OLIG2 is a useful immunohistochemical marker in differential diagnosis of clear cell primary CNS neoplasms

AIMS: To analyse OLIG2 expression in clear cell primary central nervous system (CNS) tumours to clarify the diagnostic usefulness of OLIG2 immunohistochemistry in this subset of brain tumours. METHODS AND RESULTS: We analysed OLIG2 expression in 60 oligodendroglial neoplasms (57 with and three without chromosome 1p aberration), 10 central neurocytomas, 10 clear cell ependymomas, nine dysembryoplastic neuroepithelial tumours (DNTs) and two clear cell meningiomas using immunohistochemistry. Additionally, we analysed oligodendroglial neoplasms with numerous gliofibrillary and minigemistocytic oligodendrocytes for OLIG2/glial fibrillary acidic protein (GFAP) coexpression and central neurocytoma for coexpression of neurone-specific nuclear protein (NeuN) and OLIG2 using double immunofluorescent labelling and confocal laser scanning microscopy. All oligodendroglial neoplasms and DNTs showed widespread OLIG2 expression. Eight of 10 central neurocytomas, all clear cell meningiomas and 8/10 clear cell ependymomas were negative for OLIG2. Two of 10 central neurocytomas and 2/10 clear cell ependymomas showed focal OLIG2 expression. We found prominent coexpression of GFAP and OLIG2 in gliofibrillary and minigemistocytic oligodendrocytes. Further, we found coexpression of NeuN and OLIG2 in single cells in central neurocytoma. CONCLUSIONS: Widespread OLIG2 expression discriminates oligodendroglial neoplasms or DNTs from other clear cell primary brain tumour types. In clear cell primary brain tumours lacking OLIG2 expression, differential diagnosis may require additional immunohistochemical markers.     

Immunohistochemical comparative analysis of GFAP,MAP – 2, NOGO – A,OLIG – 2 and WT – 1 expression in WHO 2016 classified neuroepithelial tumours and their prognostic value

Immunohistochemistry is routinely used in differential diagnosis of tumours of the central nervous system (CNS). The latest 2016 WHO 2016 revision now includes molecular data such as IDH mutation and 1p/19q codeletion thus restructuring glioma classification. Direct comparative information between commonly used immunohistochemical markers for glial tumours GFAP, MAP – 2, NOGO – A, OLIG – 2 and WT – 1 concerning quality and quantity of expression and their relation to the new molecular markers are lacking. We therefore compared the immunohistochemical staining results of all five antibodies in 34 oligodendrogliomas, 106 ependymomas and 423 astrocytic tumours. GFAP expression was reduced in cases with higher WHO grade, oligodendroglial differentiation and in IDH wildtype diffuse astrocytomas. By contrast MAP – 2 expression was significantly increased in diffuse astrocytomas with IDH mutation, while NOGO – A expression was not associated with any molecular marker. WT – 1 expression was significantly decreased in tumours with IDH mutation and ATRX loss. OLIG – 2 was increased in IDH-mutant grade II astrocytomas and in cases with higher proliferation rate. In univariate survival analysis high WT – 1 expression was significantly associated with worse outcome in diffuse astrocytic tumours (log rank p?<?0.0001; n?=?211; median time: 280?days vs 562?days). None of the markers was prognostic in multivariate survival analysis. Among the evaluated markers MAP – 2, OLIG – 2 and WT – 1 showed the best potential to separate between glioma entities and can be recommended for a standardized immunohistochemical panel.     

EGFR expression stratifies oligodendroglioma behavior

Epidermal growth factor receptor (EGFR) expression and signaling contribute to glioma biological features and, thus, are a target for new drug development. The role, if any, of EGFR in routine surgical neuropathological diagnostics is less clear. Herein, we describe prospective EGFR IHC analysis in an adult cohort comprising 750 infiltrative gliomas. EGFR expression increased with World Health Organization grade but did not significantly differ between grade-matched astrocytic and oligodendroglial tumors. Survival did not significantly differ by EGFR expression among astrocytic tumors adjusted for World Health Organization grade. However, grade II oligodendrogliomas with strong EGFR expression and 1p/19q codeletion showed reduced survival, compared with their codeleted counterparts with weaker EGFR expression. Surprisingly, an inverse phenomenon was found with grade III anaplastic oligodendrogliomas, in which stronger EGFR expression was a favorable marker for survival. Among all gliomas, the likelihood of EGFR amplification, as viewed by fluorescence in situ hybridization, increased with the strength of EGFR expression, and <1% of cases with weak or no EGFR immunostaining showed amplification. These data suggest that EGFR IHC is useful in certain circumstances (ie, it may help supplement 1p/19q prognostic information in oligodendroglial tumors and screen out cases that would not benefit from more costly EGFR fluorescence in situ hybridization analysis).     

MIR‐137 Suppresses Growth and Invasion,is Downregulated in Oligodendroglial Tumors and Targets CSE1L

MicroRNA‐137 (miR‐137) expression has been reported to be decreased in astrocytic tumors in two expression profiling studies but its role in the pathogenesis of oligodendroglial tumors is still limited. In this study, we demonstrate that miR‐137 expression is significantly downregulated in a cohort of 35 oligodendroglial tumors and nine glioma cell lines compared with normal brains. Lower miR‐137 expression is associated with shorter progressive‐free survival and overall survival. Restoration of miR‐137 expression in an oligodendroglial cells TC620, and also glioblastoma cells of U87 and U373 significantly suppressed cell growth, anchorage‐independent growth, as well as invasion. Demethylation and deacetylation treatments resulted in upregulation of miR‐137 expression in TC620 cells. In silico analysis showed that CSE1 chromosome segregation 1‐like (yeast) (CSE1L) is a potential target gene of miR‐137. Luciferase reporter assay demonstrated that miR‐137 negatively regulates CSE1L by interaction between miR‐137 and complementary sequences in the 3′ UTR of CSE1L. Immunohistochemistry revealed that CSE1L is upregulated in oligodendroglial tumors. Knockdown of CSE1L resulted in similar outcomes as overexpressing miR‐137 in oligodendroglioma cells and glioblastoma cells. Overall, our data suggest that miR‐137 regulates growth of glioma cells and targets CSE1L, providing further understanding in the tumorigenesis of gliomas.     

Isolation and characterization of stem cell-like precursor cells from primary human anaplastic oligoastrocytoma.

A small population of stem cell-like precursors in solid tumors are linked to histological composition, progression, angiogenesis, metastasis, recurrence and drug resistance of a variety of malignant tumors. Oligoastrocytoma is the most common brain mixed glioma composed of mixed cells of oligodendroglial and astrocytic phenotypes. Identification and characterization of stem cell-like precursors in oligoastrocytoma may shed light on the oncogenesis of this unique type of tumor and assist in the design of novel therapeutic strategy. Here, tumor stem cell-like precursors were identified from primary human anaplastic oligoastrocytomas by labeling of the tumor sections with nestin and CD133. Tumor cells were cultured in vitro in stem cell medium with growth factors and the capacity of the surviving stem cell-like precursors to form tumor spheres was tested. The tumor spheres were further injected subcutaneously into nude mice to observe the contribution of stem cell-like precursors to histological composition and tumor progression. We found that primary human oligoastrocytoma tissues contained nestin+/CD133+ stem cell-like precursors. These cells differentiated into tumor cells with both oligodendroglial and astrocytic characteristics and formed tumor spheres in vitro, which upon implantation in nude mice, grew into tumor nodules containing nestin+/CD133+ cells at levels higher than in the primary tumor tissues. This study revealed for the first time that anaplastic human oligoastrocytomas contained stem cell-like precursors, which exhibit neural stem cell properties with tumorigenicity. These stem cell-like precursors may be responsible for the oligodendroglial and astrocytic components of human oligoastrocytoma and should be considered as therapeutic targets.     

Clonality of oligoastrocytomas

Oligoastrocytomas (OA) are mixed glial tumors that show morphologic features of both oligodendrogliomas and astrocytomas. The histogenesis of these tumors remains undefined. The aim of this study was to investigate the clonality of OA on the basis of tumor-dependent genetic alterations and tumor-independent X-chromosome inactivation. We microdissected 11 biphasic OA and subjected the oligodendroglial and astrocytic components to allelic loss analysis of chromosomes 1p, 9p21, 10q, 13q, 17p, and 19q; TP53 immunohistochemical and mutation analyses; and X-linked HUMARA gene methylation study. On the basis of the genetic findings, we categorized these tumors into 3 groups. Group 1 consisted of 4 tumors that showed identical genetic aberrations in the 2 histologic elements, characterized by allelic loss on 1p and 19q. These results suggest that group 1 tumors are of monoclonal origin and share a precursor cell with oligodendrogliomas. Group 2 consisted of 5 tumors characterized by losses on 1p and 19q, with additional allelic losses on chromosomes 9p, 10q, 13q and/or 17p. Four of these tumors were of the anaplastic type. Thus, group 2 tumors may be regarded as advanced variants of group 1 OA with heterogeneous genetic changes during clonal expansion. The X-chromosome inactivation analysis confirmed the monoclonality of groups 1 and 2 OA. Group 3 consisted of two tumors that showed divergent allelic loss patterns in the 2 histologic components. Mutation and overexpression of TP53 were detectable in the astrocytic components only. These findings raise the possibility that group 3 tumors have a biclonal origin. In conclusion, our results suggest that OA are predominantly of monoclonal origin but that a small subset of tumors may be derived from different precursors.     

Supratentorial Pilocytic Astrocytomas, Astrocytomas, Anaplastic Astrocytomas and Glioblastomas are Characterized by a Differential Expression of S100 Proteins

The levels of expression of the S100A1, S100A2, S100A3, S100A4, S100A5, S100A6 and S100B proteins were immunohistochemically assayed and quantitatively determined in a series of 95 astrocytic tumors including 26 World Health Organization (WHO) grade I (pilocytic astrocytomas), 23 WHO grade II (astrocytomas), 25 WHO grade III (anaplastic astrocytomas) and 21 WHO grade IV (glioblastomas) cases. The level of the immunohistochemical expression of the S100 proteins was quantitatively determined in the solid tumor tissue (tumor mass). In addition twenty blood vessel walls and their corresponding perivascular tumor astrocytes were also immunohistochemically assayed for 10 cases chosen at random from each of the four histopathological groups. The data showed modifications in the level of S100A3 protein expression; these modifications clearly identified the pilocytic astrocytomas from WHO grade II-IV astrocytic tumors as a distinct biological group. Modifications in the level of S100A6 protein expression enabled a clear distinction to be made between low (WHO grade I and II) and high (WHO grade III and IV) grade astrocytic tumors. Very significant modifications occurred in the level of S100A1 protein expression (and, to a lesser extent, in their of the S100A4 and S100B proteins) in relation to the increasing levels of malignancy. While the S100A5 protein was significantly expressed in all the astrocytic tumors (but without any significant modifications in the levels of malignancy), the S100A2 protein was never expressed in these tumors. These data thus indicate that several S100 proteins play major biological roles in human astrocytic tumors.     

DNA hypermethylation and aberrant expression of the EMP3 gene at 19q13.3 in Human Gliomas

Allelic losses on 19q are found in the majority of oligodendroglial tumors and approximately one-third of diffuse astrocytomas. However, the tumor suppressor genes (TSG) on 19q are still elusive. Using cDNA microarray expression profiling, EMP3 at 19q13.3 was among those genes showing the most pronounced expression differences. In line with this, other authors reported EMP3 as being epigenetically silenced in neuroblastomas and astrocytomas. To further investigate EMP3 as a TSG candidate on 19q13.3, we performed molecular analysis of this gene in 162 human gliomas. Mutation analysis did not reveal EMP3 alteration in 132 gliomas. In oligodendroglial tumors, we found that aberrant methylation in the 5'-region of EMP3 was significantly associated with reduced mRNA expression and LOH 19q. In astrocytomas, EMP3 hypermethylation was also paralleled by reduced expression but was independent of the 19q status. EMP3 hypermethylation was detected in more than 80% of diffuse, anaplastic astrocytomas and secondary glioblastomas. Primary glioblastomas, however, mostly lacked EMP3 hypermethylation and frequently overexpressed EMP3. Our data corroborate that oligodendroglial and astrocytic gliomas often show EMP3 hypermethylation and aberrant expression. Furthermore, our findings suggest that primary and secondary glioblastomas are not only characterized by distinct genetic profiles but also differ in their epigenetic aberrations.     

The expression of Rho proteins decreases with human brain tumor progression: Potential tumor markers

Astrocytic tumors are the most common human brain tumors. Establishment of tumor grade is a key determinant both in the choice of a therapeutic approach and in the prognosis. The diagnosis of astrocytic tumors is currently determined following histopathological analysis. The identification of molecular markers would offer a complementary tool for characterizing tumors with respect to their clinical behavior. In this study we determined the expression levels of 3 small GTP binding proteins (RhoA, RhoB and Rac1), of their inhibitor RhoGDI and of caveolin-1 in 24 human astrocytic tumors of grades I to IV. Our results demonstrated that the expression of RhoA and RhoB decreased significantly in all brain tumors studied and was inversely related with tumor of grade II to IV malignancy. The amount of caveolin-1 immunodetected was not significantly different from normal brain samples while the Rac1 expression level was diminished in astrocytic tumors of grades III and IV. Our finding that RhoA and RhoB expression levels are correlated to tumor malignancy suggests that they may serve as novel and efficient diagnostic markers for astrocytic brain tumors of histological grade II to IV and complement currently applied histopathological analysis.     

CD133 expression and cancer stem cells predict prognosis in high-grade oligodendroglial tumors

High-grade oligodendroglial tumors, that is, anaplastic oligodendroglial tumors and glioblastomas with oligodendroglial component, differ significantly in terms of prognosis and response to chemotherapy. Differentiation might be difficult because the histological differences are vague and reliable markers are not established. We correlated the presence of putative cancer stem cells (CSC) in high-grade oligodendroglial tumors (WHO grades III and IV) with clinical outcome. Tumors with favorable prognosis neither contained CSC nor did they show CD133 expression. Tumor cells resembled lineage-restricted progenitor cells with limited proliferative capacity and differentiation profile. In contrast, CD133 expression and stem cell-like tumor cells characterized tumors with poor prognosis. They showed neurosphere-like growth, differentiated into cells of all neural lineages, and were tumorigenic in nude mice. In our series, CSC and expression of CD133 predicted the clinical course of disease better than the histological grading. To confirm these results, we retrospectively analyzed 36 high-grade oligodendroglial tumors. Again, CD133 expression indicated shorter survival and predicted clinical outcome more reliable than the histological assessment. Our data show that detection of CSC and expression of CD133 is predictive of prognosis in high-grade oligodendroglial tumors. The presence or absence of CD133(+) CSC might explain the crucial biological difference between WHO grade III and IV oligodendroglial tumors.     

Expression of mos in astrocytic tumors and its potential role in neoplastic progression

The c-mos gene and its protein product mos, components of the mitogen-activated protein kinase transduction pathway, are known to be involved in the control of meiosis and mitosis. Apart from a study on lung carcinomas, there is little information about its role in human neoplasia. The aim of this study was to investigate expression of mos in astrocytic tumors and to correlate it with accumulation of p53. We studied expression of mos in 62 cases of supratentorial astrocytic tumor. Intracytoplasmic immunostaining for mos was found in 28 (45%) cases: 3 of 20 (15%) grade 2 astrocytomas, 9 of 20 (45%) grade 3 anaplastic astrocytomas, and 16 of 22 (73%) glioblastomas. Immunopositivity for mos correlated significantly (P < 0.01) with tumor grade but not with p53 expression. In contrast to the findings in relation to lung tumors, immunopositivity for mos in astrocytic tumors did not predict recurrence-free or overall survival time. Cytoplasmic immunostaining was observed in scattered large cortical neurons adjacent to tumors, possibly due to stress-induced abortive entry into the cell cycle. The correlation of mos immunopositivity with tumor grade may reflect the expansion of more malignant mos-positive clones. This study provides evidence that mos may be involved in the neoplastic progression of a proportion of astrocytic tumors.     

Podoplanin is expressed in subsets of tumors of the central nervous system

Immunohistochemical analyses with the monoclonal antibody D2-40 were performed to ascertain the expression of podoplanin (a.k.a. T1-alpha, gp36, or aggrus) in tumors of the central nervous system (CNS) and to determine the diagnostic utility of the antibody. The analyses were performed on 325 tumors of various histologic types. The chief finding was almost constant immunoreactivity in ependymal tumors (37/40, 92.5%), choroid plexus papillomas (8/8, 100%), and meningiomas (100/100, 100%). The reactivity was considered “tissue-specific,” as the corresponding normal tissue of each tumor was also found to express podoplanin. In addition, expression, not committed to the lineages, was found in many other tumor types, including astrocytic tumors, medulloblastomas, and hemangioblastomas, with variable frequency and intensity. The way of expression was not fully understood, but the expression in astrocytic tumors seemed to be associated with pronounced fibrous properties or malignant phenotype, as was shown by high-frequent expression in pilocytic astrocytomas (12/12, 100%) and glioblastomas (29/35, 82.9%). The present study has shown that podoplanin is expressed in several types of CNS tumors with variable frequency and intensity. Given the widespread expression of podoplanin, the antibody D2-40 is of little use in diagnostic practice for CNS tumors.     

Subtyping of oligo-astrocytic tumours by comparative genomic hybridization

Oligo-astrocytic tumours (OAs) histologically show both oligodendroglial and astrocytic differentiation. Unequivocal criteria for delineation of OAs from pure oligodendroglial (Os) and astrocytic (As) tumours and for grading of OAs are lacking. Molecular genetic analysis may allow for a better characterization of OAs and thereby guide prognostic and therapeutic decisions. Comparative genomic hybridization (CGH) was performed on 39 gliomas with variable phenotypic expression of histological features characteristic of both astrocytic and oligodendroglial differentiation. The results show that OAs are genetically more heterogeneous than Os. In addition to the "-1p/-19q" and "+7/-10" subtypes that have been previously recognized, two additional genetic subtypes, "intermediate" and "other", were identified in the present study. "Intermediate" OAs likely represent progression from "-1p/-19q" tumours. The "other" subtype appears to represent an additional, heretofore unrecognized, genetic pathway(s). Application of rigorously "strict" histopathological criteria, as opposed to "relaxed" criteria, for the selection of oligo-astrocytic tumours resulted in a higher percentage of "-1p/-19q" tumours, but some "-1p/-19q" tumours might be missed. The results suggest that molecular genetic analysis is a useful and valid additional tool for the classification of gliomas, particularly for the significant subset of tumours in which subjective histopathological criteria are insufficient for an unequivocal distinction between Os, As, and mixed OAs.     

SLP-2基因在喉鳞状细胞癌和乳腺浸润性癌组织中的表达及其与预后的关系

目的 探讨两种恶性上皮性肿瘤组织--喉鳞状细胞癌(简称喉癌)和乳腺浸润性癌(简称乳腺癌)中stomatin like protein-2(SLP-2)基因在mRNA和蛋白水平的表达,及其与肿瘤的临床病理参数和预后的相关性.方法 应用逆转录聚合酶链反应(RT-PCR)检测了46对喉癌及喉正常上皮组织中SLP-2基因的表达,Western blot方法检测了其中10对标本的SLP-2蛋白表达,同时采用免疫组织化学方法分别检测了104例喉癌组织芯片和263例乳腺癌组织芯片中SLP-2蛋白的表达水平,分析SLP-2蛋白的表达与临床病理变量之间的关系.结果 RT-PCR结果显示SLP-2基因在46例喉癌中的38例肿瘤组织中的表达升高(83%,38/46),喉正常上皮组织中表达阴性.Western blot结果显示,有7例喉癌组织中SLP-2蛋白表达显著高于对应的喉正常上皮组织.喉癌组织芯片的免疫组织化学染色结果显示,与全部20例喉正常上皮组织的阴性表达(0/20)相比,SLP-2蛋白染色在104例喉癌组织中有36例出现了过表达(34.6%,36/104;P=0.000).与喉正常上皮表达相比,喉癌组织中SLP-2基因在mRNA和蛋白水平的表达均明显升高.SLP-2蛋白过表达与喉癌患者的临床分期较晚(P<0.01)和淋巴结发生转移(P=0.003)密切相关.乳腺癌组织芯片免疫组织化学染色结果显示,与在正常乳腺组织中的阴性表达(0/10)相比,SLP-2蛋白在乳腺癌组织中呈现过表达(52.5%,138/263),差异有统计学意义(P:0.000),且该蛋白的过表达与乳腺肿物的大小(P=0.020)、淋巴结转移(P<0.01)、临床分期Ⅲ期(P<0.01)以及发生远处转移(P=0.002)密切相关.此外,还与HER2/neu蛋白的表达存在显著相关性(P:0.037),生存分析表明,SLP-2蛋白过表达乳腺癌患者总生存率显著降低.多因素分析显示淋巴结状态、HER2/neu蛋白表达和SLP-2蛋白表达可能作为独立的预后因子.结论 SLP-2蛋白的过表达可能与喉癌和乳腺癌的侵袭、转移过程密切相关,并可能作为独立的预后指标提示乳腺癌患者预后不良.     

AGR2 associates with HER2 expression predicting poor outcome in subset of estrogen receptor negative breast cancer patients

Expression of the AGR2 oncogene was shown to be associated with estrogen receptor positive tumors. This gene contributes to enhanced cellular proliferation, drug resistance, metastasis development and may also serve as a predictor of poor prognosis. However, our analysis of AGR2 expression in a subset of estrogen-receptor negative tumors revealed that AGR2 could also be upregulated in hormone-independent manner. AGR2 expression was shown to be significantly increased in HER2 positive breast tumors on both the mRNA and the protein level. Moreover, in a subset of estrogen- and progesterone-receptor negative and simultaneously HER2-positive cases, increased AGR2 expression significantly correlated with worse patient prognosis. Subsequent analysis of independent data sets either collected in our institute or obtained from Oncomine cancer microarray database confirmed all these findings.