erbB2癌基因mRNA在头颈部恶性肿瘤组织中的表达研究
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摘要
erbB2癌基因mRNA在头颈部恶性肿瘤组织中的表达研究
目的 应用实时荧光定量PCR法（quantitative real-time PCR）检测头颈部恶性肿瘤组织中erbB2基因的表达。
方法 提取新鲜41例头颈部恶性肿瘤组织，16例良性肿瘤组织和20例炎性病变组织的总RNA，逆转录为cDNA，应用实时荧光定量PCR法观察肿瘤组织与炎性组织erbB2表达水平。以TBP基因（TATA box-binding protein）为内参照，得到标化的erbB2表达。
结果 恶性肿瘤组的erbB2表达（37.13±0.14）明显高于良性肿瘤组（0.95±0.13）和炎性组（1.00±0.12）（P=0.0000）。按照高出炎性组织均数5为表达阳性，22/41（54%）恶性肿瘤病人有明显的过度表达（5.39~903.89），所有良性肿瘤和炎性病例均未发现有erbB2阳性表达；按照文献进一步划分为低度表达（在2~5之间），中度表达（≥5，<10），高度表达(≥10)。炎性组中有20%（4/20）的病例有低度表达；良性肿瘤组有19%（3/16）出现低度表达；上述两组无1例有中度以上表达；在恶性组中，仅有2.4%（1/41）为低度表达，4.9%（2/41）为中度表达， 48.8%（20/41）的病例表现出高水平表达。没有发现erbB2表达与病人年龄，肿瘤分期和淋巴结转移之间有显著意义（P>0.05）。
结论 erbB2 mRNA的扩增在头颈部恶性肿瘤的发生过程中扮演了重要角色，但其与头颈恶性肿瘤预后的关系仍不清楚。新的高特异性的荧光定量PCR法检测erbB2癌基因表达，有较高的敏感性，并为头颈部恶性肿瘤的Herceptin治疗提供理论基础。
Evaluation of erbB2 Over-expression/Amplification in Head and Neck Cancer For Future Management of Applications.
Objective The study was designated to investigate the erbB2 status in head and neck cancer by a quantitative real-time PCR.
Methods Total RNA samples from the fresh tissues of 41 head and neck malignant tumors cases, 16 benign tumors and 20 inflammatory tissues were used to cDNA synthesis. The status of erbB2 were analyzed using real-time PCR. The erbB2 mRNA signal was normalized to the signal for TATA box-binding protein (TBP) mRNA.
Results The mean normalized expression of erbB2 in malignant tumors (37.13±0.14) was obviously higher than benign tumors(0.95±0.13) and inflammatory tissues (1.00±0.12), P=0.0000. Over-expression (>5 above mean for inflammatory tissues) of the erbB2 gene was observed in 22/41 (54%) malignant tumors, at 5.37~903.89 fold. No over-expression signal was observed in benign and inflammatory samples. There were 1 and 2 malignant cases showed low-level amplification (2-fold to <5-fold) and intermediate-level amplification(≥5-fold to <10-fold) respectively. Whereas 20 cases presented high-level erbB2 amplifications (up to 10-fold). Among benign tumors and inflammatory tissues, 4 and 3 cases revealed low-level amplifications respectively, none presented intermediate-and high-level amplification. There was no significant correlation between the erbB2 status and histology, clinical stage, or lymph node metastasis.
Conclusion It has been suggested that over-expression/amplification of erbB2 plays an important role in the tumorigenesis of head and neck cancer. But there was no clear relationship between the gene over-expression and poor prognosis. Real-time PCR, the new simple, rapid, semi-automated assay may be a powerful tool for gene alteration analysis in human tumors and to support future erbB2-based biological and gene therapy approaches.
目的 应用实时荧光定量PCR法（quantitative real-time PCR）检测头颈部恶性肿瘤组织中erbB2基因的表达。
方法 提取新鲜41例头颈部恶性肿瘤组织，16例良性肿瘤组织和20例炎性病变组织的总RNA，逆转录为cDNA，应用实时荧光定量PCR法观察肿瘤组织与炎性组织erbB2表达水平。以TBP基因（TATA box-binding protein）为内参照，得到标化的erbB2表达。
结果 恶性肿瘤组的erbB2表达（37.13±0.14）明显高于良性肿瘤组（0.95±0.13）和炎性组（1.00±0.12）（P=0.0000）。按照高出炎性组织均数5为表达阳性，22/41（54%）恶性肿瘤病人有明显的过度表达（5.39~903.89），所有良性肿瘤和炎性病例均未发现有erbB2阳性表达；按照文献进一步划分为低度表达（在2~5之间），中度表达（≥5，<10），高度表达(≥10)。炎性组中有20%（4/20）的病例有低度表达；良性肿瘤组有19%（3/16）出现低度表达；上述两组无1例有中度以上表达；在恶性组中，仅有2.4%（1/41）为低度表达，4.9%（2/41）为中度表达， 48.8%（20/41）的病例表现出高水平表达。没有发现erbB2表达与病人年龄，肿瘤分期和淋巴结转移之间有显著意义（P>0.05）。
结论 erbB2 mRNA的扩增在头颈部恶性肿瘤的发生过程中扮演了重要角色，但其与头颈恶性肿瘤预后的关系仍不清楚。新的高特异性的荧光定量PCR法检测erbB2癌基因表达，有较高的敏感性，并为头颈部恶性肿瘤的Herceptin治疗提供理论基础。
Evaluation of erbB2 Over-expression/Amplification in Head and Neck Cancer For Future Management of Applications.
Objective The study was designated to investigate the erbB2 status in head and neck cancer by a quantitative real-time PCR.
Methods Total RNA samples from the fresh tissues of 41 head and neck malignant tumors cases, 16 benign tumors and 20 inflammatory tissues were used to cDNA synthesis. The status of erbB2 were analyzed using real-time PCR. The erbB2 mRNA signal was normalized to the signal for TATA box-binding protein (TBP) mRNA.
Results The mean normalized expression of erbB2 in malignant tumors (37.13±0.14) was obviously higher than benign tumors(0.95±0.13) and inflammatory tissues (1.00±0.12), P=0.0000. Over-expression (>5 above mean for inflammatory tissues) of the erbB2 gene was observed in 22/41 (54%) malignant tumors, at 5.37~903.89 fold. No over-expression signal was observed in benign and inflammatory samples. There were 1 and 2 malignant cases showed low-level amplification (2-fold to <5-fold) and intermediate-level amplification(≥5-fold to <10-fold) respectively. Whereas 20 cases presented high-level erbB2 amplifications (up to 10-fold). Among benign tumors and inflammatory tissues, 4 and 3 cases revealed low-level amplifications respectively, none presented intermediate-and high-level amplification. There was no significant correlation between the erbB2 status and histology, clinical stage, or lymph node metastasis.
Conclusion It has been suggested that over-expression/amplification of erbB2 plays an important role in the tumorigenesis of head and neck cancer. But there was no clear relationship between the gene over-expression and poor prognosis. Real-time PCR, the new simple, rapid, semi-automated assay may be a powerful tool for gene alteration analysis in human tumors and to support future erbB2-based biological and gene therapy approaches.
引文
黄文生，姚连生，施产甫等，肿瘤相关基因在头颈肿瘤中的表达研究进展。南京军医学院学报，2002，24（3）：177-179.
Reis PP, Rogatto SR, Kowalski LP et al. Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer. Oncogene, 2002,21(42):6480-7.
Yamamoto T, Ikawa S, Akiyama T et al. Similarlity of protein encode by the human c-erbB-2 gene to epidermal growth factor receptor. Nature, 1988，319-230.
N Ady, L Morat, K Fizazi et al. Detection of Her-2/neu-positive circulating epithelial cells in prostate cancer patiens. Bri J Cancer, 2004,90:443-448.
Srividya Sundaresan, Penelope E.Roberts, et al. Biological response to erbB ligands in nontransformed cell lines correlates with a specific pattern of receptor expression. Endocrinology, 1998, 139(12): 4756-4764.
O’Malley FP, Parkes R, Latta E，et al. Comparison of HER2/Neu status assessed by quantitative polymerase chain reaction and immunohistochemistry. Am J Clin Pathol, 2001，115:504-11.
Lee K-F, Simon H, Chen H et al. Requirement for Neuregulin receptor erbB2 in Neural and cardiac development. Nature, 1995, 378:394–398.
郑绘霞，阙秀。乳腺癌HER-2/neu基因检测与Herceptin治疗。实用癌症杂志，2001，16（2）：222-224。
Elaine Lyon, Alison Millson, Mary C,et al. Quantification of HER-2/Neu gene amplification by competitive PCR using fluorescent melting curve analysis. Clin Chem, 2001,47:844-854.
Kimmo J. Savinainen, Outir.Saramaki, Marika J.Linja,et al. Expression and gene copy number analysis of erbB2 oncogene in prostate cancer. Am J Pathol, 2002, 16:339-345.
Jan Brahender, Kathleen D.Danenberg, Ralf Metzger et al. Epidermal growth factor receptor and HER2/neu mRNA expression in no-small cell lung cancer is correlated with survival. Clin Cancer Res, 2001, 7:1850-1855.
Kern J. A., Schwartz D. A., Nordberg J. E et al. P185 Neu expression in human lung adenocarcinomas predicts shortened survival. Cancer Res, 1990，50: 5184-5191.
Pfeiffer P., Clausen P. P., Andersen K et al. Lack of prognostic significance of epidermal growth factor receptor and the oncoprotein p185 HER-2 in patients with systemically untreated non-small cell lung cancer: an immunohistochemical study on cryosections. Br J Cancer, 1996，74: 86-91.
Philp S.B, Carl T.W. Real-time PCR technology for cancer dagnostics. Clin Chem, 2002, 48(8):1178-1185.
Stefania Gelmini, Claudio Orlando, Roberta Sestini等，Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification. Clin Chem，1997，43（5）:752-758.
S.A.Bustin et al. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinology，2000, 25:169-193.
Ian M.Mackay, Katherine E. Arden et al. Real-time PCR in virology. Nucl Aci Res，2002, 30(6): 1292-1305.
Bieche I, Onody P, Laurendeau I, et al. Real-time reverse transcription-PCR assay for future management of erbB2-based clinical application. Clin Chem, 1999, 45:1148-1156.
Katja Specht, Thomas Richter et al. Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. Am J Pathol，2001，158:419-429.
Pei-Wen Chiang, David G Beer, Wan-Li Wei et al. Detetion of erbB-2 amplifications in tumors and sera from esophageal carcinoma patients. Clin Cancer Res，1999，June（5）：1381-1386.
Korenberg J R, Hugdins L, McGiuioray B et al. Down syndrome phenotypes:the comsequences of chromosomal imbalance. Proc Natl Acad Sci， 1994，91: 4997-5001.
Chan AT, Lo YM, Zee B et al .Plasma Epstein-Barr Virus DNA and residual
diease after radiotherapy for undifferentiated Nasopharyngeal carcinoma. J Natl Cancer Inst , 2002，94(21):1614-9.
ChiYomi Egawa, Yasua MiYoshi, Kyoko Iwao et al. Quantitative analysis of estrogen receptor-αand –βmRNA expression in normal and malignant thyroid tissues by real-time PCR. Oncology, 2001,61:293-298.
Nieawenhuis EJ, Jaspars LH et al. Quantitative molecular delection of minimal residual head and neck cancer in lymph node. Clin Cancer Res，2003 9(2):755-61.
Sabine glockner, Ulrich lehman, Nadine Willke et al. Amplification of growth regulatory genes in intraductal breast cancer is associated with higher nuclear grade but not with the progression to invasiveness. Lab Invest，2001 81:565-571.
王传新，邹雄，王谦等，血清癌基因C-erbB2蛋白对恶性肿瘤诊断价值的研究。山东大学学报（医学版），2002，40（6）：552-553。
Felip E, Del Campo JM, Rugbio D, et al. Overexpression of c-erbB2 in epithelial ovarian cancer. Cancer, 1995，75:2147.
Yan J, Fang Y, Huang BJ, et al. Absence of evidence for Her-2 amplification in nasopharyngeal carcinoma. Can Gen Cyt, 2002, 132(2):116-9.
Roychowdhury DF, Tseng A, Fu KK, et al，New prognostic factors in nasopharyngeal carcinoma: Tuomor angiogenesis and C-erbB-2 expression. Cancer, 1996, 77(8):1419-1426.
顾康生，吴良秋，侯景辉等，HER-2/NEU基因在鼻咽癌中的表达及其临床意义，癌症，2001，20（8）：869-872。
鄢践，方燕等，原发性鼻咽癌患者Her2基因表达的意义。中华医学杂志，2001，81（15）：904-906。
金鸥，颜亚晖等，鼻咽癌组织c-erbB-2表达与细胞增殖预后的关系。湖南医科大学学报，1998，23：235-238。
金鸥，陈森林等，鼻咽癌及癌旁黏膜组织中c-erbB-2，EGFR mRNA的表达。湖南医科大学学报，1997，22（6），487-490。
罗扬，王金万. Herceptin治疗HER-2过度表达乳腺癌的现状。国外医学肿瘤学分册，2002，29（2）：126-128。
Agus DB, Scher HI, Higgins B et al. Response of prostate cancer to anti-Her-2/Neu antibody in androgen-dependent and -independent human xenograft models. Cancer Res 1999, 59:4761-4764.
Baselga J, Norton L, Albanell J et al. Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/Neu overexpressing human breast cancer xenografts. Cancer Res，1998，58:2825-2831.
于永强，董卫东，任重等，癌基因C-erbB2抑癌基因nm23在鼻咽癌中的表达意义。肿瘤，2002，22（4）：305-307。
赵丽纯，李佳莉等，以HER2/neu为靶点抗肿瘤治疗研究进展，国外医学遗传学分册，2002，25（1）：33-36.
Formenti SC, Spicer D, Skinner K, et al. Low HER2/Neu gene expression is associated with pathological response to concurrent paclitaxel and radiation therapy in locally advanced breast cancer. Int J Radiat Oncol Biol Phys, 2002, 52(2):397-405.
Reis PP, Rogatto SR, Kowalski LP et al. Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer. Oncogene, 2002,21(42):6480-7.
Yamamoto T, Ikawa S, Akiyama T et al. Similarlity of protein encode by the human c-erbB-2 gene to epidermal growth factor receptor. Nature, 1988，319-230.
N Ady, L Morat, K Fizazi et al. Detection of Her-2/neu-positive circulating epithelial cells in prostate cancer patiens. Bri J Cancer, 2004,90:443-448.
Srividya Sundaresan, Penelope E.Roberts, et al. Biological response to erbB ligands in nontransformed cell lines correlates with a specific pattern of receptor expression. Endocrinology, 1998, 139(12): 4756-4764.
O’Malley FP, Parkes R, Latta E，et al. Comparison of HER2/Neu status assessed by quantitative polymerase chain reaction and immunohistochemistry. Am J Clin Pathol, 2001，115:504-11.
Lee K-F, Simon H, Chen H et al. Requirement for Neuregulin receptor erbB2 in Neural and cardiac development. Nature, 1995, 378:394–398.
郑绘霞，阙秀。乳腺癌HER-2/neu基因检测与Herceptin治疗。实用癌症杂志，2001，16（2）：222-224。
Elaine Lyon, Alison Millson, Mary C,et al. Quantification of HER-2/Neu gene amplification by competitive PCR using fluorescent melting curve analysis. Clin Chem, 2001,47:844-854.
Kimmo J. Savinainen, Outir.Saramaki, Marika J.Linja,et al. Expression and gene copy number analysis of erbB2 oncogene in prostate cancer. Am J Pathol, 2002, 16:339-345.
Jan Brahender, Kathleen D.Danenberg, Ralf Metzger et al. Epidermal growth factor receptor and HER2/neu mRNA expression in no-small cell lung cancer is correlated with survival. Clin Cancer Res, 2001, 7:1850-1855.
Kern J. A., Schwartz D. A., Nordberg J. E et al. P185 Neu expression in human lung adenocarcinomas predicts shortened survival. Cancer Res, 1990，50: 5184-5191.
Pfeiffer P., Clausen P. P., Andersen K et al. Lack of prognostic significance of epidermal growth factor receptor and the oncoprotein p185 HER-2 in patients with systemically untreated non-small cell lung cancer: an immunohistochemical study on cryosections. Br J Cancer, 1996，74: 86-91.
Philp S.B, Carl T.W. Real-time PCR technology for cancer dagnostics. Clin Chem, 2002, 48(8):1178-1185.
Stefania Gelmini, Claudio Orlando, Roberta Sestini等，Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification. Clin Chem，1997，43（5）:752-758.
S.A.Bustin et al. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinology，2000, 25:169-193.
Ian M.Mackay, Katherine E. Arden et al. Real-time PCR in virology. Nucl Aci Res，2002, 30(6): 1292-1305.
Bieche I, Onody P, Laurendeau I, et al. Real-time reverse transcription-PCR assay for future management of erbB2-based clinical application. Clin Chem, 1999, 45:1148-1156.
Katja Specht, Thomas Richter et al. Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. Am J Pathol，2001，158:419-429.
Pei-Wen Chiang, David G Beer, Wan-Li Wei et al. Detetion of erbB-2 amplifications in tumors and sera from esophageal carcinoma patients. Clin Cancer Res，1999，June（5）：1381-1386.
Korenberg J R, Hugdins L, McGiuioray B et al. Down syndrome phenotypes:the comsequences of chromosomal imbalance. Proc Natl Acad Sci， 1994，91: 4997-5001.
Chan AT, Lo YM, Zee B et al .Plasma Epstein-Barr Virus DNA and residual
diease after radiotherapy for undifferentiated Nasopharyngeal carcinoma. J Natl Cancer Inst , 2002，94(21):1614-9.
ChiYomi Egawa, Yasua MiYoshi, Kyoko Iwao et al. Quantitative analysis of estrogen receptor-αand –βmRNA expression in normal and malignant thyroid tissues by real-time PCR. Oncology, 2001,61:293-298.
Nieawenhuis EJ, Jaspars LH et al. Quantitative molecular delection of minimal residual head and neck cancer in lymph node. Clin Cancer Res，2003 9(2):755-61.
Sabine glockner, Ulrich lehman, Nadine Willke et al. Amplification of growth regulatory genes in intraductal breast cancer is associated with higher nuclear grade but not with the progression to invasiveness. Lab Invest，2001 81:565-571.
王传新，邹雄，王谦等，血清癌基因C-erbB2蛋白对恶性肿瘤诊断价值的研究。山东大学学报（医学版），2002，40（6）：552-553。
Felip E, Del Campo JM, Rugbio D, et al. Overexpression of c-erbB2 in epithelial ovarian cancer. Cancer, 1995，75:2147.
Yan J, Fang Y, Huang BJ, et al. Absence of evidence for Her-2 amplification in nasopharyngeal carcinoma. Can Gen Cyt, 2002, 132(2):116-9.
Roychowdhury DF, Tseng A, Fu KK, et al，New prognostic factors in nasopharyngeal carcinoma: Tuomor angiogenesis and C-erbB-2 expression. Cancer, 1996, 77(8):1419-1426.
顾康生，吴良秋，侯景辉等，HER-2/NEU基因在鼻咽癌中的表达及其临床意义，癌症，2001，20（8）：869-872。
鄢践，方燕等，原发性鼻咽癌患者Her2基因表达的意义。中华医学杂志，2001，81（15）：904-906。
金鸥，颜亚晖等，鼻咽癌组织c-erbB-2表达与细胞增殖预后的关系。湖南医科大学学报，1998，23：235-238。
金鸥，陈森林等，鼻咽癌及癌旁黏膜组织中c-erbB-2，EGFR mRNA的表达。湖南医科大学学报，1997，22（6），487-490。
罗扬，王金万. Herceptin治疗HER-2过度表达乳腺癌的现状。国外医学肿瘤学分册，2002，29（2）：126-128。
Agus DB, Scher HI, Higgins B et al. Response of prostate cancer to anti-Her-2/Neu antibody in androgen-dependent and -independent human xenograft models. Cancer Res 1999, 59:4761-4764.
Baselga J, Norton L, Albanell J et al. Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/Neu overexpressing human breast cancer xenografts. Cancer Res，1998，58:2825-2831.
于永强，董卫东，任重等，癌基因C-erbB2抑癌基因nm23在鼻咽癌中的表达意义。肿瘤，2002，22（4）：305-307。
赵丽纯，李佳莉等，以HER2/neu为靶点抗肿瘤治疗研究进展，国外医学遗传学分册，2002，25（1）：33-36.
Formenti SC, Spicer D, Skinner K, et al. Low HER2/Neu gene expression is associated with pathological response to concurrent paclitaxel and radiation therapy in locally advanced breast cancer. Int J Radiat Oncol Biol Phys, 2002, 52(2):397-405.
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