Analysis of PTEN/MMAC1 alteration in neuroblastoma

Neuroblastoma is the most common extracranial solid tumor in children. Although it has been reported that loss of heterozygosity at various loci, including 10q, frequently occurs in neuroblastoma, a bona fide tumor suppressor gene has not been identified. Recently, a gene mapped to chromosome 10q23, PTEN/MMAC1, was identified as a tumor suppressor gene that inhibits cell survival and cell proliferation by catalyzing the dephosphorylation of phosphatidylinositol 3,4,5-triphosphate. To screen for mutations of this gene in neuroblastoma, we analyzed 11 primary neuroblastoma tumors and 16 neuroblastoma cell lines for PTEN/MMAC1 mutations and deletions. All nine exons of the PTEN/MMAC1 gene were examined using the polymerase chain reaction-single strand conformational polymorphism assay and sequencing. Only one of the cell lines showed a mutation, a 1-bp frameshift deletion in exon 7, and an allelic loss in the opposite allele was revealed by a microsatellite analysis. Our results indicate that the disruption of the PTEN/MMAC1 gene is not a frequent event in neuroblastoma, and suggest that this disruption may be responsible for malignant progression in only a limited proportion of cases of neuroblastoma.     

Epigenetic PTEN silencing in malignant melanomas without PTEN mutation

A tumor suppressor gene at 10q 23.3, designated PTEN, encoding a dual specificity phosphatase with lipid and protein phosphatase activity, has been shown to play an important role in the pathogenesis of a variety of human cancers. Germline mutations in PTEN cause Cowden syndrome (CS), which is characterized by multiple hamartomas and a high risk of breast and thyroid cancers. Frequent loss of heterozygosity at 10q is found in both early and advanced-stage sporadic melanomas; however, mutations or deletions in PTEN are detected mainly in melanoma cell lines. In this study, we examined PTEN expression in 34 unselected sporadic melanomas (4 primary melanomas, 30 metastases) using immunohistochemistry and correlated this with the results of structural studies of this gene. Immunostaining of 34 melanoma samples revealed no PTEN expression in 5 (15%) and low PTEN expression in 17 (50%), whereas the rest of the tumors (35%) had high levels of expression. Hemizygous deletion was found in 32% of the tumors but neither intragenic PTEN mutation nor biallelic deletion was found in any of the samples. Of the 5 melanomas showing no PTEN expression, 4 had no mutation or deletion of PTEN. Of the 13 tumors having weak PTEN immunoreactivity and informative loss of heterozygosity results, 6 had evidence of hemizygous allelic loss of PTEN while the remaining 7 had intact PTEN. These results strongly support PTEN as a major tumor suppressor on 10q involved in melanoma tumorigenesis and suggest an epigenetic mechanism of biallelic functional inactivation not previously observed in other cancers where PTEN might be involved.     

Detection of new PTEN/MMAC1 mutations in head and neck squamous cell carcinomas with loss of chromosome 10.

Alterations of the candidate tumor suppressor gene PTEN/MMAC1 and the cell cycle control gene p16((CDKN2/MTS-1/INK4a)) have been detected in many types of human cancer. Here, we wanted to study the role of PTEN/MMAC1 in head and neck squamous cell carcinomas (HNSCC) in correlation to mutation and methylation of p16 and to previous in situ hybridization results concerning loss of chromosomes 9 and 10. We screened for alterations of PTEN/MMAC1 and p16 in 52 HNSCC of different sites. Mutations of PTEN/MMAC1 were found in 23% of tumor samples (missense mutations in 7 carcinomas, 13%). A loss of chromosome 10 was detected in five carcinomas with missense PTEN/MMAC1 mutations (71%). The missense mutations of PTEN/MMAC1 occurred in exons 5 (five different mutations in the neighborhood of the protein tyrosine phosphatase domain), 6, 7, and 8. Only one of these mutations had been described before. In addition, in three laryngeal carcinomas (6%), missense mutations of p16 (in exon 2) were detected and 14% of carcinomas showed a methylation of p16. Our results focus on the essential but not solitary role of PTEN/MMAC1 in the tumorigenesis or progression of a subset of HNSCC.     

PTEN gene loss, but not mutation, in benign and malignant phaeochromocytomas

Mutations of the 'phosphatase and tensin homologue deleted on chromosome 10' (PTEN/MMAC1) gene have been associated with a variety of human cancers, including prostate cancer, glioblastoma, and melanoma. The gene is thought to be one of the most frequently mutated tumour suppressor genes and inactivation of PTEN is associated with disease progression and angiogenesis. High vascularization and resistance to chemo- and radio-therapy are two well-established features of phaeochromocytomas (PCCs). Furthermore, benign and malignant PCCs are found in several PTEN knockout mouse models. This study therefore evaluated whether inactivation of PTEN may be involved in the tumourigenesis of PCC in man and whether PTEN abnormalities may help to define the malignant potential of these tumours. Tumour and germline DNA was analysed from 31 patients with apparently sporadic PCC, including 14 clinically benign and 17 malignant tumours, for loss of the PTEN gene locus, mutations in the PTEN gene, and for PTEN protein expression by immunohistochemistry. Loss of heterozygosity (LOH) analysis showed loss of PTEN in four malignant tumours (40%) and in one benign tumour (14%). However, no mutations of PTEN were observed. Immunohistochemistry showed no correlation with clinical behaviour and/or LOH status. The results indicate that inactivation of the PTEN/MMAC1 gene may play a minor role in the development of malignant phaeochromocytomas.     

Intragenic PTEN/MMAC1 loss of heterozygosity in conventional (clear-cell) renal cell carcinoma is associated with poor patient prognosis.

Inactivation of the PTEN/MMAC1 tumor suppressor gene has been linked to tumor progression in several human malignancies. However, the role of PTEN/MMAC1 in the development and progression of the major renal cell carcinoma morphotypes remains controversial. We examined microdissected specimens from 80 conventional (clear cell) renal cell carcinomas (cRCC), 27 papillary renal cell carcinomas (pRCC), and 16 chromophobe renal cell carcinomas (chRCC) for loss of heterozygosity (LOH) at and around the PTEN/MMAC1 locus and for mutations in the PTEN/MMAC1 gene. The results of the molecular studies were correlated with tumor stage, grade, and patient survival. LOH at one or more of the examined loci occurred in 37.5% of cRCC, 29.6% of pRCC and 87.5% of chRCC specimens. The chRCC specimens showed increasing rates of LOH the further that a marker was located toward the q telomer of chromosome 10, consistent with nonspecific genetic disarray in genomically highly unstable tumors. No such pattern was discernible in the cRCC and pRCC. In the cRCC, LOH at intragenic PTEN/MMAC1 microsatellite markers (indicating deletional events involving the actual PTEN/MMAC1 gene) was significantly associated with tumor death, with 85.7% of such patients dying, whereas only 45.3% of patients without intragenic LOH died (P =.018). There were no PTEN/MMAC1 mutations in our specimens. We conclude that PTEN/MMAC1 inactivation may play a role in the progression of cRCC. Biallelic inactivation may preferentially occur by nonmutational mechanisms, or, alternatively, haploinsufficiency of PTEN/MMAC1 may be sufficient to affect tumor progression in cRCC.     

Loss of heterozygosity in the MXI1 gene is a frequent occurrence in melanoma.

Melanoma development and progression is thought to be the result of a multi-step accumulation of genetic damage, with loss of heterozygosity in chromosome 9p (MTS1) frequently described. In addition, chromosome 10q allelic loss has been reported, implicating the tumor suppressor gene PTEN/MMAC1 on 10q23.3. The MXI1 gene at 10q24-25 is another candidate tumor suppressor that has only rarely been studied in melanomas, with conflicting results. We used microdissection-based genotyping to investigate 29 melanomas from 20 patients for loss of heterozygosity in intragenic and flanking microsatellite markers for this latter gene. Concurrently, the MTS1 gene was similarly studied using two flanking microsatellites. Fifty-four percent (15 of 28) of the informative cases showed loss of heterozygosity for one or both MXI1 markers, as compared with 67% (16 of 24) of the informative cases for MTS1. MXI1 allelic loss was seen more frequently in recurrent/metastatic tumors (59%), as compared with in primary (33%) lesions. Eighty percent of the primary tumors showed loss of heterozygosity for MTS1, as well as 63% of recurrent/metastatic ones. We studied more than one tumor in eight patients, with those from three patients showing discordant genetic patterns. One patient showed a metastatic tumor with allelic loss for MXI1 that was not identified in the primary melanoma or a local recurrence. The other two patients showed clonal heterogeneity in MXI1 at synchronous and metachronous metastatic foci. These findings support MXI1 as a putative tumor suppressor gene involved in conventional melanoma progression. Genetic heterogeneity seen in different metastases from the same primary suggests a nonlinear pattern of chromosomal damage, with the development of multiple clones within the primary tumor, each acquiring its own metastatic potential.     

Absence of mutations of the BRAF gene and constitutive activation of extracellular-regulated kinase in malignant melanomas of the uvea

The v-raf murine sarcoma viral homolog B1 (BRAF) gene, one of the human isoforms of RAF, is activated by Ras, leading to cooperative effects in cells responsive to growth factor signals. Recently, somatic missense mutations of the BRAF gene have been detected in more than 66% of malignant melanomas of the skin. We analyzed 42 malignant melanomas of the uvea, 3 corresponding liver metastases, and 10 cutaneous melanomas for possible BRAF mutations: after microdissection, mutation analysis of BRAF and KRAS was performed. The expression of extracellular-regulated kinase 1 and 2 (ERK1/2), an important downstream point of convergence in the Ras-RAF-MEK-Erk pathway, was analyzed immunohistochemically. Interestingly, we failed to detect activating BRAF mutations in uvea melanomas and their corresponding liver metastases. There were no mutations of BRAF in corresponding non-neoplastic uvea specimens, although we detected three BRAF mutations in sporadic cutaneous melanoma that led to a substitution of valine by glutamic acid at position 599 (V599E). KRAS mutations were detected in 1 of 10 cutaneous melanoma but not in uveal or metastatic melanoma. Despite the lack of activating mutations in the BRAF gene, we identified constitutively activated ERK in almost all (86%) uveal melanoma tissues tested but not in corresponding normal retina or uveal cells. Our data indicate that BRAF gene mutations are rare to absent events in uveal melanoma. The finding of activated Erk suggests a causative role for MAPK activation in uveal melanoma independent of activating BRAF or RAS mutations.     

Mutation analysis of the putative tumor suppressor gene<Emphasis Type="Italic"> PTEN/MMAC1</Emphasis> in advanced gastric carcinomas

A novel tumor suppressor gene, PTEN/MMAC1, located on chromosome band 10q23.3, encodes a 403-amino acid, dual-specificity protein phosphatase. The defects in this gene are responsible for the development of some advanced cancers. Inactivating alterations, including mutations and deletions, in the PTEN/MMAC1 gene have been identified in several types of human cancers and cancer cell lines. To clarify the participation of the PTEN/MMAC1 gene in advanced gastric carcinogenesis, we screened their frequency of mutations in primary advanced gastric adenocarcinoma tissues. Cancer specimens and their corresponding normal tissues were obtained surgically from 60 patients with pathologically proven advanced gastric carcinoma at the Department of Surgery of Kaohsiung Medical University Hospital. All nine exons of the PTEN/MMAC1 gene were amplified using polymerase chain reaction and screened for mutations by single-strand conformation polymorphism analysis and followed by direct sequencing. After neutral polyacrylamide gel electrophoresis, 17 patients (28.3%) showed an apparent electrophoretic mobility shift between the cancer and its paired normal tissue. These results from direct sequencing indicated that mutations consisted of eight cases (47.1%) of missense mutation, five silent mutations (29.4%), two nonsense mutations (11.8%), a 12-bp deletion (5.9%), and a mutation within the splice donor site of intron 6 (5.9%). The mutation hot spots at codons 45, 66, 82 and 204 in advanced gastric cancer have not been observed previously. Based on the present analysis, our study implicated that the mutations of the PTEN/MMAC1 gene do not occur at a significant rate in human advanced gastric carcinoma, but the rare clustered mutation site (exons 2-6) perhaps suggested that PTEN/MMAC1 might contribute to the gastric carcinogenesis and its progression.     

Allelic losses on chromosome arm 10q and mutation of the PTEN (MMAC1) tumour suppressor gene in primary and metastatic malignant melanomas

Malignant melanomas frequently show loss of alleles on the long arm of chromosome 10. The PTEN (MMAC1) gene has been identified as a tumour suppressor gene at 10q23.3 that is mutated in various types of advanced human cancers. We have investigated a series of 40 sporadic melanomas from 37 patients (15 primary cutaneous melanomas and 25 melanoma metastases) for allelic losses on chromosome 10, as well as for deletion and mutation of the PTEN gene. Microsatellite analysis revealed loss of heterozygosity at loci located on 10q in tumours from 15 of 34 patients investigated (44%). Somatic PTEN mutations were identified in melanomas from 4 of 37 patients (11%), all of whom had metastatic disease. In two of these patients, the tumours had additionally lost one PTEN allele, indicating complete loss of wild-type PTEN in the tumour cells. Our findings corroborate that loss of heterozygosity on chromosome 10 is a frequent aberration in malignant melanomas and implicate PTEN as a tumour suppressor gene inactivated by somatic mutation in a fraction of these tumours. Received: 1 September 1999 / Accepted: 22 December 1999     

10q23.3 loss of heterozygosity is higher in lymph node-positive (pT2-3,N+) versus lymph node-negative (pT2-3,N0) prostate cancer

Loss of heterozygosity (LOH) in the region of 10q23.3 has been associated with multiple tumors, including glioblastoma multiforme, melanoma, endometrial carcinoma, and prostate carcinoma. The tumor suppressor gene, PTEN/MMAC1, is also located in this region, and, in addition to other tumor types (eg, glioblastoma multiforme, endometrial, and melanoma), PTEN/MMAC1 mutations have been found in prostate cancer cell lines, xenografts, and hormone refractory prostate cancer tissue specimens. The aim of this study was to evaluate LOH at 10q23.3 as a marker of cancer progression in node-positive prostate cancer. Genetic alterations in the region of 10q23.3 were assessed in 23 node-positive (pT2-3, N+) and 44 node-negative prostate (pT2-3, N0) cancers with D10S532, D10S1687, D10S541, and D10S583 flanking polymorphic genetic markers; PTENCA, a genetic marker within PTEN/MMAC1, was also tested. Using DNA from paired normal and microdissected tumor samples, LOH at microsatellite loci was determined after polymerase chain reaction amplification. LOH in at least 1 marker was identified in 14% (6 of 44) of lymph node-negative and 43% (10 of 23) of lymph node-positive prostate cancers (chi-square test, P = .007). This increase in genetic alterations in node-positive prostate cancer suggests that 10q23.3 is a marker for metastatic progression.     

Inactivation of the PTEN/MMAC1/TEP1 gene in human lung cancers

The PTEN/MMAC1/TEP1 gene has been isolated as a tumor suppressor gene that is altered in several types of human tumors including brain, breast, and prostate cancers. In the present study, we report PTEN/MMAC1/TEP1 alterations in human lung cancers. Intragenic homozygous deletions were detected in 6 (40%) of 15 small cell lung carcinoma (SCLC) cell lines and in 2 (8%) of 25 non–small cell lung carcinoma (NSCLC) cell lines. A nonsense mutation and a missense mutation were detected in 2 (8%) NSCLC cell lines. An intragenic homozygous deletion, a 1-bp frameshift mutation, and a nonsense somatic mutation were also detected in three (6%) of 47 surgical specimens. All the lung tumors with PTEN/MMAC1/TEP1 mutations were homozygous for the mutant alleles. These findings suggest that PTEN/MMAC1/TEP1 plays a role as a tumor suppressor gene in the genesis and/or progression of human lung cancer. Genes Chromosomes Cancer 22:152–156, 1998. © 1998 Wiley-Liss, Inc.     

少突胶质细胞瘤的分子遗传学、病理与临床预后分析

目的 探讨少突胶质细胞肿瘤遗传分子表型、病理和临床预后的关系。方法 对51例（对）少突胶质细胞肿瘤和外周血进行DNA的提取,变性梯度凝胶电泳（DGGE）和DNA测序检测抑癌基因TP53、PTEN／MMAC1和p18突变;多重PCR检测EGFR扩增、p16／CDKN2A和p18缺失;多因素分析预后和生存期。结果 26例少突胶质细胞瘤中TP53和p18突变各1例;未发现PTEN／MMAC1突变;19．2％EGFR扩增;27％p16／CDKN2A缺失。25例GBMO中TP53,p18和PTEN／MMAC1突变分别是24％、0和20％。44％EGFR扩增,48％p16／CDKN2A缺失。所有肿瘤均未见p18同源性缺失。结论 缺乏TP53和PTEN／MMAC1突变是少突胶质细胞肿瘤独特的分子特性。其恶化进展和生存期短与EGFR扩增密切相关。     

Heterotransplantation of human malignant melanomas to the mouse mutant nude.

In 32 experiments involving transplantation of human malignant melanomas to the mouse mutant nude, tumourtake was observed in 14 instances. The tumour tissue which took and grew after inoculation was derived from malignant melanoma metastases of the skin (10 takes from 13 transplantations), and from primary malignant melanomas of the eye (4 takes from 6 transplantations). Not one of 13 attempts to transplant primary tumours of the skin was successful. In 7 instances serial transfers of transplanted tumour were successful, the degree of success ranging from 3 to 41 passages. Some passages were wilfully interrupted but two are continuing. The transplanted tumours grow locally. Neither lymph node metastases nor organ metastases have been observed. Histological appearances of the transplanted tumours, also after serial passages, were identical with those of the original human donor material. The human tumour/nude mouse system is contrasted and compared with other heterotransplantation models.     

Retinoblastoma-binding protein 2-homolog 1: a retinoblastoma-binding protein downregulated in malignant melanomas.

In malignant melanomas, the loss of cell cycle control is thought to be due to a lack of retinoblastoma protein (pRb)-activity. Members of the previously described family of retinoblastoma-binding proteins (RBPs) are supposed to act as pRb-modulating factors. Based on RNA-fingerprinting of normal human melanocytes, we previously described a new family member with high sequence homology to the retinoblastoma-binding protein-2 (RBP-2), termed RBP2-Homolog 1 (RBP2-H1). Based on its UVB responsiveness, it was hypothesized that this gene may also play a role in melanocytic tumors. In the present study, we can confirm by real-time RT-PCR (six common melanocytic nevi, five advanced nodular melanomas and seven melanoma metastases) and immunohistochemistry (tissue microarrays: 52 melanocytic nevi, 60 melanomas, 60 metastases; and conventional sections: five common nevi, four advanced nodular melanomas, five melanoma metastases) that RBP2-H1 expression is progressively downregulated in advanced and metastatic melanomas in vivo with a certain intratumoral heterogeneity. Whereas benign melanocytic nevi are RBP2-H1 positive in about 70% of the cases, a lack of RBP2-H1 expression was found in 90% of the primary malignant melanomas and 70% of the melanoma metastases, respectively. Interestingly, a similar deficiency can be found in glioblastomas, but not epithelial cancers. In accordance to the in vivo data, established melanoma cell lines exhibit low but heterogeneous levels of RBP2-H1 expression. By co-immunoprecipitation, we provide the first evidence that a subfraction of total RBP2-H1 can bind to pRb, which makes this protein a true pRb-interacting factor. We conclude that loss of RBP2-H1 is a common finding in the progression of malignant melanomas. Since a direct interaction of RBP2-H1 and pRb seems possible, the loss of RBP2-H1 may possibly contribute to uncontrolled growth in malignant melanomas.     

Patterns of melastatin mRNA expression in melanocytic tumors

The melanocyte-specific gene Melastatin (MLSN1) shows an inverse correlation of mRNA expression with metastatic potential in human and murine cell lines in vitro. Melastatin mRNA expression in primary cutaneous melanoma also has been found to correlate with disease-free survival. The histologic patterns of Melastatin mRNA expression in nevi, primary melanoma, and melanoma metastases have not been described previously. Using in situ hybridization with (35)S-labeled probes, we examined Melastatin mRNA expression in 64 cases of normal skin, benign melanocytic nevi, primary cutaneous melanomas, and melanoma metastases. Ubiquitous melanocytic expression of Melastatin mRNA was observed in all benign melanocytic proliferations (14 of 14), although some nevi showed a gradient of reduced Melastatin expression with increased dermal depth (3 of 14). Uniform expression of Melastatin mRNA was observed in 49% of primary cutaneous melanomas (18 of 37 cases, including 1 case of in situ melanoma). Melastatin mRNA loss by a portion of the melanoma was identified in 53% of the invasive melanoma samples (19 of 36) and 100% of the melanoma metastases (11 of 11). Primary melanomas without mRNA loss ranged in thickness from 0.17 to 2.75 mm (median, 0.5 mm; mean, 0.73 mm), whereas tumors that showed Melastatin mRNA down-regulation ranged in thickness from 0.28 to 5.75 mm (median, 1.7 mm; mean, 2.13 mm). A focal aggregate or nodule of melanoma cells without detectable signal was the most commonly observed pattern of Melastatin loss (13 of 19 cases), whereas complete loss of Melastatin mRNA expression by all of the dermal melanoma cells was observed in only 4 of the 19 cases. Two invasive melanomas displayed a scattered, nonfocal pattern of Melastatin mRNA loss. Of the 11 melanoma metastases examined, 64% displayed focal Melastatin mRNA loss, and 36% had complete loss of Melastatin mRNA expression. We observed several patterns of Melastatin mRNA expression in primary melanoma that may be distinguished from expression in benign melanocytic nevi. Melastatin mRNA expression appears to correlate with melanocytic tumor progression, melanoma tumor thickness, and the potential for melanoma metastasis. HUM PATHOL 31:1346:1356.     

Expression of vitamin D receptor decreases during progression of pigmented skin lesions

1,25-dihydroxyvitamin D3 affects proliferation, differentiation, and apoptosis and protects DNA against oxidative damage with a net tumorostatic and anticarcinogenic effect. It acts through a specific nuclear receptor that is widely distributed through the body. Although a beneficial role of vitamin D in melanoma patients has been suggested, there is lack of information on the changes in the expression pattern of vitamin D receptor during progression of pigmented lesions. Using immunohistochemistry, we analyzed the expression of vitamin D receptor in 140 samples obtained form 82 patients, including 25 benign nevi, 70 primary cutaneous melanomas, 35 metastases, 5 re-excisions, and 5 normal skin biopsies. The strongest expression was observed in normal skin that significantly decreased in melanocytic proliferations with the following order of expression: normal skin > melanocytic nevi > melanomas = metastases. The vitamin D receptor expression in skin surrounding nevi and melanoma was also significantly reduced as compared to normal skin. Tumor-infiltrating and lymph node lymphocytes retained high levels of vitamin D receptor. There was negative correlation between tumor progression and vitamin D receptor expression with a remarkable decrease of the immunoreactivity in nuclei of melanoma cells at vertical versus radial growth phases and with metastatic melanomas showing the lowest cytoplasmic receptor staining. Furthermore, lack of the receptor expression in primary melanomas and metastases was related to shorter overall patients' survival. In addition, the receptor expression decreased in melanized melanoma cells in comparison to amelanotic or poorly pigmented cells. Therefore, we propose that reduction or absence of vitamin D receptor is linked to progression of melanocytic lesions, that its lack affects survival of melanoma patients, and that melanogenesis can attenuate receptor expression. In conclusion, changes in vitamin D receptor expression pattern can serve as important variables for diagnosis, predicting clinical outcome of the disease, and/or as a guidance for novel therapy of melanomas based on use of vitamin D or its derivatives.