Toshiyuki Kosuga

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.

Circulating microRNAs in plasma of patients with oesophageal squamous cell carcinoma

Background:Several recent studies demonstrated that microRNAs (miRNAs) are stably detectable in plasma/serum. We hypothesised that plasma miRNAs concentrations contributed to potential biomarkers in patients with oesophageal squamous cell carcinoma (ESCC).Methods:We selected three oncogenic miRNAs (miR-21, miR-184, miR-221) and one tumour suppressive miRNA (miR-375), which are frequently reported in squamous cell carcinoma, as candidate targets for this plasma miRNA assay. This study was divided into three steps: (1) Determination of appropriate plasma miRNAs in preliminary tests. (2) Evaluation of whether the plasma miRNA assays could monitor tumour dynamics. (3) Validation study on the clinical application of plasma miRNA assays in 50 ESCC patients and 20 healthy volunteers.Results:(1) In preliminary tests, the plasma level of miR-21 was significantly higher (P=0.0218) and that of miR-375 (P=0.0052) was significantly lower in ESCC patients than controls. (2) The high plasma miR-21 levels reflected tumour levels in all cases (100%). The plasma level of miR-21 was significantly reduced in postoperative samples (P=0.0058). (3) On validation analysis, the plasma level of miR-21 tended to be higher in ESCC patients (P=0.0649), while that of miR-375 was significantly lower (P<0.0001) and the miR-21/miR-375 ratio was significantly higher (P<0.0001) in ESCC patients than in controls. The value of the area under the receiver-operating characteristic curve (AUC) was 0.816 for the miR-21/miR-375 ratio assay. Patients with a high plasma level of miR-21 tended to have greater vascular invasion (P=0.1554) and to show a high correlation with recurrence (P=0.0164).Conclusion:Detection of circulating miRNAs might provide new complementary tumour markers for ESCC.

Survival benefits from splenic hilar lymph node dissection by splenectomy in gastric cancer patients: relative comparison of the benefits in subgroups of patients

BackgroundThe present study estimated survival benefits from lymph node dissection at the splenic hilus in advanced proximal gastric cancer patients who underwent total gastrectomy with simultaneous splenectomy, and then determined patient subgroups that received relatively high survival benefits from splenectomy.MethodsA total of 280 patients with advanced proximal gastric cancer who underwent curative total gastrectomy with simultaneous splenectomy were retrospectively analyzed. Patients with primary tumors directly invading the spleen or pancreas and those with gross metastases to the para-aortic nodes, as determined by intraoperative diagnosis, were excluded from analyses. The index of estimated benefit from lymph node dissection at the splenic hilus by splenectomy was calculated for each clinicopathological factor by multiplying the incidence of splenic hilar metastasis by the 5-year survival rate of patients with metastasis to that nodal station.ResultsThirty patients (10.7%) showed lymph node metastasis at the splenic hilus, and the 5-year survival rate of these patients was 51.3% (overall index 5.49). The index was relatively high in patient subgroups with tumors localized on the greater curvature (19.4) and Borrmann type 4 cancers (12.9), while relatively low in subgroups with encircling tumors (1.62) and tumors invading adjacent organs other than the spleen and pancreas (0).ConclusionPatients with tumors localized on the greater curvature and Borrmann type 4 cancers might obtain relatively high survival benefits from lymph node dissection at the splenic hilus by splenectomy.

Blockade of chloride ion transport enhances the cytocidal effect of hypotonic solution in gastric cancer cells

BACKGROUND Cancer cells that are exfoliated into the peritoneal cavity during surgery are viable and have the potential to produce peritoneal recurrence. Although peritoneal lavage with distilled water is applied in some cancer surgeries to kill tumor cells, there is no consensus regarding the optimal methodology and its effects. METHODS Three human gastric cancer cell lines, MKN28, MKN45, and Kato-III, were exposed to distilled water, and the resultant morphologic changes were observed using a microscope. Analysis of cell volume changes was performed using a flow cytometer. To investigate the cytocidal effects of the water, re-incubation of the cells was performed after exposing them to hypotonic solution. Additionally, the effects of 5-nitro-2-3-phenylpropylamino)-benzoic acid (NPPB), a Cl(-) channel blocker, and R(+)-[(dihydroindenyl)oxy] alkanoic acid (DIOA), a blocker of the K(+)/Cl(-) co-transporter, on the cells during their exposure to hypotonic solution were analyzed. RESULTS After the cells had been exposed to the distilled water, a rapid increase in cell volume occurred followed by cell rupture. In the MKN45 and Kato-III cells, treatment with NPPB increased cell volume by inhibiting regulatory volume decrease and enhanced the cytocidal effects of the hypotonic solution, whereas no such effects were observed in the MKN28 cells. On the other hand, treatment of the MKN28 cells with DIOA inhibited RVD and enhanced the cytocidal effects of hypotonic shock. CONCLUSION These findings support the efficacy of peritoneal lavage with distilled water during surgery for gastric cancer and suggest that the regulation of Cl(-) transport enhances the cytocidal effects of hypotonic shock.

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.

Prediction of CCND1 amplification using plasma DNA as a prognostic marker in oesophageal squamous cell carcinoma

Background:We aimed to develop a new biomarker to predict cyclin D1 (CCND1) status using plasma DNA in oesophageal squamous cell carcinoma (ESCC) patients.Methods:We evaluated the ratio of the CCND1 (11q13) dosage to the dopamine receptor D2 (DRD2; 11q22-23) dosage (C/D ratio) as CCND1 copy number. This study was divided into three steps: (1) Determination of a cutoff value for the C/D ratio in test scale; (2) Comparison of the C/D ratio in between plasma samples and cancer tissues in ESCC patients showing high plasma C/D ratio; (3) Validation study of the clinical application of the plasma C/D ratio as a diagnostic and prognostic marker, by comparing with clinicopathologic factors in 96 ESCC patients.Results:The plasma C/D ratio was significantly higher in the ESCC group than the controls (P=0.0134). A high plasma C/D ratio reflected the tumour C/D ratio, and significantly correlated with a poorer prognosis (P=0.0186). Moreover, the high C/D ratio was found to be an independent prognostic factor on multivariate analysis (P=0.0266; hazard ratio 5.988).Conclusion:Prediction of CCND1 amplification using plasma DNA is thought to be a promising prognostic biomarker in ESCC patients.

Tumor exosome-mediated promotion of adhesion to mesothelial cells in gastric cancer cells

Background Peritoneal metastasis consists of a highly complex series of steps, and the details of the underlying molecular mechanism remain largely unclear. In this study, the effects of tumor-derived exosomes (TEX) on the progression of gastric cancers were investigated in peritoneal metastasis. Results TEX were internalized in both mesothelial and gastric cancer cells in a cellular origin non-specific manner. Internalization of TEX into mesothelial cells promoted significant adhesion between mesothelial and gastric cancer cells, and TEX internalization into gastric cancer cells significantly promoted migratory ability, while internalization of mesothelial cell-derived exosomes did not. Expression of adhesion-related molecules, such as fibronectin 1 (FN1) and laminin gamma 1 (LAMC1), were increased in mesothelial cells after internalization of TEX from gastric cancer cell line and malignant pleural effusion. Methods TEX were extracted from cell-conditioned medium by ultracentrifugation. The effects of TEX on the malignant potential of gastric cancer were investigated in adhesion, invasion, and proliferation assays. PCR array as well as western blotting were performed to determine the underlying molecular mechanisms. The molecular changes in mesothelial cell after internalization of TEX derived from malignant pleural effusion were also confirmed. Conclusions TEX may play a critical role in the development of peritoneal metastasis of gastric cancer, which may be partially due to inducing increased expression of adhesion molecules in mesothelial cells.

Single-Port Mediastinoscopic Lymphadenectomy Along the Left Recurrent Laryngeal Nerve

We herein describe a single-port mediastinoscopic method for upper mediastinal dissection in esophageal cancer surgery. After the left cervical incision and lymphadenectomy, a Lap-Protector (Hakko, Tokyo, Japan) was inserted into the wound and an EZ Access port (Hakko) was attached. Esophageal mobilization with en bloc lymphadenectomy along the left recurrent laryngeal nerve was then performed using a port-in-port technique with conventional flexible laparoscopy. Carbon dioxide insufflation expanded the intramediastinal space, and minute structures in the deep mediastinum around the aortic arch, such as nerves, bronchial arteries, and lymphatic vessels, were clearly visualized, allowing lymphadenectomy to be safely and carefully performed along the nerve.

xCT, component of cysteine/glutamate transporter, as an independent prognostic factor in human esophageal squamous cell carcinoma

BackgroundxCT is a component of the cysteine/glutamate transporter, which plays a key role in glutathione synthesis. The objectives of the present study were to investigate the role of xCT in the regulation of genes involved in cell cycle progression and the clinicopathological significance of its expression in esophageal squamous cell carcinoma (ESCC).MethodsxCT expression in human ESCC cell lines was analyzed by Western blotting and immunofluorescent staining. Knockdown experiments were conducted with xCT siRNA, and the effect on cell cycle was analyzed. The cells’ gene expression profiles were analyzed by microarray analysis. An immunohistochemical analysis of 70 primary tumor samples obtained from ESCC patients that had undergone esophagectomy was performed.ResultsxCT was highly expressed in TE13 and KYSE170 cells. In these cells, the knockdown of xCT using siRNA inhibited G1-S phase progression. Microarray analysis identified 1652 genes whose expression levels in TE13 cells were altered by the knockdown of xCT. Pathway analysis showed that the top-ranked canonical pathway was the G1/S checkpoint regulation pathway, which involves TP53INP1, CDKN1A, CyclinD1/cdk4, and E2F5. Immunohistochemical staining showed that xCT is mainly found in the nuclei of carcinoma cells, and that its expression is an independent prognostic factor.ConclusionsThese observations suggest that the expression of xCT in ESCC cells might affect the G1/S checkpoint and impact on the prognosis of ESCC patients. As a result, we have a deeper understanding of the role played by xCT as a mediator and/or biomarker in ESCC.

Laparoscopic and endoscopic co-operative surgery for non-ampullary duodenal tumors.

AIM To assess the safety and feasibility of laparoscopic and endoscopic co-operative surgery (LECS) for early non-ampullary duodenal tumors. METHODS Twelve patients with a non-ampullary duodenal tumor underwent LECS at our hospital. One patient had two mucosal lesions in the duodenum. The indication for this procedure was the presence of duodenal tumors with a low risk for lymph node metastasis. In particular, the tumors included small (less than 10 mm) submucosal tumors (SMT) and epithelial mucosal tumors, such as mucosal cancers or large mucosal adenomas with malignant suspicion. The LECS procedures, such as full-thickness dissection for SMT and laparoscopic reinforcement after endoscopic submucosal dissection (ESD) for epithelial tumors, were performed for the 13 early duodenal lesions in 12 patients. Here we present the short-term outcomes and evaluate the safety and feasibility of this new technique. RESULTS Two SMT-like lesions and eleven superficial epithelial tumor-like lesions were observed. Seven and Six lesions were located in the second and third parts of the duodenum, respectively. All lesions were successfully resected en bloc. The defect in the duodenal wall was manually sutured after resection of the duodenal SMT. For epithelial duodenal tumors, the ulcer bed was laparoscopically reinforced via manual suturing after ESD. Intraoperative perforation occurred in two out of eleven epithelial tumor-like lesions during ESD; however, they were successfully laparoscopically repaired. The median operative time and intraoperative estimated blood loss were 322 min and 0 mL, respectively. Histological examination of the tumors revealed one adenoma with moderate atypia, ten adenocarcinomas, and two neuroendocrine tumors. No severe postoperative complications (Clavien-Dindo classification grade III or higher) were reported in this series, but minor leakage secondary to pancreatic fistula occurred in one patient. CONCLUSION LECS can be a safe and minimally invasive treatment option for non-ampullary early duodenal tumors.