Plasminogen activator inhibitor 1 RNAi suppresses gastric cancer metastasis in vivo.

Cancer metastasis remains the primary cause of pain, suffering, and death in cancer patients, and even the most current therapeutic strategies have not been highly successful in preventing or inhibiting metastasis. In most patients with scirrhous gastric cancer (one of the most aggressive of diffuse-type gastric cancer), recurrence occurs even after potentially curative resection, most frequently in the form of peritoneal metastasis. Given that the occurrence of diffuse-type gastric cancers has been increasing, the development of new strategies to combat metastasis of this disease is critically important. Plasminogen activator inhibitor-1 (PAI-1) is a critical factor in cancer progression; thus, PAI-1 RNAi may be an effective therapy against cancer metastasis. In the present study, we used an RNAi technique to reduce PAI-1 expression in an in vivo model system for gastric cancer metastasis. Ex vivo plasmid transfection and adenovirus infection were tested as mechanisms to incorporate specific PAI-1 RNAi vectors into human gastric carcinoma cells. Both approaches significantly decreased peritoneal tumor growth and the formation of bloody ascites in the mouse model, suggesting that this approach may provide a new, effective strategy for inhibiting cancer metastasis.

Allelic imbalance at p53 and microsatellite instability are predictive markers for resistance to chemotherapy in gastric carcinoma.

BACKGROUND: Combined treatment with 5-fluorouracil and cisplatin (FP chemotherapy) is an effective neoadjuvant regimen for gastric carcinoma. However, it is ineffective in half of all patients. This study tests the hypothesis that genetic markers might identify those patients with gastric cancer who would respond to neoadjuvant FP chemotherapy. MATERIALS AND METHODS: A total of 23 patients with gastric carcinoma were treated with neoadjuvant chemotherapy. Pretreatment biopsy specimens before neoadjuvant chemotherapy were obtained from 15 of 23 patients, and resected tumors were obtained from all 23. Genetic studies were performed to detect allelic imbalance (AI), microsatellite instability (MSI), and K-ras mutation. RESULTS: A clinical response was observed in 13 of 23 patients. Kaplan-Meier survival curve showed that clinical responder group had a significantly higher likelihood of overall survival (P = 0.0165), compared with nonresponder group. In 23 resection specimens, 10 of 23 tumors presented AI at the p53 locus and/or MSI; 8 of the 10 tumors were nonresponders, while 12 of 13 tumors without p53 AI or MSI were responders (P = 0.0007). In 15 pretreatment biopsy specimens, 8 tumors had p53 AI and/or MSI; 7 of the 8 tumors were nonresponders, while 6 of 7 tumors without p53 AI or MSI were responders to preoperative chemotherapy (P = 0.008). Tumors with AI at the p53 locus and/or MSI were significantly more resistant to neoadjuvant chemotherapy. No relationship was found between K-ras mutations and responses. CONCLUSIONS: Analysis for p53 AI and MSI might represent a clinically useful approach to predicting the response to neoadjuvant FP chemotherapy in gastric carcinoma.

Decreased expression of the adhesion molecule desmoglein-2 is associated with diffuse-type gastric carcinoma.

Desmoglein-2 (Dsg2) is one of the components of the cell-cell adherence junction. We previously reported that loss of heterozygosity at chromosome 18q12, on which the Dsg2 gene exists, is frequently found in diffuse-type gastric cancers. This study investigated the relationship between Dsg2 expression and diffuse-type gastric cancers. A total of 112 primary tumours resected from patients with gastric cancer were stained with a monoclonal antibody against Dsg2 and examined for correlations between the expression of Dsg2 and various clinicopathological factors, including loss of heterozygosity on chromosome 18q and prognosis. Dsg2 is immunolocalised at cell-cell boundaries in normal gastric mucosa. Loss of Dsg2 expression was observed in 33 of 112 gastric tumours. There was a statistically significant correlation between a decrease in Dsg2 staining and loss of tumour differentiation (P < 0.001), tumour macroscopic feature (P < 0.001) and peritoneal dissemination (P = 0.023), and Dsg2-negative staining was correlated significantly with loss of heterozygosity on chromosome 18q12 (P = 0.001). The prognosis of patients with Dsg2-negative tumours was significantly worse than that of those with Dsg2-positive tumours (log rank, P < 0.01), while multivariate analysis revealed that Dsg2 was not an independent prognostic factor. These findings suggest that decreased expression of Dsg2 is associated with diffuse-type gastric cancers and poor prognosis in gastric carcinoma.

Carcinogenesis in the remnant stomach following distal gastrectomy with billroth II reconstruction is associated with high-level microsatellite instability.

Although the gastric remnant has been reported to be at high risk for carcinogenesis, the process of carcinogenesis of gastric remnant cancer (GRC) remains unclear. In this study, genetic alterations in GRC were examined in order to investigate the carcinogenic pathways of GRC. Twenty-one patients with GRC were investigated and were compared to 36 patients with sporadic gastric cancer (GC) as a control group. Microsatellite instability (MSI) was examined using 8 primer marker sets. Immunohistochemical staining for hMLH1 and hMSH2 as the DNA mismatch repair system was performed. The high-level MSI (MSI-H) frequency (43%; 9/21) of GRC was significantly higher (p=0.001) than that of the sporadic GC (6%; 2/36). The MSI-H incidence (67%: 8/12) of GRC after gastrojejunostomy (Billroth II anastomosis) was significantly (p=0.015) higher than that (11%: 1/9) after gastroduodenostomy (Billroth I anastomosis). The MSI-H in GRC was significantly (p<0.0001) associated with lack of expression of both hMLH1 and hMSH2. The inactivation of hMLH1 or hMSH2 was significantly frequent (p=0.035) in GRC after gastrojejunostomy (58%: 7/12), compared with that in gastroduodenostomy (11%: 1/9). GRC was more closely associated with the MSI pathway than sporadic GC. Carcinogenesis in the remnant stomach following distal gastrectomy with gastrojejunostomy was found to be associated with the MSI pathway due to inactivation of the DNA mismatch repair system.

Prognostic value of microsatellite instability in resectable pancreatic cancer.

The significance of microsatellite instability (MSI) as a prognostic predictor for resectable pancreatic cancer patients was examined. Forty-six histologically confirmed pancreatic cancer patients who had undergone resection were studied. DNA was extracted from the paraffin block sections by means of the microdissection method. PCR was performed using eight microsatellite primer marker sets. The mixed PCR sample was analyzed by a genetic analyzer. The number of MSI-positive patients was eight (17.4%) as determined by assessment of microsatellite variations in three or more of the eight tested markers. Univariate analysis revealed that patients with MSI-positive tumors had significantly longer survival times than patients with MSI-negative tumors, although there were no significant differences in clinicopathological factors between the two groups (median survival term, 62 months versus 10 months, respectively; P = 0.011). According to univariate survival analysis, patients with T3/T4, N1, or M1 tumors, as classified by Union Internationale Contre le Cancer staging, had significantly shorter survival times than patients with less progressive tumors. Multivariate survival analysis indicated that MSI status had an independent predictive value (hazard ratio = 5.577; P = 0.007). The tumor-infiltrating leukocyte intensity in MSI-positive tumors was significantly larger than that in MSI-negative tumors, suggesting that MSI-positive tumors may induce stronger antitumor immunity. In conclusion, a patient with MSI-positive pancreatic cancer may have a comparatively better prognosis after resection, possibly due to intensive immunoreaction to the tumor.

Genetic alterations in adenoma-carcinoma sequencing of intraductal papillary-mucinous neoplasm of the pancreas.

We investigated the adenoma-carcinoma sequence in intraductal papillary-mucinous neoplasm from the aspect of genetic changes. The formalin-fixed paraffin-embedded tumors and surrounding normal pancreatic tissues from patients with 16 intraductal papillary-mucinous adenoma of the pancreas (IPMA) and 10 intraductal papillary-mucinous carcinoma of the pancreas (IPMC) were provided for DNA extraction after microdissection. SSCP-DNA sequencing analysis demonstrated K-ras mutations at codon 12 in 75% of IPMA and 70% of IPMC, while those at codon 13 were observed neither in IPMA nor IPMC. There were no characteristic K-ras mutation types in IPMA and IPMC and no significant differences in incidence of K-ras mutations between the two categories. The frequencies of p53 mutations analyzed by SSCP-DNA sequencing were not high in IPMA (18.8%) and IPMC (30%), showing no significant difference between them. LOHs of APC in IPMA and IPMC were infrequent (6.3 and 20%, respectively) and showed no significant difference in incidence between the two categories. The LOH frequencies of DCC in IPMA and IPMC were 31.3 and 40%, respectively, and were not statistically different from each other. Taken together, genetic changes such as K-ras, p53, APC and DCC mutations may not be associated with adenoma-carcinoma sequence in intraductal papillary-mucinous neoplasm of pancreas.

K-ras mutation influences macroscopic features of gastric carcinoma.

INTRODUCTION: Gastric carcinoma is classified morphologically as type 1 to 4. Type 1 is defined as a polypoid tumor; types 2 and 3 are defined as ulcerated tumors with polypoid growth or gastric wall infiltration, respectively, and type 4 tumors are defined as flat. This morphological classification is important because biological characteristics differ between the four morphological types, but little is known about genetic differences between them. MATERIALS AND METHODS: One hundred eight gastric tumors were classified macroscopically as type 1 to 4. Tumoral DNA was microdissected from paraffin-embedded tissue sections. PCR amplification of exon 1 of a K-ras containing codons 12 and 13 was performed. K-ras amplicons were dot-blotted onto nylon filters and hybridized with radiolabeled oligomer primers. RESULTS: A K-ras mutation was found in 20 of 108 gastric cancers. A significant relationship of K-ras mutation with polypoid cancer was found. The frequency of K-ras mutation was 6/14 (43%), 8/29 (28%), 2/11 (18%), and 4/54 (7%) in type 1 to 4 tumors, respectively. K-ras mutation was correlated with well-differentiated tumors. Of various types of K-ras mutations, 12 Asp often was seen in type 1 and 2 gastric cancers (well-demarcated, elevated tumors), while 12 Val and 12 Ser were often seen in type 3 and 4 cases (infiltrating carcinomas). CONCLUSION: K-ras mutations occur prominently in type 1 and type 2 gastric cancers.

Very low incidence of microsatellite instability in intraductal papillary-mucinous neoplasm of the pancreas.

Intraductal papillary-mucinous carcinoma (IPMC) of the pancreas, a new entity of pancreatic cancer with a favorable prognosis, has shown a gradual increase in the number of reported cases. Patients with high-frequency microsatellite instability (MSI-H) tumors have been shown to survive longer than those with low-frequency MSI (MSI-L) or microsatellite stable (MSS) tumors in colorectal and gastric cancer. We investigated whether MSI-H in patients with IPMC can contribute to a good prognosis. The formalin-fixed paraffin-embedded tumors and surrounding normal pancreatic tissues from 10 patients with IPMCs and 16 with intraductal papillary-mucinous adenomas (IPMAs) were provided for DNA extraction after microdissection. Polymerase chain reaction (PCR) was carried out using 8 microsatellite primer marker sets. The mixed PCR samples were analyzed using a genetic analyzer. MSI-H was determined by assessment of microsatellite variations in 3 or more of the 8 tested markers. Immunohistochemical staining of the MSI-responsible proteins hMLH1 and hMSH2 was conducted for both the IPMC and IPMA samples. Ten percent of IPMC harbored MSI-H tumors, whereas no MSI-H tumors were detected in the IPMAs. Thirty percent of IPMC tumors and 25% of IPMA tumors showed MSI-L. All IPMCs and IPMAs showed normal expression of both hMLH1 and hMSH2. MSI-H and loss of hMLH1 and hMSH2 are very rare events in both IPMCs and IPMAs. We conclude that a good prognosis for patients with IPMC is not associated with MSI-H.