恶性浆膜腔积液的ras基因突变和P21～（ras）蛋白表达状况及其诊断价值
摘要
Objective Serosa metastatic carcinomas usually cause malignant serous cavity effusions. The differentiation between malignant and benign effusions is sometimes difficult but very important in clinical practice. Laboratory tests are essential to the differentiation, but current available tests can not solve the problem satisfactorily. Ras gene is an important proto-oncogene, and its mutations are frequently found in many malignant tumors. Detecting ras gene mutations and expression in effusions is probably useful in differentiating malignant effusions from benign ones. In the present study, point mutations of codon 12 of K-ras and H-ras gene in the effusion supernatants were detected by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) and P21ras expressions in the effusion exfoliative cells were detected by immunocytochemistry, and their values in diagnosis of malignant effusions were evaluated.
Methods Fresh specimens of serous cavity effusions were obtained. Exfoliative cells and supernatants were collected after centrifugation, respectively. Two cell smears were prepared for Papanicolaou staining and cytological diagnosis, by which the specimens were divided into benign and malignant effusions. Genome DNA in the supernatants was conventionally extracted by proteinase K digestion followed by phenol/chloroform extraction. The DNA fragments containing codon 12 of K-ras and H-ras gene were amplified by PCR, respectively, and were cleaved by restriction endonuclease Mva I or Msp I, respectively. Digestive products were purified and identified by
means of polyacrylamide gel electrophoresis and silver staining. On the other hand, some of exfoliative cells were processed with a "standardized" procedure: removing red cells (when existing), fixed in 1% paraformaldehyde, resuspended in a solution of 1% BSA-PBS, adjusting the cell concentration to 2×106 cells per milliliter, and finally cell smears were prepared for immunocytochemistry. The primary antibody was monoclonal mouse anti-human P21ras, the second antibody was the biotinylated goat anti-mouse IgG. Streptavidin labeled with horseradish peroxidase was as the enzyme system. The color was developed with DAB. Results were observed under a light microscope.
Results A total of 108 specimens of serous cavity effusions were collected, including 53 cases of malignant effusions and 55 cases of benign effusions. PCR amplification of codon 12 of K-ras and H-ras genes was successful in 101 supernatants of the specimens (93.5%). In 52 malignant specimens, the codon 12 mutation rates of K-ras and H-ras gene were 55.8% and 30.8%, respectively. In 49 benign specimens, the mutation rates were 10.2% and 4.1%. The differences of the mutation rates of K-ras or H-ras were significant between benign and malignant effusions (P<0.001). The specificities (Sp) of K-ras and H-ras gene mutation for the diagnosis of malignant effusions were 89.8% and 95.9%, the positive predictive values (PV+) were 85.3% and 88.9%, the negative predictive values (PV-) were 65.8% and 56.6%, and the diagnostic accordance rates (DAR) were 72.3% and 62.4%.
For the immunocytochemical staining of P21ras, tumor exfoliative cells in 39 of 53 cases (73.6%) of malignant effusions were positive, and most of them expressed strongly. The positive results were observed in 12 of 55 cases (21.8%) of benign effusions, and most of them expressed weakly. The differences of positive rates and expression intensity of P21ras were significant between malignant and benign effusions (P<0.001). Sp of P21ras immunocytochemistry in diagnosis of malignant effusions was 78.2%, PV+ 76.5%, PV 75.4% and DAR 75.9%.
Combination analysis of ras gene mutation and P21ras immunocytochemical
staining in a parallel and series way, sensitivities for the diagnosis of malignant effusions were 88.7% and 49.1%, Sp 69.1% and 96.4%, PV+ 73.4% and 92.9%, PV- 86.4% and 63.9%, DAR 78.7% and 73.2%. Parallel combination analysis enhances the sensitivity and series combination analysis enhances the s
引文
1． Kuenen-Boumeester V, Loenen PV, Henzen-Logmans SC, et al. Quality control of immunocytochemical staining of effusions using a standardized method of cell processing. Acta Cytol，1996, 40(3): 475-479
2． 中国医学科学院肿瘤研究所、肿瘤医院细胞学室编. 临床肿瘤细胞学图谱. 人民卫生出版社, 1976：45-47
3． Yamashita K, Kuba T, Shinoda H, et al. Detection of K-ras point mutations in the supernatant of peritoneal and pleural effusions for diagnosis complementary to cytologic examinations. Am J Clin Path, 1998,109:704-711
4． Ward R, Hawkins N, O'Grady R, et al. Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples. Am J Pathol, 1998, 153(2):373-379
5． Knowles MA, Williamson M. Mutation of H-ras is infrequent in bladder cancer: confirmation by single-strand conformation polymorphism analysis, designed restriction fragment length polymorphisms, and direct sequencing. Cancer Res, 1993, 53(1):133-139
6． 张焜和, 吴小萍, 吕农华, 等. 应用微机处理聚丙烯酰胺凝胶电泳银染色图像结果. 医学信息, 2001, 14(5): 261-262
7． 李立明主编. 流行病学. 第四版, 北京: 人民卫生出版社, 2000: 55
8． 潘国宗，曹世植主编. 现代胃肠病学（上册）. 第一版，北京：科学出版社. 1994：289
9． 吴晓芝，富艳冰，张振华，等. 702例浆膜腔积液恶性细胞学检查分析. 中国医科大学学报，2000，10(5)：385-386
10． 付永晴，王会祥，寇学斌，等. 对胸/腹水染色体的观察研究—附114例检查报告. 中国优生与遗传杂志，1999, 7(20): 43
11． 吴广平，张振华，黄秀霞，等. 核仁组区相关蛋白颗粒研究对恶性胸/腹水的诊断价值. 中国医学检验杂志，1999, 27(4): 203-204
12． Grocker J, Macartney JC, Smith PT. Correlation between DNA flow cytometric and nucleolar organizer region data in non-Hodgkin's lymphomas. J Pathol, 1988,154:151-156
13． Prieto M，Gomez-Lechon MJ，Hoyos M，et al. Diagnosis of malignant ascites: Comparison of ascitic fibronectin, cholesterol, and serum-ascites albumin difference. Dig Dis Sci, 1988, 33(7): 833-838
14． Gupta R, Misra SP, Dwivedi M, et al. Diagnosing ascites: value of ascitic fluid total protein, albumin, cholesterol, their ratios, serum-ascites albumin and cholesterol gradient. J Gastroenterol Hepatol, 1995, 10(3): 295-299
15． 董建平，刘雅，陈立军. 腹水脱落细胞免疫组化检查在良恶性腹水鉴别中的意义. 天津医科大学学报，1999, 5(3): 71-72
16． 高中度，袁维祥，茅爱武，等. 腹水及血清CA-50测定对消化系恶性肿瘤的诊断价值. 河北医学，2000, 6(4): 291-293
17． Bast RC, Klug TL, John E, et al. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. N Engl J Med, 1983, 309:883-887
18． Halia H, Stenman UH, Seppala M, et al. Ovarian cancer antigen CA-125 in pelvic inflammatory disease and pregnancy. Cancer, 1986, 57:1327-1329
19． Molina R, Filella X, Bruix J, et al. Cancer antigen CA 125 in serum and ascetic fluid of patients with liver disease. Clin Chem,1991, 37:1379-1383
20． Zuckerman E, Lanir A, Sabo E, et al. Cancer Antigen 125: A Sensitive Marker of Ascites in Patients With Liver Cirrhosis. Am J Gastroenterol, 1999, 94:1613-1618
21． Demirkazik A, Dincol D, Hasturk S, et al. Diagnostic value of ferrin in differential diagnosis of malignant effusions. Cancer Biochem Biophys, 1998, 16(3): 243-251
Kavanaugh WM, Turch CW, William L. PTB domain binding to signaling
22． proteins through a sequence motif containing phosphotyrosine. Science, 1995, 268:1177-1179
23． Urban T, Ricci S, Danel C, et al. Detection of codon 12 K-ras mutations in non-neoplastic mucosa from bronchial carina in patients with lung adenocarcinomas. Br J Cancer, 2000, 82(2):412-417
24． Clayton SJ, Scott FM, Walker J, et all. K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification. Clin Chem, 2000, 46(12): 1929-1938
25． Li S, Rosell R, Urban A, et al. K-ras gene point mutation: a stable tumor marker in non-small cell lung carcinoma. Lung Cancer, 1994, 11(1-2): 19-27
26． Kimura W, Zhao B, Futakawa N, et al. Significance of K-ras codon 12 point mutation in pancreatic juice in the diagnosis of carcinoma of the pancreas. Hepatogastroenterology, 1999, 46(25):532-539
27． Banerjee SK, Makdisi WF, Weston AP, et al. A two-step enriched-nested PCR technique enhances sensitivity for detection of codon 12 K-ras mutations in pancreatic adenocarcinoma. Pancreas, 1997;15(1):16-24
28． Lev Z, Kislitsin D, Rennert G, et al. Utilization of K-ras mutations identified in stool DNA for the early detection of colorectal cancer. J Cell Biochem Suppl, 2000, 34:35-39
29． Jen J, Johnson C, Levin B. Molecular approaches for colorectal cancer screening. Eur J Gastroenterol Hepatol, 1998, 10(3):213-217
30． Ito Y, Kobayashi S, Taniguchi T, et al. Frequent detection of K-ras mutation in stool samples of colorectal carcinoma patients after improved DNA extraction: comparison with tissue samples. Int J Oncol, 2002, 20(6):1263-1268
Cuatrecasas M, Villanueva A, Matias-Guiu X, et al. K-ras mutations in mucinous ovarian tumors: a clinicopathologic and molecular study of 95
31． cases. Cancer, 1997, 79(8):1581-1586
32． Cuatrecasas M, Erill N, Musulen E, et al. K-ras mutations in nonmucinous ovarian epithelial tumors: a molecular analysis and clinicopathologic study of 144 patients. Cancer, 1998, 82(6):1088-1095
33． Garrett AP, Lee KR, Colitti CR, et al. k-ras mutation may be an early event in mucinous ovarian tumorigenesis. Int J Gynecol Pathol, 2001, 20(3):244-251
34． Dokianakis DN, Varras MN, Papaefthimiou M, et al. Ras gene activation in malignant cells of human ovarian carcinoma peritoneal fluids. Clin Exp Metastasis, 1999, 17(4):293-297
35． Watanabe H, Miyagi C, Yamaguchi Y, et al. Detection of K-ras point mutations at codon 12 in pancreatic juice for the diagnosis of pancreatic cancer by hybridization protsction assay: a simple method for the determination of the types of point mutations. Jpn J Cancer Res, 1996, 87:466-474
36． Tada M, Omata M, Kawai S, et al. Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma. Cancer Res, 1993,53:2472-2474
37． Wang Y, Yamaguchi Y, Watanabe H, et al. Usefulness of p53 gene mutations in the supernatant of bile for diagnosis of biliary tract carcinoma: comparison with K-ras mutation. J Gastroenterol, 2002, 37(10): 831-839
38． Nakamoto M, Teramoto H, Matsumoto S, et al. K-ras and rho A mutations in malignant pleural effusion. Int J Oncol, 2001, 19(5):971-976
39． Salbe C, Trevisiol C, Ferruzzi E, et al. Molecular detection of codon 12 K-RAS mutations in circulating DNA from serum of colorectal cancer patients. Int J Biol Markers, 2000, 15(4):300-307
Theodor L, Melzer E, Sologov M, et al. Detection of pancreatic carcinoma: diagnostic value of K-ras mutations in circulating DNA from
40． serum. Dig Dis Sci, 1999, 44(10):2014-2019
41． Castells A, Puig P, Mora J, et al. K-ras mutations in DNA extracted from the plasma of patients with pancreatic carcinoma: diagnostic utility and prognostic significance. J Clin Oncol, 1999, 17(2):578-584
42． Kondo H, Sugano K, Fukayama N, et al. Detection of point mutations in the K-ras oncogene at codon 12 in pure pancreatic juice for diagnosis of pancreatic carcinoma. Cancer, 1994, 73(6):1589-1594
43． Rodenhuis S, Slebos RJC, Boot AJM, et al. Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung. Cancer Res,1988,48:5738-5741
44． Westra WH, Slebos RC, Offerhaus GJ, et al. K-ras oncogene activation in lung adenocarcinomas from former smokers. Cancer, 1993,72:432-438
45． Mills NE, Fishman CL, Rom WN, et al. Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma. Cancer Res, 1995, 55: 1444-1447
46． 张新，徐松涛，金建军，等. 肺癌K-ras基因突变的二步PCR检测及其临床应用. 上海医科大学学报，1999, 26(5):329-332
47． Comin CE, Novelli L, Boddi V, et al. Calretinin, thrombomodulin, CEA, and CD15: a useful combination of immunohistochemical markers for differentiating pleural epithelial mesothelioma from peripheral pulmonary adenocarcinoma. Hum Pathol, 2001, 32(5):529-536
48． Carella R, Deleonardi G, D'Errico A, et al. Immunohistochemical panels for differentiating epithelial malignant mesothelioma from lung adenocarcinoma: a study with logistic regression analysis. Am J Surg Pathol, 2001, 25(1):43-50
49． Nomoto S, Nakao A, Kasai Y, et al. Peritoneal washing cytology combined with immunocytochemical staining and detecting mutant K-ras in pancreatic cancer: comparison of the sensitivity and availability of various methods. Pancreas, 1997, 14(2):126-132
50． Chieng DC, Yee H, Schaefer D, et al. Calretinin staining pattern aids in the differentiation of mesothelioma from adenocarcinoma in serous effusions. Cancer Cytopathol, 2000,90(3):194-200
51． Bedrossian CWM. Diagnostic problems in serous effusions. Diagn Cytopathol, 1998,19:131-136
52． Delahaye M, Van der Ham F, Van der Kwast TH. Complementary value of five carcinoma markers for the diagnosis of malignant mesothelioma, adenocarcinoma metastasis, and reactive mesothelium in serous effusions. Diagn Cytopathol, 1997, 17: 115-120
53． Koutselini H, Kappatou G, Yiagnisis M, et al. Immunocytochemical study RAS oncoprotein in cytologic specimens of primary lung tumors. Anticancer Res, 1990, 10(3):597-603
54． Gulbis B, Galand P. Immunodetection of the p21-ras products in human normal and preneoplastic tissues and solid tumors: a review. Hum Pathol, 1993, 24(12):1271-1285
55． Yukawa M, Fujimori T, Maeda S, et al. Comparative clinicopathological and immunohistochemical study of ras and p53 in flat and polypoid type colorectal tumours. Gut, 1994, 35(9):1258-1261
56． Tsuda T, Mochizuki M, Wakasa H. Detection of c-ras gene mutation and expression of p21 protein in dysplasias and carcinomas complicating ulcerative colitis. J Gastroenterol, 1995,30 (8):30-32
57． Kuwashima Y, Shisa H, Uehara T, et al. Immunohistochemical detection of ras p21 oncoprotein in undifferentiated and well-differentiated epithelial carcinomas of the human ovary. Anticancer Res, 1995, 15(6B): 2847-2850
58． Ieda S, Watatani M, Yoshida T, et al. Immunohistochemical analysis of p53 and ras p21 expression in colorectal adenomas and early carcinomas. Surg Today, 1996, 26(4):230-235
Terada T, Ohta T, Nakanuma Y. Expression of oncogene products,
59． anti-oncogene products and oncofetal antigens in intraductal papillary-mucinous neoplasm of the pancreas. Histopathology, 1996, 29(4): 355-361
60． Kim YB, Han JY, Kim TS, et al. Overexpression of c-H-ras p21 is correlated with vascular endothelial growth factor expression and neovascularization in advanced gastric carcinoma. J Gastroenterol Hepatol, 2000,15(12):1393-1399
2． 中国医学科学院肿瘤研究所、肿瘤医院细胞学室编. 临床肿瘤细胞学图谱. 人民卫生出版社, 1976：45-47
3． Yamashita K, Kuba T, Shinoda H, et al. Detection of K-ras point mutations in the supernatant of peritoneal and pleural effusions for diagnosis complementary to cytologic examinations. Am J Clin Path, 1998,109:704-711
4． Ward R, Hawkins N, O'Grady R, et al. Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples. Am J Pathol, 1998, 153(2):373-379
5． Knowles MA, Williamson M. Mutation of H-ras is infrequent in bladder cancer: confirmation by single-strand conformation polymorphism analysis, designed restriction fragment length polymorphisms, and direct sequencing. Cancer Res, 1993, 53(1):133-139
6． 张焜和, 吴小萍, 吕农华, 等. 应用微机处理聚丙烯酰胺凝胶电泳银染色图像结果. 医学信息, 2001, 14(5): 261-262
7． 李立明主编. 流行病学. 第四版, 北京: 人民卫生出版社, 2000: 55
8． 潘国宗，曹世植主编. 现代胃肠病学（上册）. 第一版，北京：科学出版社. 1994：289
9． 吴晓芝，富艳冰，张振华，等. 702例浆膜腔积液恶性细胞学检查分析. 中国医科大学学报，2000，10(5)：385-386
10． 付永晴，王会祥，寇学斌，等. 对胸/腹水染色体的观察研究—附114例检查报告. 中国优生与遗传杂志，1999, 7(20): 43
11． 吴广平，张振华，黄秀霞，等. 核仁组区相关蛋白颗粒研究对恶性胸/腹水的诊断价值. 中国医学检验杂志，1999, 27(4): 203-204
12． Grocker J, Macartney JC, Smith PT. Correlation between DNA flow cytometric and nucleolar organizer region data in non-Hodgkin's lymphomas. J Pathol, 1988,154:151-156
13． Prieto M，Gomez-Lechon MJ，Hoyos M，et al. Diagnosis of malignant ascites: Comparison of ascitic fibronectin, cholesterol, and serum-ascites albumin difference. Dig Dis Sci, 1988, 33(7): 833-838
14． Gupta R, Misra SP, Dwivedi M, et al. Diagnosing ascites: value of ascitic fluid total protein, albumin, cholesterol, their ratios, serum-ascites albumin and cholesterol gradient. J Gastroenterol Hepatol, 1995, 10(3): 295-299
15． 董建平，刘雅，陈立军. 腹水脱落细胞免疫组化检查在良恶性腹水鉴别中的意义. 天津医科大学学报，1999, 5(3): 71-72
16． 高中度，袁维祥，茅爱武，等. 腹水及血清CA-50测定对消化系恶性肿瘤的诊断价值. 河北医学，2000, 6(4): 291-293
17． Bast RC, Klug TL, John E, et al. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. N Engl J Med, 1983, 309:883-887
18． Halia H, Stenman UH, Seppala M, et al. Ovarian cancer antigen CA-125 in pelvic inflammatory disease and pregnancy. Cancer, 1986, 57:1327-1329
19． Molina R, Filella X, Bruix J, et al. Cancer antigen CA 125 in serum and ascetic fluid of patients with liver disease. Clin Chem,1991, 37:1379-1383
20． Zuckerman E, Lanir A, Sabo E, et al. Cancer Antigen 125: A Sensitive Marker of Ascites in Patients With Liver Cirrhosis. Am J Gastroenterol, 1999, 94:1613-1618
21． Demirkazik A, Dincol D, Hasturk S, et al. Diagnostic value of ferrin in differential diagnosis of malignant effusions. Cancer Biochem Biophys, 1998, 16(3): 243-251
Kavanaugh WM, Turch CW, William L. PTB domain binding to signaling
22． proteins through a sequence motif containing phosphotyrosine. Science, 1995, 268:1177-1179
23． Urban T, Ricci S, Danel C, et al. Detection of codon 12 K-ras mutations in non-neoplastic mucosa from bronchial carina in patients with lung adenocarcinomas. Br J Cancer, 2000, 82(2):412-417
24． Clayton SJ, Scott FM, Walker J, et all. K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification. Clin Chem, 2000, 46(12): 1929-1938
25． Li S, Rosell R, Urban A, et al. K-ras gene point mutation: a stable tumor marker in non-small cell lung carcinoma. Lung Cancer, 1994, 11(1-2): 19-27
26． Kimura W, Zhao B, Futakawa N, et al. Significance of K-ras codon 12 point mutation in pancreatic juice in the diagnosis of carcinoma of the pancreas. Hepatogastroenterology, 1999, 46(25):532-539
27． Banerjee SK, Makdisi WF, Weston AP, et al. A two-step enriched-nested PCR technique enhances sensitivity for detection of codon 12 K-ras mutations in pancreatic adenocarcinoma. Pancreas, 1997;15(1):16-24
28． Lev Z, Kislitsin D, Rennert G, et al. Utilization of K-ras mutations identified in stool DNA for the early detection of colorectal cancer. J Cell Biochem Suppl, 2000, 34:35-39
29． Jen J, Johnson C, Levin B. Molecular approaches for colorectal cancer screening. Eur J Gastroenterol Hepatol, 1998, 10(3):213-217
30． Ito Y, Kobayashi S, Taniguchi T, et al. Frequent detection of K-ras mutation in stool samples of colorectal carcinoma patients after improved DNA extraction: comparison with tissue samples. Int J Oncol, 2002, 20(6):1263-1268
Cuatrecasas M, Villanueva A, Matias-Guiu X, et al. K-ras mutations in mucinous ovarian tumors: a clinicopathologic and molecular study of 95
31． cases. Cancer, 1997, 79(8):1581-1586
32． Cuatrecasas M, Erill N, Musulen E, et al. K-ras mutations in nonmucinous ovarian epithelial tumors: a molecular analysis and clinicopathologic study of 144 patients. Cancer, 1998, 82(6):1088-1095
33． Garrett AP, Lee KR, Colitti CR, et al. k-ras mutation may be an early event in mucinous ovarian tumorigenesis. Int J Gynecol Pathol, 2001, 20(3):244-251
34． Dokianakis DN, Varras MN, Papaefthimiou M, et al. Ras gene activation in malignant cells of human ovarian carcinoma peritoneal fluids. Clin Exp Metastasis, 1999, 17(4):293-297
35． Watanabe H, Miyagi C, Yamaguchi Y, et al. Detection of K-ras point mutations at codon 12 in pancreatic juice for the diagnosis of pancreatic cancer by hybridization protsction assay: a simple method for the determination of the types of point mutations. Jpn J Cancer Res, 1996, 87:466-474
36． Tada M, Omata M, Kawai S, et al. Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma. Cancer Res, 1993,53:2472-2474
37． Wang Y, Yamaguchi Y, Watanabe H, et al. Usefulness of p53 gene mutations in the supernatant of bile for diagnosis of biliary tract carcinoma: comparison with K-ras mutation. J Gastroenterol, 2002, 37(10): 831-839
38． Nakamoto M, Teramoto H, Matsumoto S, et al. K-ras and rho A mutations in malignant pleural effusion. Int J Oncol, 2001, 19(5):971-976
39． Salbe C, Trevisiol C, Ferruzzi E, et al. Molecular detection of codon 12 K-RAS mutations in circulating DNA from serum of colorectal cancer patients. Int J Biol Markers, 2000, 15(4):300-307
Theodor L, Melzer E, Sologov M, et al. Detection of pancreatic carcinoma: diagnostic value of K-ras mutations in circulating DNA from
40． serum. Dig Dis Sci, 1999, 44(10):2014-2019
41． Castells A, Puig P, Mora J, et al. K-ras mutations in DNA extracted from the plasma of patients with pancreatic carcinoma: diagnostic utility and prognostic significance. J Clin Oncol, 1999, 17(2):578-584
42． Kondo H, Sugano K, Fukayama N, et al. Detection of point mutations in the K-ras oncogene at codon 12 in pure pancreatic juice for diagnosis of pancreatic carcinoma. Cancer, 1994, 73(6):1589-1594
43． Rodenhuis S, Slebos RJC, Boot AJM, et al. Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung. Cancer Res,1988,48:5738-5741
44． Westra WH, Slebos RC, Offerhaus GJ, et al. K-ras oncogene activation in lung adenocarcinomas from former smokers. Cancer, 1993,72:432-438
45． Mills NE, Fishman CL, Rom WN, et al. Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma. Cancer Res, 1995, 55: 1444-1447
46． 张新，徐松涛，金建军，等. 肺癌K-ras基因突变的二步PCR检测及其临床应用. 上海医科大学学报，1999, 26(5):329-332
47． Comin CE, Novelli L, Boddi V, et al. Calretinin, thrombomodulin, CEA, and CD15: a useful combination of immunohistochemical markers for differentiating pleural epithelial mesothelioma from peripheral pulmonary adenocarcinoma. Hum Pathol, 2001, 32(5):529-536
48． Carella R, Deleonardi G, D'Errico A, et al. Immunohistochemical panels for differentiating epithelial malignant mesothelioma from lung adenocarcinoma: a study with logistic regression analysis. Am J Surg Pathol, 2001, 25(1):43-50
49． Nomoto S, Nakao A, Kasai Y, et al. Peritoneal washing cytology combined with immunocytochemical staining and detecting mutant K-ras in pancreatic cancer: comparison of the sensitivity and availability of various methods. Pancreas, 1997, 14(2):126-132
50． Chieng DC, Yee H, Schaefer D, et al. Calretinin staining pattern aids in the differentiation of mesothelioma from adenocarcinoma in serous effusions. Cancer Cytopathol, 2000,90(3):194-200
51． Bedrossian CWM. Diagnostic problems in serous effusions. Diagn Cytopathol, 1998,19:131-136
52． Delahaye M, Van der Ham F, Van der Kwast TH. Complementary value of five carcinoma markers for the diagnosis of malignant mesothelioma, adenocarcinoma metastasis, and reactive mesothelium in serous effusions. Diagn Cytopathol, 1997, 17: 115-120
53． Koutselini H, Kappatou G, Yiagnisis M, et al. Immunocytochemical study RAS oncoprotein in cytologic specimens of primary lung tumors. Anticancer Res, 1990, 10(3):597-603
54． Gulbis B, Galand P. Immunodetection of the p21-ras products in human normal and preneoplastic tissues and solid tumors: a review. Hum Pathol, 1993, 24(12):1271-1285
55． Yukawa M, Fujimori T, Maeda S, et al. Comparative clinicopathological and immunohistochemical study of ras and p53 in flat and polypoid type colorectal tumours. Gut, 1994, 35(9):1258-1261
56． Tsuda T, Mochizuki M, Wakasa H. Detection of c-ras gene mutation and expression of p21 protein in dysplasias and carcinomas complicating ulcerative colitis. J Gastroenterol, 1995,30 (8):30-32
57． Kuwashima Y, Shisa H, Uehara T, et al. Immunohistochemical detection of ras p21 oncoprotein in undifferentiated and well-differentiated epithelial carcinomas of the human ovary. Anticancer Res, 1995, 15(6B): 2847-2850
58． Ieda S, Watatani M, Yoshida T, et al. Immunohistochemical analysis of p53 and ras p21 expression in colorectal adenomas and early carcinomas. Surg Today, 1996, 26(4):230-235
Terada T, Ohta T, Nakanuma Y. Expression of oncogene products,
59． anti-oncogene products and oncofetal antigens in intraductal papillary-mucinous neoplasm of the pancreas. Histopathology, 1996, 29(4): 355-361
60． Kim YB, Han JY, Kim TS, et al. Overexpression of c-H-ras p21 is correlated with vascular endothelial growth factor expression and neovascularization in advanced gastric carcinoma. J Gastroenterol Hepatol, 2000,15(12):1393-1399