Somatic mutations of the PTEN/NMAC1 gene associated with frequent chromosomal loss detected using comparative genomic hybridization in endometrial cancer.

OBJECTIVES: We analyzed the mutational status of the transforming growth factor-beta type II receptor (TGF beta RII), BAX, and PTEN/MMAC1 genes as well as microsatellite instability (MI) in 29 consecutive cases of endometrial carcinoma operated on at the Cancer Institute Hospital (Tokyo, Japan). To identify chromosomal loss associated with significant somatic mutations, we conducted comparative genomic hybridization (CGH) analysis. METHODS: We conducted a direct sequence for mutational analysis of these genes. To examine copy number loss at the chromosomal regions bearing these genes, we used CGH analysis. CGH analysis may provides a genome-wide overview about tumor-associated genomic imbalances. RESULTS: Among nine tumors that showed the MI+ phenotype, four (44%) demonstrated a significant mutation with a definite amino acid change in the PTEN/MMAC1 gene. CGH analysis demonstrated that all four tumors (100%) showed chromosomal copy number loss around the locus of this gene, whereas four (57%) of seven tumors with PTEN/MMAC1 mutations showed chromosomal loss or double mutations in MI- carcinomas. The role of TGF beta RII and BAX genes is limited as a target gene of MI+ phenotype in endometrial cancer, because several mutations of these genes were detected but a chromosomal loss was demonstrated by CGH in only one tumor in MI+ endometrial cancers with mutation. CONCLUSIONS: This report reveals, by using CGH, that most MI+ endometrial cancers with PTEN/MMAC1 mutations as well as MI- tumors showed inactivation of both alleles of this gene, which strongly suggested the involvement of this gene in carcinogenesis.

Risk of alanine aminotransferase flare-up among asymptomatic hepatitis C virus RNA carriers: a 10-year follow-up study.

AIMS: The aim of this study was to determine the cumulative rate of flare-up of serum alanine aminotransferase (ALT) level during a 10-year follow-up period, and characterize the clinical, virologic features in 120 hepatitis C virus (HCV) RNA-positive asymptomatic carriers with persistently normal ALT levels for 6 months. RESULTS: All flare-up cases occurred during the first 5 years of the present study, 27.4% of carriers showed ALT flare-up during this period, but none in the second half of the study. Multivariate analysis showed that C100-3 antibody (Ab) and anti-human T cell leukemia virus (HTLV)-I Ab were two independent and significant predictors of ALT flare-up in hepatitis C virus (HCV) RNA asymptomatic carriers (P = 0.04, P = 0.03, respectively). Liver biopsy was performed in 44 patients (11 with flare-up of ALT level, whereas 33 had normal ALT levels). Histological features of chronic hepatitis with lymphoid infiltration in the portal tracts were commonly observed in all specimens, and no differences were noted between the flare-up ALT group and the persistently normal ALT group. CONCLUSIONS: Our results indicated that flare-up of ALT levels in asymptomatic HCV-RNA carriers with normal ALT levels occurs during the first 5 years of diagnosis, and that the presence of C100-3 and anti-HTLV-I antibodies are good predictors of a transient rise in ALT.

Familial hyperparathyroidism.

Familial hyperparathyroidism (HPT) is a hereditary disease in which HPT is transmitted in an autosomal dominant fashion. It includes a variety of diseases: multiple endocrine neoplasia (MEN) type 1 and type 2, and familial isolated hyperparathyroidism (FIHPT). We screened for MEN 1 mutations by direct nucleotide sequencing of all protein-coding regions and identified the germline mutations of the MEN 1 gene in two families with familial HPT. Patients with FIHPT have multiple abnormal parathyroid glands and are prone to both recurrent and persistent HPT. They frequently present with profound hypercalcemia, in contrast to patients with MEN-associated HPT or sporadic HPT. We recommend subtotal or total parathyroidectomy plus autotransplantation in patients with MEN-associated HPT and patients with FIHPT. Because parathyroid remains or supernumerary glands are often present in the thymus or perithymic tissue, we advocate routine bilateral dissection of the central zone with bilateral cervical thymectomy.

Evaluation of adult T-cell leukemia/lymphoma incidence and its impact on non-Hodgkin lymphoma incidence in southwestern Japan.

The incidence of adult T-cell leukemia/lymphoma (ATL) and its impact on that of total non-Hodgkin lymphoma (NHL) were evaluated in Nagasaki, an area in southwestern Japan where human T-cell lymphotropic virus type I (HTLV-I) is endemic. The first study area comprised 4 towns located on the K Islands, which had a population of 26,870 in 1990. The overall HTLV-I seroprevalence estimated from the serologic survey of 18,485 subjects was 16.2%. By using the data from the Nagasaki Prefectural Cancer Registry (NPCR) and reviewing clinical and laboratory information, we identified 40 cases of ATL and 35 cases of other NHL diagnosed between 1985 and 1995. The crude annual incidence of ATL among 100,000 HTLV-I carriers aged 30 or older was estimated at 137.7 for men and 57.4 for women, with a significant sex difference after adjustment for age (rate ratio = 2.50, 95% confidence interval 1.32-4.73). The cumulative risk from 30 to 79 years of age was estimated at approximately 6.6% for men and 2.1% for women. Among the entire population, ATL accounted for 51 to 59% of the total NHL incidence, showing the strong impact of HTLV-I infection. The second study area comprised the whole of Nagasaki Prefecture (total population in 1990 = 1.56 million). Between 1985 and 1995, 989 cases of ATL and 1,745 cases of other NHL were registered in the NPCR. The world age-standardized annual incidence rate of ATL per 100,000 persons aged 30 or older was estimated at 10.5 for men and 6.0 for women, which accounted for approximately 37 to 41% of the total NHL incidence.

CD40 activation mediates p53-dependent cell cycle regulation in human multiple myeloma cell lines.

It has been reported that the activation of multiple myeloma (MM) cells by CD40 induces proliferation, growth arrest, and apoptosis. To determine whether the biologic sequelae of CD40 activation in MM cells depends on p53 function, we identified temperature-sensitive p53 mutations in the RPMI 8226 (tsp53E285K) and the HS Sultan (tsp53Y163H) MM cell lines. These cells were then used as a model system of inducible wtp53-like function because wild-type-like p53 is induced at permissive (30 degrees C) but not at restrictive (37 degrees C) temperatures. Using p21-luciferase reporter assays, we confirmed that CD40 induces p53 transactivation in RPMI 8226 and HS Sultan cells cultured under permissive, but not restrictive, conditions. Furthermore, CD40 activation of these MM cells under permissive, but not restrictive, temperatures increased the expression of p53 and p21 mRNA and protein. Importantly, CD40 activation induced the proliferation of RPMI 8226 and HS Sultan cells at restrictive temperatures and growth arrest and increased subG1 phase cells at permissive temperatures. These data confirmed that CD40 activation might have distinct biologic sequelae in MM cells, depending on their p53 status.

Genetic alterations on chromosome 17p associated with response to radiotherapy in bulky cervical cancer.

Chromosome 17 alterations are found in more cancers than those of any other chromosome, and frequently involve the p53 gene on 17p13. The aim of this study was to identify the correlations between the presence of loss of heterozygosity (LOH) and microsatellite instability (MI) on chromosome 17p13 in patients with cervical cancer and the patients' response to radiotherapy. A total of 50 patients were treated with definitive radiotherapy. We performed biopsies and took specimens from the tumour and venous blood of all patients. Tumour and normal DNAs were analysed by polymerase chain reaction for genetic losses and instability at three polymorphic microsatellite loci mapped to 17p13. Nineteen of the 50 tumours (38%) displayed a genetic alteration (GA) on 17p13, 16 (32%) were found to have LOH, and three (6%) showed MI. The sizes of the tumours of the GA-positive patients were significantly greater than those of the GA-negative patients (P = 0.009). The mean tumour diameter of all patients was 6 +/- 2.4 cm. We divided the patients into those with tumours smaller than 6 cm in diameter (n = 26) and those with tumours equal to or greater than 6 cm in diameter (n = 24). The former group survived significantly longer compared to the latter group (P = 0.0002). Among the patients with < 6 cm tumours, all six GA-positive patients are alive with no evidence of disease (NED), whereas of the 20 GA-negative patients, 18 have NED and two are alive with disease (AWD) or suffered cancer-caused death (CD). Thus, there was no correlation between GA and radiotherapy response in the tumours smaller than 6 cm. However, among the patients with > or = 6 cm tumours, two of the GA-positive patients have NED and 11 are AWD/CD, whereas seven of the GA-negative patients have NED and four are AWD/CD. Among the patients with > or = 6 cm tumours, the response to radiotherapy of the GA-positive patients were significantly poorer than those of the GA-negative patients (P = 0.02). In addition, the GA-negative patients survived significantly longer compared to the GA-positive patients (P = 0.026). The results of this study suggest that GA increases with tumour growth. Improved success in the management of bulky cervical cancer requires a better understanding of its biological behaviour.

[A case of multiple endocrine neoplasia type 2A (MEN2A) with a mutation in the RET gene].

A 44-year-old woman complained of headache and palpitation. Magnetic resonance imaging showed bilateral adrenal tumors 10 x 9 cm in size on the left side and 8 x 4 cm in size on the right side. CT scan revealed a 0.7 x 0.7 cm mass in the thyroid. Hormonal examinations showed high values of urinary cathecholamines and serum calcitonin. DNA sequence analysis of peripheral white blood cells revealed that codon 634 in exon 11 of the RET gene was mutated from TGC (Cys) to TAC (Tyr). From these findings, a diagnosis was made of MEN2A with bilateral adrenal pheochromocytomas and medullary thyroid carcinoma. Bilateral adrenalectomy and thyroidectomy were performed. The same mutation of the RET gene was detected in all her 3 children, in two of whom, early stage medullary thyroid carcinoma was detected and thyroidectomy was performed. DNA analysis of the RET gene was useful for the diagnosis of carriers of MEN2A and the early detection of medullary thyroid carcinoma.

Complete DNA sequence and characterization of a 330-kb VNTR-rich region on chromosome 6q27 that is commonly deleted in ovarian cancer.

We report the complete genomic DNA sequence and the characterization of a 330-kb region on chromosome 6q27 that is often deleted in ovarian cancers. Using computer programs to predict exonic sequences, we isolated four novel genes, HGC6.1-4, as well as the known AF-6 gene. None of the deduced products of the novel genes exhibited significant homology to previously known proteins. We also identified ten microsatellites and 12 different VNTR sequences within the target region. HGC6.3 contained a VNTR within a coding exon, each repeat consisting of 42 nucleotides; the predicted 14-amino-acid consensus unit is MTPTVFSSQHTAGG. At least nine different sizes of this VNTR locus were detected among 20 unrelated DNA samples from caucasians. The polymorphic markers and the transcript map documented here may contribute to identification of novel genes or allelic aberrations associated with the development of ovarian cancers.

Distinct P-cadherin expression in cultured normal human keratinocytes and squamous cell carcinoma cell lines.

Spatially regulated expression of E (epithelial)- and P (placental)-cadherins is crucial for maintaining normal epidermal architecture. In cutaneous squamous cell carcinomas (SCCs), aberrant P-cadherin expression is often observed in "squamoid" cancer cells, whereas E-cadherin expression in cancer cells is generally reduced. Therefore, it is plausible that SCC cells have acquired the ability to express P-cadherin and that P-cadherin plays a role in tumor progression. To address the issue, the in vitro effect of extracellular calcium on differentiation is a good model for investigating P-cadherin in normal and neoplastic skin. With elevations in extracellular calcium, human SCC cell line (DJM-1) cells initiate de novo synthesis of P-cadherin and express P-cadherin on the cell surface, whereas in normal human keratinocytes, P-cadherin expression on the cell surface is enhanced via the translocation from the cytosol to the cell membrane and/or the stabilization of P-cadherin at the cell surface. DJM-1 cells maintain P-cadherin expression on the cell surface at high levels for over 4 days after calcium elevation, whereas normal human keratinocytes cannot sustain cell surface P-cadherin when the cells are cultured in high calcium for more than 2 days. P-cadherin synthesis in DJM-1 cells is regulated at translational levels by extracellular calcium concentrations. SCC cells have the ability to produce P-cadherin by a mechanism not observed in normal keratinocytes, which might relate to the aberrant expression of P-cadherin in SCC of the skin.

Novel V184E MEN1 germline mutation in a Japanese kindred with familial hyperparathyroidism.

We studied the MEN1 gene in a kindred where three patients (the proposita and two of her sons) were affected with hyperparathyroidism. By polymerase chain reaction (PCR)-based direct sequencing of 10 exons of MEN1, a novel germline mutation was identified in the proposita. This mutation, a T-to-A transition at codon 184 in exon 3, predicts an amino acid change from valine to glutamine (V184E). PCR-single-strand conformational polymorphism (PCR-SSCP) analysis of exon 3 followed by sequencing showed the same mutation in the two sons, and in two clinically normal granddaughters of an affected son. Since the T-to-A substitution segregated with the disorder in the kindred except for the granddaughters and it was not detected in 100 alleles from 50 normal individuals, the change observed in MEN1 is not a polymorphism, but causes familial hyperparathyroidism. Thus the two grandchildren with the mutation were diagnosed as presymptomatic carriers.

Peripheral primitive neuroectodermal tumor of the ovary confirmed by CD99 immunostaining, karyotypic analysis, and RT-PCR for EWS/FLI-1 chimeric mRNA.

We report a case of peripheral primitive neuroectodermal tumor (pPNET), which belongs to the PNET/Ewing's sarcoma family, arising in the left ovary of a 29-year-old woman. Microscopically, the tumor was composed of solid nests and sheets of monotonous, primitive, small round cells with a few rosettes, making it difficult to distinguish from small cell carcinoma of the ovary. Immunohistochemically, the tumor cells showed intense cell-membranous immunoreactivity for MIC2 protein (CD99). A short-term cell culture and karyotypic analysis revealed the tumor to possess a balanced t(11;22)(q24;q12) chromosomal translocation that is highly specific for tumors of the PNET/Ewing's sarcoma family. In addition, EWS/FLI-1 chimeric mRNA that originated from the characteristic chromosomal translocation was detected by reverse transcription-polymerase chain reaction. These results confirmed the diagnostic validity of the present tumor being a pPNET, thus raising the possibility that in the past, pPNETs which have arisen in the ovary may have been mistakenly diagnosed as small cell carcinomas of the ovary.

Novel MEN1 gene mutations in familial multiple endocrine neoplasia type 1.

The recent isolation of the gene responsible for multiple endocrine neoplasia type 1 (MEN 1) has enabled direct genetic diagnosis for people with endocrine tumors and family members of affected patients. Although MEN 1 is rarely recognized in the Japanese population compared to its prevalence in Caucasians, we have previously reported a high prevalence of this disease in a limited area (Nagano Prefecture; population, 2.15 million). In this communication, we report mutations of the MEN1 gene in kindreds living in Nagano Prefecture. The absence of a common mutation among these kindreds indicates that the high prevalence of MEN 1 in this area is not due to a regional accumulation of patients descended from a common ancestor. This result implies that the prevalence of MEN 1 in other areas of Japan could also be higher than had been thought.

Recurrent pemphigus vulgaris limited to the surgical area after mastectomy.

A 45-year-old Japanese female presented with bullae and erosions on the trunk in December 1987. The histologic findings revealed a suprabasal cleft and acantholytic cells. Immunofluorescence staining showed IgG autoantibodies in the intercellular spaces. With a working diagnosis of pemphigus vulgaris, she was treated successfully with low dosage prednisolone. Adenocarcinoma of the right breast was found in March 1994, and she received a mastectomy followed by breast reconstruction that used a transverse rectus abdominis muscle flap. Six months later, vesicobullous eruptions developed and were limited to the surgical area (right chest and abdomen). The histopathologic and direct immunofluorescence findings were consistent with pemphigus vulgaris. Although these skin lesions improved with high dosages of prednisolone, she died of multiple metastases due to the breast cancer.

Complete genomic structure DNA polymorphisms, and alternative splicing of the human AF-6 gene.

In our previous work, detailed deletion mapping of ovarian cancers indicated that a 300-kb region of chromosome 6q27 was likely to contain one or more putative tumor suppressor genes associated with development of this type of cancer. DNA sequencing in the region disclosed the presence of AF-6, a gene that had been identified as the ALL-1 fusion partner involved in acute myeloid leukemias with t(6;11)(q27;q23) translocations. In the work reported here, we determined the complete genomic sequence of the AF-6 gene, including exon-intron boundaries, and found six DNA polymorphisms. One of them, an insertion/deletion polymorphism, determined the presence or absence of seven amino acids in the AF-6 product. We also identified two alternatively spliced forms of the gene; the two novel transcripts would encode additional C-terminal peptides in comparison to the reported protein. Sequencing of seven cosmid clones that covered the entire gene revealed 32 exons (not including one exon involved in the insertion/deletion polymorphism), spanning approximately 140 kb of genomic DNA. These results may contribute to an understanding of the mechanism causing chromosomal translocations in leukemic cells.

MDM2 protein overexpression inhibits apoptosis of TF-1 granulocyte-macrophage colony-stimulating factor-dependent acute myeloblastic leukemia cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth factor for acute myeloblastic leukemia (AML) cells. Murine double minute 2 (MDM2) oncoprotein, a potent inhibitor of wild-type p53 (wtp53), can function both to induce cell proliferation and enhance cell survival, and is frequently overexpressed in leukemias. Therefore, we focused on the importance of MDM2 protein in GM-CSF-dependent versus GM-CSF- independent growth of AML cells. The TF-1 AML cell line, which has both wtp53 and mutant p53 genes, showed GM-CSF-dependent growth; deprivation of GM-CSF resulted in G1 growth arrest and apoptosis. MDM2 mRNA and protein were highly expressed in proliferating TF-1 cells in the presence of GM-CSF and decreased significantly with deprivation of GM-CSF. In contrast, p53 protein increased with GM-CSF deprivation. Ectopic overexpression of MDM2 in TF-1 AML cells conferred resistance to GM-CSF deprivation, and is associated with decreased p53 protein expression. Moreover, a variant of TF-1 cells that grows in a GM-CSF-independent fashion also expressed high levels of MDM2 and low levels of p53. These results suggest that GM-CSF-independent growth of AML cells is associated with overexpression of MDM2 protein and related modulation of p53 expression.

Membranous and soluble forms of Fas antigen in cutaneous lupus erythematosus.

The role of Fas-mediated apoptosis in cutaneous lupus erythematosus (LE) is still unclear, although the Fas/FasL system has been investigated in autoimmune diseases in relation to impaired apoptosis. In order to elucidate the connections between acute cutaneous LE (ACLE) and chronic cutaneous LE (CCLE), we determined the expression of membranous Fas antigen (mFas) on peripheral blood mononuclear cells (PBMC) by flow cytometry and the levels of the soluble form of the Fas antigen (sFas) in sera. The ratio and the mean fluorescence intensity of mFas were much higher in ACLE patients than in others, including patients with CCLE and atopic dermatitis and normal healthy controls. The levels of sFas in ACLE and CCLE patients were also elevated, and there was a significant increase in sFas levels in ACLE patients over that in CCLE patients. Immunohistochemical studies revealed that Fas antigen was predominantly expressed on infiltrating cells around blood vessels and appendages in ACLE and CCLE patients. Based on these findings, it is suggested that the expression of Fas antigen is closely associated with the activation of circulating lymphocytes, especially in ACLE patients, but is not directly associated with keratinocyte damage.

Mutational analysis of the RET proto-oncogene in 71 Japanese patients with medullary thyroid carcinoma.

Multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinomas (FMTC) are caused by germline mutations in the RET proto-oncogene. To investigate the spectrum of RET mutations among Japanese patients, we screened the RET gene in 71 patients with thyroid carcinomas. The panel included representatives of 44 families carrying FMTC or MEN2, 22 sporadic medullary thyroid carcinomas (MTCs), and five MTCs without familial information. Mutations in nucleotide sequences encoding one of three specific cysteine residues in the extracellular domain of the RET protein were found in 33 of the 34 MEN2A patients and in five of the six FMTC patients examined. A mutation at codon 918, causing the substitution of threonine for methionine in the tyrosine kinase domain of the protein, was found in germline DNAs of all four patients with MEN2B and in two of the 22 patients with sporadic MTCs; codon 918 was mutated somatically in tumor DNAs from three other sporadic cases. Germline mutations of codon 768, GAG to GAC (Glu to Asp), were detected in one FMTC, in one patient with sporadic MTC, and in one of the patients without familial information. Two somatic mutations, an Asp to Gly substitution at codon 631 and a Cys to Arg substitution at codon 634, had not been reported previously. Of five germline mutations found among the 22 sporadic cases, four were confirmed as de novo mutations since in each case neither parent carried the mutation. As nearly one-fourth of the patients with sporadic MTCs carried germline mutations and 50% of their children are expected to develop MTC and other endocrine tumors, these results indicated the importance of careful clinical surveillance of family members of any patient with MTC.

Roles of E- and P-cadherin in the human skin.

The Ca(2+)-dependent cell-cell adhesion molecules, termed cadherins, are subdivided into several subclasses. E (epithelial)- and P (placental)-cadherins are involved in the selective adhesion of epidermal cells. E-cadherin is expressed on the cell surfaces of all epidermal layers and P-cadherin is expressed only on the surfaces of basal cells. Ultrastructural studies have shown that E-cadherin is distributed on the plasma membranes of keratinocytes with a condensation in the intercellular space of the desmosomes. During human skin development P-cadherin expression is spatiotemporally controlled and closely related to the segregation of basal layers as well as to the arrangement of epidermal cells into eccrine ducts. In human skin diseases E-cadherin expression is markedly reduced on the acantholytic cells of tissues in pemphigus and Darier's disease. Cell adhesion molecules are now considered to play a significant role in the cellular connections of cancer and metastatic cells. Reduced expression of E-cadherin on invasive neoplastic cells has been demonstrated for cancers of the stomach, liver, breast, and several other organs. This reduced or unstable expression of E- and P-cadherin is observed in squamous cell carcinoma, malignant melanoma, and Paget's disease, but cadherin expression is conserved in basal cell carcinoma. Keratinocytes cultured in high calcium produce much more intense immunofluorescence of intercellular E- and P-cadherin than those cells grown in low calcium. E-cadherins on the plasma membrane of the keratinocytes are shifted to desmosomes under physiological conditions, and therein may express an adhesion function in association with other desmosomal cadherins. Soluble E-cadherins in sera are elevated in various skin diseases including bullous pemphigoid, pemphigus vulgaris, and psoriasis, but not in patients with burns. Markedly high levels in soluble E-cadherin are demonstrated in patients with metastatic cancers.

E- and P-cadherin expression in tumor tissues and soluble E-cadherin levels in sera of patients with skin cancer.

The expression pattern of epithelial (E)- and placental (P)-calcium-dependent cell-cell adhesion molecules was examined immunohistochemically in various skin tumors. E- and P-cadherin expression was preserved in nodular and superficial types of basal cell carcinoma (BCC). In well-differentiated squamous cell carcinoma (SCC), E-cadherin on the cell surface of the tumor was reduced but expression of P-cadherin was preserved more frequently at the peripheral sites of the tumor than in the central sites of the tumor. Paget's cells and melanoma cells did not express E- or P-cadherins in the nest of the epidermis. Immunoreactive E-cadherin levels in sera were significantly elevated in patients with invasive Paget's disease, metastatic malignant melanoma and severe types of psoriasis and atopic dermatitis when compared with those of normal controls. Reduced or loss of cadherin in localized tumor cells may be correlated with the proliferation, and the level of soluble E-cadherin in circulation may be a marker in the extent of damaged skin by tumor and/or inflammation.