Discovery of gastric cancer specific biomarkers by the application of serum proteomics.

Current diagnostic markers for gastric cancer are not sufficiently specific or sensitive for use in clinical practice. The aims of this study are to compare the proteomes of serum samples from patients with gastric cancers and normal controls, and to develop useful tumor markers of gastric cancer by quantitative proteomic analysis. We identified a total of 388 proteins with a ≤1% FDR and with at least two unique peptides from the sera of each group. Among them, 215, 251, and 260 proteins were identified in serum samples of patients in an advanced cancer group, early cancer group, and normal control group, respectively. We selected differentially expressed proteins in cancer patients compared with those of normal controls via semiquantitative analyses comparing the spectral counts of identified proteins. These differentially expressed proteins were successfully verified using an MS-based quantitative assay, multiple reactions monitoring analysis. Four proteins (vitronectin, clusterin isoform 1, thrombospondin 1, and tyrosine-protein kinase SRMS) were shown to have significant changes between the cancer groups and the normal control group. These four serum proteins were able to discriminate gastric cancer patients from normal controls with sufficient specificity and selectivity.

Mixed Carcinoma as an Independent Prognostic Factor in Submucosal Invasive Gastric Carcinoma.

Mixed carcinoma shows a mixture of glandular and signet ring/poorly cohesive cellular histological components and the prognostic significance of each component is not fully understood. This study aimed to investigate the significance of the poorly cohesive cellular histological component as a risk factor for lymph node metastasis and to examine the diagnostic reliability of endoscopic biopsy. Clinicopathologic characteristics of 202 patients who underwent submucosal invasive gastric carcinoma resection with lymph node dissection in 2005-2012 were reviewed. Mixed carcinoma accounted for 27.2% (56/202) of cases. The overall prevalence of lymph node metastasis was 17.3% (35/202). Lymphatic invasion (P < 0.001), family history of carcinoma (P = 0.025), tumor size (P = 0.004), Lauren classification (P = 0.042), and presence of any poorly cohesive cellular histological component (P = 0.021) positively correlated with the lymph node metastasis rate on univariate analysis. Multivariate analyses revealed lymphatic invasion, family history of any carcinoma, and the presence of any poorly cohesive cellular histological component to be significant and independent factors related to lymph node metastasis. Review of preoperative biopsy slides showed that preoperative biopsy demonstrated a sensitivity of 63.6% and a specificity of 100% in detecting the presence of the poorly cohesive cellular histological component, compared with gastrectomy specimens. The presence of any poorly cohesive cellular histological component was an independent risk factor associated with lymph node metastasis in submucosal invasive gastric carcinoma. Endoscopic biopsy had limited value in predicting the presence and proportion of the poorly cohesive cellular histologic component due to the heterogeneity of mixed carcinoma.

Preoperative neutrophil-to-lymphocyte ratio is a prognostic factor for overall survival in patients with gastric cancer.

PURPOSE: Preoperative neutrophil-to-lymphocyte ratio (NLR) reflects patients' inflammation status, clinical stage, and survival in various malignancies. However, only a limited amount of information on the clinical importance and prognostic significance of NLR in gastric cancer has been reported. The objective of this study is to assess the prognostic values of preoperative NLR in patients with gastric cancer. METHODS: During the period between August 1, 2005 and December 31, 2011, we collected data from 601 patients among those who had undergone surgery for gastric cancer at the Department of Surgery, Konkuk University Medical Center. We classified the subjects into high NLR (NLR ≥ 1.7) group and low NLR (NLR < 1.7) group. RESULTS: The gender, age, and American Society of Anesthesiologists (ASA) score did not differ in the two NLR groups. The pathologic features (tumor size, histology, number of examined lymph node, T stage, N stage, and TNM stage) did not differ either, except for tumor location (P = 0.006). Multivariate analysis identified TNM stage, NLR, and age as significant prognostic factors. Gender, tumor size, location, and histology did not show their prognostic significance when we applied the Cox proportional hazard model. CONCLUSION: Our study verified that advanced stage, high preoperative NLR (NLR ≥ 1.7), and old age (≥ 70 years) are significant, independent prognostic factor for overall survival in patients with gastric cancer.

Is Helicobacter Pylori associated with Her2/neu Overexpression in Gastric Cancer Patients who Underwent Curative Resection?

BACKGROUND/AIMS: Human epidermal growth factor receptor 2 (Her2/neu) is an important target for treatment of gastric cancer using trastuzumab and Helicobacter pylori is a well-known risk factor of gastric cancer. We investigated the association of Her2/neu overexpression with Helicobacter pylori and other clinicopathologic factors in gastric cancer patients who underwent curative resection. METHODOLOGY: Her2/neu immunohistochemistry was performed in gastric cancer patients who underwent curative gastrectomy or endoscopic submucosal dissection. Analysis about its associations with clinicopathologic features such as Lauren histologic types, differentiation, Helicobacter pylori infection, location, depth of invasion, lymph node metastasis, and AJCC tumor stage was performed retrospectively. RESULTS: The incidence of Her2/neu 3+, 2+, 1+ and 0 was 8.9%, 22.1%, 4.4% and 64.6% respectively. Intestinal type of gastric cancer showed higher incidence of Her2/neu 3+ than diffuse type. The score 2+ and 3+ was more common in Helicobacter pylori-positive patients than Helicobacter pylori-negative patients (p = 0.024). 7th American joint Cancer Committee Tumor stage, depth of tumor invasion and lymph node metastasis did not show correlation with the level of Her2/neu expression. CONCLUSIONS: Helicobacter pylori-positive gastric cancer showed more Her2/neu overexpression. Helicobacter pylori eradication may be necessary to avoid Her2/neu overexpression.

Benign regional lymph nodes in gastric cancer on multidetector row CT.

BACKGROUND: Benign regional lymph nodes (LNs) are sometimes enlarged in gastric cancer patients due to reactive or inflammatory changes. Frequently these enlarged LNs can mimic LN metastasis and lead to overstaging. PURPOSE: To assess frequency of benign regional LNs in gastric cancer patients compared with that in a healthy population and to assess the frequency of benign regional LNs in gastric cancer according to T-staging. MATERIAL AND METHODS: Between August 2005 and June 2009, 177 patients with surgically proven gastric cancer without LN metastasis (TanyN0M0) who had previously undergone preoperative multidetector row CT (MDCT) and 168 healthy patients who visited the healthcare center and underwent an abdominal MDCT were included in this retrospective study. An abdominal radiologist evaluated the distribution of regional LNs and measured the short diameter of LNs ≥6 mm, ≥8 mm, and ≥10 mm. The number of enlarged benign LNs was compared between the two groups, and the distribution of LN with regard to T-stage of gastric cancer was also evaluated. RESULTS: At least one LN ≥6 mm, ≥8 mm, and ≥10 mm was detected in 64.4% (114/177), 22.0% (39/177), and 4.0% (7/177) of patients in the gastric cancer group, respectively, compared to 29.8% (50/168), 4.2% (7/168), and 0% of patients in the healthy group, respectively. The difference between the two groups was statistically significant (P <0.0001). The LN ≥8 mm was found in 14.9% (20/134) in early gastric cancer (T1), and 44.2% (19/43) in advanced cancer (T2 or higher); the difference was statistically significant (P = 0.0002). However, the frequency of LN ≥6 mm in mucosal cancer (T1a) and submucosal cancer (T2b) was not significantly different, regardless of its size. CONCLUSION: Benign regional LNs ≥6 mm are more frequently detected in gastric cancer patients than in a healthy population and in advanced gastric cancer than in early cancer.

Comparative analysis of the efficacy and safety of chemotherapy with oxaliplatin plus fluorouracil/leucovorin between elderly patients over 65 years and younger patients with advanced gastric cancer.

BACKGROUND: A chemotherapy regimen with oxaliplatin, fluorouracil, and leucovorin is commonly used to treat advanced gastric cancer (AGC). This study was designed to compare the efficacy and the safety of oxaliplatin plus fluorouracil/leucovorin administered biweekly (mFOLFOX6) between elderly patients aged over 65 years and younger counterparts with AGC. METHODS: This analysis included 82 AGC patients (≥65:31, <65:51). Patients with previously untreated chemo-naïve advanced adenocarcinoma of the stomach received oxaliplatin 85 mg/m(2), 5-FU bolus 400 mg/m(2) on day 1 and 5-FU 1,500 mg/m(2), leucovorin 75 mg/m(2) 22 h infusion on days 1 and 2 every 2 weeks. The aim of the study was to compare efficacy and safety, including response rate (RR), progression-free survival (PFS), overall survival, and grade ≥3 adverse events, between patients aged ≥65 years and patients aged <65 years. RESULTS: Median progression-free survival (PFS) was not significantly different between both groups (≥65: 5.8 months, <65: 5.7 months, respectively, HR 0.77, 95% CI: 0.44-1.16, P = 0.18). Median overall survival was not significantly different between both groups (≥65: 10.3 months, <65: 9.5 months HR 0.83, 95% CI: 0.50-1.37, P = 0.46). The rate of grade 3 or 4 neutropenia did not differ with age group (≥65: 51.6%, <65: 43.1%); nor did the rates of neutropenic fever (≥65: 16.1%, <65: 5.9%), and infection without neutropenia (≥65: 3.2%, <65: 3.9%). Rates of grade ≥3 toxicities such as thrombocytopenia, nausea/vomiting, or peripheral neuropathy were not significantly different between the two groups. CONCLUSIONS: mFOLFOX6 maintains its efficacy and safety in elderly patients aged over 65 years in comparison with AGC patients aged <65 years. Its judicious use should be considered regardless of age.

High FDG uptake in PET/CT predicts worse prognosis in patients with metastatic gastric adenocarcinoma.

PURPOSE: We evaluated the role of FDG-PET/CT in patients with metastatic gastric adenocarcinoma before palliative chemotherapy to predict prognosis and chemotherapeutic response. METHODS: The study included 35 consecutive newly diagnosed patients with metastatic gastric adenocarcinoma who underwent FDG-PET/CT before palliative chemotherapy. Maximum standardized uptake value (SUVmax) of the primary tumor was assessed to evaluate survival and chemotherapeutic response. Survival analysis was performed for time to progression and overall survival using the Kaplan-Meier method. Cox proportional hazard models were used to determine independent prognostic factors. RESULTS: All primary tumors were visualized using FDG-PET/CT (mean SUVmax = 8.1 ± 4.5, range 2.5-22.1). Sensitivity, specificity, and accuracy of FDG-PET/CT in detection of solid organ metastasis were 95.2% (20/21), 100% (14/14), and 97.1% (34/35), respectively. No significant difference of primary tumor SUVmax was found among the chemotherapeutic response groups. Univariate survival analysis demonstrated ECOG performance status (≥2), presence of solid organ metastasis, number of organs involved in distant metastasis (≥2), and SUVmax of the primary tumor (>8) as significant predictors for poor overall survival. Multivariate survival analysis showed SUVmax of the primary tumor (P = 0.048), presence of solid organ metastasis (P = 0.015), and ECOG performance status (P = 0.002) as significant independent prognostic predictors for overall survival. CONCLUSIONS: High FDG uptake of the primary tumor in patients with metastatic gastric adenocarcinoma is associated with poor overall survival. Assessment of tumor FDG uptake has limited value for prediction of chemotherapeutic response, but provides useful information regarding prognosis.

Torus hyperplasia of the pyloric antrum.

Primary or idiopathic hypertrophy of the pyloric muscle in adult, so called torus hyperplasia, is an infrequent but an established entity. It is caused by a circular muscle hypertrophy affecting the lesser curvature near the pylorus. Since most of the lesions are difficult to differentiate from tumor, distal gastrectomy is usually preformed to rule out most causes of pyloric lesions including neoplastic ones through a pathological study. A 56-yr-old man with a family history of gastric cancer presented with abdominal discomfort of 1 month duration. Upper gastrointestinal endoscopy showed a 1.0 cm sized irregular submucosal lesion proximal to the pylorus to the distal antrum on the lesser curvature. On colonoscopy examination, a 1.5 cm sized protruding mass was noticed on the appendiceal orifice. Gastrectomy and cecectomy were done, and histological section revealed marked hypertrophy of the distal circular pyloric musculature and an appendiceal mucocele. To the best of our knowledge, this is the first case of torus hyperplasia with appendiceal mucocele which is found incidentally.

Molecular identification of Helicobacter DNA in human gastric adenocarcinoma tissues using Helicobacter species-specific 16S rRNA PCR amplification and pyrosequencing analysis.

Helicobacter pylori (H. pylori) is a microaerophilic gram-negative bacterium known to be associated with chronic gastritis, peptic ulcer and gastric adenocarcinoma. In the present study, the presence of Helicobacter DNA was investigated using a Helicobacter species-specific 16S rRNA PCR amplification and pyrosequencing analysis in 51 resected gastric adenocarcinomas. DNA was extracted from paraffin-embedded tissues of resected gastric adenocarcinomas. PCR primers were designed to amplify the 133-bp PCR fragment in highly conserved regions of the 16S rRNA gene. The sequence of the PCR products was analyzed using a PSQ 96 system with SQA software. The pyrosequencing analysis of 16S rRNA showed that H. pylori was present in 47 (92.2%) of the 51 gastric adenocarcinomas. In the 4 H. pylori-negative cases, Helicobacter cinaedi (2 cases), Helicobacter mustelae (1 case) and Campylobacter hyointestinalis (1 case) were detected. Pyrosequencing technology was useful in the identification and differentiation of H. pylori from other species by analyzing the gene encoding 16S rRNA. Gastric adenocarcinoma tissues contain bacteria, and the majority are H. pylori. Helicobacter cinaedi, Helicobacter mustelae and Campylobacter hyointestinalis rarely occur. The roles of these organisms in the pathogenesis of gastric adenocarcinoma remain unclear.

Contribution of soluble intercellular adhesion molecule-1 to the migration of vascular smooth muscle cells.

Soluble intercellular adhesion molecule-1 (sICAM-1), a circulating form of ICAM-1, has been known to be involved in the development of vascular diseases that are associated with vascular smooth muscle cell migration, such as hypertension and atherosclerosis. Here we investigated the contributions of sICAM-1 in promoting vascular migration in rat aortic smooth muscle cells (RASMCs). sICAM-1 increased RASMC migration, and this response was stronger in spontaneously hypertensive rats (SHRs) than in Wistar Kyoto (WKY) rats. The CD11a, CD11b, and CD18 subunits of ICAM-1 receptors were expressed in both SHRs and WKY rats; however, the expression levels of CD18 and CD11b were greater in SHRs than in WKY rats. The neutralization of the receptor subunits with anti-CD11a and -CD18 antibodies abolished the sICAM-1-increased migration. The treatment of inhibitors of spleen tyrosine kinase (Syk) and p38 mitogen-activated protein kinase suppressed the sICAM-1-stimulated migration of RASMCs. sICAM-1 also increased the sprout formation in aortic rings on Matrigel, and this response was inhibited by treatment with these inhibitors. The results suggest that sICAM-1 play crucial roles in vascular cell function through Syk pathways, and that the altered responses of sICAM-1 in RASMCs from SHRs may be mediated by the increased expression of the CD18 receptor.

p38 mitogen-activated protein kinase contributes to angiotensin II-stimulated migration of rat aortic smooth muscle cells.

In this study, we clarified the intracellular mechanism of angiotensin II (Ang II) in promoting migration in rat aortic smooth muscle cells (RASMCs). RASMC migration was measured with the Boyden chamber assay, and the result was confirmed with an aortic sprout assay. The activities of kinases were investigated by western blot analysis. Ang II enhanced RASMC migration, which was chemotaxis directed, and induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and heat shock protein 27 (Hsp27). Ang II-enhanced cell migration was inhibited by SB203580 (a p38 MAPK inhibitor) and piceatannol (a spleen tyrosine kinase inhibitor), but only partially by PD98059 (an ERK inhibitor) and PP2 (a Src inhibitor). The Ang II-stimulated phosphorylation of p38 MAPK and Hsp27 in RASMCs was inhibited by piceatannol and SB203580. The phosphorylation of ERK1/2 stimulated by Ang II was suppressed by PD98059, piceatannol, and PP2. Ang II increased the sprout outgrowth from aortic rings and this response was attenuated by pretreatment with SB203580, PD98059, PP2, or piceatannol. These results suggest that p38 MAPK contributes to the regulation of the Ang II-induced chemotactic migration of vascular smooth muscle cells, which is mediated by Hsp27 phosphorylation.

Spleen tyrosine kinase participates in Src-mediated migration and proliferation by PDGF-BB in rat aortic smooth muscle cells.

Tyrosine kinases, Src and spleen tyrosine kinase (Syk), play crucial roles in cell responses to platelet-derived growth factor (PDGF) and may have their functional interactions. In this study, we focused on investigating the roles of Syk in the regulation of Src signaling in PDGF-mediated vascular cell responses. Migration, proliferation, and activity of kinases were determined in rat aortic smooth muscle cells (RASMCs). PDGF-BB (10 ng/mL) induced the migration and proliferation of RASMCs, which were significantly inhibited by PP2 (10 microM) and piceatannol (30 microM), inhibitors of Src and Syk, respectively. The phosphorylation of Syk induced by PDGF-BB was abolished by PP2. PDGF-BB increased the co-association of the PDGFbeta-receptor and the kinases, Src or Syk, and its maximal binding to Src was achieved in a shorter time than that to Syk. PDGF-BB stimulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2, which was inhibited by PP2 and piceatannol. PDGF-BB-induced proliferation and migration were inhibited by SB203580 (30 microM) and PD98059 (30 microM), inhibitors of p38 MAPK and ERK1/2, respectively. These results imply that Syk is regulated by Src kinase, which participates in migration and proliferation in response to PDGF-BB in RASMCs.

Analysis of peroxiredoxin decreasing oxidative stress in hypertensive aortic smooth muscle.

To determine the role of peroxiredoxin (Prx) in response to oxidative stress and during hypertension in the vasculature, we identified Prx proteins and analyzed their antioxidant effects. Rat aortic smooth muscle contains all six Prxs (I-VI). Prx I, II, and VI shifted to its acidic site on two-dimensional polyacrylamide gel electrophoresis after exposure to H(2)O(2). The total expression of Prx I and VI was increased in response to H(2)O(2). The expression of Prx I, but not that of Prx II and VI, increases and the acidic form of Prx I and the sulfonic acid form of Prx (SO(3)H-Prx) are more strongly expressed in the aortic smooth muscle of hypertensive rats than in that of normotensive control rats. Prxs were also found in the mesenteric artery, heart, and kidney. The expression levels of Prx I and VI were increased in mesenteric artery, but not heart and kidney, from hypertensive rats compared with that from normotensive rats. These results suggest that Prxs play a crucial role against oxidative stress in vascular smooth muscles during hypertension.

Sonic hedgehog expression in gastric cancer and gastric adenoma.

Hedgehog protein is essential to gastrointestinal tract development, and disruption of the hedgehog signaling pathway is associated with gastrointestinal tumorigenesis. Here, we analyzed the degree of hedgehog expression in gastric cancer and precancerous tissue. From August 2005 to May 2006, 52 gastric cancers and 16 gastric adenomas were obtained from surgically or endoscopically resected specimens. Immunohistochemical staining using sonic hedgehog (Shh) antibody was performed in cancerous and noncancerous tissue portions. Hedgehog expression was assessed based on the summed scores of the intensity and proportion of Shh staining. According to Lauren's classification, Shh expression was stronger in the intestinal type than in the diffuse type (p<0.001). Although Shh expression was not related to the location, size, metastatic status, or mucin phenotype of the gastric cancer, the expression was stronger in the tubular type of gastric cancer than in the mucinous and signet-ring cell types (p=0.001). Shh expression was stronger in gastric adenoma than in the diffuse-type gastric cancer (p<0.001), but revealed no difference with that of the intestinal-type gastric cancer (p=0.30). Shh expression was strongest in all types of intestinal metaplasia of noncancerous tissues. Shh expression is related to the intestinal type of gastric cancer. The stronger Shh expression in intestinal metaplasia and gastric adenoma indicates that hedgehog protein is involved at an early phase of gastric carcinogenesis.