Metabolic differences between primary and recurrent human brain tumors: a 1H NMR spectroscopic investigation

High-resolution proton magnetic resonance spectroscopy was performed on tissue specimens from 33 patients with astrocytic tumors (22 astrocytomas, 11 glioblastomas) and 13 patients with meningiomas. For all patients, samples of primary tumors and their first recurrences were examined. Increased anaplasia, with respect to malignant transformation, resulting in a higher malignancy grade, was present in 11 recurrences of 22 astrocytoma patients. Spectroscopic features of tumor types, as determined on samples of the primary occurrences, were in good agreement with previous studies. Compared with the respective primary astrocytomas, characteristic features of glioblastomas were significantly increased concentrations of alanine (Ala) (p = 0.005), increased metabolite ratios of glycine (Gly)/total creatine (tCr) (p = 0.0001) and glutamate (Glu)/glutamine (Gln) (p = 0.004). Meningiomas showed increased Ala (p = 0.02) and metabolite ratios [Gly, total choline (tCho), Ala] over tCr (p = 0.001) relative to astrocytomas, and N-acetylaspartate and myo-inositol were absent. Metabolic changes of an evolving tumor were observed in recurrent astrocytomas: owing to their consecutive assessments, more indicators of malignant degeneration were detected in astrocytoma recurrences (e.g. Gly, p = 0.029; tCho, p = 0.034; Glu, p = 0.015; tCho/tCr, p = 0.001) in contrast to the comparison of primary astrocytomas with primary glioblastomas. The present investigation demonstrated a correlation of the tCho-signal with tumor progression. Significantly elevated concentrations of Ala (p = 0.037) and Glu (p = 0.003) and metabolite ratio tCho/tCr (p = 0.005) were even found in recurrent low-grade astrocytomas with unchanged histopathological grading (n = 11). This may be related to an early stage of malignant transformation, not yet detectable morphologically, and emphasizes the high sensitivity of 1H NMR spectroscopy in elucidating characteristics of brain tumor metabolism.     

Frequent LOH on 22q12.3 and TIMP-3 inactivation occur in the progression to secondary glioblastomas

Frequent allelic losses on the long arm of chromosome 22 (22q) in gliomas indicate the presence of tumor suppressor gene (TSG) at this location. However, the target gene(s) residing in this chromosome are still unknown and their putative roles in the development of astrocytic tumors, especially in secondary glioblastoma, have not yet been defined. To compile a precise physical map for the region of common deletions in astrocytic tumors, we performed a high-density loss of heterozygosity (LOH) analysis using 31 polymorphic microsatellite markers spanning 22q in a series of grade II diffuse astrocytomas, anaplastic astrocytomas, primary glioblastomas, and secondary glioblastomas that had evolved from lower grade astrocytomas. LOH was found at one or more loci in 33% (12/36) of grade II diffuse astrocytomas, in 40% (4/10) of anaplastic astrocytomas, in 41% (26/64) of primary glioblastomas, and in 82% (23/28) of secondary glioblastomas. Characterization of the 22q deletions in primary glioblastomas identified two sites of minimally deleted regions at 22q12.3-13.2 and 22q13.31. Interestingly, 22 of 23 secondary glioblastomas affected shared a deletion in the same small (957 kb) region of 22q12.3, a region in which the human tissue inhibitor of metalloproteinases-3 (TIMP-3) is located. Investigation of the promoter methylation and expression of this gene indicated that frequent hypermethylation correlated with loss of TIMP-3 expression in secondary glioblastoma. This epigenetic change was significantly correlated to poor survival in eight patients with grade II diffuse astrocytoma. Our results suggest that a 957 kb locus, located at 22q12.3, may contain the putative TSG, TIMP-3, that appears to be relevant to progression to secondary glioblastoma and subsequently to the prognosis of grade II diffuse astrocytoma. In addition, the possibility of other putative TSGs on 22q12.3-13.2 and 22q13.31 that may also be involved in the development of primary glioblastomas cannot be ruled out.     

Argyrophilic nucleolar organizer region proteins (Ag-NORs) in human central nervous tumors]

A silver colloid staining technique for identifying nucleolar organizer region-associated proteins (Ag-NORs) was applied to 17 primary gliomas and 16 meningiomas. These comprised 8 glioblastomas, one pleomorphic xanthoastrocytoma, 8 benign astrocytoma, 10 nonrecurrent meningiomas and 6 recurrent meningiomas, in which the mean number of Ag-NORs' per nuclei, were 3.37, 2.34, 2.57, 2.16 and 2.80 respectively, identifying significant differences among these groups. Ag-NORs of four giant cell glioblastomas showed low numbers of Ag-NORs, suggesting better prognosis. In spite of the time consuming and complicated counting method Ag-NORs is reproductive and useful as a tool for estimating the proliferating potential of various brain tumors, and appears to widely applicable in clinical laboratories.     

星形细胞肿瘤表皮生长因子受体与p53基因的异常表达

目的研究星形细胞肿瘤中癌基因表皮生长因子受体（EGFR）过表达与抑癌基因p53突变、表达与肿瘤病理类型、恶性程度及两者的相互关系。方法对37例不同恶性程度的星形细胞肿瘤及6例正常脑组织,采用免疫组织化学、逆转录聚合酶链反应（RT—PCR）方法检测EGFR的表达;采用免疫组织化学、PCR—SSCP及DNA测序方法检测同一标本的p53基因突变和异常表达,分析它们的异常改变和内在联系。结果p53突变率在弥漫性星形细胞瘤、间变性星形细胞瘤、原发性胶质母细胞瘤、继发性胶质母细胞瘤分别为1／10,4／19（21．1％）,4／6和2／2,而EGFR过表达分别为5／10,10／19（52．6％）,5／6和2／2。随着胶质瘤级别增高,p53积聚与EGFR过表达在同一标本中发生率升高。结论在低度恶性胶质瘤中p53基因突变少见,EGFR过表达不少见;在原发性和继发性胶质母细胞瘤中p53基因突变及EGFR过表达均常见。提示p53与EGFR分子通路可能对胶质瘤的恶性进展不是相互排斥而是协同产生促进作用。     

Genetic changes of CDH1, APC, and CTNNB1 found in human brain tumors

This paper focuses on changes in E-cadherin (CDH1), adenomatous polyposis coli (APC), and beta-catenin (CTNNB1) in 50 tumors of the central nervous system. All gene products are components of adherens junctions, but are also involved in wnt signaling. The results of our analysis showed LOH of CDH1 gene in 31% of meningiomas examined (significant correlation; p=0.002). LOH was noted in a single case of germinoma, while other tumor types did not demonstrate any change in CDH1. Fourteen samples (29.2%) with changes in APC gene were observed. The changes were seen in 33.3% of glioblastomas and in 27% of meningiomas; LOH occurred in five informative astocytomas (20%) and in six informative neurinomas (17%). One oligoastrocytoma showed LOH at exon 11, and one medulloblastoma had allelic imbalance at both exons. Five samples (10%) showed heteroduplexes in exon 3 of beta-catenin. Potential mutations were confined to two meningiomas, one astrocytoma, one glioblastoma, and one germinoma. Our results suggest that genetic changes in wnt components are involved in brain tumor genesis. Changes in E-cadherin are involved in meningiomas, while changes in APC gene occur in different tumor types, with glioblastomas showing the highest percentage.     

Expression and immunohistochemical localization of cathepsin L during the progression of human gliomas

Recent studies suggest that cysteine proteinase cathepsin L is involved in the process of tumor invasion and metastasis. We examined cathepsin L activity in brain tumor tissue samples by an enzymatic assay, and cathepsin L protein content by enzyme-linked immunoadsorbent assays and Western blotting to determine whether increased levels of cathepsin L correlate with the progression of human gliomas. Native and acid-activatable cathepsin L activities were highest in glioblastomas followed by anaplastic astrocytomas and were lowest in low-grade gliomas and normal brain tissues. Significantly higher amounts of an M _r 29 000 cathepsin L were present in glioblastomas and anaplastic astrocytomas than in normal brain tissues and low-grade glioma tissue extracts. Using specific antibodies to cathepsin L, we also studied its cellular distribution by immunohistochemical procedures. Higher diffuse cathepsin L immunoreactivity was found in glioblastomas than in low-grade gliomas and normal brain tissue samples. Finally, the addition of cathepsin L antibody inhibits the invasion of glioblastoma cell lines through Matrigel invasion assay. These results suggest the expression of cathepsin L is dramatically upregulated in malignant gliomas and correlates with the malignant progression of human gliomas in vivo.     

Comparative Genomic Hybridization of Human Malignant Gliomas Reveals Multiple Amplification Sites and Nonrandom Chromosomal Gains and Losses

Nine human malignant gliomas (2 astrocytomas grade III and 7 glioblastomas) were analyzed using comparative genomic hybridization (CGH). In addition to the amplification of the EGFR gene at 7p12 in 4 of 9 cases, six new amplification sites were mapped to 1q32, 4q12, 7q21.1, 7q21.2-3, 12p, and 22q12. Nonrandom chromosomal gains and losses were identified with overrepresentation of chromosome 7 and underrepresentation of chromosome 10 as the most frequent events (1 of 2 astrocytomas, 7 of 7 glioblastomas). Gain of a part or the whole chromosome 19 and losses of chromosome bands 9pter-23 and 22q13 were detected each in five cases. Loss of chromosome band 17p13 and gain of chromosome 20 were revealed each in three cases. The validity of the CGH data was confirmed using interphase cytogenetics with YAC clones, chromosome painting in tumor metaphase spreads, and DNA fingerprinting. A comparison of CGH data with the results of chromosome banding analyses indicates that metaphase spreads accessible in primary tumor cell cultures may not represent the clones predominant in the tumor tissue     

An experimental model for anaplastic astrocytomas and glioblastomas using adult F344 rats and N-methyl-N-nitrosourea

An experimental model for induction of gliomas corresponding to human anaplastic astrocytomas and glioblastomas is reported. Eleven week old F344 and ACI rats were given 100 or 200 p.p.m. N-methyl-N-nitrosourea (MNU) solution as their drinking water for 42 weeks. Gliomas were induced at very high incidences (82.5-92.5%) in each group. Induced gliomas showed apparent evidence of morphologic malignancy by an analysis based on diagnostic criteria of human astrocytomas. All of the gliomas from the killed animals were classified histologically into subtypes according to the classification scheme used in the diagnosis of human gliomas. The majority of macrotumors more than 1 mm in diameter in both strains were diagnosed as anaplastic astrocytomas and glioblastomas. lmmunohistochemically, tumor cells in these tumors were almost negative for glial fibrillary acidic protein, while ultrastructurally neoplastic astrocytes contained glial filaments. A strain difference was observed in the ratio of histological subtypes of macrotumors. In F344 rats, astrocytic tumors diagnosed as anaplastic astrocytomas and glioblastomas of an astrocytic type formed the majority, whereas glioblastomas of mixed oligo-astrocytic type predominated in ACI rats. The results indicate that MNU-administration to adult F344 rats may provide a suitable experimental model for gliomas which occur in adult humans.     

Comparative genomic hybridization reveals recurrent enhancements on chromosome 20 and in one case combined amplification sites on 15q24q26 and 20p11p12 in glioblastomas

We examined homogenized tissue samples of biopsies from 19 astrocytomas of different grades for genetic imbalances using comparative genomic hybridization (CGH): three astrocytomas grade II, and 16 astrocytomas grade IV (glioblastoma multiforme), one of the glioblastomas representing the recurrence of a benign oligoastrocytoma. In two of three cases of astrocytoma grade II, a gain of chromosome 7 was found. The alterations in the glioblastomas were complex, and most frequently showed the characteristic gain of chromosome 7 and loss of chromosome 10. The single analyzed case of recurrence of an oligoastrocytoma was characterized by a unique CGH pattern. This tumor showed two distinct alterations: apart from an amplification on 15q24q26, we found a distinct amplification of a small region on 20p11.2p12, which has not been previously described in brain tumors. Partial or complete gains of chromosome 20 arose in six other tumors; we conclude that chromosome 20 in particular 20p11. 2p12, may harbor relevant genes for glioma progression.     

星形细胞瘤p16与Rb蛋白的表达及其相关性研究

目的检测ｐ１６与Ｒｂ蛋白在原发性星形细胞瘤中的存在状况，以探讨不同病理类型星形细胞瘤中ｐ１６与Ｒｂ蛋白表达及其相关性。方法使用抗ｐ１６及Ｒｂ蛋白抗体对１０２例星形细胞瘤手术标本进行免疫细胞化学检测。结果低度恶性星形细胞瘤（ＷＨＯⅠ～Ⅱ级）中ｐ１６及Ｒｂ蛋白均为阳性表达，在高度恶性星形细胞瘤（ＷＨＯⅢ～Ⅳ级）中其阳性表达分别为４８．１％（２６／５４）和５７．４％（３１／５４）。在３１例Ｒｂ蛋白阳性标本中，有２４例（７７．４％）显示ｐ１６蛋白表达缺失或低表达，而在２３例Ｒｂ蛋白阴性标本中却有１９例（８２．６％）显示ｐ１６蛋白阳性或强阳性。结论（１）ｐ１６及Ｒｂ蛋白参与星形细胞瘤细胞增殖过程并影响其细胞分化；（２）ｐ１６与Ｒｂ蛋白之间阳性表达的相互抑制，可能是高度恶性星形细胞瘤的标志之一。     

Expression of matrix metalloproteinases and their inhibitors in human brain tumors

Sixty human brain tumors, classified according to the New World Health Organization (WHO) classification including, grade I schwannomas, meningiomas and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas, grade IV glioblastomas, grade III anaplastic oligodendrogliomas and grade IV glioblastomas and lung and melanoma metastases were analyzed for the expression of three matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs) and for MMP activity. Some correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. MMP-9 levels were found to be significantly higher in grade III anaplastic astrocytomas and anaplastic oligodendrogliomas than those in grade I schwannomas and meningiomas. Anaplastic astrocytomas and Grade IV glioblastomas expressed significantly higher levels MMP-12 than grade I meningiomas. All sixty tumors showed a similar pattern of activity in zymography, proMMP-9 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were especially low in tumors of grade II and grade III but significantly higher in tumors of grade I, particularly in schwannomas. Taken together, these data suggest that: 1) a balance between MMPs and TIMPs has an important role to play in human brain tumors; 2) TIMP expression may be valuable markers for tumor malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phenotype vs genotype in the evolution of astrocytic brain tumors

Astrocytic brain tumors are the most frequent human gliomas and they include a wide range of neoplasms with distinct clinical, histopathologic, and genetic features. Diffuse astrocytomas are predominantly located in the cerebral hemispheres of adults and have an inherent tendency to progress to anaplastic astrocytoma and (secondary) glioblastoma. The majority of glioblastomas develop de novo (primary glioblastomas), without an identifiable less-malignant precursor lesion. These subtypes of glioblastoma evolve through different genetic pathways, affect patients at different ages, and are likely to differ in their responses to therapy. Primary glioblastomas occur in older patients and typically show epidermal growth factor receptor (EGFR) overexpression, PTEN mutations, p16 deletions, and, less frequently, MDM2 amplification. Secondary glioblastomas develop in younger patients and often contain TP53 mutations as their earliest detectable alteration. Morphologic variants of glioblastoma were shown to have intermediate clinical and genetic profiles. The giant cell glioblastoma clinically and genetically occupies a hybrid position between primary (de novo) and secondary glioblastomas. Gliosarcomas show identical gene mutations in the gliomatous and sarcomatous tumor components, which strongly supports the concept that there is a monoclonal origin for gliosarcomas and an evolution of the sarcomatous component due to aberrant mesenchymal differentiation in a highly malignant astrocytic neoplasm.     

Genetic pathways to primary and secondary glioblastoma

Glioblastoma is the most frequent and most malignant human brain tumor. The prognosis remains very poor, with most patients dying within 1 year after diagnosis. Primary and secondary glioblastoma constitute distinct disease subtypes, affecting patients of different age and developing through different genetic pathways. The majority of cases (>90%) are primary glioblastomas that develop rapidly de novo, without clinical or histological evidence of a less malignant precursor lesion. They affect mainly the elderly and are genetically characterized by loss of heterozygosity 10q (70% of cases), EGFR amplification (36%), p16(INK4a) deletion (31%), and PTEN mutations (25%). Secondary glioblastomas develop through progression from low-grade diffuse astrocytoma or anaplastic astrocytoma and manifest in younger patients. In the pathway to secondary glioblastoma, TP53 mutations are the most frequent and earliest detectable genetic alteration, already present in 60% of precursor low-grade astrocytomas. The mutation pattern is characterized by frequent G:C-->A:T mutations at CpG sites. During progression to glioblastoma, additional mutations accumulate, including loss of heterozygosity 10q25-qter ( approximately 70%), which is the most frequent genetic alteration in both primary and secondary glioblastomas. Primary and secondary glioblastomas also differ significantly in their pattern of promoter methylation and in expression profiles at RNA and protein levels. This has significant implications, particularly for the development of novel, targeted therapies, as discussed in this review.     

Expression of urokinase-type plasminogen activator receptor (uPAR) in primary central nervous system neoplasms.

The cellular receptor for urokinase-type plasminogen activator receptor (uPAR) is a member of the glycosylphosphatidylinositol (GPI) anchored protein family. It is a specific cell surface receptor for its ligand, urokinase-type plasminogen activator, which catalyzes the formation of plasmin from plasminogen to generate the proteolytic cascade and leads to the breakdown of the extracellular matrix. uPAR has been shown to correlate with a propensity to tumor invasion and metastasis in several types of non-central nervous system tumors. In this study, the authors examined the immunohistochemical expression of uPAR in 65 primary brain tumors (5 pilocytic astrocytomas, 5 diffuse astrocytomas, 6 anaplastic astrocytomas, 8 glioblastomas, 5 oligodendrogliomas, 4 oligoastrocytomas, 6 anaplastic oligoastrocytomas, 4 gangliogliomas, 4 ependymomas, 5 medulloblastomas, 6 schwannomas, 5 meningiomas, 2 atypical meningiomas). The specimens were evaluated for intensity of immunostaining (0-3 scale), cellular localization of staining, and specific or unique patterns of staining. Some degree of uPAR expression was observed in all tumors. A significant positive correlation (P = 0.0006) between tumor grade and staining intensity was identified within the astrocytoma/glioblastoma subgroup, suggesting a possible correlation with anaplastic change and propensity to tumor invasion. Expression of uPAR in nonmalignant, noninvasive tumors such as schwannoma and meningioma suggests that uPAR may have other biologic functions in addition to promotion of tumor invasion.     

Acquisition of the Glioblastoma Phenotype during Astrocytoma Progression Is Associated with Loss of Heterozygosity on 10q25-qter

Loss of heterozygosity on chromosome 10 (LOH#10) is the most frequent genetic alteration in glioblastomas and occurs in more than 80% of cases. We recently reported that PTEN (MMAC1) on 10q23.3 is mutated in approximately 30% of primary (de novo) glioblastomas but rarely in secondary glioblastomas that progressed from low-grade or anaplastic astrocytomas. Because secondary glioblastomas also show LOH#10, tumor suppressor genes other than PTEN are likely to be involved. We analyzed LOH on chromosomes 10 and 19, using polymorphic microsatellite markers in microdissected foci showing histologically an abrupt transition from low-grade or anaplastic astrocytoma to glioblastoma, suggestive of the emergence of a new tumor clone. When compared to the respective low-grade or anaplastic astrocytoma of the same biopsy, deletions were detected in 7 of 8 glioblastoma foci on 10q25-qter distal to D10S597, covering the DMBT1 and FGFR2 loci. Six of 8 foci showed LOH at one or two flanking markers of PTEN but did not contain PTEN mutations. LOH on 10p and 19q was found in only one case each. These data indicate that acquisition of a highly anaplastic glioblastoma phenotype with marked proliferative activity and lack of glial fibrillary acidic protein expression is associated with loss of a putative tumor suppressor gene on 10q25-qter.