Kazuya Sakuma

CpG island methylation status in gastric carcinoma with and without infection of Epstein-Barr virus

Purpose: EBV-associated gastric carcinoma shows global CpG island methylation of the promoter region of various cancer-related genes. To further clarify the significance of CpG island methylator phenotype (CIMP) status in gastric carcinoma, we investigated methylation profile and clinicopathologic features including overall survival in four subgroups defined by EBV infection and CIMP status: EBV-associated gastric carcinoma and EBV-negative/CIMP-high (H), EBV-intermediate (I), and EBV-negative (N) gastric carcinoma. Experimental Design: Methylation-specific PCR was applied to 106 gastric carcinoma cases. CIMP-N, CIMP-I, and CIMP-H status was determined by the number (0, 1-3, and 4-5, respectively) of methylated marker genes (LOX, HRASLS, FLNc, HAND1, and TM), that were newly identified as highly methylated in gastric cancer cell lines. The methylation status of 10 other cancer-related genes (p14, p15, p16, p73, TIMP-3, E-cadherin, DAPK, GSTP1, hMLH1, and MGMT) was also evaluated. Results: Nearly all (14 of 15) of EBV-associated gastric carcinoma exhibited CIMP-H, constituting a homogenous group (14%). EBV-negative gastric carcinoma consisted of CIMP-H (24%), CIMP-I (38%), and CIMP-N (24%). EBV-associated gastric carcinoma showed significantly higher frequencies of methylation of cancer-related genes (mean number ± SD = 6.9 ± 1.5) even if compared with EBV-negative/CIMP-H gastric carcinoma (3.5 ± 1.8). Among EBV-negative gastric carcinoma subgroups, CIMP-H gastric carcinoma showed comparatively higher frequency of methylation than CIMP-I or CIMP-N, especially of p16 and hMLH1. CIMP-N gastric carcinoma predominantly consisted of advanced carcinoma with significantly higher frequency of lymph node metastasis. The prognosis of the patients of CIMP-N was significantly worse compared with other groups overall by univariate analysis (P = 0.0313). Conclusion: The methylation profile of five representative genes is useful to stratify gastric carcinomas into biologically different subgroups. EBV-associated gastric carcinoma showed global CpG island methylation, comprising a pathogenetically distinct subgroup in CIMP-H gastric carcinoma.

p73 gene promoter methylation in Epstein‐Barr virus‐associated gastric carcinoma

To clarify the significance of p73 in Epstein‐Barr virus (EBV)‐associated gastric carcinoma (GC), the immunohistochemical expression and CpG‐island methylation of p73 were evaluated in cancer tissues and adjacent nonneoplastic tissues of GC with and without EBV infection. Loss of p73 expression by immunohistochemistry was specific to EBV‐associated GC (11/13) compared to EBV‐negative GC (3/38), which was independent of abnormal p53 expression. With methylation‐specific polymerase chain reaction (MSP), the aberrant methylation of p73 exon 1 was similarly specific to EBV‐associated GC (12/13), and also rare in EBV‐negative GC (2/38). Bisulfite sequencing for p73 exon 1 and its 5′ region confirmed the MSP results, showing uniform and high‐density methylation in EBV‐associated GC. Comparative MSP analysis of p14, p16 and p73 methylation, using 20 cases each of formalin‐fixed and paraffin‐embedded tissues of early GC with and without EBV infection, confirmed 2 types of methylation: global methylation with increased rates (p14 and p16) and specific methylation of p73 in EBV‐associated GC. In nonneoplastic mucosa, p14, p16 and p73 methylation occurred in both EBV‐associated (8/33, 6/34 and 3/38, respectively) and EBV‐negative GC (6/23, 4/35, and 1/35). p73 methylation was observed in the mucosa without H. pylori infection in all 4 samples. Loss of p73 expression through aberrant methylation of the p73 promoter occurs specifically in EBV‐associated GC, together with the global methylation of p14 and p16. A specific type of gastritis, prone to a higher grade of atrophy and p73 methylation, may facilitate the development of EBV‐associated GC.

Promoter hypermethylation of E‐cadherin and its abnormal expression in Epstein‐Barr virus‐associated gastric carcinoma

Promoter hypermethylation of various tumor‐related genes is extremely frequent in Epstein‐Barr virus (EBV)‐associated gastric carcinoma (EBVaGC). To investigate the significance of the promoter methylation in EBVaGC, we focused on one of the important proteins in the carcinogenesis of the stomach, E‐cadherin. Methylation‐specific PCR analysis (MSP) was applied to surgically resected gastric carcinomas, together with immunohistochemistry, PCR‐based analysis of mutations and allelic loss, and site‐specific MSP of E‐cadherin gene. By MSP, nearly all of the carcinomas showed aberrant methylation of E‐cadherin promoter in EBVaGC (21/22), and the frequency of this aberration was significantly higher than that in EBV‐negative gastric carcinoma (GC; 45/81; p = 0.0003). According to immunohistochemistry of E‐cadherin, the frequency of abnormal staining pattern in EBVaGC (87%) was comparable to that in the diffuse type (80%), but higher than that in the intestinal type of EBV‐negative GC (47%). Promoter methylation was well correlated with abnormal staining pattern in EBVaGC, but not in EBV‐negative GC. Neither mutation nor allelic loss of E‐cadherin was observed in EBVaGC. Methylation status of E‐cadherin within each carcinoma was heterogeneous as far as examined. Thus, in addition to the known association involving p16, we determined that promoter methylation‐mediated silencing of E‐cadherin gene was also closely associated with the development of EBVaGC, although it becomes heterogeneous within a given tumor along its progression.

Global and non-random CpG-island methylation in gastric carcinoma associated with Epstein-Barr virus

DNA hypermethylation may play a primary role in the genesis of Epstein‐Barr virus (EBV)‐associated gastric carcinoma (GC) (EB‐VaGC). Methylation‐specific PCR targeting CpG‐islands demonstrated markedly increased methylation of specific genes, such as p14, p15 and p16 genes, in EBVaGC in vivo. A high frequency of methylation was observed in an EBVaGC strain of severe combined immunodeficiency mice, and the expression of methylated genes in the strain was apparently lower than the expression of the unmethylated genes in EBV‐negative GC strains. Although over‐expression of DNA methyltransferases (DNMTs) is known to be associated with some human cancers, real‐time PCR demonstrated that DNMTs expression was suppressed in EBVaGC. The DNA methylation of specific genes, independently of DNMTs expression, may be important in the development of EBVaGC. (Cancer Sci 2003; 94: 76–80)

High-density methylation of p14ARF and p16INK4A in Epstein-Barr virus-associated gastric carcinoma.

Promoter hypermethylation of various tumor‐related genes is extremely frequent in gastric carcinoma (GC) associated with Epstein‐Barr virus (EBV). To investigate the significance of the promoter methylation in this type of GC, we examined the methylation densities of the promoter regions of p14ARF and p16INK4A in EBV‐associated (n = 7) and EBV‐negative (n = 14) GC. Bisulfite sequencing demonstrated a high frequency of concurrent methylation of p14ARF and p16INK4A promoter regions in EBVaGC. Methylation was observed in all 29 CpG sites of p14ARF and all 16 sites of p16INK4A with equally high densities. In EBV‐negative GC, the methylation profiles differed between the 2 genes. Promoter methylation was sporadic and variable in p14ARF, and only the last position of CpG in p14ARF was methylated at high frequency. High‐density methylation in p16INK4A was observed in a subset of GC, but the first position of CpG was never methylated in EBV‐negative GC. These findings suggest the presence of mechanisms of de novo and maintenance methylation specific to EBVaGC that might be associated with EBV infection.

Reduced Expression and Promoter Methylation of p16 Gene in Epstein-Barr Virus-associated Gastric Carcinoma

Epstein‐Barr virus (EBV)‐associated gastric carcinoma (EBVaGC) is a unique type of gastric carcinoma (GC), which is considered to develop in a different pathway from EBV‐negative GC. To evaluate a possible role of p16, an inhibitor of G1/S transition of the cell cycle, in the carcinogenesis of EBVaGC, pl6‐immunohistochemistry and methylation‐specific PCR analysis (MSP) were applied to surgically resected gastric carcinomas. When the percentage of p16‐positive cells in more than 1000 carcinoma cells was expressed as p16 labeling index (p16‐LI), it ranged from 2.5 to 88.1 (mean 42.9±24.4) in 70 gastric carcinomas. EBVaGC showed significantly lower values (n=15, 26.1±22.1) than EBV‐negative GC (n=55, 47.5±23.2) (P=0.0036). Fresh frozen tissues of 55 gastric carcinomas (16 EBVaGC and 39 EBV‐negative GC) were further subjected to MSP, to evaluate abnormal methylation of the promoter region of the p16 gene. The frequency of methylation was significantly higher in EBVaGC (14/16) than in EBV‐negative GC (9/39) (<0.0001). The methylation‐positive carcinomas showed significantly lower p16‐LI (35.9±21.6) than the unmethylated ones (55.2±22.7) (P=0.0014). Thus, a marked decrease of p16 expression, caused by the aberrant methylation of the p16 gene promoter, is closely associated with the development of EBVaGC.

Interleukin-1β Expression in Human Gastric Carcinoma with Epstein-Barr Virus Infection

ABSTRACT The KT tumor is a transplantable strain of a human Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), established in severe combined immunodeficiency (SCID) mice, with which the cytokine expression of EBVaGC can be investigated without interference from the infiltrating lymphocytes. As a part of a high-density oligonucleotide array (GeneChip) analysis of EBVaGC, the interleukin-1β (IL-1β) gene was the only cytokine gene that showed markedly higher expression in the KT tumor cells than in two tumor strains of EBV-negative GC. The results were confirmed by Northern blotting, Western blotting, and enzyme-linked immunosorbent assay. Furthermore, we demonstrated a positive signal for IL-1β mRNA in the carcinoma cells of a surgically resected EBVaGC, but not in EBV-negative GC, by in situ hybridization. In vitro, IL-1β increased the cell growth of a GC cell line, TMK1. Thus, IL-1β may act as an autocrine growth factor in EBVaGC.

Cancer risk to the gastric corpus in Japanese, its correlation with interleukin-1β gene polymorphism (+3953*T) and Epstein-Barr virus infection

Polymorphisms of interleukin‐1 (IL‐1) genes have been reported to modify the risk of gastric carcinoma (GC) in Caucasians. The significance of IL‐1β gene polymorphisms was evaluated in Japanese GC patients with or without infection of Helicobacter pylori and Epstein Barr virus (EBV) with special reference to the topographic features of GC. IL‐1β gene polymorphisms at positions ‐511 and +3953 were evaluated by PCR‐RFLP and a penta‐allelic polymorphism of IL‐1RA by PCR in healthy controls (n = 103) and GC (n =140; corpus 95, antrum 45). EBV‐infection was determined in the neoplastic tissues by EBER1 in situ hybridization, and H. pylori infection in nonneoplastic gastric mucosa by PCR targeting of the H. pylori urease A gene. GC consisted of EBV‐associated (n = 24) and EBV‐negative (n = 116) patients, whereas H. pylori infection was positive in 130 cases. Among IL‐1β gene polymorphisms, genotype IL‐1β+3953 C/T was more frequent in the EBV‐negative (21%) and corpus GC (23%) patients, compared to the controls (10%), respectively, although there was no genotype IL‐1β+3953 T/T in either group. Thus, the effect of IL‐1β+3953 T was statistically significant in logistic regression models adjusted for age in EBV negativity (odds ratio [OR] 2.27, 95% confidence interval [CI] 1.02–5.05) and in the corpus GC (2.70, 1.19–6.12) with highest OR 3.55 (1.54–8.23) in EBV‐negative corpus GC. There was no significant influence of IL‐1 gene polymorphism in EBV‐associated GC, but it occurred predominantly in the corpus (24/24) compared to EBV‐negative GC (71/116) (p = 0.00002). There was no correlation between H. pylori infection and IL‐1 gene polymorphism in GC. The cancer risk of the gastric corpus in Japanese is influenced by IL‐1β+3953 polymorphisms. On the other hand, the risk of EBV‐associated GC, which occurs predominantly in the corpus, is not influenced by this pro‐inflammatory polymorphism.

Clinicopathological study of lymph‐node metastasis in 1389 patients with early gastric cancer: Assessment of indications for endoscopic resection

BACKGROUND:  The endoscopic resection of early gastric cancers (EGC) is a standard technique in Japan and is increasingly used throughout the world. Further experience in the treatment of EGC and a clearer delineation of the factors related to lymph‐node metastasis would permit a more accurate assessment of endoscopic resection.

Preoperative chemotherapy for advanced esophageal cancer and relation with histological effect

The results of surgical treatment for advanced esophageal cancer remain extremely poor. Irradiation and chemotherapy are not superior to surgery. Perioperative morbidity and the influence on long-term survival of a combination of surgery and preoperative chemotherapy were investigated in patients with advanced esophageal cancer. Forty-nine patients with advanced esophageal squamous cell carcinoma were subjected to preoperative chemotherapy of cisplatin-5-fluorouracil. Fifty-seven patients were chosen as a historical control group who had not undergone chemotherapy before surgery but had the same histological stages as the chemotherapy group. The response to chemotherapy was assessed by histological studies of surgical specimens. The survival rates noted no significant difference between preoperative chemotherapy plus surgery and a resection alone. However, subclassification according to the grading of chemotherapeutic effectiveness showed that, compared with control, preoperative chemotherapy was beneficial to high responders (P=0.01), ineffective in low responders (P=0.61), and detrimental to nonresponders (P=0.03). Postoperative morbidity was significantly higher in the chemotherapy group than in the control group (P=0.02). These findings suggest that preoperative chemotherapy is necessary only for high responders and we therefore need to reliably identify non-, low, and high responders before chemotherapy to improve the survival and quality of life of patients with advanced esophageal cancer.