Immunohistochemical detection of phosphorylated JAK-2 and STAT-5 proteins and correlation with erythropoietin receptor (EpoR) expression status in human brain tumors.

Phosphorylated (activated) forms of Janus Kinase 2 (pJAK-2) and STAT-5 transcription factor (pSTAT-5), which are preferentially expressed after binding of erythropoietin (Epo) to its receptor EpoR, are known to be implicated in the molecular mechanisms controlling brain development. The purpose of this study was to investigate the expression of these proteins (pJAK-2, pSTAT-5, and EpoR) in human brain tumors compared with normal brain. Using specific antibodies and immunohistochemistry on formalin-fixed, paraffin-embedded semi-serial tissue sections a total of 87 human brain tumors and samples from normal brain tissue were studied. pJAK-2/pSTAT-5 nuclear co-expression was detected in 39% of astrocytomas, 43% of oligodendrogliomas, 50% of ependymomas, and in all (100%) of the medulloblastomas examined. In contrast, most of the meningiomas showed weak or no immunoreactivity for pJAK-2/pSTAT-5 proteins. A significant percentage of tumors exhibited pSTAT-5 immunoreactivity, being pJAK-2 immunonegative. EpoR/pJAK-2/pSTAT-5 co-expression was detected in a small percentage of astrocytomas (18%) and ependymomas (33%). Oligodendrogliomas and medulloblastomas were EpoR immunonegative. Tumor vessels exhibited EpoR, pJAK-2, and pSTAT-5 immunoreactivity. In normal brain tissue, EpoR immunoreactivity was detected in neurons and vessels whereas pSTAT-5 and pJAK-2 immunoreactivity was limited to some neurons and a few glial cells, respectively. These results indicate the existence of ligand (other than Epo)-dependent or independent JAK-2 activation that leads to constitutive activation of STAT-5 in most human brain tumors. Given the oncogenic potential of the JAK/STAT pathway, detection of different pJAK-2 and pSTAT-5 expression profiles between groups of tumors may reflect differences in the biological behavior of the various human brain tumors.

Detection of JC virus DNA sequences and expression of the viral regulatory protein T-antigen in tumors of the central nervous system.

JC virus (JCV) is a neurotropic polyomavirus infecting greater than 70% of the human population worldwide during early childhood. Replication of JCV in brains of individuals with impaired immune systems results in the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Furthermore, JCV possesses an oncogenic potential and induces development of various neuroectodermal origin tumors including medulloblastomas and glioblastomas in experimental animals. The oncogenecity of JCV is attributed to the viral early gene product, T-antigen, which has the ability to associate with and functionally inactivate well-studied tumor suppressor proteins including p53 and pRB: The observations from laboratory animal experiments have provided a rationale for examining the presence of the JCV DNA sequence and expression of the viral oncogenic protein in human brain tumors. We have examined 85 clinical specimens from the United Kingdom, Greece, and the United States, representing various human brain tumors including oligodendroglioma, astrocytoma, pilocytic astrocytoma, oligoastrocytoma, anaplastic astrocytoma, anaplastic oligodendroglioma, glioblastoma multiforme, gliomatosis cerebri, gliosarcoma, ependymoma, and subependymoma, for their possible association with JCV. We performed gene amplification techniques using a pair of primers that recognize the JCV DNA sequence, and we demonstrated the presence of the viral early sequence in 49 (69%) of 71 samples. More importantly, our results from immunohistochemistry analysis revealed expression of JCV T-antigen in the nuclei of tumor cells in 28 (32.9%) of 85 tested samples. These observations, along with earlier in vitro and in vivo data on the transforming ability of this human neurotropic virus invite additional studies to re-evaluate the role of JCV in the pathogenesis of human brain tumors.

Aberrant localization of the neuronal class III beta-tubulin in astrocytomas.

BACKGROUND: The class III beta-tubulin isotype (betaIII) is widely regarded as a neuronal marker in development and neoplasia. In previous work, we have shown that the expression of betaIII in neuronal/neuroblastic tumors is differentiation dependent. In contrast, the aberrant localization of this isotype in certain nonneuronal neoplasms, such as epithelial neuroendocrine lung tumors, is associated with anaplastic potential. OBJECTIVE: To test the generality of this observation, we investigated the immunoreactivity profile of betaIII in astrocytomas. DESIGN: Sixty archival, surgically excised astrocytomas (8 pilocytic astrocytomas, WHO grade 1; 18 diffuse fibrillary astrocytomas, WHO grade 2; 4 anaplastic astrocytomas, WHO grade 3; and 30 glioblastomas, WHO grade 4), were studied by immunohistochemistry using anti-betaIII monoclonal (TuJ1) and polyclonal antibodies. A monoclonal antibody to Ki-67 nuclear antigen (NC-MM1) was used as a marker for cell proliferation. Antibodies to glial fibrillary acidic protein (GFAP) and BM89 synaptic vesicle antigen/synaptophysin were used as glial and neuronal markers, respectively. RESULTS: The betaIII immunoreactivity was significantly greater in high-grade astrocytomas (anaplastic astrocytomas and glioblastomas; median labeling index [MLI], 35%; interquartile range [IQR], 20%-47%) as compared with diffuse fibrillary astrocytomas (MLI, 4%; IQR, 0.2%-21%) (P <.0001) and was rarely detectable in pilocytic astrocytomas (MLI, 0%; IQR, 0%-0.5%) (P <.0001 vs high-grade astrocytomas; P <.01 vs diffuse fibrillary astrocytomas). A highly significant, grade-dependent relationship was observed between betaIII and Ki-67 labeling and malignancy, but this association was stronger for Ki-67 than for betaIII (betaIII, P <.006; Ki-67, P <.0001). There was co-localization of betaIII and GFAP in neoplastic astrocytes, but no BM89 synaptic vesicle antigen/synaptophysin staining was detected. CONCLUSIONS: In the context of astrocytic gliomas, betaIII immunoreactivity is associated with an ascending gradient of malignancy and thus may be a useful ancillary diagnostic marker. However, the significance of betaIII-positive phenotypes in diffuse fibrillary astrocytomas with respect to prognostic and predictive value requires further evaluation. Under certain neoplastic conditions, betaIII expression is not neuron specific, calling for a cautious interpretation of betaIII-positive phenotypes in brain tumors.

AP-1 and heat shock protein 27 expression in human astrocytomas.

PURPOSE: In previous work we have shown that the expression of heat shock protein 27 (Hsp27) is associated with anaplastic potential of astrocytomas (Anticancer Res 1997, 17:2677-2682). Heat shock protein-coding genes have been found to have a putative AP-1 (activator protein-1)-binding site in their promoter region and the synthesis of these proteins is induced by the same extracellular stimuli that also activate AP-1 (a homo/heterodimer of members of the Jun and Fos families). In order to detect the putative relation of Hsp27 with AP-1 activation in human astrocytomas we examined eighty astrocytic tumors with different grades of malignancy for c-Jun, c-Fos, and Hsp27 expression. METHODS: Six pilocytic astrocytomas (WHO grade I), 15 diffuse fibrillary astrocytomas (WHO grade II), 19 anaplastic astrocytomas (WHO grade III), and 40 glioblastomas multiforme (WHO grade IV), were studied by immunohistochemistry using monoclonal and polyclonal antibodies directed against human Hsp27, c-Fos, and active (phosphorylated) forms of c-Jun (p-c-Jun). Monoclonal antibodies against the phosphorylated forms of the over-expressed MAP kinases JNK (c-Jun N-terminal kinase) (p-JNK) and p38 (p-p38) were also used. RESULTS: Overexpression of p-c-Jun, c-Fos and p-JNK was observed in the majority of glioblastomas (grade IV) whereas only minimal expression was noted in diffuse fibrillary astrocytomas (grade II). Hsp27 expression was observed only in the tumor specimens where c-Jun and c-Fos were co-expressed. AP-1/Hsp27 co-expression was associated with ascending grading of malignancy and it was independent of the proliferation index of the tumors. CONCLUSIONS: Our findings suggest that during malignant progression of astrocytomas there is activation of signal transduction cascade(s) culminating in AP-1 induction.

Human meningiomas: immunohistochemical localization of progesterone receptor and heat shock protein 27 and absence of estrogen receptor and PS2.

Formalin-fixed, paraffin-embedded tumor specimens from 64 human meningiomas of various histologic types were immunostained using a streptavidin-peroxidase method and monoclonal antibodies for estrogen receptors (ER) and progesterone receptors (PgR) and hormone-induced proteins PS2 and heat shock protein 27 (Hsp27). The immunohistochemical analysis was scored in a semiquantitative fashion incorporating both the intensity and distribution of specific staining (score). Strong PgR nuclear immunoreactivity (mean score, 54.2) was observed in 51 of 64 meningiomas (80%). Meningiomas with atypical features were negative for PgR. Low Hsp27 cytoplasmic immunoreactivity (mean score, 9.8)--irrespective of histologic type--was observed in 26 of 64 meningiomas (40%). Whor1 formations in transitional meningiomas were Hsp27-positive. Hsp27 immunoreactivity was observed in tumor blood vessels. All neoplasms were negative for ER and PS2. Normal arachnoid tissue was positive for PgR and negative for ER, PS2, and Hsp27. Compared with previously reported data on gliomas, the results of this study indicate that meningiomas show a clear out difference concerning the expression of PgR and Hsp27.

Does sex steroid receptor status have any prognostic or predictive significance in brain astrocytic tumors?

Formalin-fixed paraffin-embedded tumor specimens from 90 patients with supratentorial astrocytic brain tumors were immunostained following microwave pretreatment using monoclonal antibodies against estrogen receptor (ER), progesterone receptor (PgR), and PS2 protein (PS2) and the streptavidin/peroxidase method. The immunohistochemical analysis was scored in a semiquantitative fashion incorporating both the intensity and distribution of specific staining (SCORE) and the immunohistochemical results were correlated with histologic grade of tumors and patient's sex and age. Strong PgR nuclear immunopositivity was observed in 27 of 46 (59%) glioblastomas, in 9 of 20 (45%) anaplastic astrocytomas, and in 2 of 24 (8%) astrocytomas. The mean PgR score was 18.5 in glioblastomas, 11.3 in anaplastic astrocytomas, and 0.9 in astrocytomas. The expression of PgR immunoreactivity appeared to be dependent on the age and independent of the sex of patients. All tumors were negative for ER and PS2 protein. Normal astrocytes remained consistently negative for both sex steroid receptors and PS2. Our findings indicate a consistent expression of PgR in a number of poorly differentiated tumors including glioblastomas and anaplastic astrocytomas contrasting with the low PgR score values found in benign astrocytomas. The absence of ER and PS2 immunoreactivities supports the hypothesis that estrogen receptor in astrocytic tumors is probably truncated. However, regional tumor heterogeneity may be a limiting factor for the use of ER, PgR, PS2 immunohistochemistry in the evaluation of routine pathology specimens examination.

Prognostic significance of Hsp-27 in astrocytic brain tumors: an immunohistochemical study.

Formalin-fixed paraffin-embedded tumor specimens from 95 patients with supratentorial astrocytic brain tumors were immunostained by a monoclonal antibody against the heat shock protein-27 (Hsp-27) using the streptavidin/peroxidase method. The immunohistochemical analysis was scored in a semiquantitative fashion incorporating both the intensity and distribution of specific staining (score): the immunohistochemical results were correlated with the histological grade of the tumors and patients' sex and age. Furthermore, Hsp-27 expression was studied in two groups into which the patients were further divided: group (a) previously untreated patients (n = 76) whose biopsy or surgical resection was related to their initial presentation and diagnosis and group (b) patients (n = 19) with reccurent disease who underwent radiotherapy and/or chemotherapy. Strong Hsp-27 cytoplasmic immunopositivity was observed in 42 out of 51 (82%) glioblastomas, in 8 out of 20 (40%) anaplastic astrocytomas and in 2 out of 24 (8%) astrocytomas. The mean Hsp-27 score was 45.2 in glioblastomas, 6.5 in anaplastic astrocytomas and 0.4 in astrocytomas. The expression of Hsp-27 immunoreactivity appeared to be independent of the age and sex of the patients. A non significant difference was defined between untreated patients and previously treated patients. Hsp-27 immunoreactivity was observed in the microvascular endothelial proliferations and in tumor blood vessels. Normal astrocytes were Hsp-27 negative. These findings indicate that, in contrast with the low Hsp-27 expression found in benign astrocytomas, the expression of Hsp-27 in a number of poorly differentiated tumors, including glioblastomas and anaplastic astrocytomas, is consistent and independent of previous treatment of the patients. We support the involvement of Hsp-27 in the growth of astrocytic brain tumors.

Microvessel density in brain tumors.

Sections from formalin fixed paraffin embedded tumor tissue from 165 patients with brain tumors including 62 meningiomas, 80 supratentorial astrocytomas (19 astrocytomas-grade I/II, 20 anaplastic astrocytomas-grade III, 41 glioblastomas-grade IV), 7 cerebellar astrocytomas-grade I/II, one gliosarcoma, 7 oligodendrogliomas, 3 ependymomas and 5 medulloblastomas were immunostained for factor VIII-related antigen in order to highlight microvessel endothelial cells. Microvessel count (MVC; the highest number of microvessels in three areas of highest vascular density at X200 magnification) was determined and correlated with histological grade of tumors and patients' sex and age. The mean MVC was 27.9 in meningiomas. Astrocytic tumors, particularly malignant astrocytomas (grade III, IV), were highly vascular. The mean MVC as regards the supratentorial astrocytic neoplasms was 14.5 in astrocytomas (grade I/II), 42.3 in anaplastic astrocytomas (grade III) and 50.2 in glioblastomas (grade IV). All cerebellar astrocytomas studied, even though well differentiated (grade I/II), were highly vascularised tumors (MVC: 41.1. A comparison of the mean MVC vlaues showed that there was a statistically significant difference between supratentorial astrocytomas (Grade I-II) and cerebellar astrocytomas (Grade I-II) (p = 0.0004), anaplastic astrocytomas (Grade I-II) (p = 0.00004) and glioblastomas (p = 0.00001). There was no significant difference between cerebellar astrocytomas and anaplastic astrocytomas (p = 0.8) and glioblastomas (p = 0.4). Astrocytic neoplasms showed statistically significant higher mean MVC from meningiomas (p = 0.002). The mean MVC was 14.1 in oligodendrogliomas, 22.7 in ependymomas and 19.6 in medulloblastomas. In one gliosarcoma that was studied the MVC was 40. The MVC appeared to be independent of the age and sex of patients. This study supports the importance of microvessel density as a measure of angiogenesis, as well as a further morphologic feature in the classification of brain tumors. The determination of microvessel density may become useful in the planning and monitoring of anti-angiogenesis therapies of these tumors.