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Background: A wide range of response rates have been reported in HER2-positive gastric cancer (GC) patients treated with trastuzumab. Other HER2-targeted therapies for GC have yet to show efficacy in clinical trials. These findings raise question about the ability of standard HER2 diagnostics to accurately distinguish between GC patients who would and would not benefit from anti-HER2 therapies., Patients and Methods: GC patients (n = 237), including a subset from the Trastuzumab in GC (ToGA) trial were divided into three groups based on HER2 status and history of treatment with standard chemotherapy or chemotherapy plus trastuzumab. We applied mass spectrometry-based proteomic analysis to quantify HER2 protein expression in formalin-fixed tumor samples. Using HER2 expression as a continuous variable, we defined a predictive protein level cutoff to identify which patients would benefit from trastuzumab. We compared quantitated protein level with clinical outcome and HER2 status as determined by conventional HER2 diagnostics., Results: Quantitative proteomics detected a 115-fold range of HER2 protein expression among patients diagnosed as HER2 positive by standard methods. A protein level of 1825 amol/µg was predicted to determine benefit from the addition of trastuzumab to chemotherapy. Trastuzumab treated patients with HER2 protein levels above this cutoff had twice the median overall survival (OS) of their counterparts below the cutoff (35.0 versus 17.5 months, P = 0.011). Conversely, trastuzumab-treated patients with HER2 levels below the cutoff had outcomes similar to HER2-positive patients treated with chemotherapy. (Progression-free survival = 7.0 versus 6.5 months: P = 0.504; OS = 17.5 versus 12.6 months: P = 0.520). HER2 levels were not prognostic for response to chemotherapy., Conclusions: Proteomic analysis of HER2 expression demonstrated a quantitative cutoff that improves selection of GC patients for trastuzumab as compared with current diagnostic methods., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology.)

Background: The aim of this study was to compare gene copy number (GCN) and protein expression of MET and to evaluate their prognostic roles in gastric carcinomas., Methods: MET protein expression and gene amplification (GA) status were determined by immunohistochemistry (IHC) and silver in-situ hybridisation (SISH), respectively, in a large series of gastric carcinoma., Results: Protein overexpression was observed in 104 of 438 cases, with IHC 2+ in 94 and IHC 3+ in 10, and high polysomy of chromosome 7 and GA were found in 61 and 13 of 381, respectively. Direct comparison revealed a significant correlation between high level of protein expression and increased GCN. All cases with GA showed protein overexpression. Furthermore, all with IHC 3+ showed GA except 1, even which could be categorised as GA according to the ASCO/CAP guideline for human epidermal growth factor receptor 2 assessment. IHC 3+ and GA were significantly associated with poor prognosis., Conclusion: MET IHC reflects well on GA, and therefore, it could be a primary screening test for patient selection for anti-MET therapy if GA is a major determinant of drug responsiveness. Also, the prognostic role of MET indicates that anti-MET therapy is a very promising modality in adjuvant treatment for gastric cancer.

Background: We aimed to evaluate the immunologic nature of sentinel lymph nodes (SLNs) in gastric cancer patients and to determine whether it can predict non-SLN metastasis., Methods: Sentinel lymph node samples were collected from 64 gastric carcinoma patients who had undergone gastrectomy with SLN biopsy. One representative SLN sample was selected from each patient and was subjected to immunostaining for CD8, CD57, FOXP3, and DC-LAMP. The numbers of marker-positive cells in each sample were counted. The relationships between various immune cell densities and clinicopathologic parameters or metastasis status of SLNs and non-SLNs were sought., Results: High FOXP3+ Treg density of the SLN was found to be significantly associated with the presence of metastasis in either SLNs or non-SLNs. DC-LAMP+ cell density of the SLN was the highest at the isolated tumours cell level, and this decreased along with an increase in tumour metastasis in either SLNs or non-SLNs. Univariate and multivariate logistic regression models revealed that high FOXP3+ Treg density of the SLN was an independently significant predictor of non-SLN metastasis., Conclusions: This study is the first to indicate an important role of SLNs in metastatic dissemination of gastric cancer. Our findings suggest that Tregs could be a new therapeutic target for regulating the metastasis of gastric cancer.

Background: Meta-analyses of the published literature indicate that about 9% of gastric cancers contain Epstein-Barr virus (EBV), with consistent and significant differences by sex and anatomic subsite. This study aimed to identify additional determinants of EBV positivity and their joint effects., Methods: From 15 international populations with consistent laboratory testing for EBV, we pooled individual-level data for 5081 gastric cancer cases including information on age, sex, subsite, histologic type, diagnostic stage, geographic region, and period of diagnosis. First, we combined population-specific EBV prevalence estimates using random effects meta-analysis. We then aggregated individual-level data to estimate odds ratios of EBV positivity in relation to all variables, accounting for within-population clustering., Results: In unadjusted analyses, EBV positivity was significantly higher in males, young subjects, non-antral subsites, diffuse-type histology, and in studies from the Americas. Multivariable analyses confirmed significant associations with histology and region. Sex interacted with age (P=0.003) and subsite (P=0.002) such that male predominance decreased with age for both subsites. The positivity of EBV was not significantly associated with either stage or time period., Conclusion: Aggregating individual-level data provides additional information over meta-analyses. Distinguishing histologic and geographic features as well as interactions among age, sex, and subsite further support classification of EBV-associated gastric cancer as a distinct aetiologic entity.

Background: The pattern of gastric cancer in the Western world is changing, with an increased proportion of tumours in the upper stomach. The aim of this study was to investigate changes in clinicopathological features and survival of patients with resected gastric cancer at a single institution, in an area of high incidence in the Far East., Methods: Clinical features and pathological findings were compared in patients with gastric cancer who underwent gastrectomy at Seoul National University Hospital during four consecutive periods (1986-1990, 1991-1995, 1996-2000 and 2001-2006)., Results: There were 12 026 patients. The mean age increased from 53·4 years in the first period to 57·4 years in the last (P < 0·001). The proportion of patients aged 70 years or older also increased, reaching 16·1 per cent in the final period. Upper-third cancer increased from 5·3 per cent in the first period to 14·0 per cent in the fourth (P < 0·001). Early gastric cancer (pathological T1) increased continuously over the four time intervals, from 24·8 to 48·9 per cent (P < 0·001). The overall 5-year survival rate increased from 64·0 per cent in the first period to 73·2 per cent at the end (P < 0·001), and this survival improvement was apparent in patients aged 40 years or more., Conclusion: The mean age of patients with gastric cancer has increased during the past 20 years. The proportion of early gastric cancer and overall survival have gradually increased, especially in patients aged over 40 years.

Background: The underlying mechanisms involved in the activation of hypoxia-inducible factor-1 (HIF-1) in gastric cancer remain unclear. As nuclear factor-κB (NF-κB) as well as HIF-1 have been implicated in angiogenesis of various cancers, we investigated their relationship in gastric cancer., Methods: Nuclear expressions of HIF-1α and NF-κB/RelA were assessed in 251 human gastric carcinoma specimens by immunohistochemical tissue array analysis. Stable human gastric cancer cells, infected with a retroviral vector containing super-suppressive mutant form of IκBα (IκBαM), were used for animal studies as well as cell culture experiments. Xenografted tumours were measured and IκBαM effects on angiogenesis and HIF-1α activation were assessed by immunohistochemistry, western blotting, luciferase reporter assay, and semiquantitative reverse transcription-polymerase chain reaction. In addition, NF-κB effects on the HIF-1α degradation and synthesis were examined., Results: Hypoxia-inducible factor-1α activation positively correlated with RelA activation in clinical gastric cancer samples (P<0.001). The IκBαM overexpression suppressed tumour growth, microvessel density, and HIF-1α activation in xenografted tumours. Cell culture experiments showed that hypoxia-induced HIF-1α expression was reduced by NF-κB inhibition under hypoxic conditions at the translational level., Conclusion: The hypoxia-dependent activation of the NF-κB/HIF-1α/VEGF pathway contributes, at least in part, to gastric cancer promotion via enhancement of angiogenesis.

Background: This study aimed to determine the appropriate extent of lymph node (LN) dissection in gastric cancer by analysing LN metastasis patterns from prospectively collected topographical data on nodal status at Seoul National University Hospital, Korea., Methods: The metastasis rate for each LN station was analysed according to the depth of tumour invasion in patients with primary lower-third gastric cancer who underwent curative gastrectomy. The Maruyama Index of unresected disease (MI) was calculated using the WinEstimate(®) program with simulation of various extents of LN dissection., Results: LN metastasis in mucosal cancer was rare; 2·6 per cent of patients had a MI of more than 5 with simulation of D1 plus station 7 dissection, whereas 0·9 per cent had a MI above 5 with D1 plus stations 7 and 8a. In submucosal cancer, 3·3 per cent of tumours metastasized to level 2 LN stations outside the range of D1 plus stations 7, 8a and 9. The proportion of patients with a MI above 5 was 9·0 per cent with D1 plus stations 7, 8a and 9 dissection. The nodal metastasis rate was higher at level 1 and 2 for muscularis propria or deeper cancers., Conclusion: D1 dissection plus stations 7 and 8a for mucosal cancer, and D2 dissection for cancers of the muscularis propria or deeper seems appropriate. For submucosal cancer, an expanded dissection to the D2 level should be considered to ensure complete removal of metastatic LNs., (Copyright © 2010 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

The aim of this study was to analyse the impact of epidermal growth factor receptor (EGFR), thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), aurora kinase (ARK) A/B, and excision repair cross-complementing gene 1 (ERCC1) on the efficacy of adjuvant chemotherapy with 5-fluorouracil and cisplatin (FP) after curative gastric resection. Normal and cancer tissue were separately obtained from gastrectomy samples of 153 patients with AJCC stage III-IV (M0) who subsequently treated with adjuvant FP chemotherapy. TS, DPD, TP, ERCC1, and ARK proteins were measured by immunohistochemistry (IHC). EGFR expression was investigated using a standardized IHC with the EGFR PharmDx assay. Amplification of EGFR gene was analysed using fluorescent in situ hybridisation (FISH). In multivariate analysis, stage, ratio of positive to removed lymph nodes, and EGFR expression were significant prognostic factors for overall survival. Patients with higher EGFR expression had better overall survival than those with lower expression (relative risk: 0.475 (95% confidence interval, 0.282-0.791, P=0.005). Low EGFR expression might be a predictive marker for relapse in curative resected stage III-IV (M0) gastric cancer patients who received adjuvant FP chemotherapy.

This prospective study was conducted with the Korean Cancer Study Group to evaluate the efficacy and safety of cetuximab combined with modified FOLFOX6 (mFOLFOX6) as first-line treatment in recurrent or metastatic gastric cancer and to identify potential predictive biomarkers. Patients received cetuximab 400 mg m(-2) at week 1 and 250 mg m(-2) weekly thereafter until disease progression. Oxaliplatin (100 mg m(-2)) and leucovorin (100 mg m(-2)) were administered as a 2-h infusion followed by a 46-h continuous infusion of 5-fluorouracil (2400 mg m(-2)) every 2 weeks for a maximum of 12 cycles. Biomarkers potentially associated with efficacy were analysed. Among 38 evaluable patients, confirmed response rate (RR) was 50.0% (95% CI 34.1-65.9). Median time-to-progression (TTP) was 5.5 months (95% CI 4.5-6.5) and overall survival (OS) 9.9 months. Eleven patients having tumour EGFR expression by immunohistochemistry with low serum EGF and TGF-alpha levels showed a 100% RR compared to 37.0% in the remaining 27 patients (P<0.001). Moreover, ligand level increased when disease progressed in seven out of eight patients with EGFR expression and low baseline ligand level. No patient exhibited EGFR amplification or K-ras mutations. Gastric cancer patients with EGFR expression and low ligand levels had better outcomes with cetuximab/mFOLFOX6 treatment.

RUNX3 is a tumor suppressor that is silenced in cancer following hypermethylation of its promoter. The effects of hypoxia in tumor suppressor gene (TSG) transcription are largely unknown. Here, we investigated hypoxia-induced silencing mechanisms of RUNX3. The expression of RUNX3 was downregulated in response to hypoxia in human gastric cancer cells at the transcriptional level. This downregulation was abolished following treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and cytosine methylation inhibitor 5-aza-2-deoxycytidine (5-Aza), suggesting that an epigenetic regulatory mechanism may be involved in RUNX3 silencing by hypoxia. DNA methylation PCR and bisulfite-sequencing data revealed that hypoxia did not affect the methylation of RUNX3 promoter. A chromatin immunoprecipitation (ChIP) assay revealed increased histone H3-lysine 9 dimethylation and decreased H3 acetylation in the RUNX3 promoter following hypoxia. Hypoxia resulted in the upregulation of G9a histone methyltransferase (HMT) and HDAC1; additionally, overexpression of G9a and HDAC1 attenuated RUNX3 expression. The overexpression of G9a and HDAC1, but not their mutants, inhibited the nuclear localization and expression of RUNX3. Diminished mRNA expression and nuclear localization of RUNX3 during hypoxia was abolished by siRNA-mediated knockdown of G9a and HDAC1. This study suggests that hypoxia silences RUNX3 by epigenetic histone regulation during the progression of gastric cancer.

The study aims to determine whether type and density of tumour-infiltrating lymphocytes (TILs) can predict the clinical course in gastric cancer. Gastric carcinomas (n=220) were immunostained for CD3, CD8, CD20, and CD45RO and evaluated for clinicopathologic characteristics. Number of TILs that immunostained positively for each marker were counted using NIH ImageJ software. Tumours were grouped into low- and high-density groups for each marker (CD3, CD8, CD45RO). The densities of CD3(+), CD8(+), and CD45RO(+) TILs were found to be independent predictors of lymph node metastasis by multivariate analysis with odds ratios (95% CI) of 0.425 (0.204-0.885), 0.325 (0.150-0.707), and 0.402 (0.190-0.850), respectively. Kaplan-Meier survival analysis revealed that patients in the high-density groups for CD3, CD8, and C45RO had a significantly longer survival time than the patients in the corresponding low-density groups, respectively. In multivariate survival analysis, the densities of CD3(+), CD8(+), and CD45RO(+) TILs remained independent prognostic factors with hazard ratios (95% CI) of 0.549 (0.317-0.951), 0.574 (0.347-0.949), and 0.507 (0.298-0.862), respectively. In conclusion, density of TILs was found to be independently predictive of regional lymph node metastasis and patient survival in gastric cancer.

Aims: Epidermal growth factor receptor (EGFR) expression has been observed in a variety of solid tumours with the potential of new targeted therapeutic agents. The aim was to evaluate the EGFR status of gastric carcinoma (GC) using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH)., Methods and Results: The EGFR status was evaluated in GC tissues from 511 patients using IHC and FISH. In addition, the clinicopathological characteristics were examined and the results were compared with the EGFR status. One hundred and forty cases (27.4%) showed EGFR overexpression by IHC. EGFR overexpression was associated with older age (P = 0.001), moderately or poorly differentiated histology (P = 0.001) and higher stage disease (P = 0.046). Sixteen cases (3.1%) showed high polysomy and 12 cases (2.3%) had gene amplification by FISH. The correlation between IHC and FISH results was statistically significant (P < 0.001). The patients with GC who had EGFR overexpression had an unfavourable prognosis and multivariate analysis showed that EGFR overexpression was a possible independent unfavourable prognostic factor., Conclusions: EGFR overexpression was observed in a subset of cases with GC and was associated with an unfavourable prognosis. It will be important to evaluate EGFR status to interpret future clinical trials properly using EGFR targeted agents.

Lewis (Le)-associated antigens are carbohydrates that are related biochemically to the ABO blood groups, and may have a role in Helicobacter pylori adherence. To evaluate their relationship to clinicopathological outcomes, gastric Le expression, including type 1 precursor, type 1 H, Le(a), Le(b), Le(x), Le(y) and sialylated Le(a) (CA19-9), was evaluated immunohistochemically in 233 gastric biopsy specimens obtained at routine gastroscopy. Expression was also investigated in gastric tissues showing chronic gastritis, intestinal metaplasia, and carcinoma from 42 patients with gastric cancer. A polymerase chain reaction was performed for H. pylori and the bacterial babA2 gene. We identified type 1 precursor expression in 34.3%, type 1 H in 55.8%, Le(a) in 44.2%, Le(b) in 82.0%, Le(x) in 44.2%, Le(y) 56.7%, and CA19-9 in 16.3% of the 233 gastric biopsy specimens. Expression of type 1 H, Le(b), and CA19-9 was significantly associated with H. pylori infection and histological features (p < 0.05), and expression of type 1 H was an independent predictive factor for H. pylori infection by multivariate logistic regression (p = 0.020). Positivity for the babA2 genotype correlated significantly with H. pylori infection and type 1 H expression in gastric biopsy specimens (p < 0.05). The babA2 genotype was more frequent in gastric mucosa from the gastric cancer patients than in gastric biopsy specimens from routine gastroscopy (p = 0.009). In the 42 gastric cancer patients, the frequency of type 1 precursor, Le(a), and Le(x) expression was significantly higher in intestinal metaplasia and carcinoma than in chronic gastritis (p < 0.05), but the frequency of type 1 H and Le(b) expression was significantly lower in intestinal metaplasia and carcinoma (p < 0.05). In conclusion, Le expression, especially that of type 1 H, was significantly associated with clinicopathological features. In gastric cancer patients, Le expression was altered in intestinal metaplasia and carcinoma in comparison with chronic gastritis., (Copyright 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Aims: To evaluate the feasibility and accuracy of sentinel node (SN) biopsy for gastric cancer., Patients and Methods: One hundred patients with gastric cancer diagnosed as cT1 (n=80) or cT2a (n=20) were enrolled. Indocyanine green-stained SNs were analysed by hematoxylin and eosin staining (n=100) and by cytokeratin immunohistochemistry (n=50)., Results: SNs were identified in 94 of the 100 patients and the mean number of SNs was 4.4 (range, 1-12). Of these 94 patients, 14 patients had lymph node metastases. Two patients with T1 and one patient with T2 had metastases in non-SNs alone by hematoxylin and eosin staining (diagnostic accuracy =97.3% in T1 and 95.0% in T2). All three patients with a false negative result had a tumour, which was more than 4 cm in size and signet ring cell histology. In two of them, the tumour was located at lesser curvature. By immunohistochemical staining, three patients with T1 and one patient with T2 were found to have lymph node micrometastases in non-SNs alone among 45 patients (diagnostic accuracy =92.1% in T1, 85.7% in T2)., Conclusion: SN biopsy using indocyanine green can be performed rapidly and easily with a high detection rate and accuracy in patients with T1 gastric cancer. However, it should be performed with caution for large tumours with a signet ring cell histology located at lesser curvature due to the possibility of a false negative result.

Background: Transforming growth factor-beta (TGF-beta) modulates the growth and function of many cells, including those with malignant transformation. Smad proteins have been identified as major components in the intracellular signaling of TGF-beta family members., Patients and Methods: To clarify the correlations between clinicopathologic profiles and the patient's survival, the expression of common mediator Smad (Smad4) and inhibitory Smad (Smad7) were evaluated immunohistochemically in 304 consecutive gastric carcinomas using the tissue array method., Results: Positive Smad4 expression was observed in 266 (87.5%) tumors and positive Smad7 expression in 98 (32.2%) tumors. The prognosis of patients with a Smad4-positive tumor was significantly better than that of the patients with a negative tumor. The survival rate was significantly higher in patients with negative Smad7 expression than those with positive Smad7 expression. In subgroup analysis according to TNM (tumour-node-metastasis) stage, both Smad4 and Smad7 showed most significant prognostic differences in stage I gastric cancer patients. Multivariate analysis indicated that tumor size, depth of invasion, lymph node metastasis and Smad7 expression were independent prognostic factors., Conclusion: Enhanced expression of the TGF-beta signaling inhibitor Smad7 may present one of the novel mechanisms of TGF-beta resistance in human gastric carcinomas.

Aims: Gastric adenocarcinoma in young patients has been considered to differ in many ways from gastric carcinoma in older patients. This study was designed to determine the clinicopathological features and molecular mechanisms., Methods and Results: Based on 4123 patients of gastric cancer in Seoul National University Hospital, 135 patients (3.3%) were chosen by the age of 30 years or younger. Expression of E-cadherin, beta-catenin, p53 and Epstein-Barr virus (EBV) was analysed using the tissue array method in formalin-fixed paraffin-embedded specimens and microsatellite instability (MSI) was determined. As a control, 320 cases of older patients were compared. Gastric adenocarcinoma of young patients revealed significant female predominance, type IV gross type, proximal location, diffuse type and frequent lymph node metastasis. In-situ hybridization for EBV showed higher positivity in young patients (9/78, 11.5%) than in older ones, but not statistically significant. In EBV+ cases, p53 over-expression was significantly higher in young patients than older patients (P < 0.05). Alteration of E-cadherin or beta-catenin was significantly higher in younger patients than in older patients (P < 0.05). Overall survival was significantly poorer in younger patients than older ones. The frequency of MSI was rare (1.3%, P < 0.05) in young patients compared with older patients (9.3%)., Conclusions: These data indicate that gastric adenocarcinoma of young patients has a poor prognosis, possesses aggressive histopathological features, exhibits reduced expression of E-cadherin and beta-catenin, and demonstrates lower MSI than tumours in older patients.

Promoter hypermethylation of CpG islands in tumour suppressor genes can lead to transcriptional inactivation. To investigate the association between methylation and expression at O6-methylguanine-DNA methyltransferase, we performed methylation-specific PCR and immunohistochemistry in 149 gastric carcinomas. Promoter methylation was found in 14.1% of tumours and loss of expression was detected in 11.4% of tumours. To examine correlation between the O6-methylguanine-DNA methyltransferase expression and the clinical data, we investigated O6-methylguanine-DNA methyltransferase expression in 315 consecutive gastric carcinomas. A similar frequency of loss of O6-methylguanine-DNA methyltransferase expression was confirmed in these cases. The loss of O6-methylguanine-DNA methyltransferase expression was significantly associated with pTNM stage (P=0.037), tumour invasion (P=0.02), microsatellite instability (P=0.041) and overall survival (P=0.01). Among 11 gastric cancer cell lines, SNU-620 showed the loss of O6-methylguanine-DNA methyltransferase expression as well as promoter methylation. After treatment with 5-aza-2-deoxycytidine, a demethylating agent, SNU-620 re-expressed O6-methylguanine-DNA methyltransferase mRNA. In summary, we suggest that during gastric carcinogenesis, the loss of O6-methylguanine-DNA methyltransferase expression frequently occurs via the hypermethylation of the CpG islands of the promoter region, and that this is significantly associated with the clinicopathological characteristics., (Copyright 2002 Cancer Research UK)

Purpose: Aberrant methylation of the promoter CpG island of hMLH1 is associated with gene silencing in colon cancer and gastric cancer with microsatellite instabilities (MSI). We analyzed the pattern of promoter methylation causing gene silencing., Methods: Comprehensive analysis of hMLH1 promoter was performed by bisulfite genomic sequencing in human gastric cancer cell lines. Altered expression of hMLH1 was examined by the immunocytochemical staining method and RT-PCR, and microsatellite instability was examined using two representative mononucleotide repeat microsatellite markers, BAT-25 and BAT-26., Results: As a result, MSI-positive gastric cancer cell lines were methylated extensively at the overall promoter region. MSI-negative gastric cancer cell lines - except in SNU-620 - were unmethylated completely at the overall promoter region including the more upstream region in contrast to colorectal cancer cell lines. Even though SNU-620 was methylated fully at the overall promoter region - except for partial methylation at the specific region (from -270 to -199) near the transcriptional start - hMLH1 protein was expressed., Conclusion: Our data suggest that the methylation density of a specific region plays an important role in gene inactivation of hMLH1 and that the methylation status of the more upstream promoter region and exon 1 start region are not essential for gene inactivation.

Background: Nodal staging for gastric cancer according to the 1997 Union Internacional Contra la Cancrum tumour node metastasis classification is based on the number of metastatic lymph nodes. The aim of this study was to evaluate whether the number of lymph nodes examined affected staging of gastric cancer., Methods: A retrospective study was performed in 4789 consecutive patients with gastric cancer, who had undergone curative resection (R0) from 1986 to 1995. Patients were classified according to the number of nodes examined. The number of metastatic lymph nodes and stage-stratified survival were compared., Results: There were significant differences in the number of metastatic lymph nodes and survival in stage IIIA between patients with 15 or more lymph nodes and those with fewer than 15 nodes. In analysis restricted to patients with 15 or more nodes, stage-stratified survival did not vary significantly with lymph node yields for any stage except IIIB, in which there was a significant difference between the subgroup with fewer than 20 examined lymph nodes and patients with 35 or more nodes., Conclusion: The number of lymph nodes examined did not significantly affect node staging of gastric cancer as long as at least 15 nodes were examined. For stage IIIB, more than 15 lymph nodes may be required for optimal staging.

Background: Although mucin expressions appear to be correlated with prognoses in patients with various cancers, several studies have reported conflicting and inconclusive results on the prognostic significance of mucin expression in gastric carcinomas., Methods: To clarify the correlations between clinicopathologic profiles and the patients' survival, the expression of MUC1, MUC2, MUC5AC, MUC6 mucins and the p53 protein were evaluated immunohistochemically in 300 consecutive gastric carcinomas using the tissue-array method. In addition, 59 gastric adenomas and 57 adenoma-associated carcinomas were investigated., Results: MUC1 was expressed in 2 (3.4%) cases of gastric adenoma, and MUC2 in 19 (32.2%) cases of gastric adenoma, out of a total of 59 lesions. In consecutive gastric carcinomas, 24.3% of gastric carcinomas expressed MUC1, 27.3% expressed MUC2, 38.0% expressed MUC5AC and 12.7% expressed MUC6. The rate of MUC1 expression in gastric carcinomas was significantly higher than in associated gastric adenomas (P < 0.01). The patients with MUC1-positive carcinomas showed significantly poorer survival than those with MUC1-negative carcinomas. On the other hand, MUC2, MUC5AC and MUC6 expressions were not significantly associated with patient survival. Interestingly, combined evaluation revealed that the group with the MUC1-negative plus p53-negative expression pattern showed a better prognosis than the remaining cases. In contrast, the group with the MUC2-negative plus p53-positive pattern showed a worse prognosis., Conclusions: Mucin expression is altered in gastric adenoma and carcinoma, and MUC1 mucin expression is significantly associated with poorer outcome in gastric carcinomas. A MUC1-negative plus p53-negative pattern or a MUC2-negative plus p53-positive pattern may predict outcome in patients with gastric carcinomas., (Copyright 2001 American Cancer Society.)

Genomic alterations and abnormal expression of the fragile histidine triad (FHIT) gene in gastric carcinomas were examined to determine whether the FHIT gene is actually a frequent target for alteration during gastric carcinogenesis. To correlate DNA and RNA lesions of the FHIT gene with the effect on FHIT protein expression, we have investigated the FHIT gene for loss of heterozygosity (LOH), aberrant transcripts, point mutations, and protein expression in 35 gastric adenocarcinomas. Allelic loss at D3S1300 was detected in 7 of 33 (21%) informative cases. Aberrant transcripts, with deletions and/or insertions, were observed in 20 of 35 (57.1%) cases and resulted from alternative splicing through exon skipping and/or insertion of the FHIT intron 5 sequence or activation of the cryptic splice site. Point mutations were not found in the FHIT coding region but detected in noncoding exon 2, 3, 4, or 5 of eight aberrant transcripts. Significant reduction of FHIT protein expression was observed in 22 of 35 (62.9%) cases. Aberrant FHIT transcription was shown to be associated with loss of FHIT protein expression. However, aberrant FHIT transcripts themselves were not associated with any clinicopathological parameters, such as age, sex, tumor site, or clinical stage. Moreover, there was no association between the presence of LOH at D3S1300 and the expression of aberrant FHIT transcripts. Nevertheless, high frequency of aberrant FHIT transcripts, significant rate of LOH at D3S1300, and altered expression of the FHIT protein indicate that alterations of the FHIT gene can play an important role in gastric carcinogenesis., (Copyright 2001 Academic Press.)

Little is known about genetic alterations of patients who present multiple primary cancers. We hypothesized that microsatellite instability (MSI) is one of the underlying genetic factors in the development of double primary cancers in colorectal cancer patients. We examined for MSI in 41 colorectal cancer patients who presented with extra-colonic primary cancers consisted of 17 gastric and 24 non-gastric cancers. Coincident MSI+ in tumors of two organs were observed in 3 (17.7%) of 17 patients with colon and stomach cancers and 0 of 24 patients with colon and non-gastric cancers (P =.03). In 17 patients with colon and stomach cancers, 6 (31.6%) of 19 colon cancers and 3 (17.7%) of 17 gastric cancers exhibited MSI+. Among four patients with metachronous colon cancers who were identified within the 41 double primary cancer patients, two patients were associated with the MSI+ phenotype. In summary, the prevalent coincidence of MSI suggests that genetic defect of mismatch repair deficiency may be responsible for a small subset of double primary cancers of the colorectum and stomach.

There are limited reports on methylation analysis of the premalignant lesions of gastric carcinoma thus far. This is despite the fact that gastric carcinoma is one of the tumors with a high frequency of CpG island hypermethylation. To determine the frequency and timing of hypermethylation during multistep gastric carcinogenesis, non-neoplastic gastric mucosa (n = 118), adenomas (n = 61), and carcinomas (n = 64) were analyzed for their p16, human Mut L homologue 1 (hMLH1), death-associated protein (DAP)-kinase, thromobospondin-1 (THBS1), and tissue inhibitor of metalloproteinase 3 (TIMP-3) methylation status using methylation-specific PCR. Three different classes of methylation behaviors were found in the five tested genes. DAP-kinase was methylated at a similar frequency in all four stages, whereas hMLH1 and p16 were methylated in cancer samples (20.3% and 42.2%, respectively) more frequently than in intestinal metaplasia (6.3% and 2.1%, respectively) or adenomas (9.8% and 11.5%, respectively). However, hMLH1 and p16 were not methylated in chronic gastritis. THBS-1 and TIMP-3 were methylated in all stages but showed a marked increase in hypermethylation frequency from chronic gastritis (10.1% and 14.5%, respectively) to intestinal metaplasia (34.7% and 36.7%, respectively; P < 0.05) and from adenomas (28.3% and 26.7%, respectively) to carcinomas (48.4% and 57.4%, respectively: P < 0.05). The hMLH1, THBS1, and TIMP-3 hypermethylation frequencies were similar in both intestinal metaplasia and adenomas, but the p16 hypermethylation frequency tended to be higher in adenomas (11.5%) than in intestinal metaplasia (2.1%; P = 0.073). The average number of methylated genes was 0.6, 1.1, 1.1, and 2.0 per five genes per sample in chronic gastritis, intestinal metaplasia, adenomas, and carcinomas, respectively. This shows a marked increase in methylated genes from non-metaplastic mucosa to intestinal metaplasia (P = 0.001) as well as from premalignant lesions to carcinomas (P = 0.002). These results suggest that CpG island hypermethylation occur early in multistep gastric carcinogenesis and tend to accumulate along the multistep carcinogenesis.

beta-catenin serves not only as a structural component of the E-cadherin-mediated cell-cell adhesion system, but also as a signaling molecule of the Wnt/wingless pathway. Deregulated expression of beta-catenin and mutations of the gene have been identified in a number of human malignancies. To determine the role of beta-catenin defects in stomach cancer, we investigated beta-catenin exon 3 mutations and altered protein expression in 77 primary gastric carcinomas and 11 cell lines. In addition, the immunohistochemical expression pattern of beta-catenin in 303 consecutive gastric cancers was determined and their relationships with clinicopathologic features and patient outcome were investigated. This study revealed 5% (4 of 77) tumors and 27% (3 of 11) cell lines with beta-catenin gene alteration, 6 missense mutations, and 1 interstitial deletion. These genetic changes were shown to correlate closely with nuclear localization of the protein (p = 0.001). In an immunohistochemical analysis, abnormal expressions of beta-catenin, such as nuclear accumulation and loss of membranous distribution, were detected in 27% (81 of 303) of tumors overall. These altered beta-catenin expressions were more commonly observed in 37% (58 of 158) diffuse type gastric carcinomas (p < 0.001). Loss of membranous beta-catenin staining was associated with poor survival (p = 0.045). In conclusion, our results demonstrate that beta-catenin mutations are common in gastric cancer cell lines but occur infrequently in gastric carcinoma tissues. These mutations are one of the causes of the nuclear accumulation of beta-catenin. Frequent abnormalities of beta-catenin expression in gastric carcinoma support the idea that both structural and signaling functions of the protein play a critical role in gastric carcinogenesis., (Copyright 2001 Wiley-Liss, Inc.)

We tested three mononucleotide, 45 dinucleotide, and five tetranucleotide repeats in 30 gastric adenomas and 30 gastric carcinomas for microsatellite instability (MSI) in order to evaluate which microsatellites might indicate the MSI status in gastric neoplasms. Along with the increase in tested markers, the proportion of low-frequency MSI (MSI-L) tumors increased. On immunohistochemistry, MSI-L gastric neoplasms did not show any alteration in hMLH1 or hMSH2 protein expression, while most of the high-frequency MSI (MSI-H) tumors did show alterations in the above mismatch repair proteins. The above findings suggested that MSI-L tumors cannot be distinguished from microsatellite stable tumors. Two mononucleotides, BAT25 and BAT26, were sufficient for the screening of MSI. An additional three dinucleotides, D17S786, D6S105 and D19S188, were also highly sensitive and specific in identifying MSI phenotype tumors.

Synchronous gastric carcinomas are found in 4% to 10% of all gastric carcinomas, and the tumor multiplicity is believed to be related to genetic predisposition. To investigate the role of mismatch repair error in synchronous gastric carcinomas, we analyzed the microsatellite instability (MSI) status of 101 cancers from 48 gastrectomy specimens and compared them with 149 solitary gastric carcinomas. Multiple synchronous gastric carcinomas are characterized by slightly older age, predominance in males, early stage and lower lymph node metastasis. Among the 48 cases, 8 (18 lesions) were associated with a gastric adenoma (type I) and 40 (83 lesions) were not associated with a gastric adenoma (type II). The MSI+ rate was 50% in the type I and 8.4% in the type II synchronous gastric carcinomas (p < 0.001), while that of solitary gastric carcinomas was 9.4%. In addition, the frameshift mutation rates of the TGF-betaRII, BAX and hMSH3 genes in the type I synchronous carcinomas were higher than those in the type II synchronous carcinomas. These findings indicate that a defect in the mismatch repair system might play a role in the carcinogenesis of a minor subset of multiple gastric carcinomas associated with adenomas., (Copyright 2001 Wiley-Liss, Inc.)

In order to identify the significant allelic loss involving the gastric adenoma-carcinoma sequence, we used 49 genome-wide microsatellite markers in an allelotype study of 30 cases of stomach resections that harbored both adenomas and carcinomas. Frequent loss of heterozygosity was demonstrated on 12q (53.3%), 2p (50.0%), and 18p (50.0%) in adenomas and on 8q (80.0%), 2p (70.0%), 18p (66.7%), and 17p (61.9%) in carcinomas. Significant difference in the loss of heterozygosity rate between the adenoma and the carcinoma was noted on 17p. Our results suggested that the critical target of loss of heterozygosity in gastric adenomacarcinoma sequences may be the p53 gene on 17p.

We evaluated the rate of Epstein-Barr virus (EBV) infection in gastric carcinomas of Korean patients and investigated the associations between EBV infection and clinicopathological characteristics, the survival rates of patients, and p53 overexpression. EBV-encoded small RNA (EBER)-in situ hybridization and immunohistochemistry for p53 protein were done in 306 consecutive gastric carcinoma cases, of which 17 (5.6%) showed EBV infection. Of these 17 EBV-positive cases, one case strongly expressed p53 protein, while 98 (34%) of 285 EBV-negative cases overexpressed p53 (p < 0.05). The EBV-positive gastric carcinomas tended to have lymphoid stroma. They were mostly of the poorly differentiated type, negative for p53 immunoexpression, more prevalent in male patients, and diffuse according to Lauren's classification (p < 0.05). There was no significant difference in the survival rate for the EBV status. In conclusion, the EBV infection rate among gastric carcinomas in Korea is similar to that ascertained in other countries. An inverse correlation between EBV and p53 overexpression was disclosed. Further study is needed to find out whether or not two genetic changes could be functionally overlapping during gastric carcinogenesis.

Gastric cancer is the most common cause of cancer related death in Korea. Early gastric cancer (EGC), confined to mucosa or submucosa, regardless of lymph node metastasis, is known to have a favorable prognosis. From 1976 to 1995, four thousand nine hundred and twenty eight gastric cancer patients underwent operation at the Severance Hospital, Yonsei University, College of Medicine, Seoul, Korea. Of these, 1,117 patients (22.6%) were diagnosed as EGC and underwent curative operation. Clinicopathologic characteristics were reviewed and survival data was analyzed. The proportion of EGC has increased during the last two decades, from 14.9% during 1976-1985 to 25.8% for 1986-1995. EGC has a wide age distribution range from the thirties to the sixties, with highest incidence in the sixties. The male to female ratio is 1.8:1, without any significant change in last two decades. Most lesions are located in the lower third of stomach (52.3%), and the lesser curvature (52.2%) was the most frequent site in the transverse axis. Macroscopically, the depressed type was the most common (66.1%) followed by the elevated, flat and mixed types, in that order. Tumor confined to the mucosa layer was seen in 52.5%, and lymph node involvement in 11.7%. The depth of tumor invasion correlated with tumor size and regional lymph node involvement. On histopathologic examination, signet ring cell type accounted for 29.6% of all EGCs. Overall 5-year survival rate was 92.7% and the presence of lymph node metastasis significantly affected survival (84.6% versus 96.2%) (p<0.05). In conclusion, the proportion of EGC, in terms of the gastric cancers operated upon, has been increasing in Korea over the last two decades. The introduction of active diagnostic approaches and diagnostic modalities could improve early diagnosis and the cure rate of gastric cancer in Korea.

Aims: To clarify the relationship between the Epstein-Barr virus (EBV) and gastric carcinoma with lymphoid stroma (GCLS) in Koreans, and to characterize the EBV-positive GCLS., Methods and Results: EBV infection was examined using EBER in-situ hybridization and polymerase chain reaction in 45 cases of GCLS among Koreans, and in 292 consecutive cases of gastric carcinomas without lymphoid stroma (non-GCLS) as controls. EBV infection was found in 30 tumours (67%) of GCLS and 10 tumours (3.4%) of non-GCLS (P < 0.05). EBV-positive GCLS was more prevalent in males, poorly differentiated histological type and diffuse type in Lauren's classification, and tended to be located more in the middle third of the stomach than EBV-negative GCLS (P < 0.05). p53 overexpression was observed in 22% of GCLS (17% of EBV-positive GCLS and 33% of EBV-negative GCLS), and 34% of non-GCLS (EBV-positive GCLS vs. non-GCLS: P = 0.056). The survival of the patient with GCLS was not correlated with EBV infection or p53 immunoexpression (follow-up period: 11-97 months)., Conclusions: GCLS in Koreans is strongly associated with EBV infection. The prognosis in GCLS is not dependent upon either the status of EBV infection or the status of p53 immunoexpression.

The MEN1 gene locus is known to be partly responsible for the tumorigenesis of sporadic gastric neuroendocrine tumors, but the genetic events that drive the neoplastic process of this tumor remain largely unknown. In order to screen the tumor suppressor genes associated with the tumorigenesis of gastric neuroendocrine tumors, 15 neuroendocrine carcinomas and three carcinoid tumors in the stomach were analyzed for loss of heterozygosity (LOH) using 22 microsatellite markers. In our study, the gastric neuroendocrine tumors showed a high rate of LOH in chromosomes 8p (82%), 15q (58%), 17p (57%), llp (50%), 12p (50%) and 13q (50%). The mean fractional allelic loss (FAL) was higher in the neuroendocrine carcinoma components than in the adenocarcinoma components (0.42 versus 0.33, respectively). In four cases, the adenocarcinoma components showed discordant LOH patterns from those of the neuroendocrine counterparts in half of the informative chromosomes analyzed. Comparably, the gastric neuroendocrine carcinomas exhibited a higher LOH frequency on 8p and a lower LOH on 7q than did the gastric adenocarcinomas. It is suggested that chromosome 8p is the possible location of the tumor suppressor genes associated with the tumorigenesis of gastric neuroendocrine tumors.

Microsatellite instability (MSI) due to defective DNA mismatch repair (MMR) is a form of genomic instability underlying the tumorigenesis of various human neoplasms. To evaluate the roles of MSI in the pathogenesis of gastric carcinomas with squamous differentiation, 17 primary stomach cancer patients (15 adenosquamous and two squamous cell carcinomas) were examined for MSI frequency using five microsatellite markers and the criteria for MSI recommended by the National Cancer Institute Workshop. The molecular causes and consequences of MSI in these neoplasms were further researched through the immunohistochemistry of MMR proteins and the mutational analysis of cancer-associated genes targeted by MSI, respectively. Two of the 17 (12%) cases demonstrated MSI at the most examined loci and were classified as having high level MSI (MSI-H). These tumors also exhibited frame-shift mutations at mononucleotide repeats in the target genes, including TGFbetaRII, IGFIIR, BAX, and hMSH6. It is interesting to note that the mutations of the serine (AGC)13 repeats within the E2F-4 gene were found only in the squamous cell carcinoma portions of them, whereas such alterations were not detected in any of the adenocarcinomatous portions. This suggests that E2F-4 might be implicated in the transformation of adenocarcinoma into squamous cell carcinoma and further studies are needed to understand its role in squamous differentiation.

To investigate the genetic changes that occur during establishment of gastric cancer cell lines, 4 gastric cancer cell lines and their original tumor tissues were examined for microsatellite instability (MSI), loss of heterozygosity (LOH), and p53 mutation. MSI status did not change during the establishment, though the cell lines gained frameshift mutation in some of the genes with polytract coding sequences. There was no difference in p53 mutation between the cell lines and the original tumors. The frequency of LOH was similar between the cell lines and each original tumor, in the range 30.8% to 85.7%. The difference in the LOH results between the cell lines and the original tumors was not greater than the difference between the different areas of the original tumors. The above results suggested that most of the genetic alterations, such as MSI, LOH, and p53 gene mutation, were sustained during the establishment of gastric cancer cell lines. Minor genetic differences between original tumor tissues and cancer cell lines could be explained as a tumor heterogeneity because separate areas of the original tumor tissues manifest similar variations., (Copyright 2000 Wiley-Liss, Inc.)

A 42-year-old female diagnosed with tuberous sclerosis was found to have multiple polyps in the fundus of stomach. On histologic examination, the lesions were hamartomatous polyps. In tuberous sclerosis, many lesions occur in multiple organs and there are several reports about the frequent association of hamartomatous polyps of the colon. However, gastric manifestation of tuberous sclerosis has not been established probably due to its asymptomatic nature. This is the first report of multiple gastric hamartomatous polyposis in patient with tuberous sclerosis.

Silencing of p16(INK4a) by methylation of the CpG islands in the promoter region has been found to be an alternative mechanism of inactivation in several tumors. However, in gastric carcinoma, the relationship between methylation status and the transcriptional silencing of the p16 gene remains to be clarified. In this study, we investigated whether methylation of a few specific CpG sites in the promoter region could significantly down-regulate p16 activity in the tumorigenesis of gastric carcinoma. By Southern analysis and bisulfite-modified genomic sequencing of 9 gastric-carcinoma cell lines, we found that the 5 cell lines (55.5%) not expressing p16 mRNA had methylated CpG sites at the promoter region of p16. In addition, we analyzed the p16-protein expression of 28 primary gastric carcinomas and their normal counterparts by immunohistochemical staining (IHC) on paraffin sections. Loss of p16 expression was detected in 6 cases (22%). In 5 out of these 6 (83%), the actual p16 gene was inactivated by de novo methylation of the promoter sites. Taken together, these results suggest a strong correlation between de novo methylation of a few specific CpG sites and transcriptional silencing of the p16 gene in gastric carcinoma., (Copyright 2000 Wiley-Liss, Inc.)

Clinicopathological analysis, microsatellite analysis, detection of Epstein-Barr virus (EBV), and immunohistochemistry on p53 protein were performed in 26 cases of gastric remnant cancer (GRC). They were divided into two groups; Group A (n = 14) who had undergone a primary gastrectomy for benign gastric disease, and Group B (n = 12) who had undergone the same operation for gastric cancer. EBV infection was present in 29% of Group A, 8% of Group B and 6% of the conventional gastric carcinoma (CGC) (Group A vs CGC, P = 0.01). Microsatellite instability (MSI) was found in 7% of Group A, 25% of Group B, and 9% of the CGC (Group B vs CGC; P = 0.08). p53 Overexpression was observed in 46% of the GRC and 33% of the CGC. p53 Overexpression was observed in 90% of the intestinal type of GRC, but in only 20% of the diffuse type of GRC (P = 0.002). The cancer stage was a significant factor in the univariate analysis of survival (P = 0.04). In conclusion, GRC is different from CGC in terms of MSI or EBV association. The pathogenetic differences between the two groups require further investigation. EBV infection may have been involved in the carcinogenesis of Group A. MSI may be an important factor in the carcinogenesis of metachronous multiple gastric cancer (Group B).

Sixty-three cases of stomach resections harboring both adenoma and carcinoma were analyzed for microsatellite instability (MSI). The cases included 28 carcinomas arising from adenoma (Type I) and 35 carcinomas with separate adenoma (Type II). The results of MSI assessed by 49 markers were the same for BAT-26 instability. The incidence of MSI was 21% in gastric adenoma and 30% in gastric carcinoma, which is significantly higher than gastric carcinoma without associated adenoma (p < 0.01). Five of eight (63%) cases of multiple carcinomas associated with adenoma showed MSI+ in adenoma and in one or more carcinoma lesion(s). Eight of thirteen (62%) MSI+ adenomas were associated with carcinoma, whereas 20 of 50 (40%) MSI adenomas were associated with carcinoma. MSI+ adenomas of Type I showed a higher mutation rate of the TGF-beta RII gene than Type II (88% versus 40%). Gastric adenoma with TGF-beta RII gene mutation was more prone to transform into carcinoma (p = 0.03). This study revealed that gastric carcinoma arising from adenoma is frequently associated with a mismatch repair deficiency mechanism. In the gastric adenoma-carcinoma sequence, TGF-beta RII gene mutation occurred early in the adenoma stage and it persisted after malignant transformation.

Hepatocyte growth factor (HGF) can promote proliferation of many types of tumor cells including gastric cancer cells. To study the role of HGF in the progression of gastric carcinoma, HGF levels were measured by an enzyme immunoassay (EIA) system in sera of gastric cancer patients and followed up the levels after the operation. The mean serum HGF level in 212 healthy control subjects, 140 patients with primary gastric cancer, and 13 patients with recurrent gastric cancer were 0.199 +/- 0.073, 0.325 +/- 0.209, and 0.578 +/- 0.258 ng/ml, respectively. The increase of the levels was significantly correlated with the progression of tumor stage. The levels decreased to normal levels 1 month after curative resection of the tumors. However, the levels did not decrease significantly in nonresected cases. During the follow-up of the patients for several months, the level was significantly increased in recurrent gastric cancer patients, whereas there was no increase in nonrecurrent patients. In conclusion, the serum HGF levels significantly correlated with the aggressiveness of the tumors, suggesting an important role of HGF in the progression of gastric carcinoma.

Calcification of gastric carcinoma is unusual and most of the reported cases were of the mucinous type. This report describes two cases of papillo-tubular adenocarcinoma of the stomach with psammomatous calcification confined only to the papillary portion. Calcification was so heavy that specimen X-ray was able to clearly delineate its distribution. Microscopically, the calcification was confined to the papillary carcinoma area and was not found in the area of the tubular adenocarcinoma. Polymorphic calcific bodies were found in the supportive stroma of papillae and extrapapillary spaces as concentrically laminated psammoma bodies. They were also found in tumor cells as minute corpuscles. The mechanism of neoplastic mineralization in these cases seemed different from ontogenic calcification of mucinous gastric carcinoma and we postulated the mechanism of psammomatous calcification which is referred as intracellular calcification.

Gastric cancer is the most common cancer in Korea. Germline mutations of the E-cadherin gene have recently been identified in familial gastric cancer patients. We screened five Korean familial gastric cancer patients to investigate germline mutations of the E-cadherin gene. These patients fulfilled the following criteria: presence of at least two gastric cancer patients within first-degree relatives and one patient diagnosed before the age of 50 years. Abnormal band patterns were found in exons 6 and 10 in two familial gastric cancer patients by polymerase chain reaction-single strand conformation polymorphism analysis (probands from the SNU-G2 and SNU-G1001 families, respectively). DNA sequencing analysis of the E-cadherin gene of these two patients revealed missense mutations in each exon. The SNU-G2 proband harbored a missense mutation from aspartic acid (GAT) to glycine (GGT) at codon 244 in exon 6 of the E-cadherin gene, and the SNU-G1001 proband had a missense mutation from valine (GTG) to alanine (GCG) at codon 487 in exon 10. The SNU-G2 proband was diagnosed with gastric cancer at the age of 38; three brothers and two sisters had died of gastric cancer under the age of 50, and their mother had died of gastric cancer at the age of 63. The SNU-G1001 proband was diagnosed with gastric cancer at the age of 42 and one brother had died of gastric cancer at the age of 49. In summary, we found germline mutations of the E-cadherin gene in two of five Korean familial gastric cancer patients screened.

The expression of p53, p16 and RB proteins and their clinicopathologic correlation were investigated in 15 cases of primary gastric adenosquamous carcinoma and 2 cases of squamous cell carcinoma of the stomach. The male to female ratio of the patients was 13:4 and the average age was 55.7 years. None of the cases was early gastric carcinoma, and none of the adenocarcinoma components were of the diffuse or signet ring cell types. Fourteen cases showed metastasis to regional lymph nodes and/or other organs at the time of surgery. The adenocarcinoma component was metastasized to lymph nodes in 12 cases, and both adenocarcinoma and squamous cell carcinoma components were metastasized in three cases. The altered expression of p53 correlated with the advanced stage, but did not correlate with the depth of invasion, lymph node metastasis or recurrence. The altered expression of p16 and RB proteins did not correlate with any of the above clinico-pathologic factors. Both the adenocarcinoma and squamous cell carcinoma components revealed an inverse correlation between the expression pattern of p16 and RB proteins (p < 0.05). This suggests that the two proteins share a role in the carcinogenesis of these tumors. The expression pattern of p53 proteins in the adenocarcinoma and squamous cell carcinoma components was exactly the same in all of the cases. The expression patterns of p16 and RB protein were also identical in most of the cases. The expression patterns of all three proteins in the metastatic lesions were also identical to those in the primary lesions. The fact that the alteration of the three tumor suppressor gene products shares the same pattern suggests that squamous and adenocarcinoma components in the stomach originate from the same or a genetically related clone.

The accumulation of wild-type p53 protein results in two pathways, cell cycle G1 arrest by p21WAF1/CIP1/SDI1 and apoptosis inhibited by bcl2, which together carry out the tumor suppressor function. Since genetic alterations of p53 are frequently observed in gastric cancers, the expression of p21 and bcl2 may be altered in gastric carcinogenesis. We therefore analyzed normal mucosa, nondysplastic lesions, hyperplastic polyps, adenomas and carcinomas of the human stomach using immuno-histochemistry, polymerase chain reaction-single-strand conformation polymorphism and DNA sequencing. In normal gastric mucosa, the expression of p21, bcl2 and p53 was topographically restricted: a) p21 expression was limited to foveolar epithelial cells; b) bcl2 and p53 expression was confined to only a few regenerative epithelial cells of the mucous neck region. In chronic gastritis or intestinal metaplasia, topographic expression became more obvious. This topographic expression was altered in hyperplastic polyps and adenomas. Hyper-plastic polyp showed an increased p21 and p53 expression with no bcl2 expression. Where as bcl2 expression increased and extended up parabasal and superficial dysplastic epithelium, p21 expression increased and was limited to surface dysplastic epithelium. Weak p53 expression was in full thickness of dysplastic epithelium. p21 and bcl2 expression in adenoma was higher than in intestinal type of carcinoma. In carcinomas, this topography was abrogated, but p53 mutation (36%) was present. There was no relationship between p53, p21 and bcl2 expression. As a result, in normal gastric epithelial cells, there was a precisely ordered topographic pattern of p21, bcl2 and wild-type p53 expression that becomes disordered during neoplasia. These results suggest that altered cell cycle and apoptosis control by wild-type p53 and its mediators appears to be an early event in gastric carcinogenesis that may facilitate tumor progression.

This study was designed to demonstrate that differentiation of stomach cancer cells can be modified by microenvironmental change and to look for a method inducing or promoting tumor cell differentiation. To evaluate the biomorphological characterization of tumor cell differentiation in suramin-containing in vitro culture of human stomach cancer cell lines, inverted phase-contrast microscopic examination, analysis of growth curves and BrdU-positive S-phase fraction, immunocytochemical study, radioimmunoassay for CEA, transmission electron microscopic examination, DNA flow cytometry, and heterotransplantation in SCID mice were performed. Suramin inhibited tumor cell growth. Development of intracytoplasmic lumina and intercellular lumina was noted in suramin-containing culture with formation of numerous microvilli and frequent desmosomes. The amount of CEA released by a cell was increased in suramin-containing culture. Suramin inhibited heterotransplantation, and a transplant from suramin-containing culture revealed a much higher degree of differentiation than that from suramin-absent culture. Suramin induced no change in DNA ploidy pattern. Elimination of suramin from the culture medium did not reverse the tumor cell differentiation. Each stomach cancer cell line showed a different degree of responsiveness to suramin. In conclusion, this study shows that suramin inhibits growth of SNU-5 and SNU-16 cells and that suramin induces differentiation of SNU-16 cells.

Multiple gastric cancers, which constitute 4% to 10% of all gastric cancers, occur in older people and are associated with more extensive intestinal metaplasia. With regard to the genesis of multiple gastric cancers, multicentricity (independent origin) rather than multifocality (local or lateral spread of one cancer) has been the favored theory. Conventional morphologic study, however, has not been able to provide convincing evidence in support of multicentricity. The purpose of this study was to verify the multicentric origin of multiple gastric cancers at a genetic level. For this purpose, immunohistochemical and molecular techniques were used to define the mutation pattern of APC, MCC and p53 in multiple lesions of synchronous multiple gastric cancers. The study was based on a total of 30 gastric tumors from 13 patients, including 10 double tumors, 2 triple tumors, and 1 quadruple tumor. Single-strand conformation polymorphism and polymerase chain reaction direct sequencing were carried out for exons 5 to 8 of p53, and loss of heterozygosity was detected on the basis of polymerase chain reaction amplification of polymorphism in exon 10 of MCC and in exon 11 of APC. Twelve of 13 cases showed alteration in one or more genetic markers. Of these, three demonstrated a discordant mutation pattern of p53 in individual lesions, and another two revealed allelic loss of MCC in one lesion and p53 mutation in the other. In six other cases, only one lesion showed alteration of APC, MCC, or p53, and in the remaining case, one lesion carried p53 and MCC mutations and the other carried MCC loss of heterozygosity only. The results of this study showed discordance of the mutation pattern of APC, MCC, and p53 in individual lesions of multiple gastric cancers, providing genetic evidence for a multicentric origin of synchronous multiple gastric carcinomas. Collectively, these findings supported the theory of field cancerization in gastric carcinogenesis.

We report 8 newly established gastric-carcinoma cell lines (SNU-216, 484, 520, 601, 620, 638, 668, 719) from Korean patients. Morphologic study was carried out using light and electron microscopes. CEA, alpha FP, and CA 19-9 and TPA in supernatant and in cell lysate were measured by radioimmunoassay. p53 and c-Ki-ras gene mutations were screened and confirmed by sequencing. The cell lines, derived from tumors with moderate differentiation, grew as a diffuse monolayer, and those from tumors with poor differentiation and minimal desmoplasia grew exclusively as non-adherent. Out of the 8 gastric-cancer cell lines, 5 had detectable levels of CEA both in supernatant and in cell lysate; there was no expression or secretion of alpha FP in these cells; 4 cell lines showed high levels of CA 19-9 in cell pellets. All cell lines except SNU-484 had high concentrations of TPA both in cell lysate and in supernatants. p53 mutation was found in 6 cell lines (75%): 2 (SNU-216 and SNU-668) had mutations in exon 6, and other 3 in exon 8. The c-Ki-ras mutation was found in 2 cell lines (25%), SNU-601 and SNU-668. The former showed GGT-to-GAT transition mutation at codon 12, while the latter showed CAA-to-AAA transversion mutation at codon 61. DNA profiles using restriction endonuclease HinfI and polymorphic DNA probes ChdTC-15 and ChdTC-114 showed different unique patterns; which suggests that these cell lines are unique and not cross-contaminated. We believe that the newly characterized gastric-cancer cell lines presented in this paper will provide a useful in vitro model for studies related to human gastric cancer.

Objectives: We compared the ability of preoperative abdominal computed tomography (CT) with that of preoperative abdominal ultrasonography (US) in predicting the extent of tumor growth and the curative resectability of gastric cancer., Methods: Abdominal CT and US were done in 95 patients with gastric adeno-carcinoma. The radiologic findings were prospectively compared with surgical and pathologic findings., Results: The sensitivities of abdominal CT and US in detecting the perigastric lymph node involvement were 26.6% and 20%, respectively. The sensitivity of abdominal CT in predicting the pancreatic invasion (60%) was better than that of abdominal US (20%). However, there were 6 false positive diagnosis of pancreatic invasion by abdominal CT; in contrast, there was no false positive diagnosis of pancreatic invasion by abdominal US. Of 95 patients who underwent surgical exploration, 14 were found to have unresectable tumors because of extragastric organ involvement and distant metastases. Abdominal CT falsely predicted 8 of 14 cases in which curative gastric resection was impossible as resectable. Moreover, abdominal CT falsely predicted 5 of 81 cases in which curative gastric resection was possible as unresectable. Abdominal US falsely predicted 12 of 14 cases in which curative surgery was impossible as resectable., Conclusion: Preoperative abdominal Ct and US did not accurately predict the tumor extent and the curative resectability of the gastric cancer. Especially, abdominal CT finding of pancreatic invasion by gastric cancer should not be a contraindication for surgical exploration.

The stomach is the most frequent site of extranodal lymphoma and primary gastric lymphoma might be distinguished from the nodal lymphoma by its different pathogenesis and prognosis. Based on the Isaacson's classification, clinico-pathologic reviews of 38 resected primary gastric lymphomas were done. Immunohistochemical stainings for PCNA, B and T cell markers, bcl-2 and p53 were performed. Eighteen were of low grade and 20 were of high grade. There were significant differences between low and high graders in the aspect of the size, depth of lesion, gross type, immunophenotype, staining intensity for PCNA, expressions of bcl-2 and p53. The overall 2-year survival rate was 85.3%. Factors with prognostic significance on survival by univariate analyses included immunophenotype, histologic grading and PCNA staining pattern. After multivariate analyses, immunophenotype proved to be a significant factor. We think that the histologic grading by Isaacson's classification and the immunohistochemical stainings performed were useful in pathologic and/or clinical aspects. The excellent survival rate in this study was partly due to the selection of resectable cases. However, earlier diagnosis and appropriate treatment might have contributed to the improved prognosis of gastric lymphoma in recent years.

This retrospective study was designed to investigate the relationship between overexpression of the 67 kD laminin receptor (67LR) using immunohistochemistry, and several clinicopathological parameters including overall survival in human gastric adenocarcinoma. We stained paraffin-embedded sections of 93 resected primary gastric adenocarcinomas using a polyclonal antibody specific for the 67LR as well as monoclonal antibodies for p53 protein, epidermal growth factor receptor, proliferating cell nuclear antigen, carcinoembryonic antigen and chromagranin. The results showed statistically significant correlations between overexpression of the 67LR and types of early or advanced gastric carcinoma (p < 0.001), depth of invasion (p < 0.001), WHO histopathologic classification (p < 0.001), stage (p = 0.001), expression of p53 protein (p = 0.019), expression of epidermal growth factor receptor (p < 0.001) and proliferating cell nuclear antigen labeling index (p = 0.002). A lower proportion of signet ring cells revealed a higher percentage of overexpression of 67LR in both early (p < 0.002) and advanced (p < 0.001) gastric carcinomas. Intestinal type adenocarcinoma (according to Lauren's classification) revealed a higher percentage of overexpression of the 67LR than the diffuse type in both early (p = 0.057) and advanced (p < 0.001) gastric carcinomas. The correlations between overexpression of the 67LR and lymph node metastasis were statistically significant (p < 0.07). Although the overexpression of the 67LR tended to correlate with lower survival rates, the correlation was not statistically significant due to the limited sample size. Our data revealed that overexpression of the 67LR is correlated with the progression of gastric carcinoma. The expression of the 67LR may be important as one of the steps which determines invasiveness during the progression of cancer.

Background: Histamine-2 receptor antagonist (H2-RA) have been shown to improve the function of various parts of the immune system. The proposed mechanism of the immunomodulative effects of H2-RA has been considered to be the inhibition of suppressor T-lymphocyte activity, an increase in interleukin-2 production, and an enhancement of natural killer cell activity. Most of these studies were done with cimetidine. Comparative data with other H2-RA are limited and conflict on immunomodulative effects. Comparison of the actions of H2-RA on the immune system is required., Methods: We compared the immunodulative effect of the H2-RAs cimetidine, ranitidine, and famotidine on peripheral blood mononuclear cells (PBMC) in normal controls and patients with gastric cancer. DNA synthesis, cytotoxicity against K562 cells, and the levels of soluble interleukin-2 receptor (sIL-2R) in supernatant were measured after addition of the various H2-RA to PBMC cultures., Results: Subjects with gastric cancer showed significantly higher levels of suppressor lymphocyte activity than normal controls. These levels were restored to levels of normal controls by the addition of cimetidine. Statistically significant lymphoblastogenesis and cytotoxicity against K562 cells were observed only in cimetidine-treated PBMC (p < 0.05); such effects were not observed in ranitidine- or famotidine-treated PBMC. Significantly increased levels of sIL-2R were found in supernatants obtained from culture flasks treated with cimetidine and phytohemagglutinin or ranitidine and phytohemagglutinin (p < 0.01)., Conclusions: Of the three H2-RAs tested, cimetidine had the strongest and famotidine the weakest immunomodulating effect. Only cimetidine augmented the cytotoxicity and proliferative response of lymphocyte to mitogen; neither ranitidine nor famotidine had such an effect. These results might be due to their structural differences. In addition, the immunologic effects of H2-RA are unlikely to be mediated via specific interaction at the H2 receptor.