METHODS: We retrospectively analyzed 29 pGBMs (age range 3 to 18 years), operated at our institute between 2009 and 2014, and evaluated their clinical and histopathological features along with the immunohistochemical expression of clinically relevant molecular markers: H3K27M, p53, ATRX, and IDH1 (R132H), and correlated their expression with clinical features. We further assessed the prognostic value of these markers in our cohort of patients. RESULTS: The median overall survival (OS) of the cohort was 6.00 ± 0.882 months. The mean overall survival was 7.571 ± 1.118 months which was lower than in most studies. Preoperative Karnofsky Performance Score (KPS), extent of surgical resection, and adjuvant radiotherapy were found to be the clinical factors strongly influencing median survival (p < 0.05). Loss of ATRX expression was predominantly noted in hemispheric tumors (84%), while p53 staining was maximum in thalamic tumors (8 out of 9 cases). H3K27M mutant protein expression was noted in 8/9 thalamic tumors and 5/7 tumors in the brain stem-cerebellar-peduncular region. Patients with tumors showing H3K27M immunopositivity had the worst prognosis with a mean OS of 5 months ± 0.832 months, as against patients with H3K27M-immunonegative tumors, which was 10.143 ± 1.866 months(p = 0.006). Other markers like p53, ATRX, and IDH1 did not influence the prognosis in this patient cohort. ATRX loss of expression was associated with a better OS, with a trend to significance, and such an association has not been reported earlier. CONCLUSIONS: Ours is one among the few studies from India describing the clinical parameters and evaluating the key immunohistochemical markers in pGBM and deriving their prognostic significance. The study reiterates the poor prognostic significance of H3K27M immunopositivity.

MN1 rearrangement in astroblastoma: study of eight cases and review of literature.

Astroblastomas are unique tumours with unresolved issues in terms of their origin, molecular biology, clinical behaviour, and response to treatment. To decipher the characteristics of this tumour, we reviewed cases histologically diagnosed as astroblastoma in our institute over the past 8 years, with immunohistochemistry, and performed fluorescence in situ hybridisation (FISH), for the newly emerged MN1 rearrangement which was reported in central nervous system high-grade neuroepithelial tumours. The mean age at diagnosis was 18.6 years with all cases seen in females and with supratentorial localisation. The tumours showed typical circumscription and bubbly appearance on imaging. The cohort included eight cases diagnosed as astroblastoma (two low grades; six anaplastic) based on histology and proliferative index. The tumours displayed characteristic astroblastic pseudorosettes with hyalinised vascular core and variable immunopositivity for glial fibrillary acidic protein, pan cytokeratin, and epithelial membrane antigen. MN1 break-apart by FISH was found in 5/8 of our cases (62.5%), which included 2 low-grade and 3 anaplastic tumours. Tumour recurrence was noted in three cases, with MN1 alteration in two. We account for one of the few series to study the MN1 rearrangement in astroblastoma and conclude that MN1 alteration is seen in a subset of these tumours.

Chromosomal aberrations in chordoid meningioma - An analysis.

INTRODUCTION: Chordoid meningiomas (CMs) are a rare subgroup of tumors, accounting for approximately 0.5% of all meningiomas. These tumors correspond to World Health Organization (WHO) Grade II lesions and behave aggressively, with an increased likelihood of recurrence. There are only two studies that have described the genetic alterations in CMs. While a majority of meningiomas are known to have deletion at many chromosomal loci such as 22q, 18p, 14q, and 1p, which are found to be associated with initiation, progression, and malignancy of these tumors, these have not yet been studied in CMs. Thus, our aim was to evaluate the status of these four chromosomal aberrations in CMs and correlate the findings with the clinical outcome of patients. MATERIALS AND METHODS: A total of 15 cases of CM operated over a period of 12 years from 2001 to 2013 were analyzed. The archival paraffin blocks were retrieved and sections were subjected to locus-specific fluorescent in situ hybridization (FISH) using 22q12.2, 18p11.3, 14q32.2, and 1p32.3 probes. Immunohistochemistry (IHC) was done on all cases using MIB-1, vimentin, glial fibrillary acidic protein (GFAP), and epithelial membrane antigen (EMA) antibodies. RESULTS: All cases had characteristic features of CM, and were positive for EMA and vimentin and negative for GFAP. The mean labeling index for MIB-1 was 2.7 ± 0.8%. Of the 15 cases, 5 cases showed recurrence with a median follow-up period of 28 months. Patients who underwent Simpson's grade I excision did not show any relapse of the tumor. Of the 5 recurrent cases, 4 had complete deletion of all four chromosomal loci. Among the 10 nonrecurrent cases, 9 (90%) showed either partial deletion or an intact status. CONCLUSIONS: This is the first study to evaluate the combined chromosomal status of 22q, 18p, 14q, and 1p in CMs. Our study shows that there was a higher propensity of recurrence in tumors, even with complete excision, with complete deletion in all four chromosomal loci.

Prognostic significance of histomolecular subgroups of adult anaplastic (WHO Grade III) gliomas: applying the 'integrated' diagnosis approach.

AIMS: Anaplastic gliomas (AGs; WHO Grade III) include anaplastic astrocytoma (AA), anaplastic oligodendroglioma (AO) and anaplastic oligoastrocytoma (AOA) and are known to have variable prognosis. Since biomarkers have a major impact on prognosis of gliomas, we compared the prognostic significance of the established biomarkers of AGs and the 'histomolecular' subgroups based on the proposed International Society of Neuropathology-Haarlem ('ISN-Haarlem') guidelines, with the current WHO 2007 classification. METHODS: The study was carried out on formalin-fixed paraffin-embedded (FFPE) tissues from 91 adult patients with AG. Clinical, histological and molecular parameters, including 1p/19q codeletion, isocitrate dehydrogenase gene (IDH1)-R132H positivity, α thalassemia/mental retardation syndrome X-linked gene (ATRX) expression and O(6)-methylguanine-DNA-methyltransferase gene (MGMT) promoter methylation (mMGMT), were correlated with overall survival (OS) and recurrence-free survival (RFS). Subsequently, following sequencing for rare IDH mutations, we derived three 'histomolecular' subgroups based on the 'integrated' diagnosis approach proposed by 'ISN-Haarlem' guidelines and correlated this with clinical outcome. RESULTS: Gross tumour resection, administration of radiochemotherapy, 1p/19q codeletion, IDH1-R132H positivity and mMGMT were associated with favourable OS and RFS (p≤0.001), while the WHO histological subgroups were prognostically not significant. The ISN 'histomolecular' subgroups prognosticated best with AOs (IDHmut, 1p/19q codeleted, ATRX predominantly retained) having the best survival, followed by the AAs (IDHmut, ATRX loss or retained, 1p19q non-codeleted) and AA IDH wild type group having the worst OS and RFS (p=<0.001 for OS). CONCLUSIONS: Our study reiterates the prognostic significance of biomarkers, 1p/19q codeletion, IDH1-R132H positivity and mMGMT in AGs. Importantly, we show that the 'histomolecular' subgroups of AGs based on the 'integrated' diagnosis has a prognostic value, superior to the WHO histological classification.