Yujin Kim

Enteroendocrine cell counts correlate with visceral hypersensitivity in patients with diarrhoea‐predominant irritable bowel syndrome

Abstractu2002 The objective of this study was to determine whether or not the number of enteroendocrine cells (ECs) in the gut is related to visceral hypersensitivity in patients with diarrhoea‐predominant irritable bowel syndrome (D‐IBS). Twenty‐five subjects with D‐IBS (mean, 43.1u2003years; 16 women, nine men) were recruited into our study, along with 13 healthy controls (mean, 40.7u2003years; nine women, four men). Maximally tolerable pressures were evaluated via barostat testing, and the levels of ECs were immunohistochemically identified and quantified via image analysis. The numbers of ECs between the D‐IBS subjects and the controls were not significantly different in the terminal ileum, ascending colon and rectum. However, the maximally tolerable pressures determined in the D‐IBS subjects were significantly lower than those of the control subjects (Pu2003<u20030.01), and we detected a significant relationship between the maximally tolerable pressures and the numbers of ECs in the rectum (ru2003=u2003−0.37, Pu2003<u20030.01). Rectal sensitivity was enhanced to a greater degree in D‐IBS patients exhibiting an elevated level of rectal ECs. This study provides some evidence to suggest that ECs play an important role in visceral hypersensitivity.

Association of Global Levels of Histone Modifications with Recurrence-Free Survival in Stage IIB and III Esophageal Squamous Cell Carcinomas

This study was aimed at understanding the effects of histone modifications on recurrence-free survival (RFS) after esophagectomy in esophageal squamous cell carcinoma (ESCC). The acetylation of histone H3 lysine (H3K9Ac), histone H3 lysine 18 (H3K18Ac), and histone H4 lysine 12 (H4K12Ac), and the dimethylation of histone H3 lysine 9 (H3K9diMe) and histone H4 arginine 3 (H4R3diMe) were analyzed by immunohistochemistry in 237 ESCCs. The K-means clustering algorithm was used to identify unique patterns of histone modifications. At a median follow-up of 5.1 years, 109 (46%) of 237 patients had developed recurrence of disease. Mean global levels of H3K9Ac, H3K18Ac, H3K9diMe, H4K12Ac, and H4R3diMe were 81.5%, 65.1%, 80.3%, 45.9%, and 27.4%, respectively. In the analysis of individual histones, a 1% increase in the global level of H3K18Ac in pathologic stage III worsened RFS at 1.009 times [95% confidence interval (CI), 1.001-1.016; P = 0.03], after adjusting for age, sex, and operative method. Cluster analysis also showed significant effects of histone modifications on RFS. For stage IIB cancers, Cox proportional hazards analysis showed that RFS of cluster 1, with high global levels of H3K18Ac and H4R3diMe, was 2.79 times poorer (95% CI, 1.14-6.27; P = 0.008) than that of cluster 2, with low levels. RFS for stage III cancers was also poorer in cluster 1 than cluster 2 (adjusted hazard ratio, 2.42; 95% CI, 1.10-5.34; P = 0.02). In conclusion, the present study suggests that global levels of histone modifications in ESCC may be an independent prognostic factor of RFS. Cancer Epidemiol Biomarkers Prev; 19(2); 566–73

Promoter Hypermethylation of the p16 Gene Is Associated with Poor Prognosis in Recurrent Early-Stage Hepatocellular Carcinoma

Despite significant advances in the detection and treatment of hepatocellular carcinoma, the prognosis of patients with hepatocellular carcinoma remains very poor, in part due to the high incidence of recurrence. This study was aimed at identifying a prognostic indicator of recurrence in patients with hepatocellular carcinoma. We retrospectively analyzed CpG island hypermethylation of the p14, p15, p16, GSTP1, integrin α4, SYK, and CDH1 genes in fresh-frozen tissues from 265 patients with hepatocellular carcinoma using the methylation-specific PCR. The expression levels of p16 and p53 were evaluated by immunohistochemistry. CpG island hypermethylation was detected in 6% for p14, 21% for p15, 67% for p16, 75% for GSTP1, 23% for integrin α4, 12% for SYK, and 57% for CDH1. Recurrence was observed in 102 (38%) of the 265 patients. There was no association between the risk for recurrence and hypermethylation of any gene studied. However, p16 methylation was associated with a poor survival after surgery for recurrent stage I to II hepatocellular carcinomas (hazard ratio, 4.05; 95% confidence interval, 1.15-14.20; P = 0.03). In addition, the hazard of failure after recurrence was about 3.80 (95% confidence interval, 1.03-14.20; P = 0.04) times higher in patients with p16 methylation than in those without. Negative expression of p16 at a protein level was also associated with poor survival in recurrent stage I to II hepatocellular carcinomas, but p53 expression did not have a synergistic effect on the poor prognosis. In conclusion, the present study suggests that p16 methylation may be associated with a poor prognosis in recurrent early-stage hepatocellular carcinomas. (Cancer Epidemiol Biomarkers Prev 2008;17(9):2260–7)

Long-term Stress and Helicobacter pylori Infection Independently Induce Gastric Mucosal Lesions in C57BL/6 Mice

Background: Long-term psychological stresses may have a role in the pathogenesis of peptic ulcer. However, the interaction between stress and Helicobacter pylori infection in the development of peptic ulcer is not established. The aim of this study was to elucidate the roles of long-term stress and H. pylori infection in the development of gastric mucosal lesions in mice. Methods: The Sydney strain (SS1) of H. pylori was inoculated into the stomach of C57BL/6 mice. Twelve weeks later, mice with or without H. pylori infection were exposed to long-term repeated water-immersion-restraint stress (WIRS) for 12 h per day, 3 times per week, for 8 weeks. Gastric mucosal lesions were evaluated both macroscopically (ulcer index) and microscopically (Updated Sydney System). Results: The long-term WIRS induced mild inflammation, oedema, interstitial haemorrhage and superficial erosions in the stomach of mice both with and without H. pylori infection. The degree of mucosal inflammation or atrophy in H. pylori -infected mice was not influenced by the stress. In the mice without H. pylori infection, the ulcer index of the stressed mice was greater than that of non-stressed mice (1.66 ± 0.39 versus 0.17 ± 0.08, P = 0.007). In the mice with H. pylori infection, the ulcer index (mean ± s x ) of the stressed mice was also greater than that of nonstressed mice (2.31 ± 0.59 versus 0.64 ± 0.22, P = 0.027). Conclusions: The present study showed that long-term stress can induce gastric mucosal inflammation and erosions, and this effect may occur independently of H. pylori infection.

Epigenetic Inactivation of Heparan Sulfate (Glucosamine) 3-O-Sulfotransferase 2 in Lung Cancer and Its Role in Tumorigenesis

Background This study was aimed at investigating the functional significance of heparan sulfate (glucosamine) 3-O-sulfotransferase 2 (HS3ST2) hypermethylation in non-small cell lung cancer (NSCLC). Methodology/ Principal Findings HS3ST2 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using 298 formalin-fixed paraffin-embedded tissues and 26 fresh-frozen tissues from 324 NSCLC patients. MS-HRM (methylation-specific high-resolution melting) and EpiTYPERTM assays showed substantial hypermethylation of CpG island at the promoter region of HS3ST2 in six lung cancer cell lines. The silenced gene was demethylated and re-expressed by treatment with 5-aza-2′-deoxycytidine (5-Aza-dC). A promoter assay also showed the core promoter activity of HS3ST2 was regulated by methylation. Exogenous expression of HS3ST2 in lung cancer cells H460 and H23 inhibited cell migration, invasion, cell proliferation and whereas knockdown of HS3ST2 in NHBE cells induced cell migration, invasion, and cell proliferation in vitro. A negative correlation was observed between mRNA and methylation levels of HS3ST2 in 26 fresh-frozen tumors tissues (ρ = -0.51, P = 0.009; Spearman’s rank correlation). HS3ST2 hypermethylation was found in 95 (32%) of 298 primary NSCLCs. Patients with HS3ST2 hypermethylation in 193 node-negative stage I-II NSCLCs with a median follow-up period of 5.8 years had poor overall survival (hazard ratio = 2.12, 95% confidence interval = 1.25–3.58, P = 0.005) compared to those without HS3ST2 hypermethylation, after adjusting for age, sex, tumor size, adjuvant therapy, recurrence, and differentiation. Conclusions/ Significance The present study suggests that HS3ST2 hypermethylation may be an independent prognostic indicator for overall survival in node-negative stage I-II NSCLC.

Association of RASSF1A and p63 with Poor Recurrence-Free Survival in Node-Negative Stage I–II Non–Small Cell Lung Cancer

Purpose: This study was aimed at analyzing the recurrence-related prognostic significance of 12 candidate molecular biomarkers in node-negative stage I–II non–small cell lung cancer (NSCLC). Experimental Design: We retrospectively analyzed promoter methylation of eight genes using methylation-specific PCR in formalin-fixed and paraffin-embedded tissues from 328 node-negative stage I–II NSCLCs. The expression of Bcl-2, E-cadherin, p53, and p63 proteins was also assessed by immunohistochemistry. Results: Recurrence was found in 145 (44%) of 328 node-negative stage I–II NSCLCs with a median follow-up period of 6.2 years. No association was found between recurrence and alteration of individual biomarker in univariate analysis. We defined recurrently divergent groups on the basis of recursive partitioning analyses for 12 biomarkers and found a significant association of co-alteration of RASSF1A and p63 with poor recurrence-free survival (RFS). Cox proportional hazards analysis showed that hypermethylation of RASSF1A and negative expression of p63 was associated with poor RFS [HR, 1.93; 95% confidence interval (CI), 1.13–5.47; P = 0.009] compared with those without co-alteration of RASSF1A and p63, after adjusting for age, adjuvant therapy, histology, and tumor size. Random forest classifier including RASSF1A and p63 showed best performance in the prediction of recurrence in node-negative stage I–II NSCLCs: area under receiver operator characteristic curve for random forest was 0.91 and error rate for the model was 17%. Conclusion: The present study suggests that RASSF1A and p63 may be independent prognostic indicators for RFS in node-negative stage I–II NSCLCs. Clin Cancer Res; 19(5); 1204–12. ©2012 AACR.

A functional single nucleotide polymorphism at the promoter region of cyclin A2 is associated with increased risk of colon, liver, and lung cancers

The objective of this was to identify functional single nucleotide polymorphisms (SNPs) in cyclin‐dependent kinases (CDKs) and cyclins that are associated with risk of human cancer.

CpG island hypermethylation as a biomarker for the early detection of lung cancer.

Lung cancer is the most frequent cause of cancer-related deaths and causes over one million deaths worldwide each year. Despite significant strides in the diagnosis and treatment of lung cancer, the prognosis is extremely poor, with the overall 5-year survival rates still remaining around 15 %. This is largely due to occult metastatic dissemination, which appears in approximately two-thirds of patients at the time of detection. Thus, the development of efficient diagnostic methods to enable the early detection of cancer for these patients is clearly imperative.One promising approach is the identification of lung cancer-specific biomarkers at an early stage. The de novo methylation of CpG islands within the promoters of tumor suppressor genes is one of the most frequently acquired epigenetic changes during the pathogenesis of lung cancer and usually associated with transcriptional downregulation of a gene. The analysis of DNA methylation patterns in sputum, bronchial fluid, plasma, or serum could become a powerful tool for the accurate and early diagnosis of lung cancer with unparalleled specificity and sensitivity.

Cystatin M loss is associated with the losses of estrogen receptor, progesterone receptor, and HER4 in invasive breast cancer

IntroductionThis study was aimed at understanding the clinicopathological significance of cystatin M loss, and investigating possible factors responsible for cystatin M loss in breast cancer.MethodsThe expression of estrogen receptor (ER), progesterone receptor (PR), HER2, HER4, and cystatin M was retrospectively analyzed using immunohistochemistry in 117 patients with ductal carcinoma in situ (DCIS) and in 175 patients with invasive breast cancer (IBC). The methylation status of CST6 gene encoding cystatin M was evaluated using methylation-specific polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissues from 292 participants and using pyrosequencing in fresh-frozen tumor and matched normal tissues from 51 IBC patients.ResultsCystatin M loss was found in 9 (8%) of 117 patients with DCIS and in 99 (57%) of 175 with invasive breast cancer (IBC) (P < 0.0001). Cystatin M loss was found in 58 (57%) of 101 HER2-negative IBCs and in 41 (55%) of 74 HER2-positive IBCs, and this difference was not statistically significant (P = 0.97). However, cystatin M loss was significantly associated with the loss of ER (P = 0.01), PR (P = 0.002), and HER4 (P = 0.003) in IBCs. Cystatin M loss occurred in 34 (76%) of the 45 HER4-negative IBCs and in 65 (50%) of the 130 HER4-positive IBCs. Multivariate analysis showed that cystatin M loss occurred at a 3.57 times (95% CI = 1.28 to 9.98; P = 0.01) higher prevalence in the triple-negative IBCs of ER, PR, and HER4 than in other subtypes, after adjusting for age. The quantity of CST6 methylation was associated with ER loss (P = 0.0002) in IBCs but not with the loss of PR (P = 0.64) or HER4 (P = 0.87).ConclusionsThe present study suggests that cystatin M loss may be associated with the losses of ER, PR, and HER4 in IBC.

HOXA9 inhibits migration of lung cancer cells and its hypermethylation is associated with recurrence in non‐small cell lung cancer

This study was aimed at understanding the clinicopathological significance of HOXA9 hypermethylation in non‐small cell lung cancer (NSCLC). HOXA9 hypermethylation was characterized in six lung cancer cell lines, and its clinicopathological significance was analyzed using methylation‐specific PCR in 271 formalin‐fixed paraffin‐embedded tissues and 27 fresh‐frozen tumor and matched normal tissues from 298 NSCLC patients, and Ki‐67 expression was analyzed using immunohistochemistry. The promoter region of HOXA9 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5‐aza‐2′‐deoxycytidine (5‐Aza‐dC). Transient transfection of HOXA9 into H23 lung cancer cells resulted in the inhibition of cell migration but not proliferation. Conversely, sequence‐specific siRNA‐mediated knockdown of HOXA9 enhanced cell migration. The mRNA levels of HOXA9 in 27 fresh‐frozen tumor tissues were significantly lower than in matched normal tissues (Pu2009<u20090.0001; Wilcoxon signed‐rank test). HOXA9 hypermethylation was found in 191 (70%) of 271 primary NSCLCs. HOXA9 hypermethylation was not associated with tumor size (Pu2009=u20090.12) and Ki‐67 proliferation index (Pu2009=u20090.15). However, patients with HOXA9 hypermethylation had poor recurrence‐free survival (hazard ratiou2009=u20093.98, 95% confidence intervalu2009=u20091.07–17.09, Pu2009=u20090.01) in never‐smokers, after adjusting for age, sex, tumor size, adjuvant therapy, pathologic stage, and histology. In conclusion, the present study suggests that HOXA9 inhibits migration of lung cancer cells and its hypermethylation is an independent prognostic factor for recurrence‐free survival in never‐smokers with NSCLC.