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PPARγ, the gamma isoform of a family of peroxisome proliferator activated receptors, plays a key role in adipocyte differentiation. Recently, its broad expression in multiple tissues and several epithelial cancers has been shown. Further, somatic loss of function mutations in PPARγ have been found in primary colorectal carcinomas. We sought to determine if somatic high penetrance mutations in this gene might also play a role in glioblastoma multiforme (GBM). We also examined this gene to determine if common low penetrance polymorphic alleles might lend low level susceptibility to GBM in the general population. No somatic high penetrance mutations were detected in 96 sporadic GBMs. However, polymorphic alleles at codons 12 and 449 were significantly over-represented among the 27 unrelated American patients with sporadic GBM compared to 80 race matched controls. While nine (33%) were heterozygous for the P12A variant, c.34C/G (cytosine to guanine change at nucleotide 34), 12 (15%) controls were heterozygous for P12A (p<0.05). Similarly, 13 of 26 (50%) glioblastoma patients compared to 10 of 80 (12%) normal controls were found to have the heterozygous H449H polymorphism (p<0.001). The over-representation of H449H in glioblastoma patients was confirmed with a second validation set of American patients. When both American series were combined, polymorphic H449H was over-represented among cases versus controls (p<0.001) and there was a similar trend (p=0.07) for P12A. The precise mechanism for this association is unknown but these PPARγ polymorphisms may be acting in a low penetrance predisposing manner. However, these associations were not found in a German population, possibly arguing that if these variants are in linkage disequilibrium with a third locus, then this effect is relatively new, after the settlement of the American colonies.

The MEN1 gene on chromosome 11q13 is mutated in patients afflicted with multiple endocrine neoplasia syndrome type 1 (MEN1). These patients develop endocrine tumours of the pancreas, the parathyroid, and the anterior pituitary. In order to determine the role of MEN1 in sporadic pituitary adenomas, 61 pituitary adenomas were analysed from patients without evidence of a familial tumour syndrome. Single strand conformation polymorphism (SSCP) analysis was performed for the entire coding sequence of MEN1. Fragments with aberrant migration patterns were sequenced bidirectionally. Only a single somatic mutation was detected in this series. In addition, several previously reported and three novel polymorphisms were observed. Loss of heterozygosity analysis with 12 polymorphic markers, however, identified 13 pituitary adenomas with allelic deletions on chromosome 11. Allelic losses occurred significantly more often in pituitary adenomas with hormone secretion than in non-functioning adenomas. These data suggest that MEN1 mutations are rare events in sporadic pituitary adenomas. However, the discrepancy of only 1/61 adenomas with MEN1 mutation but 13/61 (22 per cent) with allelic loss on chromosome 11 may suggest the presence of a yet unknown tumour suppressor gene, relevant to the pathogenesis of sporadic pituitary adenomas. Copyright © 1999 John Wiley & Sons, Ltd.

Sporadic breast carcinoma is associated with multiple genetic alterations. The clinical relevance of these alterations, however, needs further clarification. In the present study we analyzed 266 spontaneously arising breast carcinomas for allelic losses in the BRCA1 and TP53 regions on chromosome 17, the BRCA2 region on chromosome 13, the ATM (mutated in ataxia-telangiectasia) region on chromosome 11 and on the chromosomal arms 7q and 16q. In addition the following clinical and pathological parameters were evaluated: age at diagnosis, tumor size, presence or absence of regional and distant metastases, hormone-receptor status, histopathological classification and tumor grading. The analysis of genetic and clinical observations revealed significant associations: absence of expression of the estrogen receptor was linked to a high rate of allelic losses of markers in the BRCA1, TP53 and BRCA2 regions. Expression of the progesterone receptor coincided with allelic loss on the long arm of chromosome 16. High-grade malignant lesions and ductal differentiation were frequently associated with allelic losses in the proximal portion of chromosome 17q. The accumulation of multiple allelic deletions was linked to high-grade malignant tumors, to tumor size, and to loss of expression of the estrogen receptor. Our data point to a relationship between clinically relevant prognostic factors and specific genomic deletions in the BRCA1, BRCA2 and TP53 region. Int. J. Cancer 74:322-325, 1997. © 1997 Wiley-Liss, Inc.

Inactivating mutations of the RET proto-oncogene and of one of its soluble ligand molecules, glial cell line derived neurotrophic factor (GDNF), have been found in a subset of patients with Hirschsprung disease (HSCR). However, the majority of HSCR mutations remain unidentified. As normal RET function requires a multicomponent ligand complex for activation, other members of the RET ligand complex are primary candidates for these mutations. We investigated the presence of mutations in another member of the RET signalling complex, GDNF family receptor alpha-1 (GFRα-1), in a panel of 269 independent cases of HSCR. We identified 10 polymorphisms at the GFRα-1 locus. Surprisingly, however, we did not identify any sequence variants in our HSCR population that were not also present in a normal control population. Our data suggest that mutations of the GFRα-1 gene are not a common aetiological event in HSCR.

Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we asked whether alterations in the p53 and RB pathways and the expression of six BCL-2 family proteins predicted acute cytotoxicity and clonogenic cell death induced by BCNU, vincristine, cytarabine, teniposide, doxorubicin, camptothecin or β-lapachone in 12 human malignant glioma cell lines. Neither wild-type p53 status, nor p53 protein accumulation, nor p21 or MDM-2 levels, nor differential expression of BCL-2 family proteins predicted drug sensitivity, except for an association of BAX with higher β-lapachone sensitivity in acute cytotoxicity assays. p16 protein expression was associated with high doubling time and chemoresistance. We conclude that some important molecular changes, which are involved in the development of gliomas and attributed a role in regulating vulnerability to apoptosis, may not determine the response to chemotherapy in these tumors. Int. J. Cancer (Pred. Oncol.) 79:640–644, 1998. © 1998 Wiley-Liss, Inc.

The gene mutated in ataxia telangiectasia (A-T) patients (ATM) is located on chromosome 11q22–23, a region frequently altered in mammary tumors. Patients homozygous for ATM mutations are prone to develop a variety of different neoplasms. Female heterozygotes have been reported to carry a 5- to 8-fold increased risk of breast cancer. However, germline mutations in the ATM gene are rare in women with sporadic breast carcinomas. Most of the alterations described in A-T patients result in a functionally inactive ATM protein. Moreover, it has been suggested that mutations of the ATM gene in A-T patients influence the amount of ATM mRNA and that this may affect the severity of the disease. In the present study, we have analyzed ATM transcripts in a series of 39 breast carcinomas, 14 benign breast lesions and 12 normal breast tissue samples. ATM mRNA levels were determined by semiquantitative competitive RT-PCR. Competitor RNA molecules for the ATM gene and the housekeeping gene β-2-microglobulin (B2M) were generated by PCR mutagenesis. Low concentrations of ATM transcripts were detected in breast carcinomas, intermediate levels in benign lesions and highest levels in normal breast tissue specimens (F-test, p = 0.0013). Our results indicate that reduced expression of the ATM gene may contribute to the development and/or malignant progression of breast carcinomas. Int. J. Cancer 78:306–309, 1998.© 1998 Wiley-Liss, Inc.

Recently, exclusive loss of paternal 19q alleles in six of six oligodendrogliomas has been reported, indicating that parental imprinting plays a role in these tumors. We examined a series of 10 oligodendrogliomas and 3 oligoastrocytomas with allelic losses on 1p and 19q for the parental origin of the lost alleles. Ten cases lost paternal 1p alleles and 3 cases lost maternal alleles. For 19q, six cases had loss of paternal alleles and seven cases of had loss of maternal alleles. These random distributions do not support the hypothesis that parental imprinting accounts for inactivation of the putative oligodendroglioma tumor suppressor genes. Ann Neurol 2003

Pleomorphic xanthoastrocytoma (PXA) is a rare, usually well-circumscribed and superficially located neoplasm that preferentially arises in the cerebral cortex of children and young adults. The molecular aberrations that are associated with these tumors have not been studied systematically so far. We here report on a molecular genetic analysis of 62 PXAs (46 PXAs of World Health Organization [WHO] grade II and 16 PXAs with anaplastic features) for alterations of 5 candidate genes known to be frequently aberrant in diffusely infiltrating astrocytic gliomas, i.e. TP53, CDKN2A (p16INK4a), CDK4, MDM2, and EGFR. Only 3 PXAs (5%) carried a TP53 mutation. None of the 62 PXAs had lost both copies of the CDKN2A gene. The CDK4, MDM2, or EGFR genes were not amplified in any of the tumors. Fourteen PXAs were additionally analyzed for loss of heterozygosity (LOH) at microsatellite markers located on the chromosomes/chromosomal arms 1, 8p, 9p, 10, 17, 19q, and 22q. Two PXAs (14%) had LOH at all informative markers on 9p, while 1 PXA demonstrated an interstitial area of allelic imbalance between D22S533 and D22S417 at 22q11.2–q13.3. Further analysis of 10 PXAs for inactivation of the CDKN2A, p14ARF, and CDKN2B (p15INK4b) genes on 9p21 did not reveal any homozygous deletion, mutation, promoter hypermethylation, or complete loss of mRNA expression. Taken together, our results indicate that the chromosomal and genetic aberrations in PXAs are different from those typically associated with the diffusely infiltrating astrocytic and oligodendroglial gliomas. These genetic differences likely contribute to the more favorable behavior of PXAs and may be helpful for the molecular differential diagnosis of cerebral gliomas.

Pilocytic astrocytomas classified as WHO grade I typically arise in childhood and upon complete surgical removal carry a favorable prognosis. Children with neurofibromatosis 1 (NF1) have a vastly increased risk for pilocytic astrocytomas, especially for those of the optic nerve. Using 4 intragenic NF1 microsatellite markers, we examined losses of NF1 alleles on the long arm of chromosome 17 in 12 NF1-associated and 25 sporadic pilocytic astrocytomas. The TP53 gene region on the short arm of chromosome 17 was also examined in these tumors using 3 markers. Loss of 1 NF1 allele was detected in 11 of 12 (92%) informative NF1-associated pilocytic astrocytomas. In contrast, only 1 of 24 informative (4%) sporadic pilocytic astrocytomas exhibited allelic loss in the NF1 region. Among the 11 NF1-associated tumors with NF1 loss, 5 had also lost alleles on 17p. The high rate of NF1 allele loss in NF1-associated pilocytic astrocytomas suggests a tumor initiating or promoting action of the NF1 gene in these patients. On the other hand, the much lower rate of NF1-allele loss in sporadic pilocytic astrocytomas argues for only minor importance of NF1 in that patient group. The present data support different mechanisms in the formation of NF1-associated and sporadic pilocytic astrocytomas.

Approximately 30% of human astrocytomas have been reported to display allelic loss of the long arm of chromosome 22, suggesting the presence of a chromosome 22q astrocytoma suppressor gene. To define the most likely location for this putative tumor suppressor, we performed deletion mapping on 141 tumors using 16 chromosome 22q microsatellite markers. Allelic loss of 22q was observed in 2/12 (17%) of astrocytomas, 9/29 (31%) of anaplastic astrocytomas, and 38/100 (38%) of glioblastomas, consistent with a role for chromosome 22q loss in astrocytoma progression as well as formation. Twenty-two tumors exhibited allelic loss at every informative locus, consistent with loss of the entire arm of 22q. Twenty-seven tumors showed partial deletions, with one common region of deletion at 22ql2.3–ql3.1 between markers D22S280 and D22S282, and a second candidate region at 22ql3.2 near the marker D22SU70. For the proximal candidate region, the incidence of allelic loss was similar between grades; for the distal locus, the incidence increased with grade, raising the possibility that the distal locus is involved in a later stage of astrocytoma tumorigenesis.

In contrast to astrocytic tumors, approximately two thirds of anaplastic oligodendrogliomas are reported to be chemosensitive. Relatively little is known about the genetic aberrations in oligodendroglial tumors (OTs). In order to elucidate oligodendroglial oncogenesis and to And specific genetic aberrations that may have prognostic and therapeutic implications, we performed comparative genomic hybridization (CGH) to detect chromosomal copy number changes in 17 low-grade OTs (LG-OTs) and 12 high-grade OTs (HG-OTs) lacking a prominent astrocytic component. Loss of chromosome lp (79%) and 19q (76%) were most frequently detected by CGH, all LG-OTs and 50% of the HG-OTs contained −lp (including lp36–32), −19q (including 19ql3.3), or both, and the rest of the HG-OTs showed +7, −10, or both. Since losses of lp36-32 and 19ql3.3 were mutually exclusive with +7 or −10, the HG-OTs could be divided in −lp/−19q and +7/−10 tumors. While the − 1 p/− 19q tumors can be considered as pure anaplastic oligodendrogliomas, the +7/−10 tumors may rather be glioblastomas with prominent oligodendroglial differentiation. We conclude that CGH is a powerful tool to assist in the identification of 2 major subgroups of HG-OTs with prognostic and possibly therapeutic relevance.

Chromosome 19q harbors a tumor suppressor gene that is involved in astrocytoma, oligodendroglioma and mixed glioma tumorigenesis. We had previously mapped this gene to an approximately 5 megabase region of chromosome 19ql3.2–13.3 between APOC2 and HRC. To narrow the location of this tumor suppressor further, we studied 138 gliomas for loss of allelic heterozygosity at six microsatellite polymorphisms between APOC2 and HRC, including a newly described polymorphism in the ERCC2 gene. Allelic loss occurred in 48 gliomas (35%), including 25 of 41 oligodendroglial tumors (61%). Four cases had proximal breakpoints within the APOC2-HRC region, two telomeric to ERCC2 and two telomeric to D19S219. In addition, one of the latter tumors had an interstitial deletion between D19S219 and D19S112, a distance of only 425 kilobases surrounding the DM (myotonic dystrophy) gene. These findings suggest that the glioma tumor suppressor on chromosome 19q maps to 19ql3.3, telomeric to D19S219 and perhaps centromeric to D19S112. The data exclude a number of candidate genes from 19ql3.2–13.3, including a putative phosphatase gene and the DNA repair/metabolism genes ERCC1, ERCC2 and probably LIGI.

The p53 gene is a tumor suppressor gene involved in many common malignancies, including astrocytomas. Genetic analysis of the p53 gene and immunohistochemistry of the p53 protein have each been used to screen astrocytomas. To compare these methods, we performed immunohistochemistry with the monoclonal antibody PAb 1801 and single-strand conformational polymorphism (SSCP) with sequence analysis on 34 astrocytic tumors (WHO grades II, HI and IV). Seven cases had detectable p53 protein and gene mutations, while twelve cases had neither detectable protein nor gene mutations. Four tumors had frameshift mutations in the p53 gene that were not revealed by immunohistochemistry. One tumor had a genetic polymorphism and no detectable p53 protein. Ten tumors had p53 protein accumulation but no mutations by SSCP; these cases may represent p53 mutations outside of the conserved exons or elevated levels of wild-type p53 protein. Thus, some p53 mutations are missed with PAb 1801 immunohistochemistry alone. p53 immunohistochemistry, however, may reveal p53 accumulation independent of mutations in the conserved portions of the gene. Finally, we suggest that glioblastomas with p53 mutations in the conserved region of the gene may be a subset that are more common in women and in younger patients.

The tuberous sclerosis 2 (TSC2) gene is thought to function as a growth suppressor in sporadic and TSC-associated hamartomas and tumors. Clusters of dysplastic glial cells are a common feature of conical tubers and subependymal nodules in tuberous sclerosis patients. In an effort to identify TSC2 gene alterations in sporadic gliomas, we detected a novel polymorphism adjacent to the 3'splice site of intron 4. We evaluated the distribution of this variant allele in a series of 244 patients with glial tumors, including 55 gangliogliomas. 31 pilocytic astrocytomas (WHO grade I). 50 astrocytomas (WHO grades II and III), and 108 glioblastomas (WHO grade IV). The allelic distribution in the general population was estimated by examining 381 healthy blood donors. This rare allele appeared in the control population and in the patients with astrocytic gliomas with a virtually identical frequency (8,14%. and 8.20%. respectively). The frequency of the rare allele in gangliogliomas. however. was significantly higher (15.5%; p = 0.024). The fact that both gangliogliomas and cortical tubers in tuberous sclerosis contain neuronal and astrocytic elements and may resemble each other histologically suggests that the TSC2 gene may be involved in the development of these tumors. The rare allele of the TSC2 gene emerges as a candidate for a predisposing factor for the formation of sporadic gangliogliomas.

Over the last years, distinct genetic lesions have been associated with individual tumor entities. Stereotactic biopsy has become an essential diagnostic tool in surgical neuro-oncology. In order to evaluate the potential of molecular analyses in stereotactic biopsies, we examined a series of 156 human brain tumors from patients undergoing stereotactic biopsy for molecular alterations typically seen in astrocytic gliomas and compared those results with a control group of 268 astrocytic tumors obtained at open surgery. Stereotactic biopsies of astrocytomas with borderline histopathological features between the WHO grades II and III showed a higher rate of allelic losses on chromosome 10 than those of the WHO grade II from open surgery (p = 0.011). Stereotactic biopsies of astrocytomas with borderline histopathological features between the WHO grades III and IV showed a higher rate of allelic losses on chromosome 10 than those of the WHO grade III from open surgery (p = 0.013). This indicates that stereotactic biopsies with features intermediate between grades are likely to correspond to the higher malignancy grade. Our data demonstrate that molecular genetic approaches can be successfully applied to stereotactic glioma biopsies. The difference in the distribution of malignancy associated genetic alterations between a stereotactic and openly resected group of gliomas indicates that histopathology may underestimate the malignant potential in some stereotactic specimens. We propose to further evaluate the molecular analysis of stereotactic glioma biopsies as a useful adjunct to standard histopathological procedures.