Analysis of PTEN and the 10q23 region in primary prostate carcinomas

Deletions involving chromosome 10q23 occur frequently in prostatic carcinomas. Recently, a novel tumour suppressor gene, PTEN, mapping to this interval, has been identified. Mutation or deletion of PTEN has been observed in a proportion of prostate cancer cell lines; however, primary prostate carcinomas have not been studied. We have investigated the involvement of PTEN in primary prostatic adenocarcinomas using a panel of 51 matched normal and prostate tumour DNAs. We first determined the proportion of tumours with allele loss at loci in 10q23 which span the region containing the PTEN gene. Our results show that LOH involving 10q23 is common in primary prostate carcinomas. Twenty-five of 51 (49%) tumours showed loss of heterozygosity (LOH) over the region spanning the PTEN locus. We next directly analysed the PTEN gene for mutations of the coding region using single strand conformation polymorphism (SSCP) and sequence analyses. Of those tumours with LOH, only a single tumour was found to carry a missense mutation in PTEN. No mutations in PTEN were identified in tumours without LOH. Our results suggest either that mutation of PTEN is a late event in prostate tumorigenesis, or that another tumour suppressor gene important in prostate cancer may lie close to PTEN in 10q23.     

Frequent inactivation of PTEN in prostate cancer cell lines and xenografts

Loss of chromosome 10q is a frequently observed genetic defect in prostate cancer. Recently, the PTEN/MMAC1 tumor suppressor gene was identified and mapped to chromosome 10q23.3. We studied PTEN structure and expression in 4 in vitro cell lines and 11 in vivo xenografts derived from six primary and nine metastatic human prostate cancers. DNA samples were allelotyped for eight polymorphic markers within and surrounding the PTEN gene. Additionally, the nine PTEN exons were tested for deletions. In five samples (PC3, PC133, PCEW, PC295, and PC324), homozygous deletions of the PTEN gene or parts of the gene were detected. PC295 contained a small homozygous deletion encompassing PTEN exon 5. In two DNAs (PC82 and PC346), nonsense mutations were found, and in two (LNCaP and PC374), frame-shift mutations were found. Missense mutations were not detected. PTEN mRNA expression was clearly observed in all cell lines and xenografts without large homozygous deletions, showing that PTEN down-regulation is not an important mechanism of PTEN inactivation. The high frequency (60%) of PTEN mutations and deletions indicates a significant role of this tumor suppressor gene in the pathogenesis of prostate cancer.     

Identification of PTEN/MMAC1 alterations in uncultured melanomas and melanoma cell lines

A novel tumor suppressor gene, PTEN/MMAC1, has been recently shown to be mutated in gliomas, breast, prostate, kidney cancers and melanomas. Loss-of-heterozygosity studies in melanoma have suggested the presence of at least one chromosome 10q locus lost early in tumor progression. In this study, we screened 45 melanoma cell lines and 17 paired uncultured metastatic melanoma and peripheral blood specimens for PTEN/ MMAC1 alterations using PCR-SSCP and direct sequencing. We found nine melanoma cell lines with homozygous deletions (five with intragenic loss) and four cell lines with mutations (one nonsense and one frameshift; two intronic); from among our uncultured melanoma specimens, we found one tumor with a somatic 17 bp duplication in exon 7 leading to a premature stop codon and one tumor with a possible homozygous deletion. Furthermore, we have identified a novel intragenic polymorphism within intron 4 of PTEN/MMAC1. Taken together, these data suggest that PTEN/MMAC1 may be a chromosome 10q tumor suppressor important in melanoma tumor formation or progression.     

Point mutation and homozygous deletion of PTEN/MMAC1 in primary bladder cancers

A new tumor suppressor gene PTEN/MMAC1 was recently isolated at chromosome 10q23 and found to be inactivated by point mutation or homozygous deletion in glioma, prostate and breast cancer. PTEN/MMAC1 was also identified as the gene predisposing to Cowden disease, an autosomal dominant cancer predisposition syndrome associated with an increased risk of breast, skin and thyroid tumors and occasional cases of other cancers including bladder and renal cell carcinoma. We screened 345 urinary tract cancers by microsatellite analysis and found chromosome 10q to be deleted in 65 of 285 (23%) bladder and 15 of 60 (25%) renal cell cancers. We then screened the entire PTEN/MMAC1 coding region for mutation in 25 bladder and 15 renal cell primary tumors with deletion of chromosome 10q. Two somatic point mutations, a frameshift and a splicing variant, were found in the panel of bladder tumors while no mutation was observed in the renal cell carcinomas. To screen for homozygous deletion, we isolated two polymorphic microsatellite repeats from genomic BAC clones containing the PTEN/MMAC1 gene. Using these new informative markers, we identified apparent retention at the gene locus indicative of homozygous deletion of PTEN/MMAC1 in four of 65 bladder and 0 of 15 renal cell tumors with LOH through chromosome 10q. Identification of the second inactivation event in six bladder tumors with LOH of 10q implies that the PTEN/MMAC1 gene is occasionally involved in bladder tumorigenesis. However, the low frequency of biallelic inactivation suggests that either PTEN/MMAC1 is inactivated by other mechanisms or it is not the only target of chromosome 10q deletion in primary bladder and renal cell cancer.     

Somatic mutations of the PTEN/MMAC1 gene in fifteen Japanese endometrial cancers: evidence for inactivation of both alleles

Loss of heterozygosity (LOH) of chromosome 10q is observed in approximately 40% of endometrial cancers. Mutations in PTEN/MMAC1, a gene recently isolated from the 10q23 region, are responsible for two dominantly inherited neoplastic syndromes, Cowden disease and Bannayan-Zonana syndrome. Somatic mutations of this gene have also been detected in sporadic cancers of the brain, prostate and breast. To investigate the potential role of this putative tumor suppressor gene in endometrial carcinogenesis as well, we examined 46 primary endometrial cancers for LOH at the 10q23 region, and for mutations in the entire coding region and exon-intron boundaries of the PTEN/MMAC1 gene. LOH was identified in half of the 38 informative cases, and subtle somatic mutations were detected in 15 tumors (33%). Our results suggest that of the genes studied so far in endometrial carcinomas, PTEN/MMAC1 is the most commonly mutated one, and that inactivation of both copies by allelic loss and/or mutation, a pattern that defines genes as "tumor suppressors," contributes to tumorigenesis in endometrial cancers.     

Absence of PTEN/MMAC1 germ-line mutations in sporadic Bannayan-Riley-Ruvalcaba syndrome

Bannayan-Riley-Ruvalcaba syndrome (BRRS) is a rare hamartomatous polyposis condition with features of macrocephaly, intestinal juvenile polyposis, developmental delay, lipomas, and pigmentation spots of the male genitalia. An autosomal dominant pattern of inheritance exists in some families, but others appear as sporadic cases. Germ-line mutations in PTEN, a tyrosine phosphatase and putative tumor suppressor gene, have been demonstrated in two families with BRRS, and chromatin loss at the PTEN gene locus on chromosome 10q23 has been demonstrated in two BRRS patients. Germ-line mutations in PTEN have also been described in Cowden disease and in a small number of patients with juvenile polyposis syndrome. In an attempt to assess the nature of PTEN mutations in BRRS, we analyzed three sporadic BRRS patients for chromosome 10q23 deletion or PTEN germ-line mutations. All 3 patients demonstrated no loss of parental alleles at 15 chromosome 10q23 markers that encompassed the region of PTEN. In addition, analysis of mRNA and genomic DNA revealed no nonsense, missense, or insertion/deletion mutations of PTEN. Thus, other mechanisms besides mutation of PTEN must have occurred to cause BRRS in these patients. We speculate that BRRS and juvenile polyposis syndrome may have a heterogeneous etiology to cause their syndromes.     

Recovery of respiratory parameters and oxygen consumption during the recovery phase of cardiopulmonary exercise test in patients with dilated cardiomyopathy]

Cowden syndrome (CS) or multiple hamartoma syndrome (MIM 158350) is an autosomal dominant disorder with an increased risk for breast and thyroid carcinoma. The diagnosis of CS, as operationally defined by the International Cowden Consortium, is made when a patient, or family, has a combination of pathognomonic major and/or minor criteria. The CS gene has recently been identified as PTEN, which maps at 10q23.3 and encodes a dual specificity phosphatase. PTEN appears to function as a tumour suppressor in CS, with between 13-80% of CS families harbouring germline nonsense, missense, and frameshift mutations predicted to disrupt normal PTEN function. To date, only a small number of tumour suppressor genes, including BRCA1, BRCA2, and p53, have been associated with familial breast or breast/ovarian cancer families. Given the involvement of PTEN in CS, we postulated that PTEN was a likely candidate to play a role in families with a "CS-like" phenotype, but not classical CS. To answer these questions, we gathered a series of patients from families who had features reminiscent of CS but did not meet the Consortium Criteria. Using a combination of denaturing gradient gel electrophoresis (DGGE), temporal temperature gel electrophoresis (TTGE), and sequence analysis, we screened 64 unrelated CS-like subjects for germline mutations in PTEN. A single male with follicular thyroid carcinoma from one of these 64 (2%) CS-like families harboured a germline point mutation, c.209T-->C. This mutation occurred at the last nucleotide of exon 3 and within a region homologous to the cytoskeletal proteins tensin and auxilin. We conclude that germline PTEN mutations play a relatively minor role in CS-like families. In addition, our data would suggest that, for the most part, the strict International Cowden Consortium operational diagnostic criteria for CS are quite robust and should remain in place.     

Tumour suppressor genes in the pathogenesis of human pituitary tumours

Abnormal cell proliferation is controlled by opposing actions of oncogene products (stimulatory) and tumour suppressor gene (TSG) products (inhibitory). The former are dominantly acting, i.e. only one copy needed for tumorigenesis, whilst for TSG both copies of the gene must be inactivated so these are recessive at a cellular level. For anterior pituitary tumours only one oncogene (Gsp) has been identified in a variable proportion (4-40%) of a single tumour subtype (somatotrophinomas). Contrariwise, allelic deletion studies, using a PCR-based microsatellite polymorphism analysis of DNA extracted from archival specimens, have shown significant loss of heterozygosity in 20-40% of all tumour subtypes at the locus of the putative MEN-1 gene (chr. 11q13); the retinoblastoma gene (chr. 13q 12-14), and 10q26. Moreover, these DNA microdeletions were concentrated in radiologically invasive tumours compared to noninvasive tumours (modified Hardy gdes 3 and 4 vs. 1 + 2). In addition, 50% of Cushing's adenomas showed presence of p53 immunopositivity, though no point mutations in exons 4-9 were found, by SSCP analysis, to account for this. These studies show that analysis of TSGs in pituitary adenomas may provide clues to their pathogenesis, and more importantly relate to clinical behaviour of the tumour, and hence aid decisions regarding management.     

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Prostate cancer is a major cause of cancer death among elderly men in America, Europe, and Japan. However, the molecular mechanism of carcinogenesis is not yet well characterized. Frequent loss of heterozygosity (LOH) on chromosome 10q was reported in prostate cancer, and a candidate tumor suppressor gene, PTEN, was isolated on chromosome band 10q23.3. To investigate the genetic alterations of PTEN, we examined 45 primary prostate cancer specimens. LOH at the PTEN locus was observed in two (11.1%) of 18 tumors. However, no mutations were observed in any of the primary prostate cancers. These data suggest that mutation of the PTEN gene does not play a major role in prostate carcinogenesis of Japanese patients.     

No germline mutations in the dimerization domain of MXI1 in prostate cancer clusters. The CRC/BPG UK Familial Prostate Cancer Study Collaborators. Cancer Research Campaign/British Prostate Group

There is evidence that predisposition to cancer has a genetic component. Genetic models have suggested that there is at least one highly penetrant gene predisposing to this disease. The oncogene MXI1 on chromosome band 10q24-25 is mutated in a proportion of prostate tumours and loss of heterozygosity occurs at this site, suggesting the location of a tumour suppressor in this region. To investigate the possibility that MXI1 may be involved in inherited susceptibility to prostate cancer, we have sequenced the HLH and ZIP regions of the gene in 38 families with either three cases of prostate cancer or two affected siblings both diagnosed below the age of 67 years. These are the areas within which mutations have been described in some sporadic prostate cancers. No mutations were found in these two important coding regions and we therefore conclude that MXI1 does not make a major contribution to prostate cancer susceptibility.     

Mutation analysis of the PTEN/MMAC1 gene in lung cancer

We studied PTEN/MMAC1, a newly discovered candidate tumor suppressor gene at 10q23.3, for mutations in lung cancer. One hundred and thirty-six lung cancer cell line DNAs (66 small cell lung cancers, SCLC, 61 non-small cell lung cancers, NSCLC, four mesotheliomas, five extrapulmonary small cell cancers) were analysed for PTEN/MMAC1 homozygous deletions and five (8%) SCLC lines showed homozygous deletions interrupting the PTEN/MMAC1 gene. Using single stranded conformation polymorphism (SSCP) analysis, we screened the PTEN/MMAC1 open reading frame of 53 lung cancer cell line cDNAs for point mutations and found that 3/35 SCLCs and 3/18 NSCLCs contained homozygous amino acid sequence altering mutations. Northern blot analysis revealed that expression of the PTEN/MMAC1 gene was considerably lower in all the tumor cell lines with point mutations while no expression was detected for cell lines with PTEN/MMAC1 homozygous deletions. Mutation analysis of 22 uncultured, microdissected, primary SCLC tumors and metastases showed two silent mutations, and two apparent homozygous deletions. We also discovered a processed pseudogene (PTEN2) which has 98.5% nt identity to PTEN/MMAC1, that needs to be accounted for in cDNA mutation analysis. Our findings suggest that genetic abnormalities of the PTEN/MMAC1 gene are only involved in a relatively small subset of lung cancers.     

Allelic imbalance in familial and sporadic prostate cancer at the putative human prostate cancer susceptibility locus, HPC1. CRC/BPG UK Familial Prostate Cancer Study Collaborators. Cancer Research Campaign/British Prostate Group

A recent report has provided strong evidence for a major prostate cancer susceptibility locus (HPC1) on chromosome 1q24-25 (Smith et al, 1996). Most inherited cancer susceptibility genes function as tumour-suppressor genes (TSGs). Allelic loss or imbalance in tumour tissue is often the hallmark of a TSG. Studies of allelic loss have not previously implicated the chromosomal region 1q24-25 in prostate cancer. However, analysis of tumour DNA from cases in prostate cancer families has not been reported. In this study, we have evaluated DNA from tissue obtained from small families [3-5 affected members (n = 17)], sibling pairs (n = 15) and sporadic (n = 40) prostate tumours using the three markers from Smith et al (1996) that defined the maximum multipoint linkage lod score. Although widely spaced (12-50 cM), each marker showed evidence of allelic imbalance in only approximately 7.5% of informative tumours. There was no difference between the familial and sporadic cases. We conclude that the incidence of allelic imbalance at HPC1 is low in both sporadic tumours and small prostate cancer families. In this group of patients, HPC1 is unlikely to be acting as a TSG in the development of prostate cancer.     

PTEN mutations in gliomas and glioneuronal tumors

Cytogenetic and loss of heterozygosity studies have suggested the presence of at least one tumor suppressor gene on chromosome 10 involved in the formation of high grade gliomas. Recently, the PTEN gene, also termed MMAC1 or TEP1, on chromosomal band 10q23 has been identified. Initial studies revealed mutations of PTEN in limited series of glioma cell lines and glioblastomas. In order to systematically evaluate the involvement of PTEN in gliomas, we have analysed the entire PTEN coding sequence by SSCP and direct sequencing in a series of 331 gliomas and glioneuronal tumors. PTEN mutations were detected in 20/142 glioblastomas, 1/7 giant cell glioblastomas, 1/2 gliosarcomas, 1/30 pilocytic astrocytomas and 2/22 oligodendrogliomas. No PTEN mutations were detected in 52 astrocytomas, 37 oligoastrocytomas, three subependymal giant cell astrocytomas, four pleomorphic xanthoastrocytomas, 15 ependymomas, 16 gangliogliomas and one dysembryoplastic neuroepithelial tumor. In addition, all tumors were examined for the presence of homozygous deletions of the PTEN gene; these were detected in 7 glioblastomas that did not have PTEN mutations. Therefore, PTEN mutations occur in approximately 20% of glioblastomas but are rare in lower grade gliomas. These findings confirm that PTEN is one of the chromosome 10 tumor suppressor genes involved in the development of glioblastomas.     

Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma

The MMAC1/PTEN gene, located at 10q23.3, is a candidate tumor suppressor commonly mutated in glioma. We have studied the pattern of deletion, mutation, and expression of MMAC1/PTEN in 35 unrelated melanoma cell lines. Nine (26%) of the cell lines showed partial or complete homozygous deletion of the MMAC1/PTEN gene, and another six (17%) harbored a mutation in combination with loss of the second allele. Mutations could also be demonstrated in uncultured tumor specimens from which the cell lines had been established, and cell lines derived from two different metastases from one individual carried the same missense mutation. Collectively, these findings suggest that disruption of MMAC1/PTEN by allelic loss or mutation may contribute to the pathogenesis or neoplastic evolution in a large proportion of malignant melanomas.     

Amplification and over-expression of OZF, a gene encoding a zinc finger protein, in human pancreatic carcinomas

The OZF gene encodes a protein consisting of 10 zinc finger motifs and is located on chromosome 19q3.1. We report here the amplification and over-expression of the OZF gene in pancreatic carcinomas. Increased gene copy number was detected in 3 of 12 tumour cell lines and 2 of 12 primary pancreatic carcinomas. Expression was detected in all cell lines, and the gene was over-expressed in cell lines with OZF gene amplification. Five of 8 tumours, including 2 primary tumours with OZF gene amplification, displayed high levels of OZF protein, whereas normal pancreas expressed low levels. Immuno-histochemical analysis showed that expression was restricted to tumour cells. Thus, high-level expression of OZF is frequent in pancreatic carcinomas and may contribute to the development or progression of this tumour.     

Genotypic analysis of tumor suppressor genes PTEN/MMAC1 and p53 in head and neck squamous cell carcinomas

OBJECTIVES: Tumor suppressor gene mutations in both p53 and PTEN/MMAC1 genomic DNA have been detected in many types of cancer. The purpose of this study was to investigate the presence and importance of PTEN/MMAC1 mutations in squamous cell carcinomas. METHODS: Exons of each gene were amplified after polymerase chain reaction (PCR) using genomic DNA derived from cell lines of squamous cell carcinoma of the head and neck (SCCHN) and snap-frozen biopsy specimens from primary established head and neck tumors. The amplified and purified DNA was then sequenced directly. RESULT: As anticipated, point mutations of the p53 gene were found in 80% of cell lines examined. A single base mutation in codon 151 was found in six of 10 cell lines studied. PTEN/MMAC1 gene mutations were found in neither the cell lines tested nor the tumor biopsy samples. CONCLUSION: This study, as well as a large volume of data, confirms that mutations of the p53 gene are frequent events in head and neck cancer cell lines. Although PTEN/MMAC1 gene mutations have been found in a variety of carcinomas, this gene was not found to be mutated in SCCHN cell lines or in primary squamous cell carcinomas of the head and neck. This information is useful for further studies of mutations in these cell lines.     

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Loss of heterozygosity (LOH) at chromosome band 10q23 occurs frequently in a wide variety of human tumors. A recently identified candidate tumor suppressor gene, PTEN located on 10q23, is mutated in multiple advanced cancers. To explore whether PTEN is associated with human squamous cell carcinoma of the head and neck (SCCHN), DNAs from both normal muscle and tumor tissue in 19 SCCHN were used for detecting LOH at chromosome 10q23 and mutational analysis of PTEN by direct polymerase chain reaction (PCR)-DNA sequencing. LOH at 10q23 was identified in 6/15 SCCHN. Mutation of PTEN was identified in 3/19 SCCHN. Of these 3 patients, 2 had stage IV disease; the third patient, with recurrent, metastatic and stage III disease, showed a 36 bp germline heterozygous deletion within intron 7. Furthermore, a missense mutation at codon 501 (TCT --> TTT: Ser --> Phe) in exon 8 was also found in tumor from the same patient. Our results suggest that PTEN may play a role in the genesis of some SCCHNs.