Naosuke Yokomichi

Unique Association between Global DNA Hypomethylation and Chromosomal Alterations in Human Hepatocellular Carcinoma

Global DNA hypomethylation is a characteristic feature of cancer cells that closely associates with chromosomal instability (CIN). However, the association between these characteristics during hepatocarcinogenesis remains unclear. Herein, we determined the relationship between hypomethylation and CIN in human hepatocellular carcinoma (HCC) by analyzing 179 HCCs, 178 matched non-tumor livers and 23 normal liver tissues. Hypomethylation at three different repetitive DNA (rDNA) sequences and hypermethylation of 12 CpG loci, including 11 tumor suppressor gene (TSG) promoters, were quantified using MethyLight or combined bisulfite restriction analysis. Fractional allelic loss (FAL) was used as a marker for CIN, calculated by analyzing 400 microsatellite markers. Gains and losses at each chromosome were also determined using semi-quantitative microsatellite analysis. The associations between rDNA hypomethylation and FAL, as well as between TSG hypermethylation and FAL were investigated. Significantly more hypomethylation was observed in HCC tissues than in normal liver samples. Progression of hypomethylation during carcinogenesis was more prominent in hepatitis C virus (HCV)-negative cases, which was in contrast to our previous reports of significantly increased TSG methylation levels in HCV-positive tumors. Absence of liver cirrhosis and higher FAL scores were identified as independent contributors to significant hypomethylation of rDNA in HCC. Among the chromosomal alterations frequently observed in HCC, loss of 8p, which was unique in the earliest stages of hepatocarcinogenesis, was significantly associated with hypomethylation of rDNA by multivariable analysis (p = 0.0153). rDNA hypomethylation was also associated with a high FAL score regardless of tumor differentiation (p = 0.0011, well-differentiated; p = 0.0089, moderately/poorly-differentiated HCCs). We conclude that DNA hypomethylation is an important cause of CIN in the earliest step of HCC, especially in a background of non-cirrhotic liver.

Advanced hepatocellular carcinoma with lymph node metastases showing epithelial to mesenchymal transition effectively treated with systemic chemotherapy: Report of a case

We present a case in which combination chemotherapy was used to successfully treat hepatocellular carcinoma (HCC) with rapid progression of lymph node (LN) metastases after liver resection. In addition, epithelial to mesenchymal transition (EMT) markers were examined immunohistochemically. A 43‐year‐old man who had been diagnosed with HCC showed an enlarged LN near the hepatic artery proper. After extended left lobectomy with lymphadenectomy in the hepatoduodenal ligament, he experienced rapid progression of metastases to the para‐aortic and mediastinal LN. Partial remission was achieved after induction and maintenance of combination chemotherapy using etoposide, carboplatin, epirubicin and 5‐fluorouracil. As a consequence of this treatment, the patient survived 10 months. Immunohistochemical studies demonstrated that HCC cells in the metastatic LN showed low expression of E‐cadherin and high expression of N‐cadherin and vimentin, indicating EMT. Combination chemotherapy may prove effective for patients with HCC accompanied by LN metastases that show features of EMT.

Case of emphysematous cholecystitis in a patient with type 2 diabetes mellitus associated with schizophrenia

Emphysematous cholecystitis is a rare, but life‐threatening, form of acute cholecystitis caused by gas‐forming organisms in the gallbladder. A 73‐year‐old male patient with type 2 diabetes mellitus complicated with neuropathy associated with schizophrenia was admitted to Okayama University Hospital, Okayama, Japan, because of a high fever and general malaise. On the fourth hospital day, despite normal liver function tests and little abdominal pain, his abdominal computed tomography showed huge gas formation in the gallbladder lumen along with a dilated gallbladder with a thickened wall, consistent with emphysematous cholecystitis. The patient underwent an emergency open cholecystectomy. Few abdominal symptoms appeared because of the hyposensitivity to pain caused by not only diabetic neuropathy, but also antipsychotic agents the patient was taking for schizophrenia. Emphysematous cholecystitis should be taken into consideration for the differential diagnosis of high fever in diabetic patients with schizophrenia, irrespective of the level of liver function tests and clinical symptoms.

Heterogeneity of Epigenetic and Epithelial Mesenchymal Transition Marks in Hepatocellular Carcinoma with Keratin 19 Proficiency

Objective: Keratin 19 (K19) expression is a potential predictor of poor prognosis in patients with hepatocellular carcinoma (HCC). To clarify the feature of K19-proficient HCC, we traced epigenetic footprints in cultured cells and clinical materials. Patients and Methods: In vitro, KRT19 promoter methylation was analyzed and 5-aza-2’-deoxycytidine with trichostatin A (TSA) treatment was performed. Among 564 surgically resected HCCs, the clinicopathological relevance of K19-proficent HCCs was performed in comparison with hepatocytic (HepPar-1 and arginase-1), epithelial-mesenchymal transition (E-cadherin and vimentin), biliary differentiation-associated (K7 and NOTCH-1) markers, and epigenetic markers (KRT19 promoter/long interspersed nucleotide element-1 [LINE-1] methylation status). Results: KRT19 promoter methylation was clearly associated with K19 deficiency and 5-aza-2’-deoxycytidine with TSA treatment-stimulated K19 re-expression, implicating DNA methylation as a potential epigenetic process for K19 expression. After excluding HCCs with recurrence, TNM stage as IIIB or greater, preoperative therapy, transplantation, and combined hepatocellular cholangiocarcinoma, we assessed 125 of 564 HCC cases. In this cohort, K19 expression was found in 29 HCCs (23.2%) and corresponded with poor survival following surgery (p = 0.025) and extrahepatic recurrence-free survival (p = 0.017). Compared with K19-deficient HCCs, lower KRT19 promoter methylation level was observed in K19-proficient HCCs (p < 0.0001). Conversely, HCC with genome-wide LINE-1 hypermethylation was frequently observed in K19-proficient HCCs (p = 0.0079). Additionally, K19 proficiency was associated with K7 proficiency (p = 0.043), and reduced E-cadherin and HepPar-1 expression (p = 0.043 and p < 0.0001, respectively). Conclusions: K19-proficient HCC exhibited poor prognosis owing to extrahepatic recurrence, with molecular signatures differing from those in conventional cancer stem cells, providing novel insights of the heterogeneity underlying tumor development.

225PEXTENSIVE METHYLATION OF EPIDERMAL GROWTH FACTOR-CONTAINING FIBULIN-LIKE EXTRACELLULAR MATRIX PROTEIN 1 (EFEMP1) PROMOTER COULD PREDICT MALIGNANT FORMATION IN INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS (IPMN)

ABSTRACT Introduction: Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are pancreatic cystic tumors. Genetic and epigenetic alterations are believed to be accumulated during tumor progression. Whereas the specific genetic mutations and DNA methylation for each type of lesion have been somewhat known, the fundamental understanding for the dynamics of methylation expansion across specific CpG dinucleotides in a gene promoter during carcinogenesis remains unclear. We sought to evaluate whether the expansion of methylation in the EFEMP1 promoter could serve as a predictive biomarker for malignant IPMNs. Methods: KRAS mutations and the methylation status of two discrete regions within the EFEMP1 promoter in 65 IPMN tissues, including 30 IPMNs with low- and intermediate-grade dysplasia, 12 IPMNs with high-grade dysplasia and 23 invasive IPMN, were determined using genomic sequencing and High Sensitive Assay for bisulfate modification DNA (Hi-SA) - a modified COBRA by which fluorescence labeled DNA fragments are detected using a genetic analyzer (Nagasaka T et al., JNCI 2009), respectively. Thereafter, correlation between mutant KRAS, methylation status and various clinic-pathological features were examined. Results: KRAS mutations were detected in 42.9% of low- and intermediate-grade IPMNs, 33.3% of high-grade IPMNs and 71.4% of invasive IPMNs. All mutations were confined to codon 12 of the KRAS gene, and these mutations did not correlate with a specific histological grade or type of lesions, or any of the other clinic-pathological features. On the other hand, while the methylation of the region 1 or region 2 within the EFEMP1 promoter was observed in more than 80% of non-invasive IPMNs and invasive IPMNs, simultaneous methylation of both regions occurred in 0% of noninvasive vs. 34.7% of invasive IPMNs (p = 0.00148). These results suggest that KRAS mutations and extensive methylation within the EFEMP1 promoter were one of the early and late events in cancer progression, respectively. Conclusions: The extensive methylation in EFEMP1 promoter could be a potential predictive marker for identification of invasive IPMNs, and provides an attractive rationale for developing this molecular signature as a substrate for the development of non-invasive screening for invasive IPMNs. Disclosure: All authors have declared no conflicts of interest.

220PCYTOKERATIN 19, A NOVEL PROGNOSTIC BIOMARKER FOR HEPATOCELLULAR CARCINOMA, IS REGULATED BY DNA METHYLATION

ABSTRACT Aim: The expression of CK19 protein has recently been proposed as a novel predictor for poor prognosis following curative resection in patients with hepatocellular carcinoma (HCC). While the mechanisms underlying regulation of CK19 expression in HCC remain unclear, the presence of a CpG island within its promoter region implicates DNA methylation as a potential epigenetic process in this malignancy. Methods: A panel of 564 surgically resected HCCs at Okayama University between 2000 and 2010 were analyzed for CK19 expression and its promoter methylation status. Among all HCCs, we excluded all tumors with recurrence, cancers with TNM stage ≥IIIB, lesions with preoperative therapy, transplantation, and combined hepatocellular-cholangiocarcinomas. CK19 expression was evaluated by immunohistochemistry in FFPE tissues. CK19 promoter methylation was evaluated for region 1 and region 2 by Hi-SA - a modified COBRA by which fluorescence labeled DNA fragments are detected using a genetic analyzer (Nagasaka T et al., JNCI 2009). Molecular results were correlated with clinicopathological features, followed by Kaplan-Meier survival analysis. Results: A total of 125 cases matched for the selection criteria. Among these, 29 HCCs demonstrated positive CK19 staining, and these patients corresponded with significantly poor survival following surgery (p = 0.025), and extra-hepatic metastasis-free survival (p = 0.017). Multivariate analysis revealed that CK19-positive was an independent prognostic factor for survival, following surgery (HR2.85, 95%CI 1.21-6.41, p = 0.018). Methylation analysis revealed that methylation rates at both regions of CK19 gene were statistically higher in HCCs with loss of CK19 expression compared to CK19-positive group (p = 0.0002, p Conclusions: Our data report that CK19 expression is an important prognostic biomarker for HCC. Furthermore, since CK19s expression was strongly associated with the methylation of its promoter region, our study provides novel mechanistic insights for the participation of this epigenetic alteration in the development of HCC. Disclosure: All authors have declared no conflicts of interest.

Su1995 Genetic and Epigenetic Alterations in the Netrin-1 Receptors, UNC5c and DCC, Constitutes a Previously Unrecognized Pathway in Gastric Cancer Progression

Background: Downregulated expression of the netrin-1 dependence receptors, UNC5C and DCC, is a frequent event in significant proportion of colorectal tumors. However, the tumor suppressive role of this important cancer-related pathway in other gastrointestinal cancers remains unclear. We hypothesized that similar to colorectal cancer, down-regulation of UNC5C and DCC may have an important growth-regulatory function in gastric tumorigenesis as well. Methods: We performed a series of genetic (loss of heterozygosity, LOH) and epigenetic analysis (aberrant DNA methylation) for UNC5C and DCC in a cohort of 105 sporadic gastric cancers and the corresponding normal gastric mucosa. Results: In this study, the distribution of gastric cancers as categorized by TNM staging system (UICC 7th) was as follows; 20, 24, 28, and 29 cases in stages I, II, III, and IV, respectively. Overall, 58% (48 out of 83 informative cases) and 76% (65 out of 85 informative cases) of gastric cancers demonstrated either a genetic or epigenetic alteration in the UNC5C and DCC genes, respectively. Interestingly, although epigenetic mechanisms were the primary cause for UNC5C inactivation, methylation-induced transcriptional silencing served also served as the second hit responsible for the DCC gene loss. Taken together, 52% (36 out of 69 gastric cancers with informative data) of gastric cancers showed cumulative defects in these two dependence receptors. The frequency of LOH and aberrant methylation of both dependence receptors gradually increased with progressive tumor stages. Conclusions: This is the first study that unravels the observation that majority of gastric cancers harbor defects in netrin1 receptors. Our observation that both genetic and epigenetic alterations are present early, and continue to escalate in a stepwise manner with the disease progression, highlights the fundamental importance of this growth regulatory pathway in gastric carcinogenesis.