胃癌和癌前病变组织微卫星不稳定性的研究
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摘要
目的：本研究通过在胃癌和癌前病变中的异型增生、肠化组织中检测微卫星不稳定性(microsatellite instability，MSI)的发生情况及其特点，观察MSI在胃癌发生、发展过程中的变化情况，探索这一早期分子异常事件在胃癌发生中的重要作用，为寻找胃癌早期诊断的分子标志物提供重要线索。
方法：收集100例石蜡标本，其中胃癌30例、不伴癌的异型增生和肠化分别为30例、40例，提取病变组织和相应正常组织的DNA，采用1997年美国国立癌症研究院(NCI)推荐的5个微卫星位点(Bat-25、Bat-26、D5S346、D17S250和D5S123)，应用银染PCR-SSCP技术来检测5个位点的不稳定性。根据实验结果将肿瘤分为三类：第一类：高频微卫星不稳定(high frequency MSI，MSI-H)，即突变的微卫星位点数目≥30％～40％；第二类：低频微卫星不稳定(low frequency MSI，MSI-L)：突变的微卫星位点数目低于30％；第三类：微卫星稳定(microsatellite stable，MSS)：无突变的微卫星位点。
结果：胃癌组织MSI的发生率为23.3％，其中3例表现MSI-H；4例表现MSI-L；其余23例为MSS。MSI现象在BAT-26位点的发生率最高，为13.3％，但各位点之间并无统计学差异性。MSI胃癌与MSS胃癌在性别、年龄、细胞分化程度、淋巴结转移及TNM分期方面无统计学差异，但胃窦癌MSI的发生率显著高于贲门癌(P=0.044)。异型
南京医科大学硕士学位论文
增生组织的MSI发生率为30o，其中7例为MSI－L，2例为MSI－H。2
级以上异型增生组织的 MSI阳性率高于 l级异型增生门．3OVS 25O），
但统计学分析无显著性意义。MSI与MSS的异型增生在性别、年龄方
面的差异亦无显著性。肠化组织 MSI发生率 20o，除 1例 MSI－H外，
其余7例均表现MSI工。男性的MSI发生率5o门a0），女性的MSI
发生率35o门o 0，性别差异性显著（P＝0．044）。17例轻度肠化均表
现为MSS，中度肠化生的MSI阳性率达3牛8％，两组之间MS互发生率
差异性显著（P刃．013）。
结论：MSI可能是胃癌发生、发展过程中的一种独特的分子机制，
与某种临床病理特征相关；MSI在慢性胃炎驹上的胃癌癌前病变组
织中的存在，提示MSI可能是胃癌的多步骤发生过程中的早期而重要
的分子事件；对表现为MSI的胃癌癌前病变患者加强随访观察，将可
能有助于早期胃癌诊断率的提高。
Background: Gastric cancer(GC) is one of the most common malignant neoplasms among the world. Genetic alterations have been show to play roles in gastric carcinogenesis including abnormalities in proto-oncogenes, tumor suppressor genes, cell cycle regulator genes, tissue invasion-related genes, and mismatch repair genes. lintestinal metaplasia (IM) and dysplasia are considered precursor lesion of GC, but the genetic mechanism of early gastric carcinogenesis are not well known
Objective: To observe the changeable patterns of microsatellite instability (MSI) in GC and precancerous lesions, including IM and dysplasia. And to defermine the potential significance of MSI in the multistep gastric carcinogenesis pathway.
Methods: We investigated MSI status in 30 flat dysplasias and 40 IMs without GC, and 30 GCs. Silver staining single strand conformation polymorphis polymerize chain reaction (PCR-SSCP) was used to screen 5 MSI markers , recommended by the National Cancer Institute, in formalin-fixed, paraffin-embeded tissues and corresponding normal gastric tissues. In this study, a high incidence of MSI (MSI-H) was defined as samples
containing 30% or more MSI positive loci, a low incidence of MSI (MSI-L) as samples which had less than 30% loci instability, and MSS when samples displaying no alterations in 5 loci.
Results: The abnormal shifting of the single-strand DNA was identified in 7 ( 23.3% ) out of GC, in 9 ( 30% ) out of dysplasia and in 8 ( 20% ) out of IM samples. Three ( 10% ) tumors, two ( 6.7% ) dysplasia and one ( 2.5% ) IM displayed MSI-H. MSI-L was detected in 13.3% of the tumors, in 23.3% dysplasia and in 17.5% IM samples. GC with MSI was associated with distal location of the tumors ( P=0.044 ), but no association was defined between MSI and age, gender, differentiation, lymph node metastasis and PTNM stage. Although MSI showed a trend for high-grade dysplasia, the differences were not significant. MSI was more likely observed in female with IM ( P=0.044 ) and in moderate-grade IM tissues ( 34.8% versus 0; P=0.013 ).
Conclusions: The frequency of MSI in detected GC and precancerous lesions are almost the same when compared with previously reported data. MSI occurs not only in gastric adenomas and flat dysplasia coexisting with GC, but also in dysplasia without carcinoma. The progressive accumulation of MSI in areas of dysplasia and IM may contribute to GC development, suggesting that genetic instability may be an early somatic event of multistep gastric carcinogenesis.
方法：收集100例石蜡标本，其中胃癌30例、不伴癌的异型增生和肠化分别为30例、40例，提取病变组织和相应正常组织的DNA，采用1997年美国国立癌症研究院(NCI)推荐的5个微卫星位点(Bat-25、Bat-26、D5S346、D17S250和D5S123)，应用银染PCR-SSCP技术来检测5个位点的不稳定性。根据实验结果将肿瘤分为三类：第一类：高频微卫星不稳定(high frequency MSI，MSI-H)，即突变的微卫星位点数目≥30％～40％；第二类：低频微卫星不稳定(low frequency MSI，MSI-L)：突变的微卫星位点数目低于30％；第三类：微卫星稳定(microsatellite stable，MSS)：无突变的微卫星位点。
结果：胃癌组织MSI的发生率为23.3％，其中3例表现MSI-H；4例表现MSI-L；其余23例为MSS。MSI现象在BAT-26位点的发生率最高，为13.3％，但各位点之间并无统计学差异性。MSI胃癌与MSS胃癌在性别、年龄、细胞分化程度、淋巴结转移及TNM分期方面无统计学差异，但胃窦癌MSI的发生率显著高于贲门癌(P=0.044)。异型
南京医科大学硕士学位论文
增生组织的MSI发生率为30o，其中7例为MSI－L，2例为MSI－H。2
级以上异型增生组织的 MSI阳性率高于 l级异型增生门．3OVS 25O），
但统计学分析无显著性意义。MSI与MSS的异型增生在性别、年龄方
面的差异亦无显著性。肠化组织 MSI发生率 20o，除 1例 MSI－H外，
其余7例均表现MSI工。男性的MSI发生率5o门a0），女性的MSI
发生率35o门o 0，性别差异性显著（P＝0．044）。17例轻度肠化均表
现为MSS，中度肠化生的MSI阳性率达3牛8％，两组之间MS互发生率
差异性显著（P刃．013）。
结论：MSI可能是胃癌发生、发展过程中的一种独特的分子机制，
与某种临床病理特征相关；MSI在慢性胃炎驹上的胃癌癌前病变组
织中的存在，提示MSI可能是胃癌的多步骤发生过程中的早期而重要
的分子事件；对表现为MSI的胃癌癌前病变患者加强随访观察，将可
能有助于早期胃癌诊断率的提高。
Background: Gastric cancer(GC) is one of the most common malignant neoplasms among the world. Genetic alterations have been show to play roles in gastric carcinogenesis including abnormalities in proto-oncogenes, tumor suppressor genes, cell cycle regulator genes, tissue invasion-related genes, and mismatch repair genes. lintestinal metaplasia (IM) and dysplasia are considered precursor lesion of GC, but the genetic mechanism of early gastric carcinogenesis are not well known
Objective: To observe the changeable patterns of microsatellite instability (MSI) in GC and precancerous lesions, including IM and dysplasia. And to defermine the potential significance of MSI in the multistep gastric carcinogenesis pathway.
Methods: We investigated MSI status in 30 flat dysplasias and 40 IMs without GC, and 30 GCs. Silver staining single strand conformation polymorphis polymerize chain reaction (PCR-SSCP) was used to screen 5 MSI markers , recommended by the National Cancer Institute, in formalin-fixed, paraffin-embeded tissues and corresponding normal gastric tissues. In this study, a high incidence of MSI (MSI-H) was defined as samples
containing 30% or more MSI positive loci, a low incidence of MSI (MSI-L) as samples which had less than 30% loci instability, and MSS when samples displaying no alterations in 5 loci.
Results: The abnormal shifting of the single-strand DNA was identified in 7 ( 23.3% ) out of GC, in 9 ( 30% ) out of dysplasia and in 8 ( 20% ) out of IM samples. Three ( 10% ) tumors, two ( 6.7% ) dysplasia and one ( 2.5% ) IM displayed MSI-H. MSI-L was detected in 13.3% of the tumors, in 23.3% dysplasia and in 17.5% IM samples. GC with MSI was associated with distal location of the tumors ( P=0.044 ), but no association was defined between MSI and age, gender, differentiation, lymph node metastasis and PTNM stage. Although MSI showed a trend for high-grade dysplasia, the differences were not significant. MSI was more likely observed in female with IM ( P=0.044 ) and in moderate-grade IM tissues ( 34.8% versus 0; P=0.013 ).
Conclusions: The frequency of MSI in detected GC and precancerous lesions are almost the same when compared with previously reported data. MSI occurs not only in gastric adenomas and flat dysplasia coexisting with GC, but also in dysplasia without carcinoma. The progressive accumulation of MSI in areas of dysplasia and IM may contribute to GC development, suggesting that genetic instability may be an early somatic event of multistep gastric carcinogenesis.
引文
1. Parkin DM, Pisani P, Ferlay J, et al. Expression of the world wide incidence of major cancers in 1990. Int J Cancer, 1999, 80: 827-841.
2. Craanen ME, Dekker W, Blok P, et al. Time trends in gastric carcinoma: changing patterns of type and location. Am J Gastroenterol, 1992, 87: 572-579.
3. Parsonnet J, Friedman GD, Vandersteen DP, et al. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 1991, 325:1127-1131.
4. Kinzier KW, Vogelstein B, Life (and death) in a malignant tumor. Nature, 1996, 379:19-20.
5. Thibodeau SN, Bren G, Schaid D, Microsatellite instability in cancer of the proximal colon. Science, 1993, 260: 816-819.
6. Perucho M, Microsatellite instability: the mutator that mutates the other mutator. Nat Med, 1996, 2: 630-631.
7. Thibodeau SN, French AJ, Roche PC, et al. Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes. Cancer Res, 1996, 56: 4836-4840.
8. Leach FS, Polyak K, Burrell M, et al. Expression of the human mismatch repair gene hMSH2 in normal and neoplastic tissues. Cancer Res, 1996, 56: 235-240.
9. Aaltonen LA, Peltomaki P, Leach PS, et al. Clues to the pathogenesis of familial colorectal cancer. Sience, 1993,260:812-816.
10. Ionov Y, Peinado MA, Malkhosyan S, et al. Ubiquitous somatic
mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature, 1993, 363: 558-561.
11. Keller G, Rudelius M, Vogelsang H, et al. Microsatellite instability and loss of heterozygosity in gastric carcinoma in comparison to family history. Am J Pathol, 1998, 152: 1281-1289.
12. Ottini L, Palli D, Falchetti M, et al. Microsatellite instability in gastric cancer is associated with tumor location and family history in a high risk population from Tuscany. Cancer Res, 1997, 57: 4523-4529.
13. Semba S, Yokozaki H, Yamamoto S, et al. Microsatellite instability in precancerous lesions and adenocarcinomas of the stomach. Cancer, 1996, 77 (supp18): 1620-1627.
14. Kim JJ, Beak MJ, Kim L, et al. Accumulated frameshift mutations at coding nucleotide repeats during the progression of gastric carcinoma with microsatellite instability. Lab Invest, 1999, 79:1113-1120.
15. Boland CR, Thibodeau SN, Hamilton SR, et al. A national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res, 1998, 58: 5248-5257.
16．吴在德．外科学．第五版，人民卫生出版社2002，500
17. Dietmaier W, Wallinger S, Bocker T, et al. Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression. Cancer Res, 1997, 57: 4749-4756.
18. Leung WK, Kim JJ, Kim JG, et al. Microsatellite instability in gastric intestinal metaplasia in patients with and without gastric cancer.Am J
Pathol, 2000, 156:537-543.
19. Fleisher AS, Esteller M, Tamura G, et al. Hypermethylation of the hMLH1 gene promoter is associated with microsatellite instability in early human gastric neoplasia. Oncogene, 2001, 20:329-335.
20. Halling KC, Harper H, Moskaluk CA, et al. Origin of microsatellite instability in gastric cancer. Am J Pathol, 1999, 155: 205-211.
21．王永信，柯扬，宁涛，等．中国人胃癌组织微卫星DNA的不稳定性研究．中华医学遗传学杂志，1998，15：155-157。
22. Kim HS, Lee BL, Woo DK, et al. Assesment of markers for the identification of microsatellite instability phenotype in gastric neoplasmas. Cancer Lettlers, 2001, 164: 61-68.
23. Sepulveda AR, Santos AC, Yamaoka Y, et al. Marked differences in the frequency of microsatellite instability in gastric cancer from different countries. Am J Gastroentero, 1999, 94: 3035-3038.
24. Thener CP, Campbell BS, Peel DJ, et al. Microsatellite instability in Japanese vs European American patients with gastric cancer. Arch Surg, 2002, 137: 960-966.
25. Shiao YH, Bovo D, Guido M, et al. Microsatellite instability and/or loss of heterogosity in young gastric cancer patients in Italy. Int J Cancer, 1999, 82: 232-239.
26. Wu MS, Lee CW, Chun ct, et al . Distinct clinicopathologic and genetic profiles in sporadic gastric cancer with different mutator phenotypes. Genes Chromosomes Cancer, 2000, 27:403-411.
27. Dos Santos NR, Seruca R, Constancia M, et al. Microsatellite instability at multiple loci in gastric carcinoma: clinicopathologic
implication and prognosis. Gastroenterology, 1996, 110: 38-44.
28. Yamamoto H, Piteira JP, Yoshida T, et al. Gastric cancers of the microsatellite mutator phenotype display characteristic genetic and clinical features. Gastroenterology, 1999, 116: 1348-1357.
29. Wirtz HC, Muller W, Noguchi T, et al. Prognostic value and clinicopathological profile of microsatellite instability in gastric cancer. Clin Cancer Res, 1998, 4: 1749-1754.
30. Chetty R, Naidoo R, Tarin M, et al. Chromosome 2p, 3p, 5q and 18q status in sporadic gastric cancer. Pathology, 2002, 34: 275-281.
31. Kobayashi K, Okamoto T, Takayama M, et al. Genetic instability in intestinal metaplasia is a frequent event leading to well-differentiated early adenocarcinoma of the stomach. Er J Cancer, 2000, 36: 1113-1119.
32．时俊，林庚金，钱立平，等．胃癌及肠化生组织微卫星不稳定性研究．中华消化杂志，2001，21：680-683．
33. Kim JJ, Beak MJ, Kim L, et al. Accumulated frameshift mutations at coding nucleotide repeats during the progression of gastric carcinoma with microsatellite instability. Lab Invest, 1999, 79:1113-1120.
34. Kim HS, Woo DK, Bae SI,, et al. Microsatellite instability in the adenoma-carcinoma sequence of the stomach. Lab Invest, 2000, 80: 57-64.
35. Kim YH, Kim NG, Lim JG, et al. Chromosomal alteration in paired gastric adenomas and carcinomas. Am J Pathol, 2001, 158: 655-662.
36. Lee JH, Abraham SC, Kim HS, et al. Inverse relationship between APC gene mutatio in gastric adenomas and development of adenocarcinoma. Am J Pathol, 2002, 161: 611-618.
37. To KF, Leung WK, Lee TL, et al. Promoter hypermethylation oftumor-related genes in gastric intestinal metaplasia of patients with and without gastric cancer. Int J Cancer, 2002, 102: 623-628.
38. Fleisher AS, Esteller M, Wang S, et al. Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability. Cancer Res, 1999, 59: 1090-1095.
39. Kim HS, Woo DK, Bae SI, et al. Microsatellite instability in the adenoma-carcinoma sequence of the stomach. Lab Invest, 2000, 80: 57-64.
40. Kashiwagi K, Watanabe M, Ezaki T, et al. Clinical usefulness of microsatellite instability for the prediction of gastric adenoma or adenocarcinoma in patients with chronic gastritis. Br J Cancer, 1999, 82:1814-1818.
2. Craanen ME, Dekker W, Blok P, et al. Time trends in gastric carcinoma: changing patterns of type and location. Am J Gastroenterol, 1992, 87: 572-579.
3. Parsonnet J, Friedman GD, Vandersteen DP, et al. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 1991, 325:1127-1131.
4. Kinzier KW, Vogelstein B, Life (and death) in a malignant tumor. Nature, 1996, 379:19-20.
5. Thibodeau SN, Bren G, Schaid D, Microsatellite instability in cancer of the proximal colon. Science, 1993, 260: 816-819.
6. Perucho M, Microsatellite instability: the mutator that mutates the other mutator. Nat Med, 1996, 2: 630-631.
7. Thibodeau SN, French AJ, Roche PC, et al. Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes. Cancer Res, 1996, 56: 4836-4840.
8. Leach FS, Polyak K, Burrell M, et al. Expression of the human mismatch repair gene hMSH2 in normal and neoplastic tissues. Cancer Res, 1996, 56: 235-240.
9. Aaltonen LA, Peltomaki P, Leach PS, et al. Clues to the pathogenesis of familial colorectal cancer. Sience, 1993,260:812-816.
10. Ionov Y, Peinado MA, Malkhosyan S, et al. Ubiquitous somatic
mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature, 1993, 363: 558-561.
11. Keller G, Rudelius M, Vogelsang H, et al. Microsatellite instability and loss of heterozygosity in gastric carcinoma in comparison to family history. Am J Pathol, 1998, 152: 1281-1289.
12. Ottini L, Palli D, Falchetti M, et al. Microsatellite instability in gastric cancer is associated with tumor location and family history in a high risk population from Tuscany. Cancer Res, 1997, 57: 4523-4529.
13. Semba S, Yokozaki H, Yamamoto S, et al. Microsatellite instability in precancerous lesions and adenocarcinomas of the stomach. Cancer, 1996, 77 (supp18): 1620-1627.
14. Kim JJ, Beak MJ, Kim L, et al. Accumulated frameshift mutations at coding nucleotide repeats during the progression of gastric carcinoma with microsatellite instability. Lab Invest, 1999, 79:1113-1120.
15. Boland CR, Thibodeau SN, Hamilton SR, et al. A national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res, 1998, 58: 5248-5257.
16．吴在德．外科学．第五版，人民卫生出版社2002，500
17. Dietmaier W, Wallinger S, Bocker T, et al. Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression. Cancer Res, 1997, 57: 4749-4756.
18. Leung WK, Kim JJ, Kim JG, et al. Microsatellite instability in gastric intestinal metaplasia in patients with and without gastric cancer.Am J
Pathol, 2000, 156:537-543.
19. Fleisher AS, Esteller M, Tamura G, et al. Hypermethylation of the hMLH1 gene promoter is associated with microsatellite instability in early human gastric neoplasia. Oncogene, 2001, 20:329-335.
20. Halling KC, Harper H, Moskaluk CA, et al. Origin of microsatellite instability in gastric cancer. Am J Pathol, 1999, 155: 205-211.
21．王永信，柯扬，宁涛，等．中国人胃癌组织微卫星DNA的不稳定性研究．中华医学遗传学杂志，1998，15：155-157。
22. Kim HS, Lee BL, Woo DK, et al. Assesment of markers for the identification of microsatellite instability phenotype in gastric neoplasmas. Cancer Lettlers, 2001, 164: 61-68.
23. Sepulveda AR, Santos AC, Yamaoka Y, et al. Marked differences in the frequency of microsatellite instability in gastric cancer from different countries. Am J Gastroentero, 1999, 94: 3035-3038.
24. Thener CP, Campbell BS, Peel DJ, et al. Microsatellite instability in Japanese vs European American patients with gastric cancer. Arch Surg, 2002, 137: 960-966.
25. Shiao YH, Bovo D, Guido M, et al. Microsatellite instability and/or loss of heterogosity in young gastric cancer patients in Italy. Int J Cancer, 1999, 82: 232-239.
26. Wu MS, Lee CW, Chun ct, et al . Distinct clinicopathologic and genetic profiles in sporadic gastric cancer with different mutator phenotypes. Genes Chromosomes Cancer, 2000, 27:403-411.
27. Dos Santos NR, Seruca R, Constancia M, et al. Microsatellite instability at multiple loci in gastric carcinoma: clinicopathologic
implication and prognosis. Gastroenterology, 1996, 110: 38-44.
28. Yamamoto H, Piteira JP, Yoshida T, et al. Gastric cancers of the microsatellite mutator phenotype display characteristic genetic and clinical features. Gastroenterology, 1999, 116: 1348-1357.
29. Wirtz HC, Muller W, Noguchi T, et al. Prognostic value and clinicopathological profile of microsatellite instability in gastric cancer. Clin Cancer Res, 1998, 4: 1749-1754.
30. Chetty R, Naidoo R, Tarin M, et al. Chromosome 2p, 3p, 5q and 18q status in sporadic gastric cancer. Pathology, 2002, 34: 275-281.
31. Kobayashi K, Okamoto T, Takayama M, et al. Genetic instability in intestinal metaplasia is a frequent event leading to well-differentiated early adenocarcinoma of the stomach. Er J Cancer, 2000, 36: 1113-1119.
32．时俊，林庚金，钱立平，等．胃癌及肠化生组织微卫星不稳定性研究．中华消化杂志，2001，21：680-683．
33. Kim JJ, Beak MJ, Kim L, et al. Accumulated frameshift mutations at coding nucleotide repeats during the progression of gastric carcinoma with microsatellite instability. Lab Invest, 1999, 79:1113-1120.
34. Kim HS, Woo DK, Bae SI,, et al. Microsatellite instability in the adenoma-carcinoma sequence of the stomach. Lab Invest, 2000, 80: 57-64.
35. Kim YH, Kim NG, Lim JG, et al. Chromosomal alteration in paired gastric adenomas and carcinomas. Am J Pathol, 2001, 158: 655-662.
36. Lee JH, Abraham SC, Kim HS, et al. Inverse relationship between APC gene mutatio in gastric adenomas and development of adenocarcinoma. Am J Pathol, 2002, 161: 611-618.
37. To KF, Leung WK, Lee TL, et al. Promoter hypermethylation oftumor-related genes in gastric intestinal metaplasia of patients with and without gastric cancer. Int J Cancer, 2002, 102: 623-628.
38. Fleisher AS, Esteller M, Wang S, et al. Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability. Cancer Res, 1999, 59: 1090-1095.
39. Kim HS, Woo DK, Bae SI, et al. Microsatellite instability in the adenoma-carcinoma sequence of the stomach. Lab Invest, 2000, 80: 57-64.
40. Kashiwagi K, Watanabe M, Ezaki T, et al. Clinical usefulness of microsatellite instability for the prediction of gastric adenoma or adenocarcinoma in patients with chronic gastritis. Br J Cancer, 1999, 82:1814-1818.