Atsushi Shiozaki

Clinical impact of circulating miR-18a in plasma of patients with oesophageal squamous cell carcinoma

Background:Several recent studies demonstrated that microRNAs are stably detectable in plasma/serum. We tested whether miR-18a, which is located in the miR-17-92 cluster and reported to be highly expressed in tissues of oesophageal squamous cell carcinoma (ESCC), served as a plasma biomarker in patients with ESCC.Methods:This study was divided into three steps: (1) confirmation of higher miR-18a levels in primary ESCC tissues and cell lines than normal ESCC tissues and a human fibroblast cell line. (2) Evaluation of the plasma miR-18a assay using quantitative RT–PCR by comparing results from 106 consecutive patients with ESCC and 54 healthy volunteers. (3) Evaluation of the assay for monitoring tumour dynamics in patients with ESCC.Results:(1) Expression of miR-18a was significantly higher in ESCC tissues (P=0.0020) and ESCC cell lines (P=0.0121) than normal tissues and fibroblasts. (2) Plasma concentrations of miR-18a were significantly higher in ESCC patients than healthy volunteers (P<0.0001; ESCC patients vs healthy volunteers (mean±s.d.): 11.77±13.45 vs 0.73±0.54u2009amolu2009μl−1). The value of the area under the receiver-operating characteristic (ROC) curve (AUC) was 0.9449. Furthermore, the ROC curves to detect early ESCC such as pTis-1 and pStage0-I showed AUCs of 0.9479 and 0.9642, respectively. (3) Plasma levels of miR-18a were significantly lower in postoperative samples than preoperative samples (P=0.0076).Conclusion:Plasma miR-18a may be a very useful biomarker for cancer detection and the monitoring of tumour dynamics in patients with ESCC.

Plasma microRNA profiles: identification of miR-25 as a novel diagnostic and monitoring biomarker in oesophageal squamous cell carcinoma.

Background:Recent studies have demonstrated that microRNAs are stably detectable in plasma/serum because of their binding to specific proteins or being packaged in secretory particles. This study was designed to detect novel microRNAs in plasma for cancer detection and monitoring using microRNA array-based approaches in oesophageal squamous cell carcinoma (ESCC) patients.Methods:Through the integration of two Toray 3D-Gene microRNA array-based approaches to compare plasma microRNA levels between ESCC patients and healthy volunteers and between preoperative and postoperative ESCC patients, we identified a novel plasma biomarker in ESCC.Results:(1) Eight upregulated and common microRNAs (miR-15b, 16, 17, 25, 19b, 20a, 20b, and 106a) were selected using two high-resolution microRNA array approaches. (2) Test-scale analyses by quantitative RT–PCR validated a significant higher levels of plasma miR-19b (P=0.0020) and miR-25 (P=0.0030) in ESCC patients than controls. However, a significant correlation was observed between plasma miR-19b levels and concentrations of red blood cells (P=0.0073) and haemoglobin (P=0.0072). (3) miR-25 expression was found to be significantly higher in ESCC tissues (P=0.0157) and ESCC cell lines (P=0.0093) than in normal tissues and fibroblasts. (4) In a large-scale validation analysis, plasma miR-25 levels were significantly higher in 105 preoperative (P<0.0001) ESCC patients who underwent curative oesophagectomy and 20 superficial ESCC patients who underwent endoscopic resection (P<0.0001) than in 50 healthy volunteers. (5) Plasma miR-25 levels were significantly reduced in postoperative samples than in preoperative samples (P<0.0005) and were significantly increased during ESCC recurrences (P=0.0145).Conclusions:Plasma miR-25 might be a clinically useful biomarker for cancer detection and the monitoring of tumour dynamics in ESCC patients.

Circulating miR-18a in plasma contributes to cancer detection and monitoring in patients with gastric cancer

AbstractBackgroundnRecently, circulating microRNAs have been reported to be stably detectable in plasma/serum and to function as potent non-invasive biomarkers in various cancers. We hypothesized that miR-18a could contribute to a novel plasma biomarker in patients with gastric cancer (GC).MethodsWe focused on miR-18a, which is a component of miR-17-92 cluster and has been reported as highly expressed in GC tissues. The study involved three steps: (1) confirmation of the higher miR-18a expression in primary GC tissues and GC cell lines than in normal gastric tissues and a fibroblast cell line; (2) evaluation of the plasma miR-18a assay using quantitative RT-PCR by comparing 104 GC patients and 65 healthy volunteers; (3) evaluation of monitoring tumor dynamics by the plasma miR-18a assay.Results(1) The miR-18a expressions were significantly higher in GC tissues than in normal gastric tissues (Pxa0=xa00.0286) and higher in all examined GC cell lines than in the fibroblast cell line. (2) The plasma miR-18a concentrations were significantly higher in GC patients than in healthy controls (Pxa0<xa00.0001). The value of the area under the receiver-operating characteristic curve was 0.8059. (3) The plasma miR-18a levels were significantly reduced in postoperative samples compared to in preoperative samples (Pxa0=xa00.0002). In an miR-18a overexpressing cell line, the miR-18a concentration of cultured medium increased in both cell number and time-course dependent manners, suggesting microRNA might be released from cancer cells into the surrounding environment.ConclusionsCirculating miR-18a could be a useful biomarker for screening GC and monitoring tumor dynamics.

Overexpression of YWHAZ relates to tumor cell proliferation and malignant outcome of gastric carcinoma

Background:Several studies have demonstrated that YWHAZ (14-3-3ζ), included in the 14-3-3 family of proteins, has been implicated in the initiation and progression of cancers. We tested whether YWHAZ acted as a cancer-promoting gene through its activation/overexpression in gastric cancer (GC).Methods:We analysed 7 GC cell lines and 141 primary tumours, which were curatively resected in our hospital between 2001 and 2003.Results:Overexpression of the YWHAZ protein was frequently detected in GC cell lines (six out of seven lines, 85.7%) and primary tumour samples of GC (72 out of 141 cases, 51%), and significantly correlated with larger tumour size, venous and lymphatic invasion, deeper tumour depth, and higher pathological stage and recurrence rate. Patients with YWHAZ-overexpressing tumours had worse overall survival rates than those with non-expressing tumours in both intensity and proportion expression-dependent manner. YWHAZ positivity was independently associated with a worse outcome in multivariate analysis (P=0.0491, hazard ratio 2.3 (1.003–5.304)). Knockdown of YWHAZ expression using several specific siRNAs inhibited the proliferation, migration, and invasion of YWHAZ-overexpressing GC cells. Higher expression of the YWHAZ protein was significantly associated with the lower expression of miR-375 in primary GC tissues (P=0.0047).Conclusion:These findings suggest that YWHAZ has a pivotal role in tumour cell proliferation through its overexpression, and highlight its usefulness as a prognostic factor and potential therapeutic target in GC.

Progression of remnant gastric cancer is associated with duration of follow-up following distal gastrectomy

AIMnTo re-evaluate the recent clinicopathological features of remnant gastric cancer (RGC) and to develop desirable surveillance programs.nnnMETHODSnBetween 1997 and 2008, 1149 patients underwent gastrectomy for gastric cancer at the Department of Digestive Surgery, Kyoto Prefectural University of Medicine, Japan. Of these, 33 patients underwent gastrectomy with lymphadenectomy for RGC. Regarding the initial gastric disease, there were 19 patients with benign disease and 14 patients with gastric cancer. The hospital records of these patients were reviewed retrospectively.nnnRESULTSnConcerning the initial gastric disease, the RGC group following gastric cancer had a shorter interval [P < 0.05; gastric cancer vs benign disease: 12 (2-22) vs 30 (4-51) years] and were more frequently reconstructed by Billroth-Iu2005procedure than those following benign lesions (P < 0.001). Regarding reconstruction, RGC following Billroth-II reconstruction showed a longer interval between surgical procedures [P < 0.001; Billroth-II vs Billroth-I: 32 (5-51) vs 12 (2-36) years] and tumors were more frequently associated with benign disease (P < 0.001) than those following Billroth-Iu2005reconstruction. In tumor location of RGC, after Billroth-Iu2005reconstruction, RGC occurred more frequently near the suture line and remnant gastric wall. After Billroth-II reconstruction, RGC occurred more frequently at the anastomotic site. The duration of follow-up was significantly associated with the stage of RGC (P < 0.05). Patients diagnosed with early stage RGC such as stageu2005I-II tended to have been followed up almost every second year.nnnCONCLUSIONnMeticulous follow-up examination and early detection of RGC might lead to a better prognosis. Based on the initial gastric disease and the procedure of reconstruction, an appropriate follow-up interval and programs might enable early detection of RGC.

Overexpression of TRIM44 contributes to malignant outcome in gastric carcinoma.

Recent studies have shown that some members of the tripartite motif‐containing protein (TRIM) family, which is characterized by a conserved RING finger, B‐box, and coiled‐coil domains, function as important regulators for carcinogenesis. In this study, we tested whether TRIM44 (11p13) acts as a cancer‐promoting gene through overexpression in gastric cancer. We analyzed seven gastric cancer cell lines and 112 primary tumors, which were curatively resected in our hospital between 2001 and 2003. Expression of the TRIM44 protein was detected in gastric cancer cell lines (2/7 cell lines; 29%) and primary tumor samples of gastric cancer (29/112 cases; 25%). Knockdown of TRIM44 expression using several specific siRNAs inhibited the proliferation, migration, and invasion of TRIM44‐overexpressing cells. Overexpression of the TRIM44 protein was significantly correlated with an advanced type of macroscopic appearance, lymphatic invasion, and higher recurrence rate. TRIM44‐overexpressing tumors had a worse overall rate of survival than those with non‐expressing tumors (P = 0.0038, log–rank test) in both intensity and proportion expression‐dependent manner. TRIM44 positivity was independently associated with worse outcome in multivariate analysis (P = 0.0233, hazard ratio 3.37 [1.18–9.64]). These findings suggest that TRIM44 plays a crucial role in tumor cell proliferation through its overexpression, and highlight its usefulness as a predictor and potential therapeutic target in gastric cancer.

Malignant potential in pancreatic neoplasm; new insights provided by circulating miR-223 in plasma

Background: Recent studies have identified that microRNAs are stably detectable in plasma/serum because of their binding to specific proteins or being packaged in secretory vesicles. Methods: We tested miR-223 as a candidate of novel plasma biomarker in pancreatic cancer (PCa) and intraductal papillary mucinous neoplasm (IPMN). Results: i) miR-223 expression was significantly higher in PCa tissues (p = 0.0069) than in normal tissues. ii) Plasma miR-223 levels were significantly higher in 71 PCa patients than 67 healthy volunteers (p < 0.0001). iii) Plasma miR-223 levels were significantly reduced in postoperative samples (p = 0.0297). iv) Plasma miR-223 levels tended to discriminate the malignant potential between benign IPMN and malignant IPMN (p = 0.0963), and the progressive extent of invasiveness between malignant IPMN and pancreatic invasive ductal carcinoma (PIDC) (p = 0.0004). Multivariate logistic regression analysis revealed that a low level of plasma miR-223 was an independent risk factor for PIDC (p = 0.0012, odds ratio 7.90 [95% CI: 2.06 – 41.2]). v) There was no significant correlation between plasma miR-223 levels and the number of any blood cell types in the peripheral blood. Conclusion: Plasma miR-223 might be a clinically useful biomarker for screening PCa, and predicting malignant potential of IPMN and the invasiveness of PCa.

Overexpression of SMYD2 contributes to malignant outcome in gastric cancer

Background:SET and MYND domain-containing protein 2 (SMYD2) is a lysine methyltransferase for histone H3, p53 and Rb and inhibits their transactivation activities. In this study, we tested whether SMYD2 (1q42) acts as a cancer-promoting factor by being overexpressed in gastric cancer.Methods:We analysed 7 gastric cancer cell lines and 147 primary tumor samples of gastric cancer, which were curatively resected in our hospital.Results:SET and MYND domain-containing protein 2 was detected in these cell lines (five out of seven cell lines; 71.4%) and primary tumor samples (fifty-six out of one hundred and forty-seven cases; 38.1%). Knockdown of SMYD2 using specific small interfering RNA inhibited proliferation, migration and invasion of SMYD2-overexpressing cells in a TP53 mutation-independent manner. Overexpression of SMYD2 protein correlated with larger tumor size, more aggressive lymphatic invasion, deeper tumor invasion and higher rates of lymph node metastasis and recurrence. Patients with SMYD2-overexpressing tumours had a worse overall rate of survival than those with non-expressing tumours (P=0.0073, log-rank test) in an intensity and proportion score-dependent manner. Moreover, multivariate analysis demonstrated that SMYD2 was independently associated with worse outcome (P=0.0021, hazard ratio 4.25 (1.69–10.7)).Conclusions:These findings suggest that SMYD2 has a crucial role in tumor cell proliferation by its overexpression and highlight its usefulness as a prognostic factor and potential therapeutic target in gastric cancer.

Differences of the Lymphatic Distribution and Surgical Outcomes Between Remnant Gastric Cancers and Primary Proximal Gastric Cancers

BackgroundAlthough remnant gastric cancer (RGC) following distal gastrectomy is located in the proximal stomach, little is known about the differences of the lymphatic distribution and surgical outcomes between RGC and primary proximal gastric cancer (PGC).MethodsBetween 1997 and 2008, 1,149 patients underwent gastrectomy for gastric cancer. Of these, 33 (2.9%) RGC patients and 207 (18.5%) PGC patients were treated at our department. We reviewed their hospital records retrospectively.ResultsCompared with the PGC patients, those with RGC had a slightly higher age at onset (pu2009=u20090.09), higher incidence of undifferentiated cancer (pu2009=u20090.06), higher incidence of vascular invasion (pu2009=u20090.09), and higher incidence of T4 (pu2009=u20090.07). Gastrectomy for RGC involved greater blood loss (pu2009<u20090.005), longer surgical duration (pu2009=u20090.01), combined resection, and high incidence of complications. However, the survival rate for RGC patients was similar to that for PGC patients (pu2009=u20090.67). 2) Patients with RGC had a different pattern of lymph node metastasis compared with that in PGC. Particularly in advanced RGC with pT2–T4 tumors, RGC frequently demonstrated jejunal mesentery lymph node metastases (RGC vs. PGC, 35% vs. 0%) and splenic hilar lymph node metastases (RGC vs. PGC, 17% vs. 10%). The jejunal mesentery lymph node metastases were detected only following Billroth II reconstruction (Billroth I vs. Billroth II, 0% vs. 67%).ConclusionAlthough the clinical behaviors of the two gastric cancers were different, the survival rates were similar. The pattern of metastasis indicates that the jejunal mesentery and splenic hilar lymph nodes should be specifically targeted for en bloc resection during complete gastrectomy in RGC.

Histological mixed-type as an independent prognostic factor in stage I gastric carcinoma

AIMnTo evaluate the clinicopathological features of mixed-type gastric cancer and their influence on prognosis of mixed-type stage I gastric cancer.nnnMETHODSnWe analyzed 446 patients who underwent curative gastrectomy for stage I gastric cancer between 1999 and 2009. The patients were divided into two groups: those with differentiated or undifferentiated cancer (non-mixed-type, n = 333) and those with a mixture of differentiated and undifferentiated cancers (mixed-type, n = 113).nnnRESULTSnThe overall prevalence of mixed-type gastric cancer was 25.3% (113/446). Compared with patients with non-mixed-type gastric cancer, those with mixed-type gastric cancer tended to be older at onset (P = 0.1252) and have a higher incidence of lymph node metastasis (P = 0.1476). They also had significantly larger tumors (P < 0.0001), more aggressive lymphatic invasion (P = 0.0011), and deeper tumor invasion (P < 0.0001). In addition, they exhibited significantly worse overall survival rates than did patients with non-mixed-type gastric cancer (P = 0.0026). Furthermore, mixed-type gastric cancer was independently associated with a worse outcome in multivariate analysis [P = 0.0300, hazard ratio = 11.4 (1.265-102.7)].nnnCONCLUSIONnHistological mixed-type of gastric cancer contributes to malignant outcomes and highlight its usefulness as a prognostic indicator in stage I gastric cancer.