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한국인 위 선종-암 조직에서 메틸화 생물학적 표지자의 발견과 특성 (Discovery and Characteristics of Early Methylation Biomarker in Korean Patients with Gastric Neoplasia)
Researcher who has been awarded a research grant by Humanities and Social Studies Support Program of NRF has to submit an end product within 6 months(* depend on the form of business)
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  • Researchers have entered the information directly to the NRF of Korea research support system
Project Number E00095
Year(selected) 2006 Year
the present condition of Project 종료
State of proposition 재단승인
Completion Date 2008년 02월 28일
Year type 결과보고
Year(final report) 2008년
Research Summary
  • Korean
  • 배경 및 목적: DNA메칠화 이상은 위의 암화 과정 초기에 흔히 발생하므로 위 종양의 초기 단계에서 진단과 선별검사에 유용할 수 있다. 방법: 저자 등은 7종의 위암 세포주와 24명의 위 조직(시험군; 11명 정상 위점막과 13명의 위암)에서 정량적인 pyrosequencing방법을 이용하여 44개의 후보 유전자 중에서 메칠화가 잘 발생하는 CpG islands를 동정하였다. 또한 22명의 정상 위점막, 64명의 수술절제 위암 주변 정상 위점막, 그리고 128명의 단계별 위 종양(40명의 위 이형성, 43명의 조기 위암, 35명의 진행성 위암)으로 구성된 검증군 조직에서 위 과정을 통해 선택된 위암특이 고메칠화 유전자와 유전자 전반의 저메칠화 지수로 이용되는 LINE-1의 메칠화 정도를 측정하였다. 결과: 시험군에서 7종의 유전자(ADAM23, DOK5, FOXN4, GDNF, KLF7, MLF1, RORA, SPOCK2)가 위암에서 정상에 비하여 고메칠화를 나타내었다. 검증군에서 glial cell-derived neurotrophic factor (GDNF)와 RAR-related orphan receptor A isofrom a (RORAa) 유전자 메칠화 여부는 위 이형성과 조기 위암을 포함하는 조기 위종양 진단에 있어 각각 민감도가 81%와 88%, 특이도가 95%와 100%로 가장 유용한 지표로 나타났다. 또한 위의 두 유전자를 병합한 경우 진단 특이도는 95%로 유지되면서 조기 위 종양 과 진행성 위암을 포함하는 전체 위 종양의 진단 민감도는 각각 98%와 91%로 증가하였다. 저메칠화 지표인 LINE-1의 메칠화 정도는 위암화 진행도에 따라 감소하였으나 (P < 0.001) 조기 단계에서는 뚜렷하지 않았다. 진행성 위암에서 LINE-1의 저메칠화는 궤양성 육안형태 (P = 0.014), intestinal-type (P < 0.001)과 분화암(P = 0.11)과 관련이 있었다. 결론: 메칠화 마커로서 GDNF와 RORAa 유전자 프로모터의 메칠화 여부는 위암의 선별검사로서 초기 단계 종양을 진단하는 데 유용하다.
  • English
  • Background & Aims: Aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for screening and detection of early stage gastric neoplasia (ESGN). Methods: We used a two-step selection process with 7 gastric cancer cell lines and 24 gastric tissue samples (test set; 11 nonneoplastic gastric mucosa samples and 13 gastric cancer samples) to identify preferentially methylated CpG islands among 44 candidate genes using a quantitative pyrosequencing assay. We further examined the methylation status of selected genes and LINE-1 as a global hypomethylation index in a validation set consisting of 128 gastric neoplasias (40 gastric dysplasias, 43 early gastric cancers, and 35 advanced gastric cancer samples), 64 normal gastric mucosa samples adjacent to surgically resected gastric cancers, and 22 nonneoplastic gastric mucosa samples. Results: Seven genes (ADAM23, DOK5, FOXN4, GDNF, KLF7, MLF1, RORA, and SPOCK2) showed frequent differential methylation in the test set. In the validation set, GDNF and RORAa had the highest yield in detection of ESGN (sensitivity, 81% and 88%, respectively; specificity, 95% and 100%, respectively). Also, combining the markers GDNF and RORAa significantly improved the assay sensitivity (98% in ESGN samples and 91% in all gastric neoplasia samples) while maintaining 95% specificity. We also found that LINE-1 methylation levels decreased with gastric neoplastic progression (P < .001), however this global DNA hypomethylation was not apparent in ESGN samples. Instead, in advanced gastric cancer samples, LINE-1 methylation status was related to an ulcerative morphology (P = .014), intestinal-type cancer (P < .001), and differentiated carcinoma (P = 0.11). Conclusions: These findings show that the complementary use of GDNF and RORAa as methylation markers is a sensitive and specific approach for screening in ESGN.
Research result report
  • Abstract
  • Background & Aims: Aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for screening and detection of early stage gastric neoplasia (ESGN). Methods: We used a two-step selection process with 7 gastric cancer cell lines and 24 gastric tissue samples (test set; 11 nonneoplastic gastric mucosa samples and 13 gastric cancer samples) to identify preferentially methylated CpG islands among 44 candidate genes using a quantitative pyrosequencing assay. We further examined the methylation status of selected genes and LINE-1 as a global hypomethylation index in a validation set consisting of 128 gastric neoplasias (40 gastric dysplasias, 43 early gastric cancers, and 35 advanced gastric cancer samples), 64 normal gastric mucosa samples adjacent to surgically resected gastric cancers, and 22 nonneoplastic gastric mucosa samples. Results: Seven genes (ADAM23, DOK5, FOXN4, GDNF, KLF7, MLF1, RORA, and SPOCK2) showed frequent differential methylation in the test set. In the validation set, GDNF and RORAa had the highest yield in detection of ESGN (sensitivity, 81% and 88%, respectively; specificity, 95% and 100%, respectively). Also, combining the markers GDNF and RORAa significantly improved the assay sensitivity (98% in ESGN samples and 91% in all gastric neoplasia samples) while maintaining 95% specificity. We also found that LINE-1 methylation levels decreased with gastric neoplastic progression (P < .001), however this global DNA hypomethylation was not apparent in ESGN samples. Instead, in advanced gastric cancer samples, LINE-1 methylation status was related to an ulcerative morphology (P = .014), intestinal-type cancer (P < .001), and differentiated carcinoma (P = 0.11). Conclusions: These findings show that the complementary use of GDNF and RORAa as methylation markers is a sensitive and specific approach for screening in ESGN.
  • Research result and Utilization method
  • We identified frequently methylated genes in ESGN and we suggest they could be used to screen for gastric cancer at early stages. The methylation markers generally displayed three distinct patterns during multistage gastric tumorigenesis: type 1, consistently high methylation throughout tumor progression; type 2, high methylation in early stages but low in advanced cancer; and type 3, low methylation in early-stage neoplasia but high methylation in advanced cancer. Also, the distribution of methylation levels of these genes according to tumor progression was gradual with no evidence of bimodality or drastic discontinuity. Therefore, methylation changes of these markers during gastric carcinogenesis is gene- and tumor stage-dependent and that they can serve as useful biomarkers for the different stages of gastric neoplastic progression. In particular, type 1 genes could be useful for screening while type 3 genes should be examined for prognostic value. Of the genes we studied, GDNF was the most stable biomarker, as it was methylated in more than 70% of gastric tumor patients irrespective of tumor stage. Among type 2 markers, RORAa was the most frequently methylated in ESGN. Based on this observation, we developed a combined panel of type 1 and type 2 markers because we expected this combination to cover all stages of gastric neoplastic progression. As a result, use of a panel consisting of GDNF and RORAa differentiated ESGN from NGM with a sensitivity and specificity of 98% and 95%, respectively. Also, use of this panel increased the detection sensitivity for all stages of gastric neoplasia from 78% to 91%. These two genes could, therefore, be useful to molecularly detect ESGN in biopsies, gastric washing fluids or possibly serum studies. Of interest, we found that gastric precancerous lesions and EGCs showed higher methylation levels of type 2 marker genes than did AGCs. This was consistent with previous studies showing the early occurrence of DNA hypermethylation in a subset of genes in gastric precursor lesions and is also consistent with studies in colorectal neoplasia. In contrast, we saw gradual increases in the level and frequency of methylation of type 3 marker genes reaching their peak in AGCs. Another important concern of the study analysis was to examine the role of these markers as an epigenetic field defect marker. Our previous studies demonstrated the involvement of aberrant methylation in an epigenetic field defect for colon cancer37, 38 and gastric cancer.8 In the present study, three (GDNF, RORAa, and ADAM23) out of 5 type 1 and 2 markers showed significant higher methylation in ADJ samples obtained from cancer patients than those in NGM. In particular, the mean level of RORAa methylation in ADJ samples from EGC patients was significantly higher than in those from AGC patients (12.1% vs. 7.2%, P = .032), indicating a potential field defect marker for gastric cancer risk prediction. Besides local promoter CGI hypermethylation, genomic global hypomethylation may promote cancer development through genome instability and proto-oncogene hypomethylation. However, researchers have yet to explore in detail the role of DNA hypomethylation in gastric tumorigenesis and the relationships between global hypomethylation and gastric cancer phenotypes. Of interest, LINE-1 methylation was significantly lower in AGC samples showing well to moderate differentiation or intestinal-type Lauren classification. However, this association was not clear in EGC samples, suggesting the probable presence of a distinct cancer subtype, marked by the demethylation phenotype in AGC. In summary, we identified seven methylation markers for the early detection of gastric neoplasia. Of these, combined methylation analysis using GDNF and RORA was highly sensitive and specific, suggesting that this analysis is a rational approach for EGC screening that should be tested in future studies.
  • Index terms
  • early gastric neoplasia, DNA methylation, biomarker, Pyrosequencing
  • List of digital content of this reports
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