Ryo Morimura

Circulating microRNAs in plasma of patients with gastric cancers

Background:We examined plasma microRNA (miRNA) concentrations from patients with gastric cancers (GCs) to assess their clinical application for diagnosing and monitoring diseases.Methods:We initially investigated the appropriateness of plasma miRNA assay, and then compared plasma miRNA results with the expressions in cancer tissues from eight GC patients, and also compared plasma miRNAs between pre- and post-operative paired samples from 10 GC patients. Then, plasma miRNAs (miR-17-5p, miR-21, miR-106a, miR-106b and let-7a) were analysed in 69 GC patients and 30 healthy volunteers in total.Results:The initial analysis showed that miRNAs were stable and detectable in all plasma samples, and the plasma miRNA levels reflected the tumour miRNAs in most cases. The levels of these miRNAs were significantly reduced in post-operative samples. In large-scale analysis, the plasma concentrations of miRNAs (miR-17-5p, miR-21, miR-106a, miR-106b) were significantly higher in GC patients than controls (P=0.05, 0.006, 0.008 and <0.001 respectively), whereas let-7a was lower in GC patients (P=0.002). The values of the area under the receiver-operating characteristic curve were 0.721 for the miR-106b assay and 0.879 for the miR-106a/let-7a ratio assay.Conclusion:Detection of circulating miRNAs might provide new complementary tumour markers for GC.

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.

Fluorescent detection of peritoneal metastasis in human colorectal cancer using 5-aminolevulinic acid

A precise diagnosis of peritoneal dissemination is necessary to determine the appropriate treatment strategy for colorectal cancer. However, small peritoneal dissemination is difficult to diagnose. 5-aminolevulinic acid (5-ALA) is an intermediate substrate of heme metabolism. The administration of 5-ALA to cancer patients results in tumor-specific accumulation of protoporphyrin IX (PpIX), which emits red fluorescence with blue light irradiation. We evaluated the usefulness of photodynamic diagnosis (PDD) using 5-ALA to detect the peritoneal dissemination of colorectal cancer. EGFP-tagged HT-29 cells were injected into the peritoneal cavity of BALB/c nude mice. After 2 weeks, the mice were given 5-ALA hydrochloride, and metastatic nodules in the omentum were observed with white light and fluorescence images. Twelve colorectal cancer patients suspected to have serosal invasion according to preoperative computed tomography (CT) were enrolled in this study. 5-ALA (15-20 mg per kg body weight) was administered orally to the patients 3 h before surgery. The abdominal cavity was observed under white light and fluorescence. Fluorescence images were analyzed with image analysis software (ImageJ 1.45s, National Institutes of Health, Bethesda, MD, USA). The mice developed peritoneal disseminations. The observed 5-ALA-induced red fluorescence was consistent with the EGFP fluorescent-positive nodules. Peritoneal dissemination was observed with conventional white light imaging in 8 patients. All nodules suspected as being peritoneal dissemination lesions by white light observation were similarly detected by ALA-induced fluorescence. In 1 patient, a small, flat lesion that was missed under white light observation was detected by ALA-induced fluorescence; the lesion was pathologically diagnosed as peritoneal metastasis. In the quantitative fluorescence image analysis, the red/(red + green + blue) ratio was higher in the metastatic nodules compared to the non-metastatic sites of the abdominal wall, fat and liver. We demonstrated better diagnostic accuracy using 5-ALA-PDD compared to conventional laparoscopy in patients with colorectal cancer. 5-ALA-PDD is a promising candidate method for diagnosing peritoneal dissemination of colorectal 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.

Pleural lavage with distilled water during surgery for esophageal squamous cell carcinoma.

This study aimed to investigate cytocidal effects of hypotonic shock on esophageal squamous cell carcinoma (ESCC) cell lines, and to apply pleural lavage with distilled water to surgery for ESCC. Three human ESCC cell lines, TE5, TE9 and KYSE170 were exposed to distilled water, and morphological changes in ESCC cells were closely observed under a differential interference contrast microscope connected to a high-speed digital video camera. Further, serial cell volume changes after hypotonic shock were measured using a high-resolution flow cytometer. To investigate the cytocidal effects of hypotonic shock on ESCC cells, re-incubation of ESCC cells was performed after hypotonic shock. Additionally, the effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a Cl- channel blocker, during hypotonic shock were analyzed. Video recordings by high-speed digital camera demonstrated that hypotonic shock with distilled water induced cell swelling followed by cell rupture. Measurements of cell volume changes using a high-resolution flow cytometer indicated that severe hypotonicity with distilled water increased broken fragments of ESCC cells within 5 min. Re-incubation experiments demonstrated cytocidal effects of hypotonic shock on ESCC cells. Treatment of cells with NPPB increased cell volumes by the inhibition of regulatory volume decrease, which is observed during hypotonic shock, and enhanced cytocidal effects. These findings demonstrated the cytocidal effects of hypotonic shock on ESCC cells, and clearly support the efficacy of pleural lavage with distilled water during surgery for ESCC.

Post-hepatectomy survival in advanced hepatocellular carcinoma with portal vein tumor thrombosis

AIM To analyze hepatocellular carcinoma (HCC) patients with portal vein tumor thrombosis (PVTT) using the tumor-node-metastasis (TNM) staging system. METHODS We retrospectively analyzed 372 patients with HCC who underwent hepatectomy between 1980 and 2009. We studied the outcomes of HCC patients with PVTT to evaluate the American Joint Committee on Cancer TNM staging system (7(th) edition) for stratifying and predicting the prognosis of a large cohort of HCC patients after hepatectomy in a single-center. Portal vein invasion (vp) 1 was defined as an invasion or tumor thrombus distal to the second branch of the portal vein, vp2 as an invasion or tumor thrombus in the second branch of the portal vein, vp3 as an invasion or tumor thrombus in the first branch of the portal vein, and vp4 as an invasion or tumor thrombus in the portal trunk or extending to a branch on the contralateral side. RESULTS The cumulative 5-year overall survival (5yrOS) and 5-year disease-free survival (5yrDFS) rates of the 372 patients were 58.3% and 31.3%, respectively. The 5yrDFS and 5yrOS of vp3-4 patients (n = 10) were 20.0%, and 30.0%, respectively, which was comparable with the corresponding survival rates of vp1-2 patients (P = 0.466 and 0.586, respectively). In the subgroup analysis of patients with macroscopic PVTT (vp2-4), the OS of the patients who underwent preoperative transarterial chemoembolization was comparable to that of patients who did not (P = 0.747). There was a significant difference in the DFS between patients with stage I HCC and those with stage II HCC (5yrDFS 39.2% vs 23.1%, P < 0.001); however, the DFS for stage II was similar to that for stage III (5yrDFS 23.1% vs 13.8%, P = 0.330). In the subgroup analysis of stage II-III HCC (n = 148), only alpha-fetoprotein (AFP) > 100 mg/dL was independently associated with DFS. CONCLUSION Hepatectomy for vp3-4 HCC results in a survival rate similar to hepatectomy for vp1-2. AFP stratified the stage II-III HCC patients according to prognosis.

Depleted tumor suppressor miR-107 in plasma relates to tumor progression and is a novel therapeutic target in pancreatic cancer

This study explored decreased tumor suppressor microRNA (miRNA) plasma levels in pancreatic cancer (PCa) patients to clarify their potential as novel biomarkers and therapeutic targets. We used the microRNA array-based approach to select candidates by comparing plasma levels between PCa patients and healthy volunteers. Six down-regulated miRNAs (miR-107, miR-126, miR-451, miR-145, miR-491-5p, and miR-146b-5p) were selected. Small- and large-scale analyses using samples from 100 PCa patients and 80 healthy volunteers revealed that miR-107 was the most down-regulated miRNA in PCa patients compared with healthy volunteers (P < 0.0001; area under the receiver-operating characteristic curve, 0.851). A low miR-107 plasma level was significantly associated with advanced T stage, N stage, and liver metastasis and was an independent factor predicting poor prognosis in PCa patients (P = 0.0424; hazard ratio, 2.95). miR-107 overexpression in PCa cells induced G1/S arrest with the production of p21 and inhibited cell proliferation through the transcriptional regulation of Notch2. In vivo, the restoration and maintenance of the miR-107 plasma level significantly inhibited tumor progression in mice. Depletion of the tumor suppressor miR-107 in plasma relates to tumor progression and poor outcomes. The restoration of the plasma miR-107 level might be a novel anticancer treatment strategy for PCa.

Liquid biopsy in patients with hepatocellular carcinoma: Circulating tumor cells and cell-free nucleic acids

Hepatocellular carcinoma (HCC), with its high incidence and mortality rate, is one of the most common malignant tumors. Despite recent development of a diagnostic and treatment method, the prognosis of HCC remains poor. Therefore, to provide optimal treatment for each patient with HCC, more precise and effective biomarkers are urgently needed which could facilitate a more detailed individualized decision-making during HCC treatment, including the following; risk assessment, early cancer detection, prediction of treatment or prognostic outcome. In the blood of cancer patients, accumulating evidence about circulating tumor cells and cell-free nucleic acids has suggested their potent clinical utilities as novel biomarker. This concept, so-called “liquid biopsy” is widely known as an alternative approach to cancer tissue biopsy. This method might facilitate a more sensitive diagnosis and better decision-making by obtaining genetic and epigenetic aberrations that are closely associated with cancer initiation and progression. In this article, we review recent developments based on the available literature on both circulating tumor cells and cell-free nucleic acids in cancer patients, especially focusing on Hepatocellular carcinoma.

Poor prognostic subgroup in T3N0 stage IIA gastric cancer, suggesting an indication for adjuvant chemotherapy.

Adjuvant chemotherapy following curative gastrectomy is recommended for patients with pStage II or III, except pT3 (ss), N0 gastric cancer in Japan. This study aimed to detect the poor prognostic subgroup of T3N0 gastric cancer, suggesting an indication for adjuvant chemotherapy.