Takayoshi Waki

Age-related methylation of tumor suppressor and tumor-related genes: an analysis of autopsy samples.

Age-related methylation may have the potential to behave as a mutator process. To clarify the physiological consequence of age-related methylation of tumor suppressor and tumor-related genes, we studied promoter methylation status in non-neoplastic cells of various organs obtained at autopsy by methylation-specific PCR. Promoter methylation status of APC, DAP-kinase, E-cadherin, GSTP1, hMLH1, p16, RASSF1A and RUNX3 genes, which are frequently silenced in certain human malignancies, was studied in non-neoplastic cells of the esophagus, stomach, small and large intestines, liver, pancreas, kidney and lung obtained from 38 Japanese autopsies. The tumor suppressor and tumor-related genes, except APC and RASSF1A, were generally unmethylated in samples obtained from people who were less than 32 years old (n=11). Methylated promoters were present at variable frequencies in a tissue-specific manner in samples obtained from people who were greater than 42 years old (n=27), although GSTP1 and hMLH1 methylation was absent or infrequent and lacked tissue specificity. In the majority of organs, the incidence of age-related methylation paralleled the reported methylation incidence in malignant counterparts. Thus, age-related methylation of a different set of genes is thought to constitute a field defect in different organs.

Promoter Methylation Status of E-Cadherin, hMLH1, and p16 Genes in Nonneoplastic Gastric Epithelia

Silencing of tumor suppressor and tumor-related genes by hypermethylation at promoter CpG islands is one of the major events in human tumorigenesis. Promoter methylation is also present in nonneoplastic cells as an age-related tissue-specific phenomenon that precedes the development of neoplasia. To clarify the significance of promoter methylation in nonneoplastic gastric epithelia as a precancerous signal, we investigated promoter methylation status of E-cadherin, hMLH1, and p16 genes in nonneoplastic cells of various organs obtained at autopsy, and compared the results with those of nonneoplastic epithelia of a cancerous stomach. Methylation of these genes was not seen in nonneoplastic cells of organs from people who were 22 years and younger (0%, 0 of 6). In contrast, E-cadherin and p16 were methylated in nonneoplastic gastric epithelia of persons who were 45 years or older. The numbers were 86% (12 of 14) and 29% (4 of 14), respectively. E-cadherin methylation occurred preferentially in the intestines, whereas p16 methylation was almost restricted to the stomach. For samples obtained from patients with stomach cancer, methylation was frequently observed in both neoplastic and corresponding nonneoplastic gastric epithelia: 47% (44 of 94) and 67% (63 of 94) for E-cadherin, 32% (30 of 94) and 24% (23 of 94) for hMLH1, and 22% (21 of 94) and 44% (41 of 94) for p16, respectively. hMLH1 methylation was not seen in nonneoplastic gastric epithelia from autopsy samples but occurred significantly in samples from nonneoplastic tissues of individuals with stomach cancer. Therefore, detection of hMLH1 methylation in nonneoplastic gastric epithelia may be useful for screening patients who may be at risk of developing gastric cancer.

Multiple tumor suppressor genes are increasingly methylated with age in non-neoplastic gastric epithelia

A number of tumor suppressor and tumor‐related genes are silenced by promoter hypermethylation in gastric cancer. Hypermethylation is not restricted to cancer cells, but is also present in non‐neoplastic cells during aging. Such age‐related methylation in non‐neoplastic gastric epithelia is postulated to constitute a field defect that increases the risk for development of gastric cancer. To quantitatively evaluate age‐related methylation in non‐neoplastic gastric epithelia, we used a fiber‐type DNA microarray on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the promoter CpG island of four tumor suppressor genes, lysyl oxidase (LOX), p16, RUNX3 and tazarotene‐induced gene 1 (TIG1), were amplified by PCR using Cy5 end labeled primers. Methylation rates (MRs) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Non‐neoplastic gastric mucosa was obtained from 24 non‐cancer‐bearing stomachs at autopsy. MRs ranged from 0.0% to 77.2% (mean, 15.8%) for LOX, 0.0% to 45.8% (mean, 10.0%) for p16, 0.0% to 83.8% (mean, 9.0%) for RUNX3, and 0.0% to 46.1% (mean, 6.6%) for TIG1, and significantly correlated with aging (P < 0.01). The regression curves were: y = 0.013x2 − 0.6184x + 4.0512, R2 = 0.5728 (P < 0.001) for LOX; y = 0.0107x2 − 0.6055x + 5.2943, R2 = 0.7891 (P < 0.00001) for p16; y = 0.0182x2 − 1.2234x + 11.566, R2 = 0.5595 (P < 0.001) for RUNX3; and y = 0.0068x2 − 0.3586x + 2.4306, R2 = 0.4670 (P < 0.01) for TIG1. Thus, our present results are consistent with the notion that age‐related methylation is associated with cancer susceptibility in the elderly. Quantitative analysis of DNA methylation using DNA microarrays is a promising method for risk assessment in the development of gastric cancer. (Cancer Sci 2006; 97: 1155–1158)

Promoter methylation status of DAP-kinase and RUNX3 genes in neoplastic and non-neoplastic gastric epithelia.

Silencing of tumor suppressor and tumor‐related genes by hyper‐methylation at promoter CpG islands is frequently found in human tumors, including gastric cancer. Promoter methylation is not restricted to cancer cells, and is also present in non‐neoplastic cells as an age‐related tissue‐specific phenomenon. To clarify the physiological consequence of DAP‐kinase and RUNX3 age‐related methylation in gastric epithelia, we investigated the promoter methylation status of these genes in both neoplastic and non‐neoplastic gastric epithelia obtained at autopsy and surgery, as well as in 10 gastric cancer cell lines. Methylation of DAP‐kinase and RUNX3 was detected in 10% (1/10) and 70% (7/10) of the cell lines, respectively, and was almost concordant with their expression status. Among autopsy samples, methylation of these genes was not seen in non‐neoplastic gastric epithelia from persons who were aged 22 years and below (0%; 0/4). DAP‐kinase was methylated in 87% (13/15) of non‐neoplastic gastric epithelia of persons who were aged 45 years or older, while RUNX3 methylation in non‐neoplastic gastric epithelia was restricted to individuals who were aged 77 years or older. Among samples obtained from patients with stomach cancer, methylation was observed in both the neoplastic and the corresponding non‐neoplastic gastric epithelia; 43% (40/93) and 73% (68/93) for DAP‐kinase, and 45% (42/93) and 8% (7/93) for RUNX3, respectively. Frequencies of DAP‐kinase and RUNX3 methylation differed significantly in non‐neoplastic gastric epithelia (P<0.01), although those in gastric cancers were almost the same. RUNX3 methylation is mostly cancer‐specific, except for very old individuals, and therefore may be a possible molecular diagnostic marker and malignancy predictor. (Cancer Sci 2003; 94: 360–364)

Hypermethylation of the TSLC1 Gene Promoter in Primary Gastric Cancers and Gastric Cancer Cell Lines

The TSLC1 (tumor suppressor in lung cancer–1) gene is a novel tumor suppressor gene on chromosomal region 11q23.2, and is frequently inactivated by concordant promoter hypermethylation and loss of heterozygosity (LOH) in non‐small cell lung cancer (NSCLC). Because LOH on 11q has also been observed frequently in other human neoplasms including gastric cancer, we investigated the promoter methylation status of TSLC1 in 10 gastric cancer cell lines and 97 primary gastric cancers, as well as the corresponding non‐cancerous gastric tissues, by bisulfite‐SSCP analysis followed by direct sequencing. Allelic status of the TSLC1 gene was also investigated in these cell lines and primary gastric cancers. The TSLC1 promoter was methylated in two gastric cancer cell lines, KATO‐III and ECC10, and in 15 out of 97 (16%) primary gastric cancers. It was not methylated in non‐cancerous gastric tissues, suggesting that this hypermethylation is a cancer‐specific alteration. KATO‐III and ECC10 cells retained two alleles of TSLC1, both of which showed hypermethylation, associated with complete loss of gene expression. Most of the primary gastric cancers with promoter methylation also retained heterozygosity at the TSLC1 locus on 11q23.2. These data indicate that bi‐allelic hypermethylation of the TSLC1 promoter and resulting gene silencing occur in a subset of primary gastric cancers.

Aortic aneurysm rupture as a rare complication of granulomatosis with polyangiitis: a case report

IntroductionGranulomatosis with polyangiitis is characterized by systemic inflammation of medium and small blood vessels. Aortic involvement in granulomatosis with polyangiitis is extremely rare. As far as we know this is the first reported case of successful treatment in a patient with granulomatosis with polyangiitis complicated with aortic aneurysm rupture.Case presentationWe describe a case of granulomatosis with polyangiitis in a 38-year-old Japanese man who developed an aortic aneurysm rupture 22 years after disease onset. The patient was operated on and a J-graft was inserted. He recovered uneventfully.ConclusionRecommendations in regard to, and consideration of, aortic involvement should be kept in mind in the long-term careful follow up of granulomatosis with polyangiitis.

Exploring the Limits of the Endoscopic Approach to Frontal Sinus Osteoma

Objective: We present the case of a frontal sinus osteoma in a 71-year-old Japanese woman who presented with an approximately 2-year history of head dullness. Case report: CT showed a 45×39 mm radio dense mass extending from the left to the right frontal sinus. Under general anesthesia the mass was removed by endoscopic surgery. Although the tumor was large and extended into both frontal sinuses, the tumor was completely removed, without CSF leakage or orbital complications, by using a modified Lothrop procedure. Conclusion: A unique osteoma of the frontal sinus is described. The challenges related to surgical treatment of this particular case and similar lesions are addressed.