PTEN/MMAC1 mutations in endometrial cancers

Endometrial carcinomas represent the most common gynecological cancer in the United States, yet the molecular genetic events that underlie the development of these tumors remain obscure. Chromosome 10 is implicated in the pathogenesis of endometrial carcinoma based on loss of heterozygosity (LOH), comparative genomic hybridization, and cytogenetics. Recently, a potential tumor suppressor gene, PTEN/MMAC1, with homology to dual-specificity phosphatases and to the cytoskeletal proteins tensin and auxillin was identified on chromosome 10. This gene is mutated in several types of advanced tumors that display frequent LOH on chromosome 10, most notably glioblastomas. Additionally, germ-line mutations of PTEN/MMAC1 are responsible for several familial neoplastic disorders, including Cowden disease and Bannayan-Zonana syndrome. Because this locus is included in the region of LOH in many endometrial carcinomas, we examined 70 endometrial carcinomas for alterations in PTEN/MMAC1. Somatic mutations were detected in 24 cases (34%) including 21 cases that resulted in premature truncation of the protein, 2 tumors with missense alterations in the conserved phosphatase domain, and 1 tumor with a large insertion. These data indicate that PTEN/MMAC1 is more commonly mutated than any other known gene 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.     

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.     

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

Glioblastoma multiforme, the most malignant human brain tumor, may develop de novo (primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). We present further evidence that primary and secondary glioblastomas constitute distinct disease entities which develop through the acquisition of different genetic alterations. We analyzed p53 mutations, p53 protein accumulation and epidermal growth factor receptor (EGFR) overexpression in 49 biopsies classified as primary or secondary glioblastoma according to clinical and histopathologic criteria. Patients with primary glioblastoma were selected on the basis of a clinical history of less than 3 months and histopathologic features of glioblastoma at the first biopsy (19 cases; mean age, 55 years). The diagnosis of secondary glioblastomas required at least two biopsies and clinical as well as histologic evidence of progression from low grade or anaplastic astrocytoma (30 cases; mean age, 39 years). DNA sequence analysis showed that p53 mutations were rare in primary glioblastomas (11%) while secondary glioblastomas had a high incidence of p53 mutations (67%), of which 90% were already present in the first biopsy. The incidence of p53 protein accumulation (nuclear immunoreactivity to PAb 1801) was also lower in primary (37%) than in secondary glioblastomas (97%). In contrast, immunoreactivity for the EGF receptor prevailed in primary glioblastomas (63%) but was rare in secondary glioblastomas (10%). Only one out of 49 glioblastomas showed EGFR overexpression and a p53 mutation. These data indicate that overexpression of the EGF receptor and mutations of the p53 tumor suppressor gene are mutually exclusive events defining two different genetic pathways in the evolution of glioblastoma as the common phenotypic endpoint.     

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.     

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.     

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.     

Specific P53 mutations are associated with de novo resistance to doxorubicin in breast cancer patients

The mechanisms causing resistance to chemotherapeutic drugs in cancer patients are poorly understood. Recent evidence suggests that different forms of chemotherapy may exert their cytotoxic effects by inducing apoptosis. The tumor suppressor gene P53 has a pivotal role inducing apoptosis in response to cellular damage. In vitro investigations have shown intact p53 to play a critical role executing cell death in response to treatment with cytotoxic drugs like 5-fluorouracil, etoposide and doxorubicin. Recently, mutations in the P53 gene were found to confer resistance to anthracyclines in a mouse sarcoma tumor model, and overexpression of the p53 protein (which, in most cases, is due to a mutated gene) was found to be associated with lack of response to cisplatin-based chemotherapy in non-small cell lung cancer. Previous studies have shown mutations in the P53 gene or overexpression of the p53 protein to predict a poor prognosis, but also a beneficial effect of adjuvant radiotherapy or chemotherapy in breast cancer. In this study we present data linking specific mutations in the P53 gene to primary resistance to doxorubicin therapy and early relapse in breast cancer patients.     

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.     

Association of periampullary diverticula with primary choledocholithiasis but not with secondary choledocholithiasis

The precision and the diagnostic performance of the Boehringer Mannheim CEDIA DAU LSD assay was evaluated. The assay was performed in the semi-quantitative mode on a Hitachi 917 analyzer. Within-run coefficients of variation (CVs) of the semiquantitative values for 0.25 and 1.0 ng/mL were 11.2 and 6.2%, respectively. Day-to-day CVs for the same concentrations were 12.6 and 8.6%. We analyzed 318 urine samples by CEDIA, DPC Coat-A-Count RIA and Behring EMIT II. Confirmation was performed by GC-MS, after extraction on Bond Elut Certify columns. Two hundred sixty-three samples were negative by all methods. Twenty-five samples were positive by all immunoassays, 19 of which were confirmed by gas chromatography-mass spectrometry (GC-MS). One sample was falsely negative by CEDIA. Three samples were positive by EMIT and CEDIA, but negative by RIA and GC-MS. Twenty-six samples were positive by EMIT alone, but they were not confirmed by GC-MS. The LSD CEDIA assay seems to be less specific than DPC RIA but more specific than the EMIT LSD assay.     

Homonucleotide tracts, short repeats and CpG/CpNpG motifs are frequent sites for heterogeneous mutations in the neurofibromatosis type 1 (NF1) tumour-suppressor gene

Glutamatergic synaptic potentials induced by micromolar concentrations of the potassium conductance blocker 4-aminopyridine (4-AP) were recorded intracellularly from rat neostriatal neurons in the presence of 10 microM bicuculline (BIC). These synaptic potentials originate from neostriatal cortical and thalamic afferents and were completely blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 100 microM D-2-amino-5-phosphonovaleric acid (2-APV). Their inter-event time intervals could be fitted to exponential distributions, suggesting that they are induced randomly. Their amplitude distributions had most counts around 1 mV and fewer counts with values up to 5 mV. Since input resistance of the recorded neurons is about 40 M omega, the amplitudes agree to quantal size measurements in mammalian central neurons. The action of a D2 agonist, quinpirole, was studied on the frequency of these events. Mean amplitude of synaptic potentials was preserved in the presence of 2-10 microM quinpirole, but the frequency of 4-AP-induced glutamatergic synaptic potentials was reduced in 35% of cases. The effect was blocked by the D2 antagonist sulpiride (10 microM). Input resistance, membrane potential, or firing threshold did not change during quinpirole effect, suggesting a presynaptic site of action for quinpirole in some but not all glutamatergic afferents that make contact on a single cell. The present experiments show that dopaminergic presynaptic modulation of glutamatergic transmission in the neostriatum does not affect all stimulated afferents, suggesting that it is selective towards some of them. This may control the quality and quantity of afferent flow upon neostriatal neurons.     

p16INK4 gene mutations are relatively frequent in ampullary carcinomas

A high incidence of gene mutations or deletions of p16INK4, a cell cycle regulator which inhibits the activity of cyclin-dependent kinase 4/cyclin D complex and blocks the G1-to-S transition, has been reported in pancreato-biliary tract cancers. In order to investigate p16INK4 gene alterations in sporadic ampullary carcinomas, 17 sporadic ampullary carcinomas were examined. After histological diagnosis, DNA samples extracted separately from both cancerous and normal paraffin-embedded tissues were investigated. Loss of heterozygosity (LOH) was investigated utilizing 3 microsatellite markers on 9p21-22, and a mutational analysis was performed by cloning and sequencing. LOH was observed in 3 cases (17.6%) and somatic mutations with retention of heterozygosity were found in 7 cases (41.2%). Of note was that two mutations resulted in truncated incomplete proteins and one was a point mutation at the consensus site in the conserved ankyrin repeats, which would be crucial for function. Although two-hit inactivation was not evident in any of the mutation cases and further investigation would be needed to elucidate the role of altered p16INK4, these results suggest that the p16INK4 gene mutations are relatively frequent and its inactivation might be important in ampullary carcinogenesis.     

PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells

PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27(KIP1) and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27(KIP1) and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27(KIP1).     

Allelic imbalance at NME1 in microdissected primary and metastatic human colorectal carcinomas is frequent but not associated with metastasis to lymph nodes or liver

Allelic imbalance at the NME locus on chromosome 17q21 was analyzed in colorectal cancer patients using a highly polymorphic microsatellite repeat sequence within NME1 itself. Duplicate samples of carcinoma and adjacent normal tissue was obtained by microdissection from 6 to 7-microns paraffin sections of 94 primary carcinomas (treatment years 1979-1993) and available lymph node and liver secondaries. In 55 patients informative (heterozygous) at this locus, allelic imbalance was examined in primary and secondary carcinomas. Microsatellite instability prevented assessment of allelic balance in two cases, and there was no evidence of homozygous loss at NME1 in any case analyzed. Allelic imbalance at the NME locus in carcinomas was frequent (27/53; 51%), and concordant results were obtained between primary carcinoma and secondary deposits in 30 of 33 (91%) cases. Three discordant cases showed allelic imbalance in secondary deposits but not the primary lesion. Although frequent, allelic imbalance at NME1 had no relationship to Dukes' stage at presentation or with subsequent hepatic metastasis, nor with the primary carcinoma site (proximal versus distal), tumor size, or mitotic or apoptotic index. Moreover, neither disease-free nor overall survival differed between patients with carcinomas showing NME1 allelic imbalance and patients with carcinomas that did not. Our results show that although allelic imbalance is frequent at the NME locus in primary and secondary colorectal carcinomas, there is no evidence to link this with clinical or pathological features or with metastatic potential. Microsatellite PCR and microdissection of enriched populations of carcinoma cells allowed uniformly successful analysis of samples from archival formalin-fixed paraffin-embedded tissue up to 15 years old and clear assessment of allelic imbalance in tumor specimens. Target sequences (e.g., microsatellites and minisatellites) up to approximately 200-250 bp may be reliably analyzed for allelic balance, suggesting that this method is of general utility in the genetic analysis of primary and metastatic neoplasia.     

Newborn mice develop balanced Th1/Th2 primary effector responses in vivo but are biased to Th2 secondary responses

Newborn mice are impaired in their abilities to mount protective immune responses. For decades, it was generally held that the poor responses of newborns were largely due to the developmentally immature state of the T cells. In vitro studies showing that neonatal T cells were deficient in Th1 cytokine production, proliferation, and secondary responsiveness strongly supported this idea. Recently, several studies have challenged this view; animals exposed to Ag as neonates were shown to have mature Th1 responses in adulthood. However, it is not clear whether the mature immune responses were actually mounted by T cells generated after the neonatal stage. We have reexamined this issue by analyzing the capabilities of neonatal lymph node T cells to develop into Ag-specific effector cells during the actual neonatal period. Our results demonstrate that the capacity to develop a balanced Th1/Th2 primary effector response is fully mature within the first week of life. However, while neonatal and adult primary cytokine profiles were very similar, Th2 secondary responses predominated in animals first immunized as newborns. Moreover, we have observed other differences between adults and neonatal responses, including 1) the kinetics of cytokine production and responsiveness to adjuvant during the primary response, and 2) the contribution of spleen and lymph node to secondary responses. We propose that these differences reflect developmental regulation of effector cell function that has important consequences to neonatal immune function.