Loss of Heterozygosity on Chromosome 19 in Secondary Glioblastomas (original) (raw)

Abstract

Glioblastomas develop rapidly de novo (primary glioblastomas) or slowly through progression from low-grade or anaplastic astrocytoma (secondary glioblastomas). Recent studies have shown that these glioblastoma subtypes develop through different genetic pathways. Primary glioblastomas are characterized by EGFR amplification/overexpression, PTEN mutation, homozygous p16 deletion, and loss of heterozygosity (LOH) on entire chromosome 10, whereas secondary glioblastomas frequently contain p53 mutations and show LOH on chromosome 10q. In this study, we analyzed LOH on chromosomes 19q, 1p, and 13q, using polymorphic microsatellite markers in 17 primary glioblastomas and in 13 secondary glioblastomas that progressed from low-grade astrocytomas. LOH on chromosome 19q was frequently found in secondary glioblastomas (7 of 13, 54%) but rarely detected in primary glioblastomas (1 of 17, 6%, p = 0.0094). The common deletion was 19q13.3 (between D19S219 and D19S902). These results suggest that tumor suppressor gene(s) located on chromosome 19q are frequently involved in the progression from low-grade astrocytoma to secondary glioblastoma, but do not play a major role in the evolution of primary glioblastomas. LOH on chromosome 1p was detected in 12% of primary and 15% of secondary glioblastomas. LOH on 13q was detected in 12% of primary and in 38% of secondary glioblastomas and typically included the RB locus. Except for 1 case, LOH 13q and 19q were mutually exclusive.

Introduction

Glioblastoma multiforme (WHO grade IV) is the most frequent and malignant neoplasm of the human nervous system. The majority of glioblastomas develop rapidly, with a short clinical history and no clinical or histologic evidence of a less malignant precursor lesion (primary or de novo glioblastoma). Other glioblastomas (secondary glioblastomas) develop slowly through progression from low-grade (WHO grade II) or anaplastic astrocytomas (WHO grade III). These glioblastoma subtypes develop in different age groups of patients and carry different genetic alterations (1–6). Primary glioblastomas occur in older patients and are characterized by frequent EGFR amplification/overexpression, PTEN mutation, homozygous p16 deletion, and LOH on entire chromosome 10 (1–5, 7, 8), while secondary glioblastomas develop in younger patients and show frequent p53 mutations and LOH on chromosome 10q (1, 2, 7–9).

The objective of the present study was to analyze in primary and secondary glioblastomas LOH on chromosomes 19q and 13q, which has been frequently observed in non-selected series of glioblastomas (10–21). LOH on chromosome 1p was also analyzed since simultaneous LOH on chromosomes 19q and 1p is a genetic hallmark of oligodendrogliomas (10, 15–20, 22, 23) and oligoastrocytomas (10, 15–19, 22). This study provides the evidence that tumor suppressor gene(s) located on chromosome 19q are frequently involved in the progression from low-grade astrocytoma to secondary glioblastoma.

Materials and Methods

Tumor Samples and DNA Extraction

Specimens were obtained from patients treated in the Department of Neurosurgery, University Hospital, Zurich, Switzerland between 1977 and 1993. Seventeen patients with primary glioblastoma had a preoperative clinical history of less than 3 months (mean, 1.4 months) and histologic diagnosis of a glioblastoma at the first biopsy, without any evidence of a less malignant precursor lesion. Thirteen patients with secondary glioblastoma had at least 2 biopsies, with clinical and histologic evidence of progression from low-grade astrocytoma.

For all primary glioblastomas and 1 secondary glioblastoma (case 295), DNA was extracted from frozen tissues by TRIzol® Reagent (GIBCO BRL, Cergy Parroise, France). Peripheral blood DNA was used as the normal control for these cases. Blood DNA was isolated using QIAamp DNA Blood Kit (QIAGEN, Courtaboeuf, France). For secondary glioblastomas (except for case 295), DNA was extracted from paraffin sections as described previously (9, 24). For 9 secondary glioblastomas (cases 11, 25, 26, 35, 58, 59, 60, 64, and 70), LOH analysis was carried out in comparison with low-grade astrocytoma DNA from the same patients, instead of using normal DNA as a control. In 2 secondary glioblastomas (cases 57 and 68), DNA was also extracted from the peritumoral brain tissue in paraffin sections.

LOH Analyses on Chromosomes 19, 1p, and 13q

Thirty-nine microsatellite markers (D19S216, D19S221, D19S226, D19S433, D19S425, D19S178, APOC2, D19S219, D19S412, D19S902, D19S606, D19S596, D19S246, D19S601, D19S180, and D19S418 for chromosome 19, D1S548, D1S508, D1S2667, D1S228, D1S552, D1S2734, AMY2B for chromosome 1p, and D13S175, D13S260, D13S267, D13S139, D13S218, D13S118, D13S153, D13S233, D13S227, D13S284, D13S176, D13S242, D13S131, D13S170, D13S159, D13S285 for chromosome 13q) were purchased from Research Genetics (Huntsville, AL). PCR was carried out using a Genius DNA Thermal Cycler (Techne, Cambridge, UK) in a total volume of 10 μl consisting of 10 ng genomic DNA, 2 μl of 5× PCR buffer, 6 pmol of each primer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.225 U Taq polymerase (Sigma, St. Louis, MO), 0.5 μCi of [α-33P]-dCTP (ICN Biomedicals, specific activity 3,000 Ci/mmol). Initial denaturation for 2 min at 95°C was followed by 30–40 cycles with denaturation at 94°C for 45 sec, annealing at 57°C–61°C for 45 sec, and extension at 72°C for 1 min. A final extension step for 7 min at 72°C was added. PCR products (10 μl) were mixed with an equal amount of loading dye (95% formamide, 20 mM EDTA, 0.05% xylene cyanol, and 0.05% bromophenol blue), denatured at 95°C for 5 min, and separated on 7% denaturing polyacrylamide gels at 70W, for 3.5–6.0 h. Dried gels were exposed to radiographic film (Kodak, BioMax, NY). The signal intensity of each allele on the X-ray film was analyzed using densitometry (Bio-Rad model GS-670) or Phosphorimager (Molecular Dynamics, Sunnyvale, CA). LOH was assumed when the signal intensity of the allele in the glioblastoma sample was less than 50% of that in the reference DNA (normal blood, normal brain, or low-grade astrocytoma).

Statistical Analysis

The Fisher exact test was used to examine possible associations between LOH on 19, 1p, 13q, and other genetic alterations in primary and secondary glioblastomas.

Results

LOH on Chromosome 19

A total of 480 loci were tested and 362 (75%) were found to be informative. LOH on chromosome 19q was detected in 7 of 13 (54%) secondary glioblastomas, significantly higher frequency than those in primary glioblastomas (1 of 17, 6%; p = 0.0094; Figs. 1, 2). The smallest deletion was found on 19q13.3 (between D19S219 and D19S902) in 1 secondary glioblastoma (case 35). In case 26, 1 of 2 alleles showed significant decreased intensity (<50%) when compared with the remaining allele at markers D19S433 and D19S601, suggesting that LOH had already occurred in low-grade astrocytoma (see asterisk in Fig. 1).

Allelic patterns of chromosomes 19, 1p and 13q in 17 primary and 13 secondary glioblastomas. Case numbers are indicated at the top of each column. The smallest region deleted on chromosome 19q is indicated by the bracket on the right. *LOH had already occurred at the stage of low-grade astrocytoma.

Fig. 1.

Representative results of LOH 19q in a primary glioblastoma (case 233) and a secondary glioblastoma (case 70). Microsatellite markers are indicated on the left side of each panel. LOH is indicated by arrows. N, normal (blood) DNA; II, low-grade astrocytoma (WHO Grade II); IV, glioblastoma (WHO Grade IV).

Fig. 2.

LOH on Chromosome 1p

A total of 210 loci were tested and 159 (76%) were found to be informative. Overall, 4 of 30 (13%) glioblastomas exhibited LOH for at least 1 of the 7 1p loci (Figs. 1, 3). Among these, cases 302 and 64 revealed allelic loss at only 1 marker, D1S508 and D1S548, respectively. One primary glioblastoma (case 257) and 1 secondary glioblastoma (case 72), which contain small areas of oligodendroglial components, showed LOH on both chromosomes 1p and 19.

Fig. 3.

Upper panel: Representative results of LOH 1p in a primary glioblastoma (case 233) and a secondary glioblastoma (case 64). Microsatellite markers are indicated on the left side. LOH is indicated by arrows. Lower panel: Representative results of LOH 13q in a primary glioblastoma (case 287) and 2 secondary glioblastomas (cases 64 and 68). Microsatellite markers are shown at the top of each panel. LOH is indicated by arrows. In 1 tumor (case 68), LOH 13q already occurred at the stage of low-grade astrocytoma. N, normal (blood) DNA; II, low-grade astrocytoma (WHO Grade II); IV, glioblastoma (WHO Grade IV).

LOH on Chromosome 13q

A total of 480 loci were tested and 359 (75%) were found to be informative. LOH on chromosome 13q was detected with at least 1 marker in 2 (12%) of primary and 5 (38%) of secondary glioblastomas (p = 0.1897; Figs. 1, 3). In most of these cases (5 of 7, 71%), LOH included marker D13S153, which is close to the RB locus. In 2 cases (cases 57 and 68), LOH already occurred in low-grade astrocytoma (see asterisk in Fig. 1).

Correlation between LOH on Chromosomes 19q, 1p, and 13q, and Other Genetic Alterations

There was no significant correlation between LOH on chromosomes 19q and 1p (p = 0.2835). LOH 13q and 19q was mutually exclusive except for 1 case (case 70), which showed LOH on both chromosomes. We also correlated the data of LOH on chromosomes 1p, 13q, 19q in this study with the data of LOH on chromosome 10q, 10p, and mutations in the p53 and PTEN genes previously published (3, 5, 8), but did not find any significant correlation (data not shown).

Discussion

LOH on chromosome 19q is most frequently observed in oligodendrogliomas (∼70%) and oligoastrocytomas (∼50%) (10, 15–20, 22, 23). LOH on chromosome 19q has been reported in ∼15% (11%–25%) of diffuse low-grade astrocytomas (10, 11, 16–19, 21, 22), in ∼45% (32%–50%) of anaplastic astrocytomas (10, 11, 16–19, 21, 22), and ∼25% (13%–32%) of unselected glioblastomas (10, 15–21). The observation of lower frequencies of LOH 19q in glioblastomas than in anaplastic astrocytomas suggests that a significant fraction of unselected glioblastomas does not develop through LOH 19q. Smith et al (19) reported that 14 of 48 (29%) primary glioblastomas (tumors from an initial resection) and 4 of 10 (40%) recurrent glioblastomas (tumors from a non-initial surgical resection) showed LOH on chromosome 19q. The present study corroborates these observations and provides evidence that LOH on chromosome 19q is frequently involved in the progression from low-grade astrocytomas to secondary glioblastomas, but is not typically involved in the development of primary glioblastomas. The low frequency of LOH 19q in primary glioblastomas observed in this study (6%) may reflect the stringent selection criteria used, i.e. a clinical history of less than 3 months and glioblastoma diagnosis at first biopsy.

Consistent with the previous reports (17–19), the smallest deletion on chromosome 19q found in this study was 19q13.3 (between D19S219 and D19S902). One of the candidate tumor suppressor genes identified on this region is the BAX gene (at 19q13.3, approximately 300 kb centrometic to HRC) (25). However, SSCP analyses of all 6 exons and flanking intronic sequences of the BAX gene did not show mutations in 20 gliomas with allelic loss of the other copy of 19q analyzed (26). Another candidate gene on 19q13.3 (teromeric to D19S219 and centromeric to D19S112) was identified to code for a serine-threonine phosphatase that is almost identical to PPP5C (PP5) (27). However, no mutation was detected in this gene in malignant gliomas (27). Further deletion mapping revealed a minimal, 900 kb region between D19S412 and STD, but so far, the putative tumor suppressor gene has not been identified (18).

We have recently shown that LOH on chromosome 10q25-qter is associated with acquisition of glioblastoma phenotype, but LOH on chromosome 19 was found in only 1 of 5 cases with foci showing a sudden histological transition from low-grade or anaplastic astrocytoma to glioblastoma (9). Although there is no direct evidence, the higher frequency of LOH 19q in this study (54%) could suggest that in the pathway leading to secondary glioblastomas, LOH 19q occurs after LOH on chromosome 10.

LOH on chromosome 1p has been reported in <10% of low-grade astrocytomas, ∼20% of anaplastic astrocytomas (16, 19, 20, 22, 28, 29), and ∼10% of glioblastomas (16, 19, 20, 28, 29). Cancer-associated genes on this chromosome include p73 (30) at 1p36.33, E2F2 (31), TR2 (32), TNFR2 (33), DR3 (34), and DR 5 (35) at 1p36, and RAD54 (36) and p18 (INK4C) (37) at 1p32. The p73 gene encodes a protein that shares a substantial homology with the p53 tumor suppressor protein, but p73 mutations have not been found in neuroepithelial, colorectal, lung, or prostate tumors (38–42). Loss of the 1p36 locus is most frequently found in oligodendrogliomas (∼70%) and oligoastrocytomas (∼50%) (16, 19, 20, 22, 23). Oligodendrogliomas with LOH on 1p often show concomitant LOH on 19q (16, 23), suggesting a cooperation of these 2 genetic alterations. In the present study on glioblastomas, there was no significant association between LOH on chromosomes 1p and 19. It is of interest to note that 1 primary and 1 secondary glioblastoma containing LOH on both 19q and 1p contained small areas with histologic features of oligodendroglial differentiation.

LOH on chromosome 13q has been found in ∼20% of low-grade astrocytomas (12–14), ∼25% of anaplastic astrocytomas (12–14), and ∼35% of glioblastomas (12–14, 43). The retinoblastoma gene (RB) is located at 13q14.2 (44) and the breast cancer susceptibility locus 2 (BRCA2) on 13q12.1 (45, 46). Mutations in the RB gene have been found in only 10% of glioblastomas (12–14, 47), suggesting the presence of other tumor suppressor gene on this chromosome. Except for 1 glioblastoma (case 70), LOH on chromosome 13q and 19q were mutually exclusive, which could indicate that the respective gene products are functionally related.

In conclusion, we show that the putative tumor suppressor gene located on chromosome 19q13.3 is operative in the progression of low-grade astrocytomas to secondary glioblastoma.

Acknowledgments

This work was supported by a grant from the Foundation for promotion of Cancer Research, Japan.

References

Subsets of glioblastoma multiforme defined by molecular genetic analysis

Brain Pathol

1993

;

–

Pathways leading to glioblastoma multiforme: A molecular analysis of genetic alterations in 65 astrocytic tumors

J Neurosurg

1994

;

427

–

Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas

Brain Pathol

1996

;

217

–

Alterations of cell cycle regulatory genes in primary (de novo) and secondary glioblastomas

Acta Neuropathol

1997

;

303

–

PTEN (MMAC1) mutations are frequent in primary glioblastomas (de novo) but not in secondary glioblastomas

J Neuropathol Exp Neurol

1998

;

684

–

Primary and secondary glioblastomas: From concept to clinical diagnosis

Neuro-Oncology

1999

;

–

Incidence and timing of p53 mutations during astrocytoma progression in patients with multiple biopsies

Clin Cancer Res

1997

;

523

–

Loss of heterozygosity on chromosome 10 is more extensive in primary (de novo) than in secondary glioblastomas

Lab Invest

2000

;

–

Acquisition of the glioblastoma phenotype during astrocytoma progression is associated with LOH on chromosome 10q25-qter

Am J Pathol

1999

;

155

387

–

Deletion mapping of chromosome 19 in human gliomas

Int J Cancer

1994

;

676

–

Loci associated with malignant progression in astrocytomas: A candidate on chromosome 19q1

Cancer Res

1994

;

1397

–

1401

CDKN2/p16 or RB alterations occur in the majority of glioblastomas and are inversely correlated

Cancer Res

1996

;

150

–

The retinoblastoma gene is involved in malignant progression of astrocytomas

Ann Neurol

1994

;

714

–

Human glioblastomas with no alterations of the CDK2A (p16 INK4A, MTS1) and CDK4 genes have frequent mutations of the retinoblastoma gene

Oncogene

1996

;

1065

–

Region-specific loss of heterozygosity on chromosome 19 is related to the morphologic type of human glioma

Genes Chromosomes Cancer

1995

;

277

–

Shared allelic losses on chromosomes 1p and 19q suggest a common origin of oligodendroglioma and oligoastrocytoma

J Neuropathol Exp Neurol

1995

;

–

Chromosome 19q deletions in human gliomas overlap telomeric to D19S219 and may target a 425 kb region centromeric to D19S112

J Neuropathol Exp Neurol

1995

;

622

–

Refined deletion mapping of the chromosome 19q glioma tumor suppressor gene to the D19S412-STD interval

Oncogene

1996

;

2483

–

Localization of common deletion regions on 1p and 19q in human gliomas and their association with histological subtype

Oncogene

1999

;

4144

–

Dynamics of genetic alterations associated with glioma recurrence

Genes Chromosomes Cancer

1998

;

153

–

Molecular genetic analysis as a tool for evaluating stereotactic biopsies of glioma specimens

J Neuropathol Exp Neurol

1999

;

–

Molecular genetic evidence for subtypes of oligoastrocytomas

J Neuropathol Exp Neurol

1997

;

1098

–

1104

Specific genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendrogliomas

J Natl Cancer Inst

1998

;

1473

–

Primitive neuroectodermal tumors after prophylactic central nervous system irradiation in children

Association with an activated K-ras gene. Cancer

1992

;

2385

–

Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death

Cell

1993

;

609

–

The BAX gene maps to the glioma candidate region at 19q13.3, but is not altered in human gliomas

Cancer Genet Cytogenet

1996

;

136

–

Cloning of a highly conserved human protein Serine-Threonine phosphatase gene from the Glioma candidate region on chromosome 19q13.3

Genomics

1995

;

533

–

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

Int J Cancer

1994

;

172

–

Allelic status of chromosome 1 in neoplasms of the nervous system

Cancer Genet Cytogenet

1995

;

160

–

Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers

Cell

1997

;

809

–

The retinoblastoma protein binds to a family of E2F transcription factors

Mol Cell Biol

1993

;

7813

–

A newly identified member of the tumor necrosis factor receptor superfamily with a wide tissue distribution and involvement in lymphocyte activation

J Biol Chem

1997

;

272

14272

–

The gene for the type II (p75) tumor necrosis factor receptor (TNF-RII) is localized on band 1p36.2-p36.3

Hum Genet

1991

;

623

–

TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas(Apo-1/CD95)

Immunity

1997

;

–

KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene

Nat Genet

1997

;

141

–

Characterization of the human homologue of RAD54: A gene located on chromosome 1p32 at a region of high loss of heterozygosity in breast tumors

Cancer Res

1997

;

2378

–

A p18 mutant defective in CDK6 binding in human breast cancer cells

Cancer Res

1996

;

4586

–

Expression level, allelic origin, and mutation analysis of the p73 gene in neuroblastoma tumors and cell lines

Cell Growth Differ

1998

;

897

–

903

Genomic organization and mutation analysis of p73 in oligodendrogliomas with chromosome 1 p-arm deletions

Genomics

1998

;

359

–

Mutational analysis of the p73 gene localized at chromosome 1p36.3 in colorectal carcinomas

Int J Oncol

1998

;

319

–

Search for mutations and examination of allelic expression imbalance of the p73 gene at 1p36.33 in human lung cancers

Cancer Res

1998

;

1380

–

Mutation, allelotyping, and transcription analyses of the p73 gene in prostatic carcinoma

Cancer Res

1998

;

2076

–

Molecular genetic correlates of p16, cdk4, and pRb immunohistochemistry in glioblastomas

J Neuropathol Exp Neurol

1998

;

122

–

A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma

Nature

1986

;

323

643

–

Consistent loss of the wild type allele in breast cancers from a family linked to the BRCA2 gene on chromosome 13q12-13

Oncogene

1995

;

1673

–

Different tumor types from BRCA2 carriers show wild-type chromosome deletions on 13q12-q13

Cancer Res

1995

;

4830

–

Alterations of retinoblastoma, p53, p16(CDKN2), and p15 genes in human astrocytomas

Cancer

1996

;

287

–