Tomohiro Arita

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.

Detection of gastric cancer-associated microRNAs on microRNA microarray comparing pre- and post-operative plasma.

Background:Recently, it was reported that plasma microRNAs (miRNAs) are low-invasive useful biomarkers for cancer. We attempted to isolate gastric cancer (GC)-associated miRNAs comparing pre- and post-operative paired plasma, thereby excluding the possible effects of individual variability.Methods:This study was divided into four steps: (1) microarray analysis comparing pre- and post-operative plasma; (2) validation of candidate miRNAs by quantitative RT–PCR; (3) validation study of selected miRNAs using paired plasma; and (4) comparison of the levels of selected miRNAs in plasma between healthy controls and patients.Results:From the results of microarray analysis, nine candidate miRNAs the levels of which were markedly decreased in post-operative plasma were selected for further studies. After confirmation of their post-operative marked reduction, two candidate miRNAs, miR-451 and miR-486, were selected as plasma biomarkers, considering the abundance in plasma, and marked decrease in post-operative samples. In validation, the two miRNAs were found to decrease in post-operative plasma in 90 and 93% of patients (both P<0.01). In comparison with healthy controls, the levels of both miRNAs were found to be significantly higher in patients, and the area under the curve values were high at 0.96 and 0.92.Conclusion:Plasma miR-451 and miR-486 could be useful blood-based biomarkers for screening GC.

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.

Plasma level of metastasis‐associated lung adenocarcinoma transcript 1 is associated with liver damage and predicts development of hepatocellular carcinoma

Recent studies have shown that metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) was overexpressed in many human solid cancers, however, its roles in plasma of hepatocellular carcinoma (HCC) patients were unclear. The aim of this study was to investigate the significance of plasma MALAT1 levels in HCC patients. Plasma samples were collected from pre‐operative HCC, hepatic disease patients, and healthy controls, and tissue samples from HCC patients and colorectal cancer patients with liver metastasis. Plasma and tissue MALAT1 levels were measured. Plasma MALAT1 levels were progressively and significantly higher in HCC patients than hepatic disease patients, and higher in hepatic disease patients than healthy controls. The expression of MALAT1 in HCC tissue was slightly higher than that in paired non‐cancerous liver tissue, but not significant. The expression of MALAT1 in the non‐cancerous liver tissue of 20 HCC patients was significantly higher than that in normal liver tissue of 13 colorectal cancer patients. In contrast, plasma MALAT1 levels were significantly low in HCC patients with hepatitis B infection, and significantly high in patients with liver damage B or liver cirrhosis. In a receiver–operator curve analysis of HCC and hepatic disease patients, the cut‐off value of plasma MALAT1 was 1.60 and the area under the curve was 0.66. Plasma MALAT1 levels were not correlated with α‐fetoprotein or protein induced by vitamin K absence II, whereas sensitivity and specificity for the detection of HCC with the combination of MALAT1, α‐fetoprotein, and protein induced by vitamin K absence II were 88.6% and 75%, respectively. In conclusion, the plasma MALAT1 level is associated with liver damage, and has clinical utility for predicting development of HCC.

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.

HER2 amplification detected in the circulating DNA of patients with gastric cancer: a retrospective pilot study

BackgroundWe used real-time quantitative polymerase chain reaction (rqPCR) to detect human epidermal growth factor receptor 2 (HER2) amplification in the circulating cell-free DNA (cfDNA) of patients with gastric cancer (GC), which shows the spatial and temporal intrinsic heterogeneity of HER2 expression/copy number during progression, for liquid biopsy and treatment monitoring.MethodsWe first enrolled 52 patients with advanced GC who underwent surgery and 40 healthy volunteers. For patients with GC, plasma cfDNA was obtained before surgery (43 patients) and during postoperative treatment (nine of 43 patients). After ribonuclease P RNA component H1 (RPPH1) had been selected as a reference gene for HER2 CN assessment by rqPCR in GC tumours and plasma, plasma HER2-to-RPPH1 ratios were determined retrospectively in a development cohort and an additional independent validation cohort.ResultsThe HER2-to-RPPH1 ratio of GC tissues determined by rqPCR was concordant with routinely determined HER2 status. The plasma HER2-to-RPPH1 ratio was significantly higher for patients with HER2-positive tumours than for those with HER2-negative tumours. The sensitivity and specificity of the plasma HER2-to-RPPH1 ratio test were 0.539 and 0.967, respectively, in the development cohort, and 0.667 and 1.000, respectively, in the validation cohort. HER2 amplifications acquired and lost during tumour progression and treatment, respectively, were apparently detected by repeated assessments of plasma HER2-to-RPPH1 ratios during postoperative treatment.ConclusionOur preliminary data demonstrated the potential clinical use of circulating cfDNA to detect HER2 amplification as a therapeutic marker to detect and monitor HER2 CN status for effective molecular targeted therapy in patients with GC.

Circulating microRNA profiles in plasma: identification of miR-224 as a novel diagnostic biomarker in hepatocellular carcinoma independent of hepatic function

Aims This study was designed to identify novel microRNAs (miRNAs) in plasma for detecting and monitoring hepatocellular carcinoma (HCC), independent of hepatic function and background liver diseases with different etiologies. Results (1) Four oncogenic miRNAs (miR-151, 155, 191 and 224) with high expression in HCC tissues were selected as candidates. (2) Quantitative RT-PCR using plasma samples from 107 HCC patients and 75 healthy volunteers revealed a significantly higher level of plasma miR-224 in HCC patients than in healthy volunteers according to a small-scale analysis (P < 0.0001), two independent large-scale cohort analysis (P < 0.0001, AUC 0.908). (3) miR-224 expression was significantly higher in HCC tissues and HCC cell lines than in normal hepatic tissues and fibroblasts, respectively. (P = 0.0011, 0.0150) (4) Plasma miR-224 reflected tumor dynamics; preoperative plasma levels of miR-224 were significantly reduced in postoperative samples (P = 0.0058), and plasma miR-224 levels were significantly correlated with paired miR-224 levels in HCC tissues (P = 0.0005). (5) Furthermore, plasma miR-224 levels significantly discriminated HCC patients from patients with chronic liver disease (P = 0.0008). A high plasma miR-224 level was significantly correlated with larger tumor size (P = 0.0005) and recurrences (P = 0.0027). The plasma miR-224 level could accurately detect small tumors less than 18 mm preoperatively. Methods We performed a systematic review of the NCBI database and selected candidate miRNAs reported as highly expressed in HCC tissue. Conclusions Plasma miR-224 may be a sensitive biomarker for screening HCC and monitoring tumor dynamics.