Osamu Dohi

Defective expression of polarity protein PAR-3 gene ( PARD3 ) in esophageal squamous cell carcinoma

The partition-defective 3 (PAR-3) protein is implicated in the formation of tight junctions at epithelial cell–cell contacts. We investigated DNA copy number aberrations in human esophageal squamous cell carcinoma (ESCC) cell lines using a high-density oligonucleotide microarray and found a homozygous deletion of PARD3 (the gene encoding PAR-3). Exogenous expression of PARD3 in ESCC cells lacking this gene enhanced the recruitment of zonula occludens 1 (ZO-1), a marker of tight junctions, to sites of cell–cell contact. Conversely, knockdown of PARD3 in ESCC cells expressing this gene caused a disruption of ZO-1 localization at cell–cell borders. A copy number loss of PARD3 was observed in 15% of primary ESCC cells. Expression of PARD3 was significantly reduced in primary ESCC tumors compared with their nontumorous counterparts, and this reduced expression was associated with positive lymph node metastasis and poor differentiation. Our results suggest that deletion and reduced expression of PARD3 may be a novel mechanism that drives the progression of ESCC.

Epigenetic silencing of miR-335 and its host gene MEST in hepatocellular carcinoma.

MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of target genes. Some tumor-suppressive miRNAs are known to be epigenetically silenced by promoter DNA methylation in cancer. In the present study, we aimed to identify miRNA genes that are silenced by DNA hypermethylation in hepatocellular carcinoma (HCC). We screened for miRNA genes with promoter DNA hypermethylation using a genome-wide methylation microarray analysis in HCC cells. It was found that miR-335, which is harbored within an intron of its protein-coding host gene, MEST, was downregulated by aberrant promoter hypermethylation via further methylation assays, including methylation-specific PCR, combined bisulfite and restriction analysis, bisulfite sequencing analysis and 5-aza-2′-deoxycytidine treatment. The expression levels of miR-335 significantly correlated with those of MEST, supporting the notion that the intronic miR-335 is co-expressed with its host gene. The levels of miR-335/MEST methylation were significantly higher in 18 (90%) out of 20 primary HCC tumors, compared to their non-tumor tissue counterparts (P<0.001). The expression levels of miR-335 were significantly lower in 25 (78%) out of 32 primary HCC tumors, compared to their non-tumor tissue counterparts (P=0.001). Furthermore, the expression levels of miR-335 were significantly lower in HCC tumors with distant metastasis compared to those without distant metastasis (P=0.02). In conclusion, our results indicate that expression of miR-335 is reduced by aberrant DNA methylation in HCC.

SOX2 identified as a target gene for the amplification at 3q26 that is frequently detected in esophageal squamous cell carcinoma

SOX2 is a transcription factor with a high-mobility group DNA-binding domain that functions as a master regulator during embryogenesis and organogenesis. We investigated DNA copy number aberrations in esophageal squamous cell carcinoma (ESCC) cell lines using a high-density oligonucleotide microarray and found frequent amplification at the chromosomal region 3q26. The estimated extent of the minimal overlapping region of amplification was 1.3 Mb. This chromosomal region includes a single gene, SOX2. The SOX2 protein was overexpressed in cell lines in which the gene was amplified. Knockdown experiments showed that SOX2 promotes proliferation of ESCC cells. Genes potentially modulated by SOX2 were determined by expression array analyses combined with small interfering RNA cell-transfection studies. A copy number gain of SOX2 (>2-fold) was observed in 6 of the 40 primary ESCCs (15%). Immunohistochemical study revealed that expression of the SOX2 protein was significantly elevated in 62 of the 89 ESCC tumors (70%), compared with their nontumorous counterparts, and that upregulated expression of SOX2 was associated with poor differentiation of ESCC. Our results suggest that SOX2 is likely to be a target of the 3q26 amplification and may therefore be involved in the development or progression of ESCC.

Alterations of the SWI/SNF chromatin remodelling subunit-BRG1 and BRM in hepatocellular carcinoma.

The SWI/SNF chromatin remodelling complex, which contains either brahma‐related gene‐1 (BRG1) or brahma (BRM) as the catalytic ATPase, functions as a master regulator of gene expression.

PEG10 is a probable target for the amplification at 7q21 detected in hepatocellular carcinoma

DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines were investigated using a high-density oligonucleotide microarray, and a novel amplification at the chromosomal region 7q21 was detected. Molecular definition of the amplicon indicated that PEG10 (paternally expressed gene 10), a paternally expressed imprinted gene, was amplified together with CDK14 (cyclin-dependent kinase 14; previously PFTAIRE protein kinase 1, PFTK1) and CDK6 (cyclin-dependent kinase 6). An increase in PEG10 copy number was detected in 14 of 34 primary HCC tumors (41%). PEG10, but not CDK14 or CDK6, was significantly overexpressed in 30 of 41 tumors (73%) from HCC patients, compared with their nontumorous counterparts. These results suggest that PEG10 is a probable target, acting as a driving force for amplification of the 7q21 region, and may therefore be involved in the development or progression of HCCs.

A Scoring System to Stratify Curability after Endoscopic Submucosal Dissection for Early Gastric Cancer: "eCura system".

Objectives:Although radical surgery is recommended for patients not meeting the curative criteria for endoscopic submucosal dissection (ESD) of early gastric cancer (EGC) because of the potential risk of lymph node metastasis (LNM), this recommendation may be overestimated and excessive. We aimed to establish a simple scoring system for decision making after ESD.Methods:This multicenter retrospective study consisted of two stages. First, the risk-scoring system for LNM was developed using multivariate logistic regression analysis in 1,101 patients who underwent radical surgery after having failed to meet the curative criteria for ESD of EGC. Next, the system was internally validated by survival analysis in another 905 patients who also did not meet the criteria and did not receive additional treatment after ESD.Results:In the development stage, based on accordant regression coefficients, five risk factors for LNM were weighted with point values: three points for lymphatic invasion and 1 point each for tumor size >30 mm, positive vertical margin, venous invasion, and submucosal invasion ≥500 μm. Then, the patients were categorized into three LNM risk groups: low (0–1 point: 2.5% risk), intermediate (2–4 points: 6.7%), and high (5–7 points: 22.7%). In the validation stage, cancer-specific survival differed significantly among these groups (99.6, 96.0, and 90.1%, respectively, at 5 years; P<0.001). The C statistic of the system for cancer-specific mortality was 0.78.Conclusions:This scoring system predicted cancer-specific survival in patients who did not meet the curative criteria after ESD for EGC. ESD without additional treatment may be an acceptable option for patients at low risk.

Genome-wide DNA methylation analysis in hepatocellular carcinoma

Epigenetic changes as well as genetic changes are mechanisms of tumorigenesis. We aimed to identify novel genes that are silenced by DNA hypermethylation in hepatocellular carcinoma (HCC). We screened for genes with promoter DNA hypermethylation using a genome-wide methylation microarray analysis in primary HCC (the discovery set). The microarray analysis revealed that there were 2,670 CpG sites that significantly differed in regards to the methylation level between the tumor and non-tumor liver tissues; 875 were significantly hypermethylated and 1,795 were significantly hypomethylated in the HCC tumors compared to the non‑tumor tissues. Further analyses using methylation-specific PCR, combined with expression analysis, in the validation set of primary HCC showed that, in addition to three known tumor-suppressor genes (APC, CDKN2A, and GSTP1), eight genes (AKR1B1, GRASP, MAP9, NXPE3, RSPH9, SPINT2, STEAP4, and ZNF154) were significantly hypermethylated and downregulated in the HCC tumors compared to the non-tumor liver tissues. Our results suggest that epigenetic silencing of these genes may be associated with HCC.

Linked color imaging improves endoscopic diagnosis of active Helicobacter pylori infection.

Background and study aims: Linked color imaging (LCI) is a new image-enhanced endoscopy technique using a laser light source to enhance slight differences in mucosal color. The aim of this study was to compare the usefulness of LCI and conventional white light imaging (WLI) endoscopy for diagnosing Helicobacter pylori (H. pylori). Patients and methods: We retrospectively analyzed images from 60 patients examined with WLI and LCI endoscopy between October 2013 and May 2014. Thirty patients had H. pylori infections, and other thirty patients tested negative for H. pylori after eradication therapy. Four endoscopists evaluated the 2 types of images to determine which was better at facilitating a diagnosis of H. pylori infection. Results: H. pylori infection was identified with LCI by enhancing the red appearance of the fundic gland mucosa. The accuracy, sensitivity, and specificity for diagnosing H. pylori infection using WLI were 74.2 %, 81.7 %, and 66.7 %, respectively, while those for LCI were 85.8 %, 93.3 %, and 78.3 %, respectively. Thus, the accuracy and sensitivity for LCI were significantly higher than those for WLI (P = 0.002 and P = 0.011, respectively). The kappa values for the inter- and intraobserver variability among the 4 endoscopists were higher for LCI than for WLI. Conclusions: H. pylori infection can be identified by enhancing endoscopic images of the diffuse redness of the fundic gland using LCI. LCI is a novel image-enhanced endoscopy and is more useful for diagnosing H. pylori infection than is WLI.

Recognition of Endoscopic Diagnosis in Differentiated-Type Early Gastric Cancer by Flexible Spectral Imaging Color Enhancement with Indigo Carmine

Background/Aims: To evaluate the usefulness of flexible spectral imaging color enhancement with indigo carmine (I-FICE) in early gastric cancer (EGC) demarcation. Methods: The study participants were 29 patients with differentiated-type EGC. The endoscope was fixed and images of the same area of EGC demarcations in each lesion were obtained using four different methods (WLE, flexible spectral imaging color enhancement (FICE), CE, and I-FICE). FICE mode at R 550 nm (Gain: 2), G 500 nm (Gain: 4), and B 470 nm (Gain: 4) was used. Four endoscopists ranked the images obtained by each method on the basis of the ease of recognition of demarcation using a 4-point system. We calculated the standard deviation of pixel values based on L*, a*, and b* color spaces in the demarcation region (Lab-SD score). Results: The median ranking score for I-FICE images was significantly higher than that obtained from the other methods. Further, the average Lab-SD score was significantly higher for I-FICE images than for images obtained by the other methods. There was a good correlation between the ranking score and Lab-SD score. Conclusion: EGC demarcations were most easily recognized both subjectively and objectively using I-FICE image, followed by CE, FICE and WLE images.

L-menthol improves adenoma detection rate during colonoscopy: a randomized trial.

BACKGROUND AND STUDY AIMS Colonoscopy is one of the most reliable methods for the detection of colorectal neoplasms. However, colonic peristalsis during colonoscopy results in some neoplastic lesions being hidden from view and commonly requires an intravenous or intramuscular injection of antispasmodic agents, which may sometimes causes unexpected adverse reactions. The aim of this study was to evaluate the efficacy of L-menthol spray as an antiperistaltic agent and its effect on adenoma detection. PATIENTS AND METHODS This was a prospective, randomized, single-blind placebo-controlled trial. A total of 226 patients who were scheduled to undergo colonoscopy were randomly assigned to receive either 20 mL of 1.6 % L-menthol (n = 118) or placebo (n = 108). Both treatments were sprayed locally onto the colonic mucosa via an endoscope. The adenoma detection rate (ADR) and the proportion of patients with no peristalsis were the primary and secondary outcomes, respectively. RESULTS The ADR was significantly higher in the L-menthol group than in the placebo group (60.2 % vs. 42.6 %; P = 0.0083). The proportion of patients with no peristalsis after treatment with L-menthol was significantly higher than in the placebo group (71.2 % vs. 30.9 %; P < 0.0001). There were no adverse effects in either group. CONCLUSIONS The results suggest that the suppression of colonic peristalsis by L-menthol sprayed directly onto the colonic mucosa improves the ADR. CLINICAL TRIAL REGISTRATION ID: UMIN 000007972.