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.

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.

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.

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.

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.