Microvessel density, lymphovascular density, and lymphovascular invasion in primary cutaneous melanoma-correlation with histopathologic prognosticators and BRAF status.

The relationship between microvessel density (MVD), lymphovascular density (LVD), and lymphovascular invasion (LVI) in primary cutaneous melanoma (PCM) remains unclear. Given this, a total of 102 PCMs were assessed for MVD (vascular endothelial growth factor receptor 2 and Endocan), LVD (D2-40), and LVI (immunostaining with D2-40/S-100 and hematoxylin and eosin); tumoral S-100A13, vascular endothelial growth factor receptor 2, and Endocan; and BRAF status. LVD was associated with MVD (P = .01). MVD was higher in PCMs with depth greater than or equal to 2 mm and ulceration (P = .04, .05), whereas LVD was higher in PCMs with depth greater than or equal to 2 mm and mitoses (P = .03, .02). After adjusting for MVD and LVD, only ulceration was associated with LVI (P < .02). A BRAF mutation was seen in 30.4% cases, and when present, both LVD and host response (P = .0008 and .04, respectively) were significantly associated with MVD. Immunostaining with S-100A13 was noted in 99% of cases and a significant association noted only with ulceration (P = .05). Immunostaining increased LVI positivity (46.5% versus 4.9% by hematoxylin and eosin, P < .0001). MVD and LVD are not associated with LVI, appear to be closely related with each other, and are associated with select markers of poor prognosticative value. The association between a host response and LVD and MVD in PCMs with a BRAF mutation suggests that they exhibit potential for strategizing immunotherapies.

Severe hypoglycemia and cognitive decline in older people with type 2 diabetes: the Edinburgh type 2 diabetes study.

OBJECTIVE: People with type 2 diabetes are at increased risk of age-related cognitive decline and dementia. Hypoglycemia is a candidate risk factor, but the direction of association between episodes of severe hypoglycemia and cognitive decline in type 2 diabetes remains uncertain. RESEARCH DESIGN AND METHODS: In the Edinburgh Type 2 Diabetes Study, cognitive function was assessed in 831 adults with type 2 diabetes (aged 60-75 years) at baseline and after 4 years. Scores on seven neuropsychological tests were combined into a standardized general ability factor g. Self-reported history of severe hypoglycemia at baseline (history of hypoglycemia) and at follow-up (incident hypoglycemia) was recorded. RESULTS: A history of hypoglycemia was reported by 9.3% of subjects, and 10.2% reported incident hypoglycemia. Incident hypoglycemia was associated with poorer cognitive ability at baseline (age- and sex-adjusted odds ratio for lowest tertile of g 2.04 [95% CI 1.25-3.31], P = 0.004). Both history of hypoglycemia and incident hypoglycemia were also associated with greater cognitive decline during follow-up (mean follow-up g adjusted for age, sex, and baseline g -0.25 vs. 0.03 [P = 0.02] and -0.28 vs. 0.04 [P = 0.01], respectively), including after addition of vascular risk factors and cardiovascular and microvascular disease to the models (-0.23 vs. 0.03 [P = 0.04] and -0.21 vs. 0.05 [P = 0.03], respectively). CONCLUSIONS: The relationship between cognitive impairment and hypoglycemia appeared complex, with severe hypoglycemia associated with both poorer initial cognitive ability and accelerated cognitive decline.

Immunophenotypic shift of CD4 and CD8 antigen expression in primary cutaneous T-cell lymphomas: a clinicopathologic study of three cases.

Primary cutaneous T-cell lymphomas (CTCL) comprise a heterogeneous group of neoplasms with diverse clinical behavior. Mycosis fungoides (MF) is the most common type of CTCL. Immunophenotypical shift during progression of the disease is a rare event and its significance is unknown. We present three primary CTCL cases that showed an immunophenotypical shift and poor prognosis. Conventional hematoxylin/eosin and immunohistochemical-stained sections were examined in all the cases. Molecular analysis for rearrangement of the T-cell receptor (TCR) gene was performed in two cases. One case was classified as MF, while the other two lacked epidermotropism, and were considered primary cutaneous peripheral T-cell lymphoma (PTCL), NOS. Two cases were CD3+/CD4+ and one case was CD3+/CD8+ at diagnosis. The first two patients suffered many relapses and eventually, new CTCL lesions with a CD3+/CD8+ phenotype were observed. Both cases revealed identical clonal TCR rearrangements on the initial and late lesions, supporting the interpretation of a single clonal proliferation with different phenotypes. The third case progressed with skin recurrences and pulmonary lesions with a predominant CD3+/CD4+/CD8- phenotype. All cases manifested poor prognosis and two patients died of lymphoma. Immunophenotypical shift between CD4 and CD8 in CTCL seems to be a rare phenomenon that may be associated with disease progression.

The influence of DNA repair on neurological degeneration, cachexia, skin cancer and internal neoplasms: autopsy report of four xeroderma pigmentosum patients (XP-A, XP-C and XP-D).

BACKGROUND: To investigate the association of DNA nucleotide excision repair (NER) defects with neurological degeneration, cachexia and cancer, we performed autopsies on 4 adult xeroderma pigmentosum (XP) patients with different clinical features and defects in NER complementation groups XP-A, XP-C or XP-D. RESULTS: The XP-A (XP12BE) and XP-D (XP18BE) patients exhibited progressive neurological deterioration with sensorineural hearing loss. The clinical spectrum encompassed severe cachexia in the XP-A (XP12BE) patient, numerous skin cancers in the XP-A and two XP-C (XP24BE and XP1BE) patients and only few skin cancers in the XP-D patient. Two XP-C patients developed internal neoplasms including glioblastoma in XP24BE and uterine adenocarcinoma in XP1BE. At autopsy, the brains of the 44 yr XP-A and the 45 yr XP-D patients were profoundly atrophic and characterized microscopically by diffuse neuronal loss, myelin pallor and gliosis. Unlike the XP-A patient, the XP-D patient had a thickened calvarium, and the brain showed vacuolization of the neuropil in the cerebrum, cerebellum and brainstem, and patchy Purkinje cell loss. Axonal neuropathy and chronic denervation atrophy of the skeletal muscles were observed in the XP-A patient, but not in the XP-D patient. CONCLUSIONS: These clinical manifestations and autopsy findings indicate advanced involvement of the central and peripheral nervous system. Despite similar defects in DNA repair, different clinicopathological phenotypes are seen in the four cases, and therefore distinct patterns of neurodegeneration characterize XP-D, XP-A and XP-C patients.

Gastrointestinal histopathology in chronic granulomatous disease: a study of 87 patients.

Gastrointestinal (GI) involvement in chronic granulomatous disease (CGD), a rare genetic immunodeficiency, mimics other inflammatory bowel diseases. We report GI pathology from 87 CGD patients seen at the NIH Clinical Center, with vague to severe clinical symptoms, in whom biopsies (313) had been evaluated (esophagus [23], stomach [71], small bowel [52] including duodenum [39], ileum [12], and jejunum [1], and colon [167]). Additionally reviewed was GI tissue from 15 autopsies. In our patient cohort, the mean age was 22 years (age range, 3 to 44 y; 2:1 male to female ratio). There were pathologic changes in 83/87 (95%) patients; with colon being the most commonly involved site and esophagus the least. There were microgranulomas in 53/87 (61%), pigmented macrophages in 64/87 (74%), tissue eosinophilia in 31/87 (36%), and chronic and/or acute inflammation in 57/87 (66%) patients. A subset of patients had villous shortening in the duodenum (8/39) and ileum (5/12). We identify microgranulomas in 76/167 (46%) colon, 12/52 (23%) small bowel, and 4/71 (6%) gastric biopsies; pigmented macrophages in 109/167 (65%) colon and 7/52 (13%) small bowel biopsies and 14/15 autopsies; chronic and/or acute inflammation in 97/167 (58%) colon, 13/52 (25%) small bowel, 42/71 (59%) gastric, and 5/23 (22%) esophageal biopsies; tissue eosinophilia in 43/167 (26%) colon, 7/52 (13%) small bowel, and 2/71 (3%) gastric biopsies. Only 4/87 (5%) patients had normal histology. No infectious etiology was identified in the majority of inflammatory lesions. We found that mild to severe GI pathology was common in CGD. In addition, microgranulomas, pigmented macrophages, and eosinophilia are not associated with acute (neutrophilic) inflammation.

NY-ESO-1 expression in synovial sarcoma and other mesenchymal tumors: significance for NY-ESO-1-based targeted therapy and differential diagnosis.

A promising targeted therapy against NY-ESO-1 (CTAG 1B) using genetically modified T-cells in synovial sarcomas was recently demonstrated in a clinical trial at the NCI. To investigate the role of NY-ESO-1 immunohistochemistry in patient selection and gain better insight into the incidence of NY-ESO-1 expression in synovial sarcomas and other mesenchymal tumors, we evaluated NY-ESO-1 expression by immunohistochemistry in 417 tumors. This collection of samples included: 50 SS18/SSX1/2 fusion positive synovial sarcomas, 155 gastrointestinal stromal tumors (GIST), 135 other spindle cell sarcomas as well as 77 other sarcomas (chondrosarcoma, osteosarcoma, dedifferentiated liposarcoma, alveolar soft part sarcoma, rhabdomyosarcoma, angiosarcoma, malignant mesothelioma, and Ewing's sarcoma). We report that 76% of synovial sarcomas expressed NY-ESO-1 in a strong and diffuse pattern (2-3+, >50-70% of tumor cells). In contrast, only rare cases of other spindle cell mesenchymal tumor expressed NY-ESO-1 (GIST (2/155), malignant peripheral nerve sheath tumors (1/34), and dermatofibrosarcoma protuberans (2/20)). Individual cases of other sarcomas (angiosarcoma, malignant mesothelioma, chondrosarcoma, osteosarcoma, dedifferentiated liposarcoma, alveolar soft part sarcoma, and Ewing's sarcoma) were positive for NY-ESO-1. However, no positive cases were identified amongst our cohort of leiomyosarcomas (0/24), hemangiopericytoma/solitary fibrous tumors (0/40), and cellular schwannomas (0/17). In summary, we find that NY-ESO-1 is strongly and diffusely expressed in a majority of synovial sarcomas, but only rarely in other mesenchymal lesions. Beyond its role in patient selection for targeted therapy, immunohistochemistry for NY-ESO-1 may be diagnostically useful for the distinction of synovial sarcoma from other spindle cell neoplasms.

KBA62 and PNL2: 2 new melanoma markers-immunohistochemical analysis of 1563 tumors including metastatic, desmoplastic, and mucosal melanomas and their mimics.

Identification of metastatic melanoma can be difficult because of its considerable morphologic variation and mimicry of a wide variety of other tumors. The more melanoma-specific melanoma markers, MelanA/MART-1, HMB45, and tyrosinase, used in addition to S100 protein, all have limitations in sensitivity and specificity. In this study, we evaluated 2 new melanoma markers, monoclonal antibodies KBA62 and PNL2 to yet unidentified antigens, using a large panel of metastatic melanomas (n=214), desmoplastic melanomas (n=34), gastrointestinal mucosal melanomas (n=54), benign nevi (n=27), clear cell sarcomas (n=16), and nonmelanocytic tumors (n=1218). Immunoreactivity for KBA62 and PNL2 was found in all pigmented nevi and in 86% and 90% of metastatic melanomas, respectively. Mucosal melanomas showed a similar rate of PNL2 immunoreactivity but somewhat less frequent KBA62 positivity (72%). In addition, KBA62 was found to be a sensitive diagnostic marker for desmoplastic melanoma (28 of 34; 82%), whereas PNL2 was only rarely positive (2 of 34; 6%). KBA62-positive normal tissues included pericytes, vascular and parenchymal smooth muscles, and basal cells of complex epithelia, including myoepithelia, whereas PNL2 labeled only melanocytes and neutrophils. Among nonmelanocytic tumors, those that were KBA62 positive were nodular fasciitis, leiomyoma and leiomyosarcoma, gastrointestinal stromal tumors, benign and malignant nerve sheath tumors, synovial sarcoma, and subsets of various carcinomas, especially those with squamous cell/stratified epithelial differentiation. PNL2 positivity in nonmelanocytic tumors was more restricted but occurred consistently in angiomyolipoma and other perivascular epitheloid cell tumor and in chronic myeloid leukemia tissue infiltrates. KBA62 may assist in the identification of desmoplastic melanomas, but its widespread occurrence in nonmelanomas limits utility. PNL2 is highly specific for melanomas but lacks reactivity with desmoplastic melanomas. It is also an excellent supplementary marker for perivascular epitheloid cell tumor at various sites.

Calcium Intake among Myanmar Residing in Bago, Kayin, and Yangon Areas.

A cross-sectional survey combined with 24-hour dietary recall and food diary was undertaken to assess the calcium intake of the Myanmar population. The study was conducted from November 2003 to October 2005. A total of 886 subjects of both sexes aged above 2 years from three States and Divisions (Bago, Kayin, and Yangon) of Myanmar were included in the study. The major measures were mean daily calcium intake (mg/day) and major sources of calcium in the diet. Overall mean calcium intake was 197+13mg/day (2-9 years), 421+2mg/day (10-19 years), 399+21 mg/day (20-49 years), and 383+25mg/day (>50 years) for males, while the corresponding values for females were 207+17 mg/day, 366+19 mg/day, 387+16 mg/day, and 327 +19 mg/day. Calcium intake was less than 80% of the recommended dietary allowances (RDA) for Myanmar for ages 2-9 years and 10-29 years in all the study areas, and for the 50 years and above age group in Yangon. Fish paste was found to be the major source of calcium. Milk and milk products contributed very little to total calcium intake, contributing 2.1% for residents in Yangon, 5.1% in Pa-an and none in Bago. Consumption of calcium rich foods, particularly milk and milk products, should be encouraged among the Myanmar people. Towards this end, appropriate nutrition education materials should be developed for promotional purposes.

ADAMTSL3/punctin-2, a gene frequently mutated in colorectal tumors, is widely expressed in normal and malignant epithelial cells, vascular endothelial cells and other cell types, and its mRNA is reduced in colon cancer.

ADAMTSL3/punctin-2 is a secreted glycoprotein that resembles the ADAMTS proteases. Recently, identification of frequent ADAMTSL3 mutations in colorectal cancer suggested it might have a regulatory role in cellular homeostasis in colorectal epithelium or in pathways to colorectal malignancy. Here, we used in situ hybridization to validate ADAMTSL3 antibodies for IHC of a variety of normal and malignant tissues, including colon cancer. Quantitative real-time PCR (RTQ-PCR) was used to compare mRNA expression levels in colon carcinoma (n = 10) and adjacent normal colon. ADAMTSL3 is expressed in epithelial cells of the colon, fallopian tube, skin, breast, prostate, epididymis, liver, pancreatic islets and bile ducts, as well as by vascular endothelial cells, smooth muscle cells, fibroblasts, cortical and ganglionic neurons and cardiac myocytes. Malignant epithelial cells in colon cancer, as well as breast, prostate, renal and skin tumors expressed ADAMTSL3. Normal colon showed stronger immunostaining of surface than basal crypt epithelium and staining of a variety of cells within the lamina propria and submucosa. Colon carcinomas demonstrated weaker staining in tumor cells than normal colon epithelium and weak stromal staining. RTQ-PCR comparison of ADAMTSL3 mRNA in colon carcinoma and adjacent normal colon demonstrated a statistically significant reduction in the tumors, possibly reflecting their decreased stromal content and lack of complete differentiation of tumor samples. The major findings of these studies are that ADAMTSL3 is expressed in numerous tissues, suggesting a broader regulatory role than in colorectal epithelium alone, and that colorectal cancer has both structural mutations as well as decreased expression of ADAMTSL3.

Differential expression of claudin-2 in normal human tissues and gastrointestinal carcinomas.

Claudins are involved in the formation of tight junctions in epithelial and endothelial cells. Claudins form a family of 24 members displaying organ- and tissue-specific patterns of expression. In the present study, we evaluated the specificity of the claudin-2 expression in various normal human tissues and gastrointestinal cancers by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. In 14 various normal tissues, claudin-2 mRNA was expressed in the kidney, liver, pancreas, stomach, and small intestine; the highest level of which was detected in the kidney. Colorectal cancers (CRCs) expressed claudin-2 mRNA at high levels. Immunohistochemical analysis of claudin-2 in 146 gastric cancers (GCs) and 99 CRCs demonstrated claudin-2 expression in 2.1% of GCs and 25.3% of CRCs, respectively. There was no obvious correlation between claudin-2 expression and clinicopathological parameters of CRCs. These results suggest that the expression of claudin-2 may involve organ specificity, and increased expression of claudin-2 may participate in colorectal carcinogenesis.

Genes involved in invasion and metastasis of gastric cancer identified by array-based hybridization and serial analysis of gene expression.

Gastric cancer (GC) is still a serious health problem and remains the second most common type of fatal cancer worldwide. Comprehensive gene expression analyses may be useful to identify genes associated with invasion/metastasis in GC. Among them, array-based hybridization and serial analysis of gene expression (SAGE) are currently the most common approaches. Over the past 3 years, several large-scale gene expression studies with array-based hybridization and SAGE have been performed and several genes have been identified. This review describes genes associated with invasion/metastasis in GC which have been identified by array-based hybridization and SAGE. We compared the expression levels of the genes identified by array-based hybridization with our SAGE data. In addition, expression of the candidate genes obtained by SAGE was further investigated by quantitative RT-PCR of 40 GC samples. MIA and GW112 were overexpressed in 10 (25%) and 22 (55%) of 40 GC samples, and the overexpression of these two genes was associated with tumor stage, respectively. We also discuss the significance of HMGB1/amphoterin in invasion and metastasis of GC.

No evidence of correlation between the single nucleotide polymorphism of DNMT3B promoter and gastric cancer risk in a Japanese population.

DNA methylation is a major epigenetic modification in humans, and aberrant DNA methylation may play an important role in the development of various cancers through the silencing of some tumor suppressor genes. DNMT3B is required for the establishment and maintenance of genomic methylation patterns. The -149 C/T single nucleotide polymorphism (SNP) in the promoter of DNMT3B has been identified. This SNP influences DNMT3B promoter function, with the T allele having greater activity than the C allele, and is associated with an increased risk of lung cancer. The purpose of our study was to investigate the correlation between the DNMT3B promoter polymorphism and the development and progression of gastric cancer. We analyzed the SNP of the DNMT3B promoter in 152 gastric cancer patients and 247 controls from a Japanese population using PCR-RFLP and sequencing analysis, and also studied the association between the genotypes of DNMT3B and clinicopathological parameters among cases. Allelic difference was not found between gastric cancer patients and control subjects at the target site, -149 bp from the transcriptional start site in the DNMT3B gene promoter. Only the T/T genotype was detected in all gastric cancer patients and control subjects. We concluded that there was no association between SNP of the DNMT3B promoter and gastric cancer risk in a Japanese population.

DNA methylation of genes linked with retinoid signaling in gastric carcinoma: expression of the retinoid acid receptor beta, cellular retinol-binding protein 1, and tazarotene-induced gene 1 genes is associated with DNA methylation.

BACKGROUND: Hypermethylation of CpG islands has been associated with silencing of various tumor suppressor genes, and the retinoid acid receptor beta (RARbeta), cellular retinol-binding protein 1 (CRBP1), and tazarotene-induced gene 1 (TIG1) genes have been associated with retinoic acid signaling. To the authors' knowledge, little is known regarding the involvement of these three genes in gastric carcinoma (GC). In this study, the authors investigated the methylation status of these genes and analyzed the role of their DNA methylation in GC. METHODS: DNA methylation of 3 retinoic acid-associated genes was analyzed in 42 samples of GC from 42 patients and in 8 GC cell lines by methylation-specific polymerase chain reaction (PCR) analysis. The mRNA expression levels for these three genes were measured by quantitative reverse transcription-PCR. RESULTS: In 7 of 8 GC cell lines, the CRBP1 gene was hypermethylated, and CRBP1 transcription was inactive. In 6 of 8 GC cell lines, the TIG1 gene was hypermethylated, and TIG1 transcription was inactive. Treatment with demethylating agent 5-aza-2'-deoxycytidine restored both CRBP1 and TIG1 transcription. DNA methylation of the RARbeta, CRBP1, and TIG1 genes was detected in 15 of 42 GC samples (36%), 14 of 42 GC samples (33%), and 4 of 42 GC samples (10%), respectively, and in 6 of 30 samples (20%), 0 of 30 samples (0%), and 1 of 30 samples (3%) of corresponding nonneoplastic mucosa. None of the 10 normal gastric mucosa samples from young, healthy individuals demonstrated hypermethylation of any of these genes. DNA methylation of each gene was associated significantly with low mRNA expression of the respective gene. Twenty-four of 42 GC samples (57%) demonstrated hypermethylation of at least 1 of the 3 genes. However, no significant, concordant hypermethylation of these genes was observed. CONCLUSIONS: The results suggested that gastric carcinogenesis involves transcriptional inactivation by aberrant DNA methylation of genes related to retinoid signaling.

DNA methylation of genes linked to retinoid signaling in squamous cell carcinoma of the esophagus: DNA methylation of CRBP1 and TIG1 is associated with tumor stage.

Hypermethylation of CpG islands is associated with the silencing of various tumor suppressor genes. Retinoic acid receptor-beta (RAR-beta), cellular retinol-binding protein 1 (CRBP1), and tazarotene-induced gene 1 (TIG1) have been linked to retinoic acid signaling. Little is known about the involvement of these three genes in esophageal squamous cell carcinoma (ESCC). In this study, we investigated the methylation status of these genes and analyzed the role of methylation of their DNA in ESCC. Methylation-specific polymerase chain reaction (PCR) was performed to study the methylation of CpG islands in 28 ESCC (stages I, II, and III) and 10 samples of corresponding non-neoplastic mucosa. The mRNA expression levels of the three genes were measured by quantitative reverse transcription-PCR. DNA hypermethylation of RAR-beta was found in seven (25.0%) of the 28 ESCC, of CRBP1 in five (17.9%), and of TIG1 in five (17.9%). DNA methylation of RAR-beta was identified in one of 10 samples of corresponding non-neoplastic mucosa (10.0%), whereas no DNA methylation of CRBP1 or TIG1 was detected. In total, at least one of the three genes was hypermethylated in 12 (42.9%) ESCC. Reduced expression of RAR-beta, CRBP1, and TIG1 was found in 14 (50.0%), 15 (53.6%), and 13 (46.4%) ESCC, respectively. DNA methylation of each gene was significantly associated with reduced expression of the respective mRNA. No correlation was found between the DNA methylation status of RAR-beta and clinicopathological factors such as depth of invasion, lymph node metastasis, or tumor stage. In contrast, DNA methylation of both CRBP1 and TIG1 was observed only in stage III ESCC. These results show that inactivation of the retinoic acid signaling-associated genes RAR-beta, CRBP1, and TIG1 by DNA methylation occurs frequently in ESCC.

Expression and localization of Reg IV in human neoplastic and non-neoplastic tissues: Reg IV expression is associated with intestinal and neuroendocrine differentiation in gastric adenocarcinoma.

Regenerating islet-derived family, member 4 (Reg IV) is a candidate marker for cancer and inflammatory bowel disease. In the present study, immunohistochemical analysis of Reg IV was performed in various human neoplastic (n = 289) and non-neoplastic tissues. In the stomach, foveolar epithelium was negative for Reg IV, whereas goblet cells of intestinal metaplasia and neuroendocrine cells at the base of intestinal metaplasia expressed Reg IV. Neuroendocrine cells of the small intestine and colon showed strong expression of Reg IV, whereas goblet cells of the small intestine and colon showed weak or no expression of Reg IV. Insulin-producing beta cells of the endocrine pancreas were positive for Reg IV. Among 143 gastric adenocarcinomas, Reg IV expression was detected in 42 (29.4%) and was associated with both the intestinal mucin phenotype and neuroendocrine differentiation. No association was found between Reg IV expression and clinical characteristics such as tumour stage and patient prognosis. Of 36 colorectal adenocarcinomas, 13 (36.1%) were positive for Reg IV, which was associated with tumour stage (p = 0.0379, Fisher's exact test). Expression of Reg IV was detected in 14 (93.3%) of 15 colorectal carcinoid tumours. Reg IV expression was also detected in 5 (21.7%) of 23 ductal adenocarcinomas of the pancreas. In contrast, lung cancers (n = 30) and breast cancers (n = 30) did not express Reg IV. This is the first immunohistochemical analysis of the expression and distribution of Reg IV protein in human tumours. These data suggest that Reg IV is expressed by gastrointestinal and pancreatic tumours, including adenocarcinomas and carcinoid tumours, and that Reg IV is associated with intestinal and neuroendocrine differentiation of the stomach and gastric carcinoma.

DNA methylation profiles of differentiated-type gastric carcinomas with distinct mucin phenotypes.

Gastric carcinomas (GC) are classified into four phenotypes according to mucin expression. Previous studies revealed the association of distinct genetic profiles in GC with mucin phenotypic expression; however, the roles of epigenetic changes, such as DNA methylation, are poorly understood. We examined whether the phenotypic expression of GC was associated with DNA methylation of hMLH1, MGMT, p16(INK4a), RAR-beta or CDH1. Expression of HGM, M-GGMC-1, MUC2, and CD10 was analyzed immunohistochemically in 33 advanced GC with differentiated histology. HGM was expressed in 14 (42.4%) cases, M-GGMC-1 in five (15.2%) cases, MUC2 in 15 (45.5%) cases and CD10 in 18 (54.5%) cases. DNA methylation was detected in five (15.2%) cases for hMLH1, 11 (33.3%) cases for MGMT, 13 (39.4%) cases for p16(INK4a), 17 (51.5%) cases for RAR-beta and 14 (42.4%) cases for CDH1 by bisulfite-polymerase chain reaction and methylation-specific polymerase chain reaction. DNA methylation of hMLH1 occurred more frequently in MUC2-negative GC than in MUC2-positive GC (P = 0.0488, Fisher's exact test). In contrast, MGMT was more frequently methylated in MUC2-positive GC than in MUC2-negative GC (P = 0.0078, Fisher's exact test). There was no correlation between gastric or intestinal-markers and methylation of the p16(INK4a), RAR-beta and CDH1 genes. These results indicate that DNA methylation of specific genes, such as hMLH1 and MGMT, may be involved partly in the distinct phenotypic expression of GC.

Molecular-pathological prognostic factors of gastric cancer: a review.

Invasion and metastasis are critical determinants of cancer morbidity. Genes and molecules participating in these steps must be regarded as potential prognostic factors. Growth factors and their receptors, cell-cycle regulators, cell-adhesion molecules and matrix-degrading enzymes are those to be used as prognostic factors, including epidermal growth factor (EGF), EGF receptor, K-sam, HER-2, interleukin (IL)-8, vascular endothelial growth factor (VEGF), cyclin E, p27, E-cadherin, CD44v6, matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Alterations in epigenetics, such as aberrant DNA methylation and histone modification that are, in part, associated with the tumor progression of gastric cancer, can be candidate prognostic factors. The number of methylated genes may serve as a marker of tumor progression. Genetic polymorphism not only affects cancer susceptibility but also influences malignant phenotype; examples include single-nucleotide polymorphism in the HER-2 and MMP-9 genes. Comprehensive gene expression analyses are useful to search for novel genes related to invasion and metastasis and potential prognostic factors. Serial analysis of gene expression (SAGE) has identified several these genes, such as CDH17, APOE, FUS, COL1A1, COL1A2, GW112, and MIA. Overexpression of MIA is found to be associated with poor prognosis. Microarray analysis has great potential for identifying the characteristics of individual cancers, from the view point of gene expression profiles. A combination of these examinations can not only foretell a patient's prognosis but can also give information directly connected with personalized cancer medicine and prevention.

Histone H3 acetylation is associated with reduced p21(WAF1/CIP1) expression by gastric carcinoma.

Histone acetylation appears to play an important role in transcriptional regulation. Inactivation of chromatin by histone deacetylation is involved in the transcriptional repression of several tumour suppressor genes, including p21(WAF1/CIP1). However, the in vivo status of histone acetylation in human cancers, including gastric carcinoma, is not well understood. This study shows that histone H3 in the p21(WAF1/CIP1) promoter region is hypoacetylated and that this hypoacetylation is associated with reduced p21(WAF1/CIP1) expression in gastric carcinoma specimens. Chromatin immunoprecipitation assays revealed that histone H3 was hypoacetylated in the p21(WAF1/CIP1) promoter and coding regions in 10 (34.5%) and 10 (34.5%) of 29 gastric carcinoma specimens, respectively. Hypoacetylation of histone H4 in the p21(WAF1/CIP1) promoter and coding regions was observed in 6 (20.7%) and 16 (55.2%) of 29 gastric carcinoma specimens, respectively. p21(WAF1/CIP1) mRNA levels were associated with histone H3 acetylation status in the p21(WAF1/CIP1) promoter region (p = 0.047) but not p53 mutation status (p = 0.460). In gastric carcinoma cell lines, expression of p21(WAF1/CIP1) protein was induced by trichostatin A, a histone deacetylase inhibitor. This induction was associated with hyperacetylation of histone H3 in the p21(WAF1/CIP1) promoter region. Hyperacetylation of histone H4 in the p21(WAF1/CIP1) promoter region did not appear to be associated with increased expression. Induction of p21(WAF1/CIP1) protein expression was associated with hyperacetylation of histones H3 and H4 in the p21(WAF1/CIP1) coding region. Expression of a dominant-negative mutant of p53 reduced expression of p21(WAF1/CIP1) protein. Histone H4 acetylation in both the promoter and coding regions of the p21(WAF1/CIP1) gene in cells expressing dominant-negative p53 was less than half of that in cells expressing wild-type p53, whereas histone H3 acetylation in both the promoter and coding regions was slightly reduced (by approximately 20%) in cells expressing the dominant-negative p53. These findings provide evidence that alteration of histone acetylation occurs in human cancer tissue specimens such as those from gastric carcinoma.

Gene expression profile of gastric carcinoma: identification of genes and tags potentially involved in invasion, metastasis, and carcinogenesis by serial analysis of gene expression.

Gastric carcinoma (GC) is one of the most common malignancies worldwide. To better understand the genetic basis of this disease, we performed serial analysis of gene expression (SAGE) on four primary GC samples and one associated lymph node metastasis. We obtained a total of 137,706 expressed tags (Gene Expression Omnibus accession number GSE 545, SAGE Hiroshima gastric cancer tissue), including 38,903 that were unique. Comparing tags from our GC libraries containing different stages and different histologies, we found several genes and tags that are potentially involved in invasion, metastasis, and carcinogenesis. Among these, we selected 27 genes and measured mRNA expression levels in an additional 46 GC samples by quantitative reverse transcription-PCR. Frequently overexpressed genes (tumor/normal ratio > 2) were COL1A1 (percentage of cases with overexpression, 78.3%), CDH17 (73.9%), APOC1 (67.4%), COL1A2 (58.7%), YF13H12 (52.2%), CEACAM6 (50.0%), APOE (50.0%), REGIV (47.8%), S100A11 (41.3%), and FUS (41.3%). Among these genes, mRNA expression levels of CDH17 and APOE were associated with depth of tumor invasion (P = 0.0060 and P = 0.0139, respectively), and those of FUS and APOE were associated with degree of lymph node metastasis (P = 0.0416 and P = 0.0006, respectively). In addition, mRNA expression levels of FUS, COL1A1, COL1A2, and APOE were associated with stage (P = 0.0414, P = 0.0156, P = 0.0395, and P = 0.0125, respectively). Quantitative reverse transcription-PCR analysis also showed a high level of REGIV expression (>100 arbitrary units) in 14 of 46 GC samples (30.4%) but not in noncancerous tissues. We detected V5-tagged RegIV protein in the culture media of cells transfected with pcDNA-RegIV-V5 by Western blot. Our results provide a list of candidate genes that are potentially involved in invasion, metastasis, and carcinogenesis of GC. REGIV may serve as a specific biomarker for GC.