Expression of tyrosine kinases FAK and Pyk2 in 331 human astrocytomas

The progression of malignancy from astrocytomas to glioblastomas remains clinically as well as histopathologically unpredictable. The focal adhesion kinase (FAK) and the proline-rich tyrosine kinase (Pyk2) show a high expression in glioma cell lines and have an influence on increased cell proliferation and migration of glioma cells in vitro and in vivo. The aim of this study was to correlate the coexpression of FAK and Pyk2 to the WHO grade of malignancy in human astrocytomas. Immunohistochemical staining scores of FAK and Pyk2 were analyzed in 331 astrocytomas and correlated to each other and to the WHO grade. Significant coexpression of FAK and Pyk2 in astrocytomas was demonstrated. Pyk2 expression occurred much more frequently and with higher expression scores within the different WHO grades. Beyond this, a significant correlation between the WHO grade of malignancy of astrocytomas and the expression of FAK, as well as of Pyk2, was detected. This connection and the roles of these two tyrosine kinases in the progression of tumors should be confirmed by further studies.     

Cell death and oxidative stress in gliomas

In gliomas, apoptosis and necrosis are determined by a number of promoting and inhibiting factors including oxidative cell stress mediated by nitric oxide synthases (NOS) and reduced by superoxide dismutases. Therefore, in 46 gliomas (including astrocytomas, oligodendrogliomas, oligo-astrocytomas, and glioblastomas), the relationship of apoptosis and necrosis and the expression of apoptosis-promoting (p53, bax, Fas, Fas-L) and inhibiting (bcl-2) factors as well as of different isoforms of NOS (NOSb, NOSe, NOSi) and manganese superoxide dismutase (MnSOD) were studied. Apoptosis was measured in situ by the TUNEL method while expression profiles of apoptosis-related and oxidative stress-associated factors were determined by immunohistochemistry. As a defining criterion, necrosis was restricted to glioblastomas while apoptosis increased with tumour malignancy (P=0.017) in all types of gliomas. Glial tumour cells displayed upregulation of bax, Fas, Fas-L, p53, and bcl-2 but with no significant correlation with malignancy. There was also a strong expression of NOS isoforms with upregulation of NOSe in all and of NOSb and NOSi in nearly 50% of the tumour specimens but only NOSb expression correlated significantly with tumour malignancy (P=0.004). Likewise, MnSOD was strongly expressed in all gliomas but was not correlated with tumour grade. There was a wide variability of expression in each tumour type without significant correlation between apoptosis and expression of apoptosis-associated or oxidative stress-related factors indicating that the network of regulating factors may be too complex for clear associations.     

Increased endothelial expression of transglutaminase in glioblastomas

D. A. Hilton, S. Love and R. Barber (1997) Neuropathology and Applied Neurobiology , 23, 507–511
Increased endothelial expression of transglutaminase in glioblastomas
Transglutaminases are a family of calcium-dependent enzymes that catalyse the formation of covalent cross-links between proteins. They have several diverse functions and are thought to be involved in cell differentiation, apoptosis and blood coagulation. We have investigated the expression of tissue transglutaminase in five fibrillary astrocytomas, five anaplastic astrocytomas and seven glioblastomas by immunohistochemistry. Strongly labelled tumour cells were seen in most of the fibrillary and anaplastic astrocytomas and all of the glioblastomas. Labelling was particularly prominent in the pseudopalisading tumour cells that surrounded foci of necrosis and apoptosis in glioblastomas. Most of the immunostained cells did not themselves show morphological features of apoptosis. In addition, apoptotic cells were demonstrated using in situ end-labelling and by in situ hybridization with digoxigenin-labelled poly(A) oligonucleotide probes. Apoptotic cells demonstrated by both of these methods were most numerous in anaplastic astrocytomas and glioblastomas. However, their distribution did not correlate with that of the tumour cells showing transglutaminase labelling. Strong transglutaminase labelling was also observed in the endothelial cells of vessels showing microvascular proliferation in all of the glioblastomas studied. In contrast, endothelial transglutaminase labelling was weak or absent in lower grade astrocytic tumours. Enhanced expression of transglutaminase by endothelial cells in glioblastomas may contribute to the high prevalence of vascular thrombosis and necrosis in these tumours.     

不同病理级别胶质瘤p57^kip2/p21^cip1mRNA的表达变化

目的探讨不同病理级别人脑神经胶质瘤组织中p57^kip2/p21^cip1mRNA的表达变化及其关系。方法采用实时荧光定量PCR检测p57^kip2/p21^cip1mRNA在68例脑胶质瘤组织和16例非肿瘤脑组织中的表达水平。结果①p57^kip2mRNA在脑胶质瘤中的表达水平显著低于非肿瘤脑组织（P〈0.01）,且其表达水平与肿瘤病理级别呈显著负相关（rs=-0.495;P〈0.01）。②p21^cip1mRNA在脑胶质瘤中的表达水平与非肿瘤脑组织相比无显著差异（P〉0.05）,但其表达水平与肿瘤病理级别呈显著负相关（rs=-0.615;P〈0.01）。结论p57^kip2/p21^cip1的异常表达可能与人脑胶质瘤的发生和发展有密切相关,其表达水平可反映肿瘤的恶性程度。     

Immunohistochemical expression of P-glycoprotein and glutathione S-transferases in cerebral gliomas and response to chemotherapy

The expression of the drug resistance-related proteins glutathione S-transferases (GST) and P-glycoprotein (Pgp) was analyzed quantitatively in samples of 53 astrocytic gliomas (eight WHO grade 1, 11 WHO grade 2, 9 WHO grade 3 and 25 glioblastomas, WHO grade 4). Sections of these tumors were immunohistochemically stained with antibodies to Pgp (MDR1-gene product) and to GST subclasses alpha, mu and pi. Pgp expression was not detected in tumor cells of the majority of low-grade astrocytomas (69%) and the percentage of Pgp stained cells generally increased with tumor grade. However, 4 of the 34 malignant gliomas were negative. Many neoplastic cells of most tumors were dominantly stained for GST-pi. The other two subclasses were expressed in a less consistent fashion with no linear correlation to grading. Grade 2 astrocytomas exhibited the highest percentage of cells with GST expression. GST-alpha was absent in 9 tumors, GST-mu in 8 and GST-pi in 4. Four tumors showed no expression of any GST subclass or Pgp in neoplastic cells. Of 13 patients 5 with a more favorable clinical course after radiation and chemotherapy had a lower percentage of neoplastic cells immunostained for Pgp and the three GST subclasses than 8 patients with a worse clinical course. These results suggest a relationship between expression of drug resistance-related proteins in gliomas and response to chemotherapy with ACNU/VM26. Received: 18 November 1996 / Revised: 5 May 1997 / Revised, accepted: 16 June 1997     

Pitfalls in the assessment of MGMT expression and in its correlation with survival in diffuse astrocytomas: proposal of a feasible immunohistochemical approach

Immunohistochemical studies showed that O₆-methylguanine-DNA methyltransferase (MGMT) protein expression is negatively associated with survival in glioblastomas treated with alkylating agents in accordance with previous results of methylation-specific PCR. Implementation of this data in routine clinical diagnostics is limited due to often inappropriate study designs, e.g. pooling of tumor entities, WHO grades or primary and secondary glioblastomas, disregard concerning the infiltration zone or various epidemiological factors. The aim of our study was to evaluate MGMT expression and its prognostic value taking into consideration the aforementioned deficiencies. For this, 162 astrocytic tumors WHO II–IV (36 diffuse astrocytomas WHO II, 51 anaplastic astrocytomas, 75 primary glioblastomas) as well as 25 glioblastoma infiltration zones and 19 glioblastoma relapses were analyzed for immunohistochemical MGMT protein expression using tissue microarray technique. Expression of MGMT significantly decreased from WHO grade II (25.6%) to glioblastoma (16.8%, p = 0.01) with lowest levels in grade III tumors (10.2%, II/III p < 0.0001). Significant negative associations of MGMT and survival were detected for WHO grade II and IV (p = 0.003 and 0.013). The optimal cut-off value of MGMT positive nuclei in primary glioblastomas discriminating patients with significantly different survival rates was at 15% (Log–Rank p = 0.0002). Individual relapse tumors showed changes of MGMT expression to a varying degree. The infiltration zone demonstrated a significant increase of MGMT (p < 0.0001). We conclude that immunohistochemical MGMT assessment has potential as a powerful diagnostic tool but analysis should only be performed in a grade dependent manner, before radio-/chemotherapy and with special attention to the infiltration zone of diffuse astrocytomas.     

Expression of survivin,an inhibitor of apoptosis protein,in tumors of the nervous system

Survivin is an inhibitor of apoptosis protein that blocks apoptosis by binding to caspases-3 and -7. It is highly expressed in less-differentiated embryonic cells and rapidly dividing tumors, but not in terminally differentiated adult tissues. Elevated survivin levels are found in malignant systemic tumors, and are associated with chemo-resistance, radiation resistance, and poor prognosis. However, expression of survivin in primary nervous system tumors has not been previously characterized. Immunohistochemistry using anti-human survivin antibody (SURV11-A) was performed on formalin-fixed, paraffin-embedded archival tissue from 112 primary central nervous system tumors. Survivin immunoreactivity was seen in most diffuse astrocytomas [WHO II (2/4), III (3/3), IV (9/10), giant-cell glioblastoma (1), and gliosarcoma (1)]. The intensity and degree of survivin expression showed trends with tumor grade, with glioblastomas having the highest positivity. Pilocytic astrocytomas (5) and pleomorphic xanthoastrocytoma (1) were positive to a lesser degree. In oligodendrogliomas (6) and mixed oligo-astrocytomas [grade II (5), II-III (3), and III (7)], oligodendroglial elements appear to be negative compared to positive mini-gemistocytic oligodendrocytes. Ependymomas [grade II (6) and grade III (1)] were positive. Medulloblastomas (5) and retinoblastoma (1/4) showed focal positivity. All meningiomas [grade I (12), II (9), III (4), and grade I (3) and II (5) with frank brain invasion] were intensely positive. All schwannomas (11) and neurofibromas (6) were intensely positive. Thus, survivin is expressed in the majority of the primary nervous system tumors, particularly in glioblastomas, meningiomas, schwannomas and neurofibromas. Overexpression of survivin in meningiomas and benign peripheral nerve sheath tumors contrasts with previous reports relating it to rapid division and poor prognosis.     

Expression pattern of the water channel aquaporin-4 in human gliomas is associated with blood-brain barrier disturbance but not with patient survival

Aquaporin-4 (AQP4), the most prominent CNS water channel, is restricted to the glia limitans and astrocytic endfeet. We previously showed the loss of spatial AQP4 expression in glioblastomas and a redistribution across the cell surface. However, opposing AQP4 functions have been described: protective in vasogenic but detrimental in cytotoxic brain edema. Thus, specific AQP4 induction to prevent or reduce vasogenic edema is suggested. To elucidate the AQP4 role in brain tumors, we investigated 189 WHO grade I-IV gliomas by immunohistochemistry and the prognostic significance for patients' survival. In gliomas, a remarkable de novo AQP4 redistribution was observed in comparison with normal CNS tissue. Surprisingly, the highest membraneous staining levels were seen in pilocytic astrocytomas WHO grade I and grade IV glioblastomas, both significantly higher than in WHO grade II astrocytomas. AQP4 up-regulation was associated with brain edema formation; however, no association between survival and WHO grade-dependent AQP4 expression was seen. Hence, AQP4 redistribution may go along with other tumor properties, such as vascular proliferation and resulting blood-brain barrier disturbance, features usually prominent in pilocytic astrocytomas WHO I and glioblastomas WHO grade IV. In summary, our findings question the protective role of AQP4 in vasogenic brain edema. Although AQP4 was associated with brain edema formation, one has to question the suitability of AQP4 induction as a promising approach in vasogenic brain edema prevention and treatment. In addition, our results provide unexpectedly high AQP4 levels in pilocytic astrocytomas and present AQP4 as tumor progression marker in WHO grade II-IV astrocytomas.     

Expression and activity of l-isoaspartyl methyltransferase decrease in stage progression of human astrocytic tumors

Protein l-isoaspartyl methyltransferase (PIMT) functions as a repair enzyme that acts upon damaged proteins bearing abnormal aspartyl residues. We previously reported that PIMT expression and activity are reduced by half in human epileptic hippocampus. Here we investigated PIMT regulation in astrocytic tumors, which are the most common human brain tumors. PIMT expression and enzyme activity were significantly decreased in all grades of human astrocytic tumors. More precisely, PIMT levels were significantly lower by 76% in pilocytic astrocytomas (grade I), 46% in astrocytomas (grade II), 69% in anaplastic astrocytomas (grade III), and a marked 80% in glioblastomas (grade IV) as compared to normal brains. RT-PCR analysis showed that levels of type I PIMT mRNA were up-regulated while those of type II PIMT mRNA were down-regulated in glioblastomas. Furthermore, the reduced PIMT levels correlated closely with a decrease in the number of neuron cells in astrocytic tumors as assessed by measuring the neuron-specific enolase level. Many proteins with abnormal aspartyl residues accumulated in brain tumors and some were specific to individual grades of astrocytic tumors. Similar results were obtained, either by measuring the reduction in PIMT activity and expression or by measuring the formation of abnormal proteins, in an orthotopic rat brain tumor model implanted with invasive CNS-1 glioma cells. The novelty of these findings was to provide the first evidence for a marked reduction of PIMT expression and activity during stage progression of astrocytic tumors in humans.     

Immunohistochemical analysis of cyclin A in astrocytic tumours

Cyclins are important regulators of the cell cycle; there is increasing evidence that some cyclins are positively involved in carcinogenesis. Amplification and translocation of the cyclin genes and overexpression of their mRNAs and proteins have been observed in a variety of tumours. We studied cyclin A protein in astrocytic tumours by immunohistochemical analysis. Immunohistochemistry with microwave antigen retrieval was carried out on formalin fixed, paraffin embedded material from 15 glioblastomas (WHO grade IV), 10 anaplastic astrocytomas (WHO grade III), seven diffuse low grade astrocytomas (WHO grade II) and nine pilocytic astrocytomas (WHO grade I) using antibodies against cyclin A and a proliferation marker MIB1. Staining for these antibodies was seen mainly in the tumour cell nuclei; 66% of all cases showing staining for cyclin A and 95% of all cases staining for MIB1. Mean labelling indices (LI) for cyclin A were higher in glioblastoma (mean LI-6.7) and anaplastic astrocytoma (mean LI-5.9) than low grade diffuse astrocytoma (mean LI-1.7) and pilocytic astrocytoma (mean LI −0.12), although there was no clear cut off point between the various tumour types. A good correlation was seen between labelling indices of cyclin A and MIB1 (Pearson correlation coefficient r =0.59, P <0.0001). Cyclin A is variably expressed in astrocytic tumours, either reflecting increased tumour proliferation (cyclin A being an integral component of the cell cycle), an alteration of its gene, protein upregulation or regulation of apoptosis. The genetic basis of expression of cyclin A in astrocytic tumours remains to be determined.     

Magnetic resonance spectroscopy in intracranial tumours of glial origin

Background and purposeTo determine in vivo magnetic resonance spectroscopy (MRS) characteristics of intracranial glial tumours and to assess MRS reliability in glioma grading and discrimination between different histopathological types of tumours.Material and methodsAnalysis of spectra of 26 patients with glioblastomas, 6 with fibrillary astrocytomas, 4 with anaplastic astrocytomas, 2 with pilocytic astrocytoma, 3 with oligodendrogliomas, 3 with anaplastic oligodendrogliomas and 17 control spectra taken from healthy hemispheres.ResultsAll tumours’ metabolite ratios, except for Cho/Cr in fibrillary astrocytomas (p = 0.06), were statistically signiflcantly different from the control. The tumours showed decreased Naa and Cr contents and a high Cho signal. The Lac-Lip signal was high in grade III astrocytomas and glioblastomas. Reports that Cho/Cr ratio increases with glioma's grade whereas Naa/Cr decreases were not confirmed. Anaplastic astrocytomas compared to grade II astrocytomas had a statistically significantly greater ml/Cr ratio (p = 0.02). In pilocytic astrocytomas the Naa/Cr value (2.58 ± 0.39) was greater, whilst the Cho/Naa ratio was lower (2.14 ± 0.64) than in the other astrocytomas. The specific feature of oligodendrogliomas was the presence of glutamate/glutamine peak Glx. However, this peak was absent in two out of three anaplastic oligodendrogliomas. Characteristically, the latter tumours had a high Lac-Lip signal.ConclusionsMRS in vivo cannot be used as a reliable method for glioma grading. The method is useful in discrimination between WHO grade I and WHO grade II astrocytomas as well as oligodendrogliomas from other gliomas.     

Co-expression of Fas and Fas ligand in human non-astrocytic glial tumors

The study of Fas (APO-1/CD95) and Fas ligand (FasL) expression in human glial neoplasms has been primarily restricted to astrocytomas. Using immunohistochemistry, we analyzed 35 non-astrocytic glial neoplasms (12 WHO grade II ependymomas, 15 grade II oligodendrogliomas, and 8 grade III oligodendrogliomas) for these factors. A significant correlation was found between Fas and FasL expression within the same tumors (P = 0.001). Western blotting was used to corroborate these findings in 7 of these tumors (3 ependymomas and 4 oligodendrogliomas). Theoretically, the co-expression of Fas and FasL should render gliomas susceptible to suicidal and fratricidal elimination. Their progressive nature, however, suggests that gliomas have acquired mechanisms to prevent Fas-mediated apoptosis. Expression of the anti-apoptotic protooncogene bcl-2 is one potential way in which Fas/FasL-bearing gliomas can escape this fate. Although expression of Bcl-2 protein was found in 6 of 12 (50%) grade II ependymomas, 5 of 15 (33%) grade II oligodendrogliomas, and 6 of 8 (75%) grade III oligodendrogliomas, no correlation between Fas/FasL co-expression and Bcl-2 production could be demonstrated, indicating that protection from Fas-mediated apoptosis probably involves other mechanisms. Received: 10 November 1998 / Revised: 15 March 1999 / Accepted: 24 March 1999     

DMBT1 homozygous deletion in diffuse astrocytomas is associated with unfavorable clinical outcome

Primary glioblastomas develop with a short clinical history, without evidence for less malignant precursor lesions, while secondary glioblastomas slowly develop via progression from diffuse astrocytoma (WHO grade II) or anaplastic astrocytoma (WHO grade III). The time until progression and the clinical outcome of diffuse astrocytomas vary significantly. We have shown that IDH1 mutations reliably distinguish between primary glioblastomas (without IDH1 mutations) and secondary glioblastomas (with IDH1 mutations). The most frequent genetic alteration shared by primary and secondary glioblastomas is loss of heterozygosity at 10q (up to 60% of cases). Here, we first assessed The Cancer Genome Atlas data to identify gene loss at 10q in glioblastomas with or without IDH1 mutations. Using log-ratio thresholds of -1.0, 10 genes were identified; with the log-ratio thresholds of -2.0, only the DMBT1 (deleted in malignant brain tumor 1) gene at 10q26.13 remained as a deleted gene in glioblastomas with or without IDH1 mutations (12.5% vs 8.0%). We then analyzed a total of 404 gliomas by differential polymerase chain reaction and found a DMBT1 homozygous deletion at a similar frequency in primary and secondary glioblastomas (19.6% vs 20.8%). A fraction (11.3%) of diffuse astrocytomas showed a DMBT1 homozygous deletion that was significantly associated with a shorter overall survival (52.8 vs 84.0 months; p = 0.003). These results indicate that a DMBT1 homozygous deletion is present in a fraction of diffuse astrocytomas and that it is associated with an unfavorable clinical outcome.     

Analysis of the IDH1 codon 132 mutation in brain tumors

A recent study reported on mutations in the active site of the isocitrate dehydrogenase (IDH1) gene in 12% of glioblastomas. All mutations detected resulted in an amino acid exchange in position 132. We analyzed the genomic region spanning wild type R132 of IDH1 by direct sequencing in 685 brain tumors including 41 pilocytic astrocytomas, 12 subependymal giant cell astrocytomas, 7 pleomorphic xanthoastrocytomas, 93 diffuse astrocytomas, 120 adult glioblastomas, 14 pediatric glioblastomas, 105 oligodendrogliomas, 83 oligoastrocytomas, 31 ependymomas, 58 medulloblastomas, 9 supratentorial primitive neuroectodermal tumors, 17 schwannomas, 72 meningiomas and 23 pituitary adenomas. A total of 221 somatic IDH1 mutations were detected and the highest frequencies occurred in diffuse astrocytomas (68%), oligodendrogliomas (69%), oligoastrocytomas (78%) and secondary glioblastomas (88%). Primary glioblastomas and other entities were characterized by a low frequency or absence of mutations in amino acid position 132 of IDH1. The very high frequency of IDH1 mutations in WHO grade II astrocytic and oligodendroglial gliomas suggests a role in early tumor development.