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Aims: The mucin phenotype is associated with clinicopathological findings and tumorigenesis in gastric cancer (GC). The aim was to search for a novel marker regulating the intestinal phenotype of GC., Methods and Results: We performed microarray analyses, and GJB6 (encoding connexin 30) was identified as a gene associated with the intestinal phenotype. Immunostaining of connexin 30 in 169 GC cases revealed that 47 (28%) cases were positive for connexin 30, while connexin 30 was negative in nonneoplastic gastric tissue. Connexin 30-negative GC cases showed more advanced T grade, N grade, and tumor stage than connexin 30-positive GC cases. Six (13%) GC cases positive for connexin 30 were histologically of the differentiated type. In addition, the expression of gastric and intestinal phenotypes of GC was examined by immunostaining for MUC5AC, MUC6, MUC2, and CD10. Connexin 30 expression occurred more frequently in the intestinal phenotype (48%) than in other phenotypes (21%) of GC., Conclusion: These results indicate that the expression of connexin 30 is a novel differentiation marker mediating the biological behavior of intestinal phenotype GC., (Copyright © 2010 S. Karger AG, Basel.)

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. We previously performed Serial Analysis of Gene Expression (SAGE) on four primary gastric cancer samples and identified several gastric cancer-specific genes. Of these genes, olfactomedin 4 (OLFM4, also known as GW112 or hGC-1) is a candidate gene for cancer-specific expression. In the present study, we examined the expression and distribution of olfactomedin 4 in CRC by immunohistochemistry. Of the 176 CRC cases, 59 (34%) were positive for cytoplasmic staining of olfactomedin 4. Olfactomedin 4-positive CRC cases showed earlier T classification (P=0.0180), N classification (P=0.0149) and stage (P=0.0144) than olfactomedin 4-negative CRC cases. In the 176 CRC patients, those with olfactomedin 4-positive CRC had a better survival rate than patients with olfactomedin 4-negative CRC (P=0.0092). Multivariate analysis indicated that T classification, M classification and negative olfactomedin 4 expression were independent predictors of survival in patients with CRC. In addition to cytoplasmic staining of olfactomedin 4, stromal staining at the invasive front was observed. In total, 29 (16%) of the 176 CRC cases were positive for stromal olfactomedin 4; however, stromal olfactomedin 4 staining was not correlated with any clinicopathologic characteristic or with patient survival. These results indicate that olfactomedin 4 is a valuable marker for long-term survival in patients with CRC.

Gastric cancer (GC) is 1 of the most common human cancers. Early detection remains the most promising approach to improving long-term survival of patients with GC. We previously performed Serial Analysis of Gene Expression (SAGE) on 4 primary GCs and identified several GC-specific genes including Reg IV. Of these genes, olfactomedin 4 (OLFM4, also known as GW112 or hGC-1) is a candidate gene for cancer-specific expression. In this study, we examined the expression of olfactomedin 4 in human GC by immunohistochemistry. We also assessed serum olfactomedin 4 levels in GC patients by enzyme-linked immunosorbent assay. 94 (56%) of 167 GC cases were positive for olfactomedin 4 by immunostaining. Olfactomedin 4 staining was observed more frequently in stage I/II cases than in stage III/IV cases. The serum olfactomedin 4 concentration in presurgical GC patients (n = 123, mean +/- SE, 36.3 +/- 3.5 ng/mL) was significantly higher than that in healthy individuals (n = 76, 16.6 +/- 1.6 ng/mL). In patients with stage I GC, the sensitivity of serum olfactomedin 4 (25%) and Reg IV (35%) was superior to that of CA19-9 (5%) or CEA (3%). Furthermore, in patients with stage I GC, the combination of olfactomedin 4 and Reg IV elevated the diagnostic sensitivity to 52%. These results suggest that serum olfactomedin 4 is a useful marker for GC and its measurement alone or in combination with Reg IV has utility in the early detection of GC.

Background: The human homologue of Drosophila prune (PRUNE, which encodes h-prune) protein interacts with glycogen synthase kinase 3 and promotes cell motility. The aim of our study was to investigate the impact of immunohistochemically detected h-prune expression on the survival of patients with esophageal squamous cell carcinoma (ESCC)., Methods: Immunohistochemical staining of h-prune was performed for 205 surgically resected specimens of ESCC., Results: In total, 43 (21%) of 205 ESCC cases were positive for h-prune. h-prune-positive ESCC cases showed a more-advanced T stage (P < 0.0001), N stage (P < 0.0001), and tumor stage (P < 0.0001) than h-prune-negative ESCC cases. In the group of 116 stage II and III ESCC cases, recurrence of ESCC was frequently found in h-prune-positive cases. In patients with lung recurrence, the tumors were more likely to be h-prune positive than h-prune negative. Univariate analysis revealed that T stage (P < 0.0001), N stage (P < 0.0001), tumor stage (P < 0.0001), and h-prune staining (P < 0.0001) were significant prognostic factors for survival. Multivariate analysis indicated that N stage (P = 0.0182) and h-prune staining (P < 0.0001) were independent predictors for survival., Conclusions: These results indicate that immunostaining of h-prune is useful to identify patients at high risk for recurrence or poor prognosis associated with ESCC.

Gastric cancer is the fourth most common malignancy in the world, and mortality due to gastric cancer is second only to that from lung cancer. 'Transcriptome dissection' is a detailed analysis of the entire expressed transcripts from a cancer, for the purpose of understanding the precise molecular mechanism of pathogenesis. Serial analysis of gene expression (SAGE) is a suitable technique for performing transcriptome dissection. Gastric cancers of different stages and histology were analyzed on SAGE, and one of the largest gastric cancer SAGE libraries in the world was created (GEO accession number GSE 545). Through SAGE, many candidate genes have been identified as potential diagnostic and therapeutic targets for the treatment of gastric cancer. Regenerating islet-derived family, member 4 (Reg IV) participated in 5-fluorouracil (5-FU) resistance and peritoneal metastasis, and its expression was associated with an intestinal phenotype of gastric cancer and with endocrine differentiation. GW112 expression correlated with advanced tumor stage. Measurement of Reg IV and GW112 levels in sera indicated a sensitivity of 57% for detection of cancer. SPC18 participated in tumor growth and invasion through transforming tumor growth factor-alpha upregulation. Palate, lung, and nasal epithelium carcinoma-associated protein (PLUNC) was a useful marker for gastric hepatoid adenocarcinoma. Expression of SOX9, HOXA10, CDH17, and loss of claudin-18 expression were associated with an intestinal phenotype of gastric cancer. Information obtained from transcriptome dissection greatly contributes to diagnosis and treatment of gastric cancer.

To elucidate the mechanism of radiation-induced cancers, molecular analysis of cancers in atomic-bomb survivors is important. In our study, we developed a custom oligonucleotide array of 208 genes. We analyzed gene expression profiles of gastric cancers (GCs) from atomic-bomb survivors and identified 9 genes with significantly lower expression in GCs from exposed patients than in GCs from nonexposed patients. Among these 9 genes, expression of versican and osteonectin was investigated in greater detail using immunohistochemistry in 116 GCs from 64 exposed and 52 nonexposed patients who developed GC after the bombing. In the Stage I/II GCs, the clinicopathologic, phenotypic and proliferative characteristics of GCs from exposed and nonexposed patients did not differ significantly; however, versican and osteonectin were expressed at much lower levels in the area of tumor-associated stroma of exposed patients than in nonexposed patients (p = 0.026 and p = 0.024, respectively). These results suggest that the characteristics of tumor-associated stromal cells differ between GCs from exposed and nonexposed patients.

Regenerating islet-derived family, member 4 (Reg IV) is a candidate marker for cancer and inflammatory bowel disease and is associated with neuroendocrine and intestinal differentiation. We have reported that 14% of prostate cancer (PCa) cases are positive for Reg IV by immunohistochemistry. In the present study, we performed immunohistochemical analysis of Reg IV in other major urological cancers, including 101 renal cell carcinoma (RCC), and 95 urothelial carcinoma (UC) of urinary bladder by immunohistochemistry. We also investigated neuroendocrine differentiation by chromogranin A and synaptophysin staining along with intestinal differentiation by MUC2 staining. Immunohistochemical analysis of Reg IV revealed no expression of Reg IV in RCC, and only one case (1%) of UC expressed Reg IV. Neither neuroendocrine nor intestinal differentiation was found in RCC. Among 95 UC cases, neuroendocrine differentiation was detected in 13 cases (14%), and intestinal differentiation was observed in 33 cases (35%). In one Reg IV-positive UC case, MUC2 staining was observed. Since Reg IV expression was frequently found in PCa, we also measured Reg IV levels in sera from patients with PCa by enzyme-linked immunosorbent assay. The serum Reg IV concentration in PCa patients (n=38, mean +/- SE, 1.69+/-0.16 ng/ml) was significantly higher than that in control individuals (n=40, 1.28+/-0.11 ng/ml, P=0.0199, Mann-Whitney U test). The sensitivity and specificity for detection of PCa were 34% (13/38) and 90% (36/40), respectively. These results suggest that among major urologic cancers, Reg IV is expressed frequently in PCa, and that serum Reg IV represents a novel biomarker for PCa.

To elucidate the mechanism of radiation-induced cancers, molecular analysis of cancers in atomic bomb (A-bomb) exposure is important. DNA double-strand breaks (DSBs) are thought to be caused by the deleterious effects of ionizing radiation, and gammaH2AX (serine 139 phosphorylated form of histone H2AX) is reported to be a significant marker for DSBs. In the present study, we performed immunohistochemical analysis of gammaH2AX in gastric cancers (GCs) from 66 exposed and 47 non-exposed patients who developed GC after the bombing. Of the 47 GCs from non-exposed patients, 6 (13%) cases showed nuclear positive staining for gammaH2AX, whereas of the 66 GCs from exposed patients, 20 (30%) cases were positive (P=0.0405). However, among stage I GC, there was no significant difference in gammaH2AX expression frequency between exposed patients and non-exposed patients. Among exposed patients, stage II-IV cases were more frequently positive for gammaH2AX than stage I cases (P=0.0197). Among GCs from non-exposed patients, gammaH2AX staining showed no significant association with Lauren's classification, depth of invasion, lymph node metastasis or TNM stage. These results suggest that the characteristics of tumor cells differ between GCs from exposed and non-exposed patients. DSBs may be involved in progression of GC in exposed patients.

Signet-ring cell carcinoma (SRCC) is a unique subtype of adenocarcinoma that is characterized by abundant intracellular mucin accumulation and a crescent-shaped nucleus displaced toward one end of the cell. Identification of an SRCC's primary site is important for better planning of patient management because the treatment and prognosis differs markedly depending on the origin of the SRCC. In the present study, we analyzed the immunohistochemical characteristics of 94 cases of SRCC, including 21 cases of gastric SRCC, 16 of colorectal SRCC, 10 of breast SRCC, and 47 of pulmonary SRCC, with antibodies against Reg IV and claudin-18, which we previously identified as gastric cancer-related genes. We also tested known markers cytokeratin 7, cytokeratin 20, MUC2, MUC5AC, caudal-related homeobox gene 2 (CDX2), thyroid transcription factor-1, mammaglobin, gross cystic disease fluid protein 15, and estrogen receptor. All 21 cases of gastric SRCC and 16 cases of colorectal SRCC were positive for Reg IV, and the remaining SRCCs were negative. Eighteen of 21 (86%) gastric SRCCs and 6 of 16 (38%) colorectal SRCCs were positive for claudin-18, whereas another SRCCs were negative. In conclusion, Reg IV staining and claudin-18 staining can aid in diagnosis of gastrointestinal SRCC.

Gastric cancer is one of the most common malignancies worldwide. In this study, we screened for genes upregulated in gastric cancer by comparing gene expression profiles from serial analysis of gene expression and microarray and identified the palate, lung, and nasal epithelium carcinoma-associated protein (PLUNC) gene. Immunostaining for PLUNC in 140 gastric cancer cases revealed strong and extensive staining of PLUNC in hepatoid adenocarcinoma of the stomach, whereas 7% of conventional gastric cancer cases showed focal immunostaining of PLUNC. Gastric hepatoid adenocarcinoma is an extrahepatic tumor characterized by morphologic similarities to hepatocellular carcinoma. To investigate the utility of PLUNC immunostaining in the diagnosis of gastric hepatoid adenocarcinoma, six cases of gastric hepatoid adenocarcinoma (six primary tumors and two associated liver metastases) were studied further. PLUNC staining was observed in all six primary hepatoid adenocarcinomas. PLUNC staining was observed in both the hepatoid adenocarcinoma and tubular/papillary adenocarcinoma components of primary tumors, although PLUNC staining was preferentially localized in tubular/papillary adenocarcinoma components. Staining of PLUNC was also detected in both liver metastases. PLUNC staining was not observed in 52 cases of primary hepatocellular carcinoma or in normal adult or fetal liver. These results indicate that PLUNC is a novel marker that distinguishes gastric hepatoid adenocarcinoma from primary hepatocellular carcinoma.

Melanoma-associated antigen (MAGE) genes are cancer-testis antigen genes that serve as immunotherapy targets in several human cancers. Previous studies have revealed that the forced expression of MAGE genes induces a paclitaxel-resistant phenotype. In the present study, we examined whether the expression of MAGE-A1 could predict the response of advanced and recurrent gastric cancers (GCs) to taxan (doce-taxel or paclitaxel)-based chemotherapy. The expression of MAGE-A1 was analyzed by immunostaining in 41 primary GC samples. DNA demethylation was assessed by methylation-specific polymerase chain reaction and the effect of the forced expression of MAGE-A1 on drug resistance to taxan drugs was monitored by MTT assay. The expression of MAGE-A1 in primary GC was observed in 4 (9.8%) of 41 cases. All 4 patients with MAGE-A1-positive GC showed progressive disease, whereas MAGE-A1 expression was not detected in any of the 23 patients showing partial response (P=0.0302). There was no association between MAGE-A1 gene demethylation and response to chemotherapy (P=0.7245). The forced MAGE-A1 expression in the TMK-1 GC cell line increased the sensitivity to paclitaxel and docetaxel. These results suggest that although MAGE-A1 does not participate directly in the drug-resistant phenotype, the expression of MAGE-A1 could be a marker for the prediction of resistance to taxan-based chemotherapies in patients with GC.

The human homolog of the Drosophila prune protein (from PRUNE, which encodes h-prune), which interacts with glycogen synthase kinase 3, promotes cellular motility. H-prune also interacts with nm23-H1, a suppressor of cancer metastasis. It has been reported that stimulation of cellular motility by h-prune is enhanced by its interaction with nm23-H1 in breast cancer cells. In the present study, we examined the expression of h-prune and nm23-H1 during tumor progression in gastric cancer (GC). PRUNE mRNA was overexpressed in 12 (32%) of 38 GC cases by quantitative reverse transcription-polymerase chain reaction. PRUNE mRNA levels correlated significantly with advanced T grade, N grade and tumor stage. Immunohistochemical analysis revealed that 43 (30%) of 143 GC cases were positive for h-prune, and h-prune-positive GC cases showed more advanced T grade, N grade and tumor stage than h-prune-negative GC cases. One hundred and twenty-four (87%) of 143 GC cases were positive for nm23-H1, and nm23-H1 was expressed in almost all (42 cases, 98%) h-prune-positive GC cases. Many GC cases positive for both h-prune and nm23-H1 showed more advanced T grade, N grade and tumor stage than other type GC cases. Patients with h-prune-positive GC had a significantly worse survival rate than patients with h-prune-negative GC. These findings indicate that overexpression of h-prune is associated with tumor progression and aggressiveness of GC. nm23-H1 may enhance motility of cancer cells by interacting with h-prune.

Acute arterial occlusion as a result of embolization from a malignant tumor is rare and infrequently reported. Recently a 70-year-old man initially presented with acute arterial occlusion of the left lower limb. He underwent embolectomy, and embolus was diagnosed as a squamous cell carcinoma histologically. Subsequent upper gastrointestinal endoscopy showed superficial esophageal cancer in the thoracic esophagus. Although scheduled to undergo chemotherapy, he died of acute pneumonia on the 81st day from onset. Autopsy showed superficial esophageal cancer, measuring 1.5 cm in diameter, and widespread tumor extension into arterial vessels such as the left femoral artery, the superior mesenteric artery, bilateral intrapulmonary arteries and veins such as bilateral brachiocephalic vein to the supra vena cava, despite small tumor size and shallow tumor invasion depth of the submucosal layer. This case is particularly interesting because the cancer manifested as arterial cancer embolism.

Objective: Regenerating islet-derived family, member 4 (regenerating gene type IV, Reg IV) is overexpressed in colorectal cancer (CRC). The aim of this study was to investigate the diagnostic utility of Reg IV determination in sera from patients with CRC., Methods: We examined the expression and distribution of Reg IV in CRC by immunohistochemistry and determined Reg IV levels in sera from patients with CRC by enzyme-linked immunosorbent assay., Results: Immunostaining revealed that 23 of 80 (29%) CRC cases were positive for Reg IV. CRC cases with metastatic recurrence in the liver showed more frequently Reg IV staining than those without (p = 0.0102). Patients with CRC showing Reg IV staining had a significantly worse survival than those without Reg IV staining (p = 0.0117). Preoperatively, serum Reg IV concentrations were not elevated in CRC patients at stage 0-III, being in contrast to the significantly increased preoperative levels in stage IV CRC patients with liver metastasis., Conclusion: These results suggest that Reg IV is a prognosticator for poor survival. Serum Reg IV concentration may predict CRC recurrence in the liver., ((c) 2008 S. Karger AG, Basel)

Objectives: RHOC, a member of the RAS-related small GTPase protein family, regulates cytoskeletal structures and has the potential to transform cultured cells. It has recently been reported that RHOC contributes to the metastatic phenotype of melanoma cells. The purpose of this study was to clarify its biological relevance to gastric carcinogenesis and metastasis., Methods: We examined the expression of RHOC by quantitative RT-PCR in 51 cases of gastric carcinoma tissues from prior surgical cases (intestinal type: 24 cases, diffuse type: 27 cases) and in 8 gastric carcinoma cell lines., Results: RHOC expression levels in primary tumors were significantly higher in cases with metastasis than in those without metastasis (p = 0.0202; Mann-Whitney U test). RHOC expression levels in primary tumor and their metastatic tumors were significantly higher than their corresponding nonneoplastic mucosa (p = 0.0357, and 0.0173, respectively; Wilcoxon signed rank test). RHOC mRNA expression was confirmed in the gastric carcinoma cell lines., Conclusion: Our findings suggest that elevated expression of the RHOC gene may be involved in the metastasis of gastric carcinomas and may be a good genetic marker for the prediction of a metastatic potential., (Copyright 2004 S. Karger AG, Basel)

BRG1, a component of the SWI/SNF complex, regulates gene transcription through chromatin remodeling. Certain human cancer cell lines have been shown to contain homozygous deletions or mutations, half of which are concentrated in exons 4 and 10, resulting in aberrant BRG1 expression. We examined the expression of BRG1 in 38 gastric carcinomas and corresponding nonneoplastic mucosa by using the quantitative real-time RT-PCR method. Twenty-three carcinomas (61%) showed increased BRG1 expression in tumor tissue in comparison with that in nonneoplastic mucosa. The T/N ratio (the expression level of BRG1 mRNA in tumor tissues relative to those in corresponding nonneoplastic mucosa) in advanced cases of gastric carcinoma (stages III and IV) was significantly higher than that in cases of stage I and II carcinoma (p = 0.029). Furthermore, gastric carcinomas with lymph node metastasis showed a tendency to express BRG1 at a higher level than gastric carcinomas without metastasis (p = 0.097). We also searched for genetic alterations of the BRG1 gene in 8 gastric carcinoma cell lines and 33 primary gastric carcinomas by PCR-SSCP analysis. No SSCP variants in exons 4, 10 and 16 of the BRG1 gene were found in both gastric carcinoma cell lines and primary gastric carcinomas. These results suggest that, although genetic abnormality of BRG1 might be rare, an increased expression of BRG1 might be associated with the development and progression of gastric carcinoma., (Copyright 2002 S. Karger AG, Basel)

Hypermethylation of CpG islands in the promoter region is associated with the silencing of a variety of tumor suppressor genes. DNA repair genes human Mut L homologue 1 (hMLH1) and O(6)-methylguanine-DNA methyltransferase (MGMT) have been shown to be hypermethylated in certain carcinomas. We studied DNA methylation of CpG islands in hMLH1 and MGMT in 50 gastric carcinomas and 10 intestinal metaplastic mucosa samples. We analyzed the methylation status of hMLH1 and MGMT using methylation-specific polymerase chain reaction and DNA sequencing analysis. We measured protein levels of hMLH1 using Western blot and immunohistochemical analysis. CpG island hypermethylation of hMLH1 and MGMT was detected in 11 (22%) and 8 (16%) of the 50 gastric tumors, respectively. Hypermethylation of the promoter was more common in intestinal-type gastric carcinomas than in poorly diffuse-type gastric carcinomas (p = 0.016 and 0.021, respectively; Fisher's exact test). However, hMLH1 promoter hypermethylation did not coincide with MGMT promoter hypermethylation except in 1 patient. Hypermethylation of the hMLH1 promoter but not the MGMT promoter occurred in intestinal metaplastic mucosae. Immunohistochemical analysis revealed a corresponding reduction in hMLH1 protein expression in some of the intestinal metaplastic mucosae. Our results suggest that at least two types of promoter methylation participate in the development of gastric carcinoma. Tumor-specific promoter hypermethylation of hMLH1 may be an early event in carcinogenesis in the stomach., (Copyright 2002 S. Karger AG, Basel)