Masayoshi Nakanishi

Tumour-amplified kinase BTAK is amplified and overexpressed in gastric cancers with possible involvement in aneuploid formation

Our recent analysis of gastric cancers using comparative genomic hybridization (CGH) revealed a novel high frequent copy number increase in the long arm of chromosome 20. Tumour-amplified kinase BTAK was recently cloned from breast cancers and mapped on 20q13 as a target gene for this amplification in human breast cancers. In the study presented here, we analysed BTAK copy-number and expression, and their relation to the ploidy pattern in 72 primary gastric cancers. Furthermore, wild-type BTAK and its deletion mutants were transfected to gastric cancers to examine changes in cell proliferation and DNA ploidy pattern. Evaluation of 72 unselected primary gastric cancers found BTAK amplification in 5% and overexpression in more than 50%. All four clinical samples with BTAK amplification showed aneuploidy and poor prognosis. Transfection of BTAK in near-diploid gastric cancers induced another aneuploid cell population. In contrast, the c-terminal-deleted mutant of BTAK induced no effect in DNA ploidy pattern and inhibited gastric cancer cell proliferation. These results suggest that BTAK may be involved in gastric cancer cell aneuploid formation, and is a candidate gene for the increase in the number of copies of the 20q, and thus may contribute to an increase in the malignant phenotype of gastric cancer.

Amplification and over-expression of the AIB1 nuclear receptor co-activator gene in primary gastric cancers.

Our analysis of chromosomal aberrations in primary gastric cancers using comparative genomic hybridization has revealed novel, high and frequent copy number increases in the long arm of chromosome 20, indicating that this region contains novel amplified genes involved in gastric cancer progression. AIB1, a member of the steroid receptor co‐activator‐1 family, has been cloned on 20q12 as a candidate target gene for this amplification in human breast cancers. In this study, we examined the numbers of AIB1 copies as well as their expression and relation to clinico‐pathological features in 72 primary gastric cancers. AIB1 amplification was observed in 7% and over‐expression in 40% of the specimens. AIB1 amplification always coincided with its over‐expression, but several cases showed AIB1 over‐expression without amplification, suggesting that expression of AIB1 is regulated not only by gene amplification but also by other mechanisms, such as transcriptional activation, in human gastric cancer. Gastric cancers with AIB1 amplification showed extensive lymph node metastases, liver metastases and poorer prognosis compared to those without amplification. Our results suggest that amplification and over‐expression of AIB1 are likely to increase the number of malignant phenotypes of gastric cancers and that it can be expected to be useful as a marker of poor prognosis. Int. J. Cancer 89:217–223, 2000.

Chromosomal aberrations in colorectal cancers and liver metastases analyzed by comparative genomic hybridization

Comparative genomic hybridization (CGH) was used to screen for changes in the number of DNA sequence copies in 30 primary colorectal cancers and 16 liver metastases, to identify regions that contain genes important for the development and progression of colorectal cancer. In primary colorectal cancer, we found frequent gains at 7p21 (36.7%), 7q31‐36 (30%), 8q23‐24 (43.0%), 12p (30%), 14q24‐32 (33.3%), 16p (40.0%), 20p (33.3%), 20q (63.3%) and 21q (36.3%), while loss was often noted at 18q12‐23 (36.7%). In metastatic tumors, there were significantly more gains and losses of DNA sequences than in primary tumors, with gains at 8q23‐24 (found in 62.5% of recurrences vs. 43.0% of primary tumors), 15q21‐26 (37.5% vs. 20.0%), 19p (43.8% vs. 20.0%) and 20q (81.3% vs. 63.3%) and losses at 18q12‐23 (50.0% vs. 36.7%). The pattern of genetic changes seen in metastatic tumors, with frequent gains at 8q23‐24 and 20q and loss at 18q12‐23, suggests the progression of colorectal cancer. We investigated a clinical follow‐up study for all patients examined by CGH and directed our attention to the genetic changes consisting of gains at 8q and 20q. The incidence of liver metastases was higher in patients with primary colorectal cancer with these genetic changes. Gains at 8q and 20q might be useful to identify patients at high risk for developing liver metastases.

Differential gene expression profiles of gastric cancer cells established from primary tumour and malignant ascites

Advanced gastric cancer is often accompanied by metastasis to the peritoneum, resulting in a high mortality rate. Mechanisms involved in gastric cancer metastasis have not been fully clarified because metastasis involves multiple steps and requires a combination of altered expressions of many different genes. Thus, independent analysis of any single gene would be insufficient to understand all of the aspects of gastric cancer peritoneal dissemination. In this study, we performed a global analysis of the differential gene expression of a gastric cancer cell line established from a primary main tumour (SNU-1) and of other cell lines established from the metastasis to the peritoneal cavity (SNU-5, SNU-16, SNU-620, KATO-III and GT3TKB). The application of a high-density cDNA microarray method made it possible to analyse the expression of approximately 21 168 genes. Our examinations of SNU-5, SNU-16, SNU-620, KATO-III and GT3TKB showed that 24 genes were up-regulated and 17 genes down-regulated besides expression sequence tags. The analysis revealed the following altered expression such as: (a) up-regulation of CD44 (cell adhesion), keratins 7, 8, and 14 (epitherial marker), aldehyde dehydrogenase (drug metabolism), CD9 and IP3 receptor type3 (signal transduction); (b) down-regulation of IL2 receptor γ, IL4-Stat (immune response), p27 (cell cycle) and integrin β4 (adhesion) in gastric cancer cells from malignant ascites. We then analysed eight gastric cancer cell lines with Northern blot and observed preferential up-regulation and down-regulation of these selected genes in cells prone to peritoneal dissemination. Reverse transcriptase–polymerase chain reaction confirmed that several genes selected by DNA microarray were also overexpressed in clinical samples of malignant ascites. It is therefore considered that these genes may be related to the peritoneal dissemination of gastric cancers. The results of this global gene expression analysis of gastric cancer cells with peritoneal dissemination, promise to provide a new insight into the study of human gastric cancer peritoneal dissemination.

HGF regulates VEGF expression via the c-Met receptor downstream pathways, PI3K/Akt, MAPK and STAT3, in CT26 murine cells

In the present study, we assessed the involvement of hepatocyte growth factor (HGF)/c-Met signalling with vascular endothelial cell growth factor (VEGF) and hypoxia inducible factor (HIF)-1α expression in the downstream pathways phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) in CT26 cells, to determine the mechanisms of the potent anti-angiogenic effect of NK4. We established genetically modified CT26 cells to produce NK4 (CT26-NK4). VEGF expression in subcutaneous CT26 tumours in vivo and in culture supernatants in vitro was determined by ELISA. HIF-1α expression in nuclear extracts was evaluated by western blot analysis. VEGF and HIF-1α mRNA levels were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The DNA binding activity of HIF-1α was evaluated using an HIF-1α transcription factor assay kit. Our results demonstrated that VEGF expression was reduced in homografts of CT26-NK4 cells, compared to those of the control cells. In vitro, VEGF expression, which was induced by HGF, was inhibited by anti-HGF antibody, NK4 and by kinase inhibitors (PI3K, LY294002; MAPK, PD98059; and STAT3, Stattic). HGF‑induced HIF‑1α transcriptional activity was also inhibited by the kinase inhibitors. Real-time RT-PCR demonstrated that HGF‑induced HIF‑1α mRNA expression was not inhibited by LY294002 and PD98059, but was inhibited by Stattic. These data suggest that the PI3K/Akt, MAPK and STAT3 pathways, downstream of HGF/c‑Met signalling, are involved in the regulation of VEGF expression in CT26 cells. HGF/c‑Met signalling may be a promising target for anti-angiogenic strategies.

Application of Polyethylene Glycolic Acid Felt with Fibrin Sealant to Prevent Postoperative Pancreatic Fistula in Pancreatic Surgery

ObjectiveThe purpose of this nonrandomized retrospective study was to report our new procedures using polyethylene glycolic acid (PGA) felt with fibrin sealant to prevent severe pancreatic fistula in patients undergoing pancreatic surgery.MethodsFrom 2000 to 2008, 54 and 63 patients underwent pancreaticoduodenectomy (PD) and distal pancreatectomy (DP), respectively. Of those patients, we applied PGA felt with fibrin sealant to 18 PD patients and 26 DP patients. In PD patients, the PGA felt was wrapped around the pancreatic suture site, while in DP patients, the PGA felt was wrapped around the predictive division site. The pancreaticojejunostomy site in PD patients and the cut stump in DP patients were coated with fibrin sealant. We compared the occurrence rates for severe postoperative pancreatic fistula (POPF) that occurred after PD or DP both with and without our new procedures.ResultsBefore introduction of our procedures, severe POPF developed in 14 of 36 PD patients (39%) and 10 of 37 DP patients (27%). In contrast, after introduction of our procedures, the incidence of POPF was only one in both of 18 PD (6%; P = 0.016) and 26 DP (4%; P = 0.017) patients.ConclusionIn summary, our procedure using PGA felt with fibrin sealant may reduce the risk of severe POPF.

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.

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

AIM To re-evaluate the recent clinicopathological features of remnant gastric cancer (RGC) and to develop desirable surveillance programs. METHODS Between 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. RESULTS Concerning 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-I procedure 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-I reconstruction. In tumor location of RGC, after Billroth-I reconstruction, 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 stage I-II tended to have been followed up almost every second year. CONCLUSION Meticulous 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.

Risk Factors and Management of Postoperative Bile Leakage after Hepatectomy without Bilioenteric Anastomosis

Background/Aims: Bile leakage frequently causes major complications after hepatic resection. We investigated perioperative risk factors and management of postoperative bile leakage after hepatic resection without extrahepatic biliary resection and reconstruction. Methods: We included 247 consecutive patients who underwent elective hepatic resection without bilioenteric anastomosis at our institution between 2002 and 2009. Perioperative risk factors, including patient and surgical variables, were evaluated using univariate and logistic regression analyses. Results: Postoperative bile leakage occurred in 26 patients (10.5%). The surgical drain was retained in 6 patients (23%); 9 (35%) underwent drain salvage and 11 (42%) underwent percutaneous puncture under computed tomography or ultrasound guidance. Eight patients underwent endoscopic nasobiliary drainage (ENBD) for postoperative bile leakage, and bile leakage healed at a median interval of 19.5 days after ENBD. By univariate analysis, postoperative bile leakage was associated with central bisectionectomy, surgical time and intraoperative blood loss. Logistic regression analysis identified central bisectionectomy as an independent risk factor for postoperative bile leakage (p = 0.0003, odds ratio 16.724). Conclusion: Meticulous procedures are necessary during parenchymal hepatic resection, especially during central bisectionectomy. Drain management should be precise in the case of postoperative bile leakage. We believe ENBD may rapidly cure postoperative major bile leakage.

5-Aminolevulinic acid-mediated photodynamic therapy using light-emitting diodes of different wavelengths in a mouse model of peritoneally disseminated gastric cancer

BACKGROUND 5-Aminolevulinic acid (5-ALA) is a precursor of the strong photosensitizer, protoporphyrin IX, in cancer cells. The efficacy of 5-ALA-mediated photodynamic therapy (ALA-PDT) using light-emitting diodes (LEDs) was evaluated in a mouse model of peritoneally disseminated gastric cancer. MATERIALS AND METHODS The effects of violet (peak wavelength 410 nm), green (peak wavelength 525 nm), and red (peak wavelength 635 nm) LEDs on reactive oxygen species generation and ALA-PDT cytotoxicity were measured in vitro. 5-ALA was intraperitoneally injected into a mouse xenograft model of peritoneally disseminated enhanced green fluorescent protein-expressing MKN-45 cells, followed by irradiation of micrometastatic nodules on the omentum and evaluation of the necrotic areas of these nodules. RESULTS ROS generation and the cytotoxic effects of ALA-PDT were highest for the violet and lowest for the red LEDs. The necrotic areas of nodules were significantly larger after irradiation with each LED than in the control mice. The violet and green LEDs had the same anticancer effects, which were significantly greater than those of the red LED. CONCLUSIONS ALA-PDT using LEDs was effective in treating peritoneally disseminated gastric cancer. The differences in the anticancer effects among the three light sources indicate the necessity of selecting the light source with the optimal wavelength most effective for in vivo clinical applications.