Takayuki Nobuoka

Upregulation of miR-196a and HOTAIR Drive Malignant Character in Gastrointestinal Stromal Tumors

Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although both offer considerable theranostic potential. In this study, we identified frequent upregulation of miR-196a and lincRNA HOTAIR in high-risk gastrointestinal stromal tumors (GIST). Overexpression of miR-196a was associated with high-risk grade, metastasis and poor survival among GIST specimens. miR-196a genes are located within the HOX gene clusters and microarray expression analysis revealed that the HOXC and HOTAIR gene were also coordinately upregulated in GISTs which overexpress miR-196a. In like manner, overexpression of HOTAIR was also strongly associated with high-risk grade and metastasis among GIST specimens. RNA interference-mediated knockdown of HOTAIR altered the expression of reported HOTAIR target genes and suppressed GIST cell invasiveness. These findings reveal concurrent overexpression of HOX genes with noncoding RNAs in human cancer in this setting, revealing miR-196a and HOTAIR as potentially useful biomarkers and therapeutic targets in malignant GISTs.

Reconsideration of Postoperative Oral Intake Tolerance After Pancreaticoduodenectomy: Prospective Consecutive Analysis of Delayed Gastric Emptying According to the ISGPS Definition and the Amount of Dietary Intake

Objective:A prospective consecutive study was planned to evaluate the postpancreaticoduodenectomy (PD) oral intake tolerance. The occurrence of delayed gastric emptying (DGE), as defined by the International Study Group of Pancreatic Surgery (ISGPS), and the amount of dietary intake were analyzed. The risk factors for low oral intake tolerance were additionally determined. Summary Background Data:The causation of DGE after PD is still unclear. Several possible factors have been discussed, such as reconstruction methods and other complications. However, none of them has followed the definition of ISGPS. Methods:Between 2003 and 2007, 101 consecutive patients underwent PD-related surgery, and standard operative procedure was performed on 85 patients. Perioperative data were prospectively collected in all patients, and the patients postoperative dietary intake was recorded for all meals until discharge. As an indicator of early postoperative oral intake tolerance, we added up the dietary intake from postoperative day 1 to 21 and called this value the total amount of dietary intake (TDI). The postoperative outcomes were compared between non-DGE and DGE. The high-low of TDI values was also analyzed. Multivariate analysis for factors associated with the occurrence of DGE and TDI was performed. Results:The occurrence of DGE as defined by ISGPS was 42%. The postoperative outcomes of DGE patients were significantly poor compared with those of non-DGE patients. TDI values were significantly low in DGE patients, and non-DGE patients with low TDI values showed a significantly extended duration of parenteral nutrition and postoperative hospitalization. Operative bleeding (>1,000 mL) and pancreatic fistulas were likely to be associated with DGE occurrence. Gender (women), BMI (>25 kg/m2), postoperative intraabdominal infection, and DGE were significantly associated with low TDI values. Conclusions:The ISGPS definition of DGE seemed feasible for patient management. TDI values provided additional information for analyzing postoperative oral intake tolerance, especially when describing the differences among non-DGE patients. Substantial risk factors for low oral intake tolerance were high BMI, postoperative intraabdominal infection, and DGE.

A Novel Correlation between LINE-1 Hypomethylation and the Malignancy of Gastrointestinal Stromal Tumors

Purpose: Gastrointestinal stromal tumors (GIST) are the most important mesenchymal tumors of the gastrointestinal tract. The vast majority of GISTs exhibit activating mutations of KIT or PDGFRA, but epigenetic alteration of GISTs is largely unknown. In this study, we aimed to clarify the involvement of DNA methylation in GIST malignancy. Experimental Design: A total of 106 GIST specimens were studied. Levels of LINE-1 methylation were analyzed using bisulfite pyrosequencing. In addition, methylation of three other repetitive sequences (Alu Yb8, Satellite-α, and NBL2) was similarly analyzed, and CpG island hypermethylation was analyzed using MethyLight. Array-based comparative genomic hybridization (array CGH) was carried out in 25 GIST specimens. Results: LINE-1 hypomethylation was significantly correlated with risk, and high-risk GISTs exhibited significantly lower levels of LINE-1 methylation than low-risk (61.3% versus 53.2%; P = 0.001) or intermediate-risk GISTs (60.8% versus 53.2%; P = 0.002). Hypomethylation of Satellite-α and NBL2 was also observed in high-risk GISTs. By contrast, promoter hypermethylation was relatively infrequent (CDH1, 11.2%; MLH1, 9.8%; SFRP1, 1.2%; SFRP2, 11.0%; CHFR, 9.8%; APC, 6.1%; CDKN2A, 0%; RASSF1A, 0%; RASSF2, 0%) and did not correlate with LINE-1 methylation or risk. Array CGH analysis revealed a significant correlation between LINE-1 hypomethylation and chromosomal aberrations. Conclusions: Our data suggest that LINE-1 hypomethylation correlates significantly with the aggressiveness of GISTs and that LINE-1 methylation could be a useful marker for risk assessment. Hypomethylation may increase the malignant potential of GISTs by inducing accumulation of chromosomal aberrations. Clin Cancer Res; 16(21); 5114–23. ©2010 AACR.

Transcriptional Control of Tight Junction Proteins via a Protein Kinase C Signal Pathway in Human Telomerase Reverse Transcriptase-Transfected Human Pancreatic Duct Epithelial Cells

In human pancreatic cancer, integral membrane proteins of tight junction claudins are abnormally regulated, making these proteins promising molecular diagnostic and therapeutic targets. However, the regulation of claudin-based tight junctions remains unknown not only in the pancreatic cancer cells but also in normal human pancreatic duct epithelial (HPDE) cells. To investigate the regulation of tight junction molecules including claudins in normal HPDE cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture. The hTERT-transfected HPDE (hTERT-HPDE) cells were positive for the pancreatic duct epithelial markers such as CK7, CK19, and carbonic anhydrase isozyme 2 and expressed epithelial tight junction molecules claudin-1, -4, -7 and, -18, occludin, JAM-A, ZO-1, ZO-2, and tricellulin. By treatment with fetal bovine serum or 12-O-tetradecanoylphorbol 13-acetate (TPA), the tight junction molecules were up-regulated at the transcriptional level via a protein kinase C (PKC) signal pathway. A PKC-alpha inhibitor, Gö6976, prevented up-regulation of claudin-4 by TPA. Furthermore, a PKC-delta inhibitor, rottlerin, prevented up-regulation of claudin-7, occludin, ZO-1, and ZO-2 by TPA. By GeneChip analysis, up-regulation of the transcription factor ELF3 was observed in both fetal bovine serum- and TPA-treated cells. Treatment with small interfering RNAs of ELF3 prevented up-regulation of claudin-7 by TPA. These data suggest that tight junctions of normal HPDE cells were at least in part regulated via a PKC signal pathway by transcriptional control.

Current Status of Surgery for Pancreatic Cancer

Background: In Japan the annual incidence of pancreatic cancer has increased over the last decade, but no advancement has been made in the long-term prognosis after resection. The significant differences in the surgical procedures between Western countries and Japan have been discussed. Therefore, an adequate comparison and analysis of the data from Japan, Europe and the USA is required. This review evaluates many important published reports from Japan which influence surgical procedure. Methods: Several important highlights and controversies regarding the concept of surgical treatment and surgical procedure are discussed comparing the results in Japan with those in Western countries. Results: No significant difference in diagnostic strategy using various imaging methods was observed between Japan and Europe. The stage classification for pancreatic cancer by the Japanese Pancreatic Society (JPS) seems to be superior to others, because the results on long-term prognosis after pancreatectomy of cases with pancreatic head cancer, diagnosed as tubular adenocarcinoma, has been arranged logically. Pancreatectomy with extended radical dissection is recommended in Japan, but several clinical studies from Europe and the USA suggest that this is ineffective. The basic concepts of this controversy have recently come closer altogether. Scientific clinical trials for instance on the necessity of adjuvant treatment, etc., are now on-going. Conclusion: The characteristics on diagnosis and treatment of pancreatic cancer in Japan are described. The JPS registration system for pancreatic cancer can provide much more information, i.e. dependency on diagnostic methods, highly frequent sites of lymph node and of distant metastases, the prognosis of small pancreatic cancers, etc. The indication for any surgical treatments should be limited to cases with the possibility of cancer free margins.

Serum Hyaluronate Level for Predicting Subclinical Liver Dysfunction after Hepatectomy

The serum hyaluronate (HA) level reflects sinusoidal endothelial cell function correlated with liver function. We have reviewed multiple liver function indicators from 37 patients who underwent hepatectomy for various liver diseases. The serum HA level was well correlated with the indocyanine green retention rate at 15 minutes (ICGR15), lectin-cholesterol (LCAT), hepatocyte growth factor (HGF), liver uptake ratio of technetium-99m galactosyl human serum albumin (99mTc-GSA) at 15 minutes (HH15), prealbumin, and hepatic uptake ratio of 99mTc-GSA at 15 minutes (LHL15). In addition, the model for end-stage liver disease (MELD) score at 7 days after operation was well correlated with serum HA, ICGR15, HH15, and LHL15. In patients who showed serum an HA level of = 100 ng/ml before hepatectomy, the MELD score had significantly deteriorated by 7 days after hepatectomy. Of the 20 patients who showed a serum HA level < 100 ng/ml before hepatectomy, 11 had high serum HA after hepatectomy. The bilirubin level 7 days after operation in this group was much higher than that for patients who maintained a serum HA level < 100 ng/ml after hepatectomy. In addition, the serum HGF level before hepatectomy in this group was significantly lower. We concluded that the serum HA level is a reliable indicator when evaluating liver function and predicting liver dysfunction after hepatectomy. Furthermore, patients with a significantly low HGF level who have a normal HA level are susceptible to liver dysfunction after hepatectomy.

Growth of hepatic angiomyolipoma indicating malignant potential.

To the Editor, Hepatic angiomyolipoma (HAML) has been considered a benign rare tumor since Ishak first described it in 1976. There have been a number of reports of HAML that investigated its clinical and pathological features. Although the progression of radiological imaging techniques has resulted in a high detection rate and diagnostic accuracy of the liver tumor, definitive diagnosis of HAML still requires histological confirmation. Reactivity of HMB-45 confirms the definitive diagnosis of HAML and once it is determined, surgical treatment has been avoided. Unlike renal angimyolipomas, well known to possess malignant potential, little information about malignant HAML has been available since Dalle et al. first described its existence in 2000. Subsequently, awareness of malignant HAML with a metastatic character has increased. Although all of these reports supported the existence of malignant HMAL, growth activity of HAML has never been reported. A 49-year-old woman was found to have a liver tumor in the right lobe on a medical examination. Computed tomography (CT) images with contrast medium revealed double hypervascular liver tumors (Fig. 1a,b) and a fine needle biopsy resulted in a definite diagnosis

Portal Blood Flow Regulates Volume Recovery of the Rat Liver after Partial Hepatectomy: Molecular Evaluation

Background/Aim: Liver regeneration is a finely tuned process that is closely regulated by multiple cell cycle steps. Although the portal blood flow affects liver regeneration, the molecular mechanism by which the blood flow regulates gene expression and liver function is largely unknown. The aim of this study was to investigate the molecular effect of portal blood flow on hepatocyte proliferation and gene regulation during liver regeneration. Materials and Methods: We developed a simple surgical rat model to investigate the relation between portal blood flow and liver regeneration by partially ligating the portal trunk with 8-0 Proline sutures under microscopy to reduce the blood flow by 40%. We investigated recovery of liver volume, DNA synthesis, and gene expression associated with cell cycle regulators, comparing partially hepatectomized (PH) rats without (PH group; n = 30) and with partial portal ligation (PHPL group; n = 30) for 7 days after the operation. Results: The hepatic tissue blood flow and the recovery ratio between liver weight and body weight in the PHPL group were significantly lower than in the PH group after hepatectomy. The peak 5-bromo-2′-deoxyuridine labeling index in the PHPL group was delayed and weak compared with the PH group. The expression of CT-1 and cyclin D, E, and B mRNAs indicated that the liver regeneration in the PHPL group was delayed and weak. In addition, there was reciprocal expression of C/EBPα and C/EBPβ mRNAs, an observation supported by their nuclear protein levels. Furthermore, the cytochrome P-450 protein level in the PHPL group was higher than that in the PH group 1 day after hepatectomy. Conclusion: The portal blood flow regulates the activity of liver regeneration and the gene expression associated with cell cycle regulators, while the functions are maintained.

Biliary tract malignancy and chronic inflammation from the perspective of pancreaticobiliary maljunction

This is a brief review of pancreaticobiliary maljunction. The basic treatment for this condition includes either cholecystectomy or extrahepatic bile duct resection. When the condition is accompanied by malignancies, a radical operation should be the first treatment option. Knowledge on molecular pathogenesis is gradually increasing. However, studies should be expanded to include larger patient cohorts, and other types of molecules should be carefully investigated and analyzed.

Efficacy of Enteral Supplementation Enriched with Glutamine, Fiber, and Oligosaccharide on Mucosal Injury following Hematopoietic Stem Cell Transplantation

The combination of glutamine, fiber and oligosaccharides (GFO) is thought to be beneficial for alleviating gastrointestinal mucosal damage caused by chemotherapy. A commercial enteral supplementation product (GFO) enriched with these 3 components is available in Japan. We performed a retrospective study to test whether oral GFO decreased the severity of mucosal injury following hematopoietic stem cell transplantation (HSCT). Of 44 HSCT patients, 22 received GFO and 22 did not. Severity of diarrhea/mucositis, overall survival, weight loss, febrile illness/documented infection, intravenous hyperalimentation days/hospital days, engraftment, acute and chronic GVHD, and cumulative incidence of relapse were studied. Sex, age, performance status, diagnosis, disease status, and treatment variables were similar in both groups. There were fewer days of diarrhea grade 3-4 in patients receiving GFO than in those who did not (0.86 vs. 3.27 days); the same was true for days of mucositis grade 3-4 (3.86 vs. 6.00 days). Survival at day 100 was 100% in the GFO group, but only 77.3% for the patients not receiving GFO (p = 0.0091, log-rank test). Weight loss and the number of days of intravenous hyperalimentation were better in the GFO group (p < 0.001 and p = 0.0014, respectively). Although not significant, less gut bacterial translocation with Enterococcus species developed in the GFO group (p = 0.0728) than in the non-GFO group. Other outcomes were not affected. To the best of our knowledge, this is the first comparative clinical study of GFO supplementation to alleviate mucosal injury after allo-HSCT. We conclude that glutamine, fiber and oligosaccharide supplementation is an effective supportive therapy to decrease the severity of mucosal damage in HSCT.