Ja-Mun Chong

Microsatellite instability in the progression of gastric carcinoma

Seventy-six gastric carcinomas were analyzed with regard to whether or how microsatellite instability was associated with the development of the carcinoma. Microsatellite instability occurred as a late genetic alteration, with an incidence significantly higher in the advanced stage (17 of 51) than in the early stage (3 of 25; P < 0.05). Chromosomal losses on 5q and 17p, detected by polymerase chain reaction-restriction fragment length polymorphism, more frequently accompanied microsatellite instability (9 of 15 and 8 of 11, respectively), compared with carcinomas which lacked instability (5 of 28 and 9 of 30, respectively; P < 0.01 and P < 0.05, respectively). Epstein-Barr virus was observed in only 8 of 76 carcinomas, none of which was associated with microsatellite instability. No significant correlation was found between instability and the familial tendency to develop gastric carcinomas. Our results suggest that microsatellite instability might play a role in the progression of gastric carcinomas but not in Epstein-Barr virus-associated gastric carcinomas.

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.

C-kit Gene Abnormalities in Gastrointestinal Stromal Tumors (Tumors of Interstitial Cells of Cajal)

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the GI tract, and expresses KIT and CD34 in most cases. Gain‐of‐function mutation of the c‐kit proto‐oncogene has been described, but its significance in GIST has not yet been fully evaluated. Mutation in exon 11 of the c‐kit gene was determined by both polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) analysis and direct sequencing in primary and metastatic GISTs and esophageal leiomyomas in Japanese subjects. C‐kit gene mutation was identified in 15 of 48 primary GISTs (31%), four of seven metastatic GISTs, but none of the leiomyomas. Three mutations were mis‐sense point mutations, and 16 were in‐frame deletions of 3–48 bp. C‐kit gene mutation was observed equally in low‐ and high‐risk groups, and was not related to any clinical and pathologic factors, phenotypes or Ki‐67 labeling index (LI) of tumor cells. In five of 15 deletion mutations (four in primary tumors and one in a metastatic tumor), the mutations were present at the distal location of exon 11 of the c‐kit gene, which was a minor mutation in previous reports from Finland and the USA. C‐kit gene mutations in GIST are not always related to a poor prognosis, but further comparative studies are necessary in Western and Japanese populations.

Embryonic form of smooth muscle myosin heavy chain (SMemb/MHC-B) in gastrointestinal stromal tumor and interstitial cells of Cajal.

Myosin heavy chain (MHC) isoform expression was evaluated by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) to clarify a possible link between gastrointestinal stromal tumor (GIST) and interstitial cells of Cajal (ICCs) in the gastrointestinal (GI) tract. Using monoclonal antibodies against MHC isoforms, 18 of 27 GISTs (67%) showed immunoreactivity for non-smooth-muscle myosin or the embryonic form of MHC (SMemb), but only one tumor showed immunoreactivity for smooth muscle cell (SMC)-specific isoforms (SM1 and SM2). Co-expression of KIT or CD34, which is also expressed in GIST and ICCs, was demonstrated in 18 (100%) and 16 SMemb-positive tumors (89%), respectively. Otherwise, the expression of SMemb in GIST was not correlated with the patients age or sex, tumor size, histological grade of GIST, or expression of mesenchymal cell markers, such as alpha-smooth muscle actin (alpha-SMA) or S100 protein. By double-fluorescence immunostaining of the tunica muscularis of the GI tract wall, co-expression of KIT, CD34, and SMemb was demonstrated in ICCs, which were negative for SM1 and SM2. RT-PCR analysis confirmed that GIST expressed SMemb-mRNA, which lacked neuronal cell-specific inserts of 30 bp. These facts further strengthen the current hypothesis that GIST is a tumor of ICCs.

Drastic genetic instability of tumors and normal tissues in Turcot syndrome

Turcot syndrome is characterized by an association of malignant brain tumors and colon cancer developing in the patients teens. Since the mechanism of carcinogenesis in Turcot syndrome is still unclear, we analysed genetic changes in tumors from a Turcot patient with no family history of the condition. All tumors, including one astrocytoma, three colon carcinomas, and two colon adenomas, exhibited severe replication error (RER), and all colon tumors showed somatic muations at repeated regions of TGFβRII, E2F-4, hMSH3, and/or hMSH6 genes. Somatic APC mutations were detected in three of three colon carcinomas, and somatic p53 mutations were detected in the astrocytoma and two of three colon carcinomas, both of which showed two mutations without allele loss. We also found that normal colon mucosa, normal skin fibroblasts and normal brain tissue from this patient showed respective high frequencies of RER, in contrast to usual HNPCC patients in which RER was very rare in normal tissues. These results suggest that extreme DNA instability in normal tissues causes the early development of multiple cancer in Turcot syndrome. A missense mutation (GAG to AAG) at codon 705 of hPMS2 gene was detected in one allele of this patient, which was inherited from his mother without tumors. Additional unknown germline mutation may contribute to the genetic instability in normal tissues.

Allelic Loss of 14q and 22q, NF2 Mutation, and Genetic Instability Occur Independently of c-kit Mutation in Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. Since c‐kit mutation occurs only in one‐third of GIST, there might be other molecular mechanisms. Loss of heterozygosity (LOH), microsatellite instability (MSI) and NF2 gene mutation were investigated in 22 GISTs (9 low‐risk and 13 high‐risk tumors). LOH and MSI were evaluated using 41 markers on 21 chromosomal arms, and NF2 gene mutation was examined by PCR‐SSCP. High frequency of LOH was observed on 14q (9/19, 47%), and 22q (17/22, 77%). The frequencies were similar in low‐risk and high‐risk tumors, and were unrelated with gastric or intestinal origin. Two other abnormalities, additional LOH on other chromosomes and MSI at more than two loci, were characteristic of the high‐risk tumors (P < 0.05). NF2 gene mutation was identified in two cases showing 22q‐LOH (8 bp deletion on the splice donor site of exon 7, and 1 bp insertion at position 432 of exon 4, which resulted in nonsense mutation). There was no significant correlation between these results and c‐kit gene mutation, which was observed in 8 of 22 tumors. Suppressor genes on 14q and 22q may be involved, independently of c‐kit gene mutation, in the development of GIST. NF2 contributes as a tumor suppressor in a small subset of GIST. These abnormalities are presumably followed by increased genetic instability.

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.