Molecular abnormalities of chromosome-19 in malignant gliomas - preferential involvement of the 19q13.2-q13.4 region

A deletion mapping analysis of chromosome 19 was performed on a series of 101 samples derived from malignant gliomas. A total of 35 tumors displayed different deletions for the loci studied (D19S21, D19S11, D19S74, D19S7, D19S8, CKM, and D19S22). In most instances, losses involving the long arm markers of chromosome 19 were observed, and only four samples were characterized by losses on the short arm. No tumor was found displaying loss of both short and long arm markers. The higher frequency of deletions was detected in tumors with a major oligodendroglial component: 76% of samples included in this group displayed losses at 19q. Among the astrocytic tumors, the frequency of 19q alterations varied as follows: 11% in pilocytic astrocytomas, 17% in astrocytomas grade II, 10% in anaplastic astrocytomas and 21% in glioblastoma multiforme. No ependymoma was found displaying allele loss on chromosome 19. The common region of overlap for the 19q deletions observed involves primarily the distal portion of the long arm, 19q13.2-q13.4. In agreement with previous reports, these data suggest the non-random involvement of a tumor suppressor gene located at 19q13 in the genesis or progression of malignant gliomas.     

Evidence for a tumor suppressor gene on chromosome 19q associated with human astrocytomas, oligodendrogliomas, and mixed gliomas.

Previous studies have shown frequent allelic losses of chromosomes 9p, 10, 17p, and 22q in glial tumors. Other researchers have briefly reported that glial tumors may also show allelic losses of chromosome 19, suggesting a putative tumor suppressor gene locus on this chromosome (D. T. Ransom et al., Proc. Am. Assoc. Cancer Res., 32:302, 1991). To evaluate whether loss of chromosome 19 alleles is common in glial tumors of different types and grades, we performed Southern blot restriction fragment length polymorphism analysis for multiple chromosome 19 loci in 122 gliomas from 116 patients. Twenty-nine tumors had loss of constitutional heterozygosity of 19q, and four tumors had partial deletions of 19q. Allelic losses on 19q were restricted to grade III anaplastic astrocytomas (4/9) and grade IV glioblastomas (11/46), grade II oligodendrogliomas (2/5) and grade III anaplastic oligodendrogliomas (2/2), and grade II (5/8) and grade III (5/7) mixed oligoastrocytomas. These data demonstrate genetic similarities between astrocytomas, oligodendrogliomas, and mixed glial tumors and indicate the presence of a glial tumor suppressor gene on chromosome 19q.     

Molecular pathogenesis of pediatric astrocytic tumors

Astrocytomas are the most common pediatric brain tumors, accounting for 7%-8% of all childhood cancers. Relatively few studies have been performed on their molecular properties; therefore, classification of pediatric astrocytic tumors into genetic subtypes similar to that of adult tumors remains to be defined. Here, we report an extensive characterization of 44 pediatric astrocytomas--16 diffuse astrocytomas (WHO grade II), 10 anaplastic astrocytomas (WHO grade III), and 18 glioblastomas (WHO grade IV)--in terms of genetic alterations frequently observed in adult astrocytomas. Some form of p53 mutation was found in three diffuse astrocytomas, in three anaplastic astrocytomas, and in six glioblastomas examined; PTEN mutations were detected only in two glioblastomas. EGFR amplification was detected in only one anaplastic astrocytoma and two glioblastomas, but no amplification was observed for the PDGFR-alpha gene. Loss of heterozygosity (LOH) on 1p/19q and 10p/10q was less common in pediatric astrocytic tumors than in those seen in adults, but the frequency of LOH on 22q was comparable, occurring in 44% of diffuse astrocytomas, 40% of anaplastic astrocytomas, and 61% of glioblastomas. Interestingly, a higher frequency of p53 mutations and LOH on 19q and 22q in tumors from children six or more years of age at diagnosis was found, compared with those from younger children. Our results suggest some differences in children compared to adults in the genetic pathways leading to the formation of de novo astrocytic tumors. In addition, this study suggests potentially distinct developmental pathways in younger versus older children.     

Molecular analysis of chromosomh 1 abnormalities in human gliomas reveals frequent loss of 1p in oligodendroglial tumors

Alterations of the short arm of chromosome I are recurrently found in cytogenetic analysis of malignant gliomas, and deletions of Ip36-p32 region characterize at least the higher-grade tumors, glioblsstoma multiforme. Molecular analysis of tumor-derived and normal genomic DNA from 57 cases of gliomas, using a panel of chromosome I-specific DNA probes showed LOH in 16 tumors. Allelic losses on I p were primarily restricted to glioblastoma multiforme (2/II) and to tumors with a major oligodendroglial component: grade II oligodendrogliomas (6/6), grade III anaplastic oligodendrogliomas (5/6) and grade II-III mixed oligo-astrocytomas (2/3). Losses for Iq markers were detected in only I tumor (glioblastoma multiforme). Our data suggest that anomalies of Ip primarily characterize oligodendrogliomas, whereas they are rare events in astrocytic tumors and indicate that a tumor-suppressor gene on I p36-p32 is involved in the development of brain tumors with oligodendroglial differentiation. © 1994 Wiley-Liss, Inc.     

Reclassification of oligoastrocytomas by loss of heterozygosity studies

Oligoastrocytomas (OAs) are WHO grade II or III tumors composed of a mixture of 2 neoplastic cell types morphologically resembling the cells in oligodendrogliomas and diffuse astrocytomas. Investigations on the genetic profile of OAs may yield important information for their classification and help for their clinical management. We have studied, in 94 OAs (46 WHO grade II and 48 WHO grade III), the patterns of loss of heterozygosity (LOH) of 4 genomic regions: 1p, 19q, 17p and 10q. Results were as follows: LOH 1p was present in 46% of the tumors; LOH 19q in 45%; LOH 17p in 22%; LOH 10q in 16%. LOH 1p and 19q were associated in 32%, other LOH associations were rare (<3%). Patients had a median follow-up of 30 months. Patients without LOH on 1p had shorter progression free survival than patients with LOH on 1p: 30 vs. 132 months, p < 0.0001. MRI indicated that tumors without LOH on 1p were often temporal (p < 0.02), and showed signal inhomogeneity on T1 and T2 images (p < 0.02) and contrast enhancement (p < 0.04). Thus, LOH on 1p identifies two subgroups of OAs. OAs without LOH on 1p behave like WHO grade II or III diffuse astrocytomas: they have shorter survival, MRI characteristics implying malignancy and genetic alterations associated with tumor progression. OAs with LOH on 1p, on the other hand, behave like WHO grade II or III oligodendrogliomas with 1p loss: they are associated with longer survival and do not have MRI or genetic alterations associated with malignancy. These findings suggest that the definition of OAs or mixed gliomas could be reshaped in agreement with the genetic information.     

Calcification on CT is a simple and valuable preoperative indicator of 1p/19q loss of heterozygosity in supratentorial brain tumors that are suspected grade II and III gliomas

Gliomas with 1p/19q loss of heterozygosity (LOH) are known to be associated with longer patient survival and higher sensitivity to treatment than tumors without 1p/19q LOH. This study was designed to clarify whether the preoperative finding of calcification on CT was correlated with 1p/19q LOH in patients with suspected WHO grade II and III gliomas. This study included 250 adult patients who underwent resection for primary supratentorial tumors at Tokyo Women’s Medical University Hospital. The tumors were suspected, based on MRI findings, to be WHO grade II or III gliomas. The presence of calcification on the patients’ CT images was qualitatively evaluated before treatment. After surgery, the resected tumors were examined to determine their 1p/19q status and mutations of IDH1 and p53. The presence of calcification was significantly correlated with 1p/19q LOH (P < 0.0001), with a positive predictive value of 91 %. The tumors of all the 78 patients with calcification were diagnosed as oligodendroglial tumors. Seventy of these patients showed classic oligodendroglial features, while 8 patients showed non-classic features. Calcification on CT is a simple and valuable preoperative indicator of 1p/19q LOH in supratentorial brain tumors that are suspected to be WHO grade II and III gliomas.     

Prognostic significance of multiple genetic lesions on chromosomes 19, 10, and 17 in oligodendrogliomas

Patients diagnosed with oligodendrogliomas/oligoastrocytomas and with somatic loss of genes on chromosome 19q13.2-q13.3 survived for >5-6 years, a survival period typical of the tumors of oligodendroglial origin. One patient with oligoastrocytoma, harboring allelic loss on chromosome 10p in the tumor DNA, had a recurrence five years later with progression to anaplastic astrocytoma. However, another patient with oligoastrocytoma, whose tumor suffered multiple genetic lesions on chromosomes 19q13.2-13.3, 10q22-24, and 17p13.1 (a point mutation in the p53 gene), had two subsequent recurrences as anaplastic astrocytomas and a survival period of 29 months. Our data suggest that in tumors of oligodendroglial origin the inactivation of a tumor suppressor gene on chromosome 10, especially in conjunction with other genetic aberrations, is indicative of aggressive clinical course.     

Loss of heterozygosity on chromosome 10q associated with malignancy and prognosis in astrocytic tumors, and discovery of novel loss regions

Certain tumor suppressor genes (TSG) residing on human chromosome 10q are implicated in astrocytic tumors. We thoroughly examined loss of heterozygosity (LOH) on chromosome 10q in astrocytic tumors to determine the extent of deletion and their relation to prognostic variables of patients. We analyzed 63 astrocytic tumors, including 9 diffuse astrocytomas, 36 anaplastic astrocytomas, and 18 glioblastomas. DNAs from tumors and leukocytes were analyzed for LOH at 18 microsatellite loci by polymerase chain reaction using fluorescence-labeled primers. Then correlation between LOH and clinicopathological variables was examined statistically. Twenty-four (66.7%) anaplastic astrocytomas and 15 (83.3%) glioblastomas had at least one LOH on chromosome 10q. However, diffuse astrocytomas exhibited no LOH. Nineteen tumors (10 anaplastic astrocytomas and 9 glioblastomas) were believed to have a total loss of one chromosome 10. Analyses on 20 tumors with interstitial LOH revealed that most of the high LOH regions matched the location of known TSGs, while some novel LOH regions were found preferentially in anaplastic astrocytoma. The median survivals of the total, partial, and no loss groups were 10.1, 14.8, and 46.8 months, respectively, indicating a significant difference in the survivals of these groups (P=0.0289). Thus, analyzing chromosome 10q loss is helpful for diagnosing malignancy in astrocytic tumors and for predicting patients' survival. Our data also suggested that there are novel TSGs for anaplastic astrocytoma at 10q24 and 10q26.     

A region of common deletion in 22q13.3 in human glioma associated with astrocytoma progression

Loss of heterozygosity for chromosome 22 (LOH 22) occurs in gliomas of all malignancy grades. Neurofibromatosis type 2 (NF2) patients are at increased risk of developing a glioma. However, the NF2 gene in 22q12.2 is not involved in glioma tumorigenesis. To detect additional regions on chromosome 22 that may harbor tumor suppressor genes important in glioma tumorigenesis, we determined LOH 22 profiles for 159 gliomas using 32 markers. LOH 22 was found in 46 tumors (29%). Thirteen tumors displayed partial LOH 22, from which we deduced a region of common deletion between markers D22S928 and D22S1169 in 22q13.3. LOH of at least this region was detected in 13% of the astrocytomas (As), in 20% of the anaplastic astrocytomas (AAs) and in 35% of the glioblastomas multiforme (GBMs). The significant increased frequency of LOH 22q13.3 in the highest malignancy grade (GBM vs. A and AA, p = 0.02) indicates that loss of this region is associated with astrocytoma progression.     

Genomic alterations in low-grade,anaplastic astrocytomas and glioblastomas

To extend our understanding of potential stepwise genetic alterations that may underlie tumor progression from low-grade astrocytomas to glioblastomas, histopathologic and comparative genomic hybridization analyses were performed on tumor specimens from 68 primary lesions, including 40 glioblastomas, 10 anaplastic and 18 low-grade astrocytomas. The number of aberrations per case increased towards the higher grade tumors (grade II: 1.66±1.49; grade III: 2.80±1.68; grade IV: 3.02±1.07; F=6.955, p=0.002). A gain of 7/7q was common and the most frequently seen aberration in low-grade astrocytomas, whereas loss of 10q was the most frequently seen anomaly in anaplastic astrocytomas and glioblastomas. Chromosome 7p amplification was only detected in glioblastomas. Chromosome 10/10q deletion and combination of lp, 19q and 17p deletions were specific to high-grade astrocytic tumors. Sequences of chromosome 7 and 10 seem to have pivotal roles in the biology of human gliomas. The genomic copy deletions of chromosomes lp and 19q might provide an alternative mechanism in the genesis of astrocytomas.     

Mutation in the PTEN/MMAC1 gene in archival low grade and high grade gliomas

The PTEN gene, located on 10q23.3, has recently been described as a candidate tumour suppressor gene that may be important in the development of advanced cancers, including gliomas. We have investigated mutation in the PTEN gene by direct sequence analysis of PCR products amplified from samples microdissected from 19 low grade (WHO Grade I and II) and 27 high grade (WHO grade III and IV) archival, formalin-fixed, paraffin-embedded gliomas. Eleven genetic variants in ten tumours have been identified. Eight of these are DNA sequence changes that could affect the encoded protein and were present in 0/2 pilocytic astrocytomas, 0/2 oligoastrocytomas, 0/1 oligodendroglioma, 0/14 astrocytomas, 3/13 (23%) anaplastic astrocytomas and 5/14 (36%) glioblastomas. PTEN mutations were found exclusively in high grade gliomas; this finding was statistically significant. Only two of the PTEN genetic variants have been reported in other studies; two of the genetic changes are in codons in which mutations have not been found previously. The results of this study indicate that mutation in the PTEN gene is present only in histologically more aggressive gliomas, may be associated with the transition from low histological grade to anaplasia, but is absent from the majority of high grade gliomas.     

Loss of 14q Chromosome in Oligodendroglial and Astrocytic Tumors

Loss of chromosome 14q has been investigated in 142 gliomas. Loss of heterozygosity (LOH) at one or more microsatellite has been found in 8/30 grade II (27%) and 2/21 grade III (10%) oligodendrogliomas, 3/9 grade II (33%) and 5/15 grade III (33%) oligoastrocytomas, 0/9 grade II (0%) and 1/7 grade III (14%) astrocytomas, 11/51 glioblastomas (22%). Two minimal regions were identified on 14q21.2-14q24.3 (between D14S288 and D14S74) and 14q31.3-14q32.1 (between D14S74 and D14S65). Loss of 14q was not correlated to survival, histological grading and subtype or other genetic alterations, except for 1p deletions. Taken together, these data suggest that LOH14q is an early alteration involving 20% of glioma.     

Genetic heterogeneity and progression in different areas within high-grade diffuse astrocytoma

The primary objective of this study was to determine loss of heterozygosity (LOH) in various portions of 9 high-grade diffuse astrocytomas, 8 glioblastomas and 1 anaplastic astrocytoma. LOH was observed on chromosomes 9, 10, 17 and 19 in 8, 3, 4 and 2 cases, respectively. Genetic heterogeneity and a multistep process were identified in 4 glioblastomas explaining the diverse morphological characteristics, a common feature of diffuse astrocytomas. In 2, 2, 3 and 1 cases, the allele losses were found within part of grade IV astrocytomas but not grades II or II/III, on chromosomes 9, 10, 17 and 19, respectively. In one of these, while genetic heterogeneity was observed on chromosome 17 between the area of grade II and grade IV, 9pLOH was found within both areas and occurred on the same allele. The other 5 cases did not demonstrate genetic heterogeneity and the LOH was on the same allele, irrespective of grade, suggesting clonal origin. In conclusion, at the molecular level, the diverse morphological features of astrocytoma develops by a multistep mechanism of genetic alterations from one cell via low-grade and more malignant tumors towards glioblastoma.     

Multiple deleted regions on the long arm of chromosome 6 in astrocytic tumours

Chromosome 6 deletions are common in human neoplasms including gliomas. In order to study the frequency and identify commonly deleted regions of chromosome 6 in astrocytomas, 159 tumours (106 glioblastomas, 39 anaplastic astrocytomas and 14 astrocytomas malignancy grade II) were analysed using 31 microsatellite markers that span the chromosome. Ninety-five per cent of cases with allelic losses had losses affecting 6q. Allelic losses were infrequent in astrocytomas malignancy grade II (14%) but more usual in anaplastic astrocytomas (38%) and glioblastomas (37%). Evidence for clonal heterogeneity in the astrocytomas and anaplastic astrocytomas was frequently observed (i.e. co-existence of subpopulations with and without chromosome 6 deletions). Clonal heterogeneity was less common in glioblastomas. Five commonly deleted regions were identified on 6q. These observations suggest that a number of tumour suppressor genes are located on 6q and that these genes may be involved in the progression of astrocytic tumours.     

胶质瘤染色体1p和19q杂合性缺失与O6-甲基鸟嘌呤DNA甲基转移酶p53和Ki-67蛋白表达的关系

目的 探讨胶质瘤染色体1p和19q杂合性缺失(LOH)与O6-甲基鸟嘌呤DNA甲基转移酶(MGMT)、p53和Ki-67蛋白表达的关系.方法 采集146例胶质瘤(45例少突胶质细胞瘤、42例少突星形细胞瘤和59例星形细胞瘤)的肿瘤组织和血液标本,采用聚合酶链反应结合变性高效液相色谱技术检测染色体1p和19q LOH,免疫组化法检测肿瘤组织中MGMT、p53和Ki-67蛋白的表达,并进一步分析其与胶质瘤临床病理特征的关系.结果 少突胶质细胞肿瘤和星形细胞瘤中,1p LOH的发生率分别为59.8％和33.9％,差异有统计学意义(P=0.002);1p和19q LOH的发生率分别为42.5％和16.9％,差异有统计学意义(P=0.001).MGMT低表达和Ki-67高表达多发生于少突胶质细胞肿瘤中,发生率分别为65.5％和54.0％,而p53高表达多发生于星形细胞瘤和少突星形细胞瘤中,发生率为75.2％.在87例少突胶质细胞肿瘤中,1p LOH和MGMT蛋白低表达多发生于Ⅱ级少突胶质细胞肿瘤中,发生率分别为72.5％和87.5％,而p53和Ki-67蛋白高表达多发生于Ⅲ级少突胶质细胞肿瘤中,发生率分别为83.0％和76.6％.1p和19q LOH在非颞叶和颞叶肿瘤的发生率分别为55.6％和21.2％(P=0.002).1p LOH与19q LOH、MGMT蛋白表达与p53蛋白表达、MGMT蛋白表达与Ki-67蛋白表达、1p和19q LOH与p53蛋白表达、1p LOH与Ki-67蛋白表达均有关(均P＜0.05).结论 1p和19q LOH及MGMT、p53和Ki-67的蛋白表达与胶质瘤的临床病理学特征有关,检测其LOH状态和表达水平对胶质瘤的诊断和治疗具有指导作用.     

Molecular markers of prognosis in astrocytic tumors

BACKGROUND: Astrocytoma is a primary brain tumor that affects 20,000 Americans each year. To date, only age and histologic grade stand out as independent predictors of survival. There is now increased interest in the use of molecular markers as objective standards against which to establish diagnosis and grade. METHODS: The study evaluated human glioma tumor suppressor genes and associated loci in fresh snap-frozen gliomas from 63 males and 37 females, with a median age of 42 years, including 19 low-grade astrocytomas. The tumor samples were selected so that about equal numbers of glioblastomas from younger and older patients were represented in the series. Methods for suppressor gene and genetic loci evaluation included loss of heterozygosity (LOH) analysis, multiplex polymerase chain reaction analysis, and gene sequencing. RESULTS: Low-grade astrocytomas had the least number of molecular abnormalities. LOH on 9p and/or CDKN2A deletion occurred more often in glioblastomas (P < 0.001), LOH on 17p/TP53 mutations occurred more frequently in anaplastic astrocytomas (AAs; P = 0.112), and LOH on 10q/PTEN mutation frequency was similar in glioblastomas and AAs (P < 0.001). Poorer survival was associated significantly with the occurrence of either deletion of p16 (P = 0.031), LOH on 9p (P = 0.016), or LOH on 10q (P = 0.0007). The absence of LOH on 17p and the presence of PTEN mutation were associated marginally with survival. Even though TP53 mutations were more frequent among younger patients with glioblastoma, they had no statistically significant effect on survival after adjustment for age (P = 0.62). In all multivariate models, age and grade were the only significant predictors of survival or were nearly significant predictors of survival. CONCLUSIONS: The results suggest that LOH on 9p and p16 deletions may prove to be objective standards for the diagnosis of patients with high-grade gliomas, although the absence of these abnormalities is nonprognostic.     

Genomic copy number changes of DNA repair genes ERCC1 and ERCC2 in human gliomas

Abnormalities of the genomic region of chromosome 19q13.2–13.4 are a common occurrence in brain malignancies and contain a possible tumor suppressor gene involved in gliomas. Since abnormalities of DNA repair are associated with malignancy, we assessed DNA status of the nucleotide excision repair genes located in this area, viz. ERCC1 and ERCC2.Radiodensitometry was used to assess gene copy number in samples obtained from brain tumor specimens from 24 patients. Nine tumors were of lower grade histology (3 pilocytic astrocytomas, 2 gangliogliomas, 4 astrocytomas); 15 tumors were pathologiclly higher grade (4 anaplastic astrocytomas, 11 glioblastomas). Tumor samples were obtained prior to radiation or chemotherapy. Abnormalities of gene copy number of ERCC1 and ERCC2 were observed in 11/24 specimens (46%). Whereas increased and decreased copy numbers were observed for ERCC1, only decreases in copy number of ERCC2 were seen. Three tumors (all lower grade) showed concurrent allelic loss of ERCC1 and ERCC2. Abnormalities of copy number for these genes were not associated with response to subsequent therapy nor survival. However, allelic loss of ERCC2 was associated with younger age at diagnosis when compared to those specimens which did not show loss. There were no significant differences between lower grade and higher grade tumors with respect to these investigations.Abnormalities in copy number of ERCC1 and ERCC2 are common in glial tumors. Further study of this genomic region is necessary to define the importance of these observations in tumor pathophysiology and treatment.     

Absence of mutation of the p73 gene in astrocytic neoplasms

In subgroups of astrocytic neoplasms, including glioblastoma (GBM), mutations of the p53 tumour suppressor gene lead to loss of growth-suppressive properties. A p53-related gene termed p73 has recently been identified; its gene product shows structural and functional similarities to p53. After being mapped to chromosome region 1p36, p73 was proposed to act as a tumour suppressor gene, as this region is frequently deleted in a variety of human cancers, including astrocytic tumours. To determine whether p73 is involved in astrocytoma/GBM development, we analysed 10 pilocytic astrocytomas, 15 WHO grade II astrocytomas, 15 WHO grade III anaplastic astrocytomas, and 20 GBM for p73 gene alterations. In parallel, we used six polymorphic markers to determine the allelic status of region 1p36 in this tumour series. Although loss of heterozygosity was evidenced in 12 of 60 cases (20% of samples), PCR-SSCP and direct sequencing failed to detect any gene mutation in the entire coding region and intronic sequences of p73. Eight tumours displayed five distinct polymorphic nucleotide changes, also present in the corresponding normal DNA. These variations consisted of T-->C variation, with no change in Thr173; C-->T transition, with no change in His197; exon 9 simultaneous double change C-->T and T-->C , with no variations in Ala336 and His349, respectively, and C-->T change at exon 9/-24 position of intron 8. These results suggest that, in astrocytic gliomas, p73 may not play a major role as a tumour suppressor, but the relatively high incidence of LOH confirms the presence at 1p36 of an as yet unidentified gene of this category, with a key function in astrocytoma/GBM progression.