Shoji Hirajima

Clinical impact of circulating miR-221 in plasma of patients with pancreatic cancer

Background:Several recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in plasma/serum. We tested miR-221 and miR-375, which are frequently reported to be highly and poorly expressed in pancreatic cancer (PCa), as candidates for plasma biomarkers in PCa.Methods:This study was divided into three parts: (1) Confirmation of higher miR-221 levels in primary PCa tissue and cell lines than normal pancreatic tissues. (2) Evaluation of plasma miR-221 and miR-375 concentrations by comparing results from 47 consecutive PCa patients and 30 healthy volunteers. (3) Evaluation of the assay for monitoring tumour dynamics in PCa patients.Results:(1) Expression of miR-221 was significantly higher in PCa tissues and cell lines than normal pancreatic tissues. (2) Plasma miR-221 concentrations were significantly higher in PCa patients than that in benign pancreatic tumours (P=0.016) and controls (P<0.0005), while plasma miR-375 concentrations tended to be lower in PCa patients (P=0.064), and the miR-221/miR-375 ratio was significantly higher (P<0.0001) in PCa patients than in controls. (3) Plasma miR-221 concentrations were significantly reduced in postoperative samples (P=0.018). Furthermore, PCa patients with high plasma miR-221 concentrations had significant correlation with distant metastasis (P=0.041), and non-resectable status (P=0.021).Conclusion:Plasma miR-221 could be a useful biomarker for cancer detection, monitoring tumour dynamics and predicting malignant outcomes in PCa patients, and may contribute to clinical decision making in PCa treatments.

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±0.54 amol μl−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.

Prognostic impact of circulating miR-21 and miR-375 in plasma of patients with esophageal squamous cell carcinoma

Background: miR-21 and miR-375 are reported to be highly and poorly expressed in esophageal squamous cell carcinoma (ESCC) tissues, respectively. Recently, we demonstrated that circulating miR-21 and miR-375 were stably detectable in plasma and reflected tumor dynamics as a tumor marker for ESCC. We hypothesized that these plasma miRNA concentrations contributed to prognostic markers in patients with ESCC. Methods: Between 2008 and 2010, 50 preoperative plasma samples were collected from consecutive patients with ESCC, who underwent curative esophagectomy in our hospital. We examined the association between plasma miRNA concentrations and prognosis retrospectively. Results: i) The postoperative cause-specific survival rate of patients with high plasma miR-21 concentration tended to be poorer than low group (3-yr survival rate: 53.4 and 81.5%, p = 0.1038), while that of high plasma miR-375 group was better than low group (3-yr survival rate: 100 and 65.2%). ii) Patients with high miR-21 and low miR-375 concentrations in plasma had significantly poorer prognosis than other patients (3-yr survival rate: 48.4 and 83.1%, p = 0.039). Multivariate analysis revealed that the presence of high miR-21 and low miR-375 concentrations in plasma was an independent prognostic factor (p = 0.029, hazard ratio 3.8 (1.14-12.5)). Conclusion: Circulating miR-21 and miR-375 could be reliable prognostic markers for ESCC. These plasma markers might facilitate clinical decision-making to select prospective candidates, which need meticulous follow-up for early detection of recurrences and additional treatments such as neo-adjuvant chemotherapy and postoperative chemotherapy in 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.

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.

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.

Plasma microRNA profiles: identification of miR-744 as a novel diagnostic and prognostic biomarker in pancreatic cancer

Background:This study aims to explore novel microRNAs in plasma for screening cancer and predicting clinical outcomes in pancreatic cancer (PCa) patients using a microRNA array-based approach.Methods:We used the Toray 3D-Gene microRNA array-based approach to compare plasma levels between PCa patients and healthy volunteers.Results:(1) Six oncogenic microRNAs (miR-615-5p, -744, -575, -557, -675, and -550a) with high expression in plasma were selected. (2) By quantitative RT–PCR using plasma samples from 94 PCa patients and 68 healthy volunteers, a significantly higher level of plasma miR-744 in PCa patients than in healthy volunteers was validated in small-scale analysis (P=0.0038), two independent cohort analyses, and large-scale analysis (P<0.0001, AUC 0.8307). (3) miR-744 expression was significantly higher in PCa tissues (P=0.0069) and PCa cell lines (P=0.0074) than in normal tissues and fibroblasts, respectively. Preoperative plasma level of miR-744 was significantly reduced in postoperative samples (P=0.0063). (4) A high level of plasma miR-744, which was correlated with lymph node metastasis (P=0.0407) and recurrences (P=0.0376), was an independent poor prognostic factor of PCa patients after pancreatectomy (P=0.0007, HR 21.2 (3.17–436)). Furthermore, a high level of plasma miR-744 contributed to poorer progression-free survival of non-operable PCa patients who underwent gemcitabine-based chemotherapy (P=0.0533). Overexpression of miR-744 in PCa cells induced significant chemoresistance to gemcitabine in vitro.Conclusions:Plasma miR-744 might be useful biomarker for screening PCa, monitoring, and predicting poor prognosis and chemoresistance in PCa patients.

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.

Liver metastasis is the only independent prognostic factor in AFP-producing gastric cancer

AIM To investigate differences between common gastric cancer and α-fetoprotein (AFP)-producing gastric cancer according to the presence or absence of liver metastasis. METHODS Between 1997 and 2011, 1299 patients underwent gastrectomy for gastric cancer (GC) at our institute and their hospital records were reviewed retrospectively. Patients were immunohistochemically divided into two groups: 23 patients (1.8%) with AFP-producing GC and 1276 patients (98.2%) without it. RESULTS AFP-producing GC patients had a significantly higher incidence of deeper tumors, venous invasion, lymphatic invasion, lymph node metastasis, and liver metastasis and a poorer prognosis (P < 0.005) than those without AFP-producing GC. However, multivariate analysis revealed that AFP-positivity was not an independent prognostic factor. The prognosis of AFP-producing GC was similar to that of AFP-non producing GC according to the presence or absence of liver metastasis. Concerning recurrence, 47.8% of patients (11/23) with AFP-producing GC and 20.0% of patients (256/1276) without AFP-producing GC exhibited recurrence. Liver metastasis [90.9% (10/11)] was the most prevalent in AFP-producing GC patients. Multivariate analysis revealed that liver metastasis was the only independent prognostic factor in AFP-producing GC (HR = 17.6, 95%CI: 2.1-147.1; P = 0.0081). CONCLUSION AFP-producing GC is similar to common GC without liver metastasis, which should be specifically targeted in an effort to improve the prognosis of AFP-producing GC patients.