Gastric Carcinoma Samples Research Articles

Expression and significance of TWIST basic helix-loop-helix protein over-expression in gastric cancer

TWIST protein has been implicated in neoplastic transformation and development of some cancers. In this study, we aimed to investigate the expression of TWIST in gastric cancer and its clinical significance. A total of 76 cases of archival gastric cancer tissues were immunohistochemically evaluated for TWIST expression, and its expression was correlated with clinicopathological parameters. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the mRNA of TWIST in four gastric cancer cell lines and a normal immortalised gastric epithelial cell line (GES-1). The expression of TWIST protein in these cell lines and 14 pairs of fresh gastric carcinoma and adjacent normal tissue samples was detected by Western blotting. TWIST expression increased in diffuse-type gastric carcinoma compared with intestinal-type gastric carcinoma (26/42, 61.9% versus 9/34, 26.5%, p<0.05). TWIST expression was significantly increased in 35 (46.1%) of the 76 cancers and correlated with lymph node metastasis (node positive rate 60.4%; node negative rate 21.4%; p<0.05). The expression of TWIST protein was higher in 9/14 (64.3%) fresh cancer tissues compared with adjacent normal tissues. The expression of mRNA and protein of TWIST in gastric cancer cell lines was up-regulated compared with that in GES-1. TWIST was highly expressed in gastric cancer. Its up-regulation was associated with the neoplastic transformation and subsequent development of gastric cancer. Therefore, TWIST may be a useful prognostic marker and target for gastric cancer therapy.

Vascular endothelial growth factor and endoglin (CD-105) in gastric cancer

Vascular endothelial growth factor (VEGF) overexpression has been associated with advanced stage and poor survival in several cancers. Additionally, CD-105 (endoglin) was proposed as a marker of neovascularization in solid malignancies. The aim of the present study was to (1) evaluate the VEGF and CD-105 expression in gastric carcinoma, (2) determine the role of VEGF gene sequence variations in VEGF expression in gastric carcinoma, and (3) correlate the results of VEGF and CD-105 expression with other standard prognostic parameters, such as size, grade, stage of the disease, metastases, and patient survival. VEGF and CD-105 expression were evaluated in 100 unrelated gastric cancer patients using immunohistochemistry. For the genotyping, DNA was isolated from the blood of the gastric cancer patients and from 100 healthy individuals. The genotyping was performed by polymerase chain-restriction fragment length polymorphism analysis. VEGF protein was strongly expressed in the cytoplasm of 36% of the gastric carcinoma samples tested. In all cases, high VEGF expression was accompanied with high endoglin expression. Our results revealed no statistical significant association of any VEGF gene polymorphism with the VEGF and endoglin expression. The correlation of VEGF/CD-105 expression with the clinicopathological parameters of gastric cancer showed that the high expression of VEGF/CD015 was correlated only with lymph node metastasis (P = 0.028). The Kaplan-Meier survival curves have shown a clear association of overall survival after diagnosis of gastric cancer with high VEGF, as well as high CD-105 expression. Our results support that VEGF and CD-105 are closely relevant to lymph node metastasis and act as two valuable indicators of prognosis.

Detection of let-7a microRNA by real-time PCR in gastric carcinoma

To establish an accurate and rapid stem-loop reverse transcriptional real-time PCR (RT-PCR) method to quantify human let-7a miRNA in gastric cancer. According to the sequence of let-7a miRNA, the stem-loop reverse transcriptional primer, the primers and quantitative MGB probes of real-time PCR were designed and synthesized. The dynamic range and the sensitivity of quantitative reverse transcriptional real-time PCR were determined. The levels of let-7a miRNA were examined in 32 gastric carcinoma samples by stem-loop RT-PCR method. The dynamic range and sensitivity of the let-7a miRNA quantification scheme were evaluated, the result showed the assay could precisely detect 10 copies of mature let-7a miRNA in as few as 0.05 ng of total RNA of gastric mucosa. The results of specificity analysis showed no fluorescence signal occurred even though 50 ng of human genomic DNA was added to the reverse transcription (RT) reaction. The expression level of let-7a miRNA in gastric tumor tissues was significantly lower compared to normal tissues in 14 samples from 32 patients. The stem-loop RT-PCR is a reliable method to detect let-7a miRNA which may play an important role in the development of gastric carcinoma.

Epstein-Barr virus (EBV) infection and p53 protein expression in gastric carcinoma

In the presented studies p53 protein expression was evaluated in samples of gastric carcinoma originating from 32 selected adult patients (with documented diagnosis of adenocarcinoma of the stomach and without the presence of Helicobacter pylori infection). Among the patients 14 individuals carried EBV-positive gastric carcinoma (group 1) while the 18 remaining patients carried EBV-negative gastric carcinoma (group 2). EBV infection was detected testing the tissue material for the presence of EBER by RNA in situ hybridization (ISH) and testing sera of the patients for EBV-specific antibodies. Expression of p53 protein was analysed using immunohistochemistry. Presence of p53 protein was noted in 9 (64.3%) cases of EBV-positive gastric cancer (group 1) and in 10 (55.5%) cases of EBV-negative gastric cancer (group 2). No significant differences were detected in the frequencies of p53 protein expression in the two studied groups. The results permit to conclude that abnormalities in p53 in gastric cancer are independent of EBV infection, even if EBV may participate in development of the tumour.

Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis

To investigate the expression of co-stimulatory molecule B7-H3 in gastric carcinoma and adenoma tissue as well as normal gastric tissue and to explore the relationship between B7-H3 expression and pathological features and prognosis of gastric carcinoma. B7-H3 expression was detected in 102 samples of human gastric carcinoma and 10 samples of gastric adenoma and 10 samples of normal gastric tissue by immunohistochemical assay. Correlation between the expression of B7-H3 and the patients' age, sex, gastric carcinoma locus, tumor size, tissue type, tumor infiltration depth, differentiation degree, lymph node metastasis, and survival time was analyzed. B7-H3 was expressed in all gastric adenoma samples and in 58.8% samples of gastric carcinoma. B7-H3 expression in gastric carcinoma samples was not related with the patients' age, sex, lymph node metastasis, and tumor size (P>0.05), but with the survival time, infiltration depth of tumor and tissue type. Detection of B7-H3 expression in gastric carcinoma tissue is beneficial to the judgment of the prognosis of gastric carcinoma patients and the choice of treatment.

Comparison of gene expression profiles between primary tumor and metastatic lesions in gastric cancer patients using laser microdissection and cDNA microarray

To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection (LMD) in combination with cDNA microarray. Normal gastric tissue samples from 30 healthy individuals, 36 cancer tissue samples from primary gastric carcinoma and lymph node metastasis tissue samples from 58 patients during gastric cancer resection were obtained using LMD in combination with cDNA microarray independently. After P27-based amplification, aRNA from 36 of 58 patients (group 1) with lymph node metastasis and metastatic tissue specimens from the remaining 22 patients (group 2) were applied to cDNA microarray. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical assay verified the results of microarray in group 2 and further identified genes differentially expressed in the progression of gastric cancer. The expression of 10 genes was up-regulated while the expression of 15 genes was down-regulated in 22 gastric carcinoma samples compared with that of genes in the normal controls. The results were confirmed at the level of mRNA and protein, and suggested that four genes (OPCML, RNASE1, YES1 and ACK1) could play a key role in the tumorigenesis and metastasis of gastric cancer. The expression pattern of 3 genes (OPCML, RNASE1 and YES1) was similar to tumor suppressor genes. For example, the expression level of these genes was the highest in normal gastric epithelium, which was decreased in primary carcinoma, and further decreased in metastatic lymph nodes. On the contrary, the expression pattern of gene ACK1 was similar to that of oncogene. Four genes were further identified as differentially expressed genes in the majority of the cases in the progression of gastric cancer. LMD in combination with cDNA microarray provides a unique support foe the identification of early expression profiles of differential genes and the expression pattern of 3 genes (OPCML, RNASE1 and YES1) associated with the progression of gastric cancer. Further study is needed to reveal the molecular mechanism of lymph node metastasis in patients with gastric cancer.

DNA methylation of genes linked with retinoid signaling in gastric carcinoma

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

Aberrant CpG island methylation in early-onset sporadic gastric carcinoma

Gastric carcinoma more commonly affects older patients, and it is thought that cases of early-onset gastric carcinoma may develop with a different molecular profile different from that of carcinoma occurring at a later age. We assayed the methylation status and genetic changes in genes associated with the APC-beta-catenin axis and the mismatch repair system in relatively early-onset gastric carcinoma samples to determine their association with gastric carcinogenesis. Tumor and normal tissue DNA samples were obtained from 40 patients with early-onset (< 50 y) gastric carcinomas and assayed for APC and CTNNB1 mutations, microsatellite instability, and methylation of the promoters of the hMLH1, TIMP3, THBS1, DAP- K, GSTP1 , APC, and MINT2. Promoter methylation at these seven loci ranged from 12.5 to 62%, with 38/40 tumors (95%) showing promoter methylation at more than one locus. The CpG island methylation phenotype (CIMP) was classified as high in 16 tumors (40%), low in 22 tumors (55%), and negative in 2 tumors (5%). Two concurrent missense mutations (E1685G, R1763L) in the APC mutation cluster region were detected in two tumors, nine tumors showed loss of APC heterozygosity (LOH), and two showed both LOH and promoter methylation. Our results indicate that, unlike in colorectal carcinoma, APC and CTNNB1 mutations do not appear to be highly implicated in early-onset gastric carcinogenesis. In contrast, our data show that promoter methylation is a prevalent phenomenon in early-onset gastric carcinoma and may be related to gastric carcinogenesis.

Clinical significance of expression of apoptotic signal proteins in gastric carcinoma tissue

To evaluate the expressions of apoptotic signal proteins FADD, TRADD, FasL, Fas, and NFkappaB in gastric carcinoma tissues and their clinical significance. Western blot immune trace method was adopted to detect the expressions of apoptotic signal proteins FADD, TRADD, FasL, Fas, and NFkappaB in 55 tissue specimens of gastric carcinoma. Five apoptotic signal proteins had different expressions in the gastric carcinoma samples and their expressions were not correlated to age (P = 0.085). Expressions of the FADD, FasL, Fas, and NFkappaB proteins reduced with increase of the volume of tumor with the exception of increased expression the TRADD protein (64.7-71.1%, P = 0.031). With gradual increase of the malignancy of gastric carcinoma tissues, expressions of the FADD, FasL, and Fas proteins decreased (78.6-28.0%, P = 0.008; 78.6-65.9%, P = 0.071; 100.0-46.3%, P = 0.014), while expressions of the TRADD and NFkappaB proteins increased (42.9-78.1%, P = 0.063; 78.6-79.1%, P = 0.134). With gradual increase of serum CEA, expression of the FADD protein decreased (62.5-34.0%, P = 0.073), but expressions of the TRADD, FasL, Fas, and NFkappaB proteins increased (0.0-80.8%, P = 0.005; 62.5-70.2%, P = 0.093; 0.0-70.2%, P = 0.003; 62.5-80.9%, P = 0.075). When compared to the tissues of gastric carcinoma without metastasis, the positive rate of expressions of the FADD and FasL proteins increased, whereas expressions of the TRADD, FADD, and NFkappaB proteins decreased. There was no significant difference between them (P = 0.095). Gastric carcinoma is endurable to Fas-related apoptosis and apoptotic signal proteins are differently expressed in gastric carcinoma.

Expression of nitric oxide synthase in human gastric carcinoma and its relation to p53, PCNA

To investigate the expression of NOS in gastric carcinoma, and to explore the relationship between the expression of nitric oxide synthases (NOS) and p53, PCNA, pathological features and clinical staging of gastric cancer. The activity of NOS protein was investigated in 85 samples of human gastric carcinoma and 25 samples of normal gastric mucosal tissue by biochemical assay. We then examined the expression of NOS, p53, PCNA in 85 samples of human gastric cancer was examined by immunohistochemistry, and NOS mRNA expression in 85 gastric cancer tissue specimens by in situ hybridization. Biochemical assay showed that the activity of NOS was significantly higher in gastric carcinoma than in normal gastric mucosal tissues (t = 0.4161, P<0.01). Immunohistochemistry revealed that endothelial nitric oxide synthase (eNOS) expressed in all samples of normal gastric mucosa, but only 6 cases of 85 gastric cancer specimens showed weak positive immunohistochemical reactions to eNOS (20%). Inducible nitric oxide synthase (iNOS) was expressed strongly in human gastric carcinoma (81.2%). In situ hybridization analysis showed that iNOS mRNA expression was significantly stronger than eNOS mRNA expression in gastric cancer tissue (chi(2) = 10.23, P<0.01). The expression of iNOS in gastric cancer was associated with differentiation, clinical stages or lymph node metastases (r = 0.3426, P<0.05). However, iNOS expression did not correlate with histological classifications and morphological types. The expression of iNOS was significantly correlated with p53 or PCNA expression (r = 0.3612, P<0.05). The expression of neuronal nitric oxide synthase (nNOS) was not examined by immunohistochemistry and in situ hybridization in gastric cancer specimens and normal gastric mucosa. In human gastric cancer, there is an enhanced expression of iNOS, but not of eNOS. NOS promotes the proliferation of tumor cells and plays an important role in gastric cancer spread. Inactivation of antioncogene p53 and overexpression of iNOS might play a synergetic role in the process of carcinogenesis of human gastric carcinoma.

Effect of Helicobacter pylori infection on p53 expression of gastric mucosa and adenocarcinoma with microsatellite instability.

To investigate the relationship between Helicobacter pylori (H pylori) infection, microsatellite instability and the expressions of the p53 in gastritis, intestinal metaplasia and gastric adenocarcinoma and to elucidate the mechanism of gastric carcinogenesis relating to H pylori infection. One hundred and eight endoscopic biopsies and gastric adenocarcinoma were available for the study including 33 cases of normal, 45 cases of gastritis, 30 cases of intestinal metaplasia, and 46 cases of gastric adenocarcinoma. Peripheral blood samples of these patients were also collected. H pylori infection and p53 expressions were detected by means of streptavidin-peroxidase (SP) immunohistochemical method. Microsatellite loci were studied by PCR-SSCP-CE using the markers BAT-26, D17S261, D3S1283, D2S123, and D3S1611. MSI was defined as the peak shift in the DNA of the gastric tissue compared with that of the peripheral blood samples. Based on the number of mutated MSI markers, specimens were characterized as high MSI (MSI-H) if they manifested instability at two or more markers, low MSI (MSI-L) if unstable at only one marker, and microsatellite stable (MSS) if they showed no instability at any marker. H pylori infection was detected in the samples of gastritis, intestinal metaplasia, and gastric adenocarcinoma and the infection frequencies were 84.4%, 76.7%, and 65.2%, respectively, whereas no H pylori infection was detected in the samples of normal control. There was a significant difference in the infection rates between gastritis and carcinoma samples (P = 0.035). No MSI was detected in gastritis samples, one MSI-H and two MSI-L were detected among the 30 intestinal metaplasia samples, and 12 MSI-H and 3 MSI-L were detected in the 46 gastric carcinomas. In those gastric carcinomas, the MSI-H frequency in H pylori-positive group was significantly higher than that in H pylori-negative group. No p53 expression was detected in the normal and gastritis samples from dyspeptic patients. P53-positive immunohistochemical staining was detected in 13.3% of intestinal metaplasia samples and in 43.5% of gastric carcinoma samples. The levels of p53 in H pylori-positive samples were higher than those in the negative group when the carcinoma samples were subdivided into H pylori-positive and -negative groups (P = 0.013). Eight samples were detected with positive p53 expression out of the 11 MSI-H carcinomas with H pylori infection and no p53 expression could be seen in the H pylori-negative samples. H pylori affect the p53 pattern in gastric mucosa when MMR system fails to work. Mutations of the p53 gene seem to be an early event in gastric carcinogenesis.

Relationship between expression and distribution of cyclooxygenase-2 and bcl-2 in human gastric adenocarcinoma

To explore expression and distribution features of COX-2 and bcl-2 in human gastric adenocarcinoma tissues and to study its biological significance. Totally 36 human gastric carcinoma samples were enrolled in this study (cardiac adenocarcinoma 16 cases, distal gastric adenocarcinoma 20 cases). The expressions of COX-2 and bcl-2 in cancerous tissues and corresponding para-cancerous tissues were investigated by immunohistochemistry using COX-2 polyclonal antibody and bcl-2 monoclonal antibody. The normal gastric mucosa tissues were used as control. The expressions of COX-2 and bcl-2 in gastric carcinoma were significantly higher than that in the para-cancerous tissues (77.8% vs 47.2%, P<0.01, 80.56% vs 58.33%, P<0.05). The expression of COX-2 in cardiac adenocarcinoma was remarkably higher than that in the distal gastric carcinoma (93.8% vs 65.0%, P<0.01). The expression of COX-2 was mainly localized in the cytoplasm of tumor cells and partly in the nucleus. There is a transition of the COX-2 cytoplasmic positivity to nucleic in tumor cells with the increase of gastric carcinoma pathological grade. Interstitial macrophages, fibroblasts and vascular endothelial cells also expressed COX-2. The tissues with higher expression of COX-2 also expressed high level of bcl-2 protein. Abnormal expression pattern of COX-2 within the tissues of human gastric cancer is correlated with tumor location and lymph node metastasis. COX-2 may regulate expression of apoptosis suppressor gene (bcl-2) through interaction of tumor cells and stromal cells and play an important role in the generation and development of tumors, which will be of great help in developing new methods for antitumor therapy.

Diffuse Expression of hRFI is Correlated with Blood Vessel Invasion in Gastric Carcinoma

hRFI, which has a relatively high homology to XIAP, is preferentially expressed in esophageal and colorectal carcinomas, and is involved in the initial tumor formation in the colorectal adenoma-carcinoma sequence. Furthermore, its diffuse expression is associated with colorectal carcinogenesis. However, hRFI expression in gastric carcinomas has not been evaluated so far. We performed immunohistochemical staining on 76 gastric carcinoma samples using the antibody to hRFI and also analyzed the correlation between the staining pattern of hRFI and the clinico-pathological characteristics. All of the samples were stained focally (31 cases, 40.8%) or diffusely (45 cases, 59.2%) in the cancerous region. On the contrary, most of the normal gastric region showed no staining, except for a few cases that showed slight immunoreactivity in speckles. Furthermore, the proportion of blood vessel involvement was significantly higher in carcinomas with diffuse hRFI expression (28/45, 62.2%) than in carcinomas with focal expression (7/31, 22.6%) (P < 0.001). Liver metastasis was consistently observed in five cases (11.1%) in diffuse, but only one (3.3%) in focal type during the average follow up period of 5 years. However, the 3-year survival rate did not show significant difference between these different staining patterns of hRFI. These results suggest that the detection of the expression pattern of hRFI in gastric carcinomas can be another useful predictor of liver recurrence, especially when combined with other factors.

Interleukin-6 increases vascular endothelial growth factor and angiogenesis in gastric carcinoma.

Interleukin-6 (IL-6) is a proinflammatory cytokine associated with the disease status of gastric carcinoma (GC). Vascular endothelial growth factor (VEGF) is a potent tumor angiogenic factor in GC. In this study, we attempted to clarify whether IL-6 can regulate VEGF and angiogenesis in GC. GC samples from 54 surgical specimens were subjected to immunohistochemical examination of IL-6, VEGF, and tumor microvessels, and results showed that IL-6 was positively correlated with VEGF expression and tumor vasculature. We determined VEGF expression in four GC cell lines by ELISA, revealing that GC cells can produce significant amount of VEGF with increasing dose and duration of IL-6 stimulation. Next, a luciferase reporter gene assay was employed to determine the signaling pathway driving the VEGF promoter by IL-6, which showed that the JAK/STAT pathway is involved in the stimulation of VEGF gene expression. The effects of IL-6 on angiogenesis in vitro and in vivo were evaluated by HUVEC studies and the Matrigel plug assay, respectively. Results showed that IL-6 effectively promoted HUVEC proliferation and tube formation in vitro and Matrigel plug vascularization in vivo, primarily by inducing VEGF in GC. This study provides evidence that the multifunctional cytokine, IL-6, may induce VEGF expression which increases angiogenesis in gastric carcinogenesis.

Interleukin-6 Increases Vascular Endothelial Growth Factor and Angiogenesis in Gastric Carcinoma

Interleukin-6 (IL-6) is a proinflammatory cytokine associated with the disease status of gastric carcinoma (GC). Vascular endothelial growth factor (VEGF) is a potent tumor angiogenic factor in GC. In this study, we attempted to clarify whether IL-6 can regulate VEGF and angiogenesis in GC. GC samples from 54 surgical specimens were subjected to immunohistochemical examination of IL-6, VEGF, and tumor microvessels, and results showed that IL-6 was positively correlated with VEGF expression and tumor vasculature. We determined VEGF expression in four GC cell lines by ELISA, revealing that GC cells can produce significant amount of VEGF with increasing dose and duration of IL-6 stimulation. Next, a luciferase reporter gene assay was employed to determine the signaling pathway driving the VEGF promoter by IL-6, which showed that the JAK/STAT pathway is involved in the stimulation of VEGF gene expression. The effects of IL-6 on angiogenesis in vitro and in vivo were evaluated by HUVEC studies and the Matrigel plug assay, respectively. Results showed that IL-6 effectively promoted HUVEC proliferation and tube formation in vitro and Matrigel plug vascularization in vivo, primarily by inducing VEGF in GC. This study provides evidence that the multifunctional cytokine, IL-6, may induce VEGF expression which increases angiogenesis in gastric carcinogenesis.

Expression of integrin-linked kinase is closely correlated with invasion and metastasis of gastric carcinoma.

Integrin-linked kinase (ILK), a serine/threonine protein kinase, implicates in cellular control of cell-matrix interactions and cell proliferation that is associated with a highly invasive phenotype of certain tumors. To study whether ILK is involved in the development and progression of gastric carcinoma, we examined the expression of ILK in gastric carcinoma cell lines, primary gastric carcinomas and corresponding non-neoplastic mucosa using reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry and analyzed the relationship with clinicopathological parameters. The expression of ILK mRNA was detected in 4 of 5 gastric carcinoma cell lines and 22 of 35 (63%) microdissected tumor samples of primary gastric carcinoma using RT-PCR. The incidence of cases with ILK mRNA expression was significantly higher in scirrhous and intermediate type (82%) than in medullary type (44%) (P=0.0204). Significant association was detected between ILK mRNA expression and presence of nodal metastasis (P=0.0388). Immunohistochemically, strong expression of ILK protein (over 50% of tumor cells were positive) was detected in 69% (84/122) of primary gastric carcinomas, whereas no ILK expression was found in non-neoplastic gastric epithelia. Strong expression of ILK protein was significantly associated with scirrhous and intermediate type (P=0.0217), deep invasion of tumor cells in gastric wall (P=0.0006), and presence of nodal metastasis (P=0.0176). These results strongly suggest that ILK may participate not only in stomach carcinogenesis especially of scirrhous and intermediate types but also in invasion and metastasis of gastric carcinoma. ILK might be a novel molecular marker for aggressive gastric cancer.

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity and low-density lipoprotein receptor expression in diffuse-type and intestinal-type human gastric cancer.

Cancer cells require more cholesterol than normal cells. This requirement may be satisfied by higher hydroxymethylglutaryl coenzyme A reductase activity or low-density lipoprotein receptor expression or both. We investigated hydroxymethylglutaryl coenzyme A reductase activity and low-density lipoprotein receptor expression in samples of gastric carcinoma and normal mucosa obtained from 30 patients. The samples of gastric carcinoma were divided into two categories according to Lauren's classification: diffuse-type and intestinal-type. Hydroxymethylglutaryl coenzyme A reductase activity was measured by radiometric assay and low-density lipoprotein receptor expression was measured by enzyme-linked immunosorbent assay. Hydroxy-methyl-glutaryl-coenzyme A reductase activity was significantly higher in neoplastic tissue than in normal mucosa, both in diffuse-type and intestinal-type gastric cancer. Low-density lipoprotein receptor levels were significantly lower in neoplastic tissue than in normal mucosa of diffuse-type gastric cancer, whereas no significant difference was present between neoplastic tissue and normal mucosa in intestinal-type gastric cancer. Hydroxymethylglutaryl coenzyme A reductase activity did not differ between intestinal-type and diffuse-type neoplastic samples, while low-density lipoprotein receptor levels were significantly higher in intestinal-type than in diffuse-type gastric cancer tissue. These findings show different behavior of cholesterol metabolism in the two types of gastric carcinoma and, therefore, suggest that different therapeutic strategies should be adopted.

Prognostic significance of tissue-type plasminogen activator (tPA) content in gastric cancer and surrounding mucosa.

We analyzed the tPA content in primary gastric carcinomas and surrounding mucosa in order to assess the relationship between tPA content, clinicopathological tumor characteristics, and estrogen and progesterone receptor content. We evaluated the prognostic value of this serine protease in gastric cancer patients. 122 resected gastric neoplasms and 95 adjacent mucosa samples were studied. The tPA content was measured in cytosol by an ELISA method. Cytosolic ER and PgR were measured with a solid phase enzyme immunoassay. Cytosolic tPA levels in neoplastic tissues (median 1.0 ng/mg prot) were significantly lower (p=0.002) than those found in paired mucosa samples (median 2.3 ng/mg prot). There was no significant association between tPA levels and clinicopathological parameters or PgR content, but tPA levels were significantly correlated with ER content. The intermediate-tPA-content group, corresponding to samples with between 0.3 and 1.70 ng/mg protein, proved to have a significantly high risk of relapse. We found a wide variability in tPA levels in gastric carcinoma and adjacent mucosa samples, with significantly decreased levels in tumors and a significantly positive relationship between tPA levels and ER status. There was a non-monotonic relationship between tPA levels and prognosis in patients with gastric cancer.

Somatic β-catenin mutation in gastric carcinoma – an infrequent event that is not specific for microsatellite instability

We screened 90 cases of gastric carcinoma (GCA) samples for β-catenin exon 3 mutation and assessed its possible relationship with microsatellite instability (MSI). Three mutations were detected in two samples, including a single mutation in an intestinal type and double mutations in a diffuse type GCA. One of the mutations found in the diffuse type GCA sample was a non-sense mutation at codon 68 (CAG→TAG). This novel mutation was predicted to disrupt the binding of β-catenin to α-catenin and may be related to the diffuse type morphology. The other two mutations were missense mutations involved or related to the GSK-3β phosphorylation site, which have been reported previously. No MSI can be demonstrated in the two cases with β-catenin mutation. Our results suggested that β-catenin mutation was infrequent in GCA and appeared not specific for MSI.

Comparative Genomic Hybridization Reveals Differences in DNA Copy Number Changes between Sporadic Gastric Carcinomas and Gastric Carcinomas from Patients with Hereditary Nonpolyposis Colorectal Cancer

Comparative genomic hybridization was used to search for DNA copy number changes in samples of gastric cancer from 12 hereditary nonpolyposis colon cancer (HNPCC) patients and in samples of sporadic gastric carcinoma from 13 patients. The gastric cancer samples from HNPCC patients showed gains affecting 19q, Xp, and whole chromosome 22, each in a single patient. Neither high-level amplifications nor losses of DNA copy number were detected. On the other hand, 10 of the 13 (77%) sporadic gastric carcinoma samples had multiple DNA copy number changes. The most frequent gains occurred with minimal common overlapping regions at 1q22–q31, 8q23–qter, 17p11.2–q22, and 20q, all at a frequency of 31%. High-level amplifications were also seen at 17q21 in three cases (23%). Losses were rare, and the most frequent loss was with a minimal common overlapping region at 4q32 (23%). This suggests that multiple DNA copy number changes are needed for the development of sporadic gastric carcinoma but not for gastric carcinoma in HNPCC patients.