逆转录环介导等温扩增CK-19基因判断乳腺癌哨兵淋巴结转移的研究
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摘要
乳腺癌患者的腋窝淋巴结状况是一个重要的治疗及预后指标,腋窝淋巴结的清除被当作乳腺癌手术治疗的一个标准步骤。目前的研究显示哨兵淋巴结活检(sentinel lymph node biopsy,SLNB)能够替代腋窝淋巴结清扫术,如果哨兵淋巴结阳性,则行腋窝淋巴结清扫,所以对哨兵淋巴结的检测非常重要,本实验的目的是建立一种快速、准确、客观的判断乳腺癌哨兵淋巴结转移的检测方法,以指导临床治疗、提高患者的生活质量。
方法:
1、通过美蓝染色法获取30例早期乳腺癌患者的哨兵淋巴结,将淋巴结均分为四份(a、b、c、d),a、c送常规病理学检查,b、d标号放置于-80℃冰箱冻存。
2、培养乳腺癌细胞系及淋巴瘤细胞系的细胞,进行环介导的等温扩增反应检测CK-19基因表达的差异性。
3、在环介导的等温扩增反应基础上加入逆转录酶,对病理学阳性的淋巴结进行检测,验证该方法的可行性。
4、优化条件、缩短反应时间,并批量验证。
结果:
1、30例患者共检出哨兵淋巴结76枚,其中阳性淋巴结23枚,平均每例检出2.53枚(76/30),阳性率30.3%(23/76)。
2、CK-19基因在乳腺癌等上皮来源的肿瘤中特异性表达,在正常淋巴结中检测到CK-19基因表明有乳腺癌肿瘤细胞转移。
3、三枚阳性淋巴结行逆转录环介导的等温扩增反应均能检测到CK-19基
因,表明有肿瘤转移,与病理切片HE染色检查结果吻合。4、优化实验条件后可以使总体反应时间小于1h,批量验证结果显示逆转录环介导等温扩增CK-19基因检测哨兵淋巴结的阳性率为44.7%,病理切片HE染色为30.3%,相比之下前者提高了14.4%。
结论:
通过逆转录环介导的等温扩增反应扩增CK-19基因,可以快速、准确、客观地判断乳腺癌患者哨兵淋巴结状况,具备良好的应用前景。
The axillary lymph node condition of breast cancer patient is a vital treatment and prognosis indicator,the clearation of axillary lymph node is considered standard procedure of breast cancer's surgical treatment。Present research reveals that sentinel lymph node biopsy can substitute axillary lymph node dissection,if the sentinel lymph node is positive,the axillary lymph node is cleared ,so the examination of sentinel lymph node is very important,the purpose of this experiment is to establish a rapid、accurate and objective examination method of judgeing breast cancer sentinel lymph node metastasis,helping instruct the clinical treatment and improve the quality of life。
Method:
1、Get the sentinel lymph node of 30 early breast cancer patients by the method of methylene blue,divide the lymph node into four parts(a、b、c、d) equally,a and c are used for conventional pathology examination,b and d are marked and frozed under the condition of -80℃。
2、Culture the cell of breast cancer cell line and lymphoma cell line,detect the variability of CK-19 expession by LAMP。
3、Add the reverse transcriptase at the base of LAMP,detcet the lymph node which pathology proves positive,verify the possibility of this way。
4、Improve the method of experiment,shorten the time of reaction,batch confirmation。
Result:
1、There are 76 sentinel lymph nodes detected from thirty patients,which the positive is 23,the average number is 2.53(76/30), the positive incidence is 30.3%(23/76)。
2、The breast cancer which origins from epidermis specifically express CK-19,the detection of CK-19 from normal lymph node means breast cancer metastasis。
3、CK-19 can be detected from 3 positive lymph nodes by RT-LAMP,meaning tumor metastasis,the result accords with pathological section HE chromoscopy examination。
4、The total reaction can be finished in 1 hour by improving experiment method,this experiment reveals that the positive incidence of sentinel lymph node which is detected by RT-LAMP is 44.7%, increasing 14.4% in contrast with pathological section HE chromoscopy examination's 30.3%。
Conclusion:
The reaction of RT-LAMP detecting CK-19 can rapidly、accurately and objectively judge the condition of sentinel lymph node,showing favourable application prospect。
方法:
1、通过美蓝染色法获取30例早期乳腺癌患者的哨兵淋巴结,将淋巴结均分为四份(a、b、c、d),a、c送常规病理学检查,b、d标号放置于-80℃冰箱冻存。
2、培养乳腺癌细胞系及淋巴瘤细胞系的细胞,进行环介导的等温扩增反应检测CK-19基因表达的差异性。
3、在环介导的等温扩增反应基础上加入逆转录酶,对病理学阳性的淋巴结进行检测,验证该方法的可行性。
4、优化条件、缩短反应时间,并批量验证。
结果:
1、30例患者共检出哨兵淋巴结76枚,其中阳性淋巴结23枚,平均每例检出2.53枚(76/30),阳性率30.3%(23/76)。
2、CK-19基因在乳腺癌等上皮来源的肿瘤中特异性表达,在正常淋巴结中检测到CK-19基因表明有乳腺癌肿瘤细胞转移。
3、三枚阳性淋巴结行逆转录环介导的等温扩增反应均能检测到CK-19基
因,表明有肿瘤转移,与病理切片HE染色检查结果吻合。4、优化实验条件后可以使总体反应时间小于1h,批量验证结果显示逆转录环介导等温扩增CK-19基因检测哨兵淋巴结的阳性率为44.7%,病理切片HE染色为30.3%,相比之下前者提高了14.4%。
结论:
通过逆转录环介导的等温扩增反应扩增CK-19基因,可以快速、准确、客观地判断乳腺癌患者哨兵淋巴结状况,具备良好的应用前景。
The axillary lymph node condition of breast cancer patient is a vital treatment and prognosis indicator,the clearation of axillary lymph node is considered standard procedure of breast cancer's surgical treatment。Present research reveals that sentinel lymph node biopsy can substitute axillary lymph node dissection,if the sentinel lymph node is positive,the axillary lymph node is cleared ,so the examination of sentinel lymph node is very important,the purpose of this experiment is to establish a rapid、accurate and objective examination method of judgeing breast cancer sentinel lymph node metastasis,helping instruct the clinical treatment and improve the quality of life。
Method:
1、Get the sentinel lymph node of 30 early breast cancer patients by the method of methylene blue,divide the lymph node into four parts(a、b、c、d) equally,a and c are used for conventional pathology examination,b and d are marked and frozed under the condition of -80℃。
2、Culture the cell of breast cancer cell line and lymphoma cell line,detect the variability of CK-19 expession by LAMP。
3、Add the reverse transcriptase at the base of LAMP,detcet the lymph node which pathology proves positive,verify the possibility of this way。
4、Improve the method of experiment,shorten the time of reaction,batch confirmation。
Result:
1、There are 76 sentinel lymph nodes detected from thirty patients,which the positive is 23,the average number is 2.53(76/30), the positive incidence is 30.3%(23/76)。
2、The breast cancer which origins from epidermis specifically express CK-19,the detection of CK-19 from normal lymph node means breast cancer metastasis。
3、CK-19 can be detected from 3 positive lymph nodes by RT-LAMP,meaning tumor metastasis,the result accords with pathological section HE chromoscopy examination。
4、The total reaction can be finished in 1 hour by improving experiment method,this experiment reveals that the positive incidence of sentinel lymph node which is detected by RT-LAMP is 44.7%, increasing 14.4% in contrast with pathological section HE chromoscopy examination's 30.3%。
Conclusion:
The reaction of RT-LAMP detecting CK-19 can rapidly、accurately and objectively judge the condition of sentinel lymph node,showing favourable application prospect。
引文
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[12] Menes TS,Tartter PI,Mizrachi H,et al. Touch preparation or frozen section for intraoperative detection of sentinel lymph node metastases from breastcancer[J].Ann Surg Oncol,2003,10(10):1166—1170.
[13] Turner RR,Ollila DW,Krashe DL, et a1.Histopathological validation of the sentinel lymph node hypothesis for breast cancer.Ann Surg,1997,226:271-278.
[14] Smidt ML,Besseling R, Wauters CA,et a1.Intraoperative scrape cytology of the sentinel lym ph node in patients with breast cancer.Br J Surg,2002,89:1290—1293.
[15] Salem AA,Douglas-Jones AG,Sweefland HM,et a1.Intraoperaive evaluation of axillary sentinel lymph nodes using touch imprint cytology and immunohistochemistry :A protocolof rapid immunostaining of touch imprints.Eur J Surg Oncol,2003,29:25—28.
[16] Peley G,Toth J,Csuka,et a1.Immunohistochemistry and reverse transcriptase polymerase chain reaction on sentinel lymph nodes call improve the accuracy of nod al staging in breast cancer patients.Int J Biol Markers,2001,16:227—232.
[17] American Joint Committ on Cancer.Cancer Staging Atlas.New York,NY,Springer,2006.
[18] Whiting S H,Champoux J j.Properties of strand displacement synthesis by Moloney murine leukemia virus reverse transcriptase:mechanistic imp1ications[J].J Mol Biol,1998,278(3):559-577.
[19] Tsujimoto M, Nakabayashi K, Yoshidome K, Kanedo T, Iwase T, Akiyama F, Kato Y, Tsuda H, Ueda S, Sato K, Tamaki Y, Noguchi S,et al. One-step nucleic acid amplification (OSNA) for intraoperative detection of lymph node metastasis in breast cancer patients. Clin Can Res 2007;13:4807–16.
[20] Cox C E, Kiluk J V, Riker A I, et al. Significance of sentinel lymph node micrometastases in human breast cancer. J Am Coll Surg, 2008, 206: 261 - 268.
[21] S.Alberti,P.Querzoli,M.Pedriali,etal.Axillary lymph node nanometastases are prognostic factors for metastatic relapse in breast cancer patients. J Clin Oncol 2006:610.
[22] David J.Dabbs,Mark Fung,Douglas Landsittel, etal. Sentinel Lymph Node Micrometastasis as a Predictor of Axillary Tumor Burden. Breast J 2004;10:101-105.
[23] Kristine E.Calhoun,Nora M.Hansen,Roderick R.Turner etal.Nonsentinel node metastases in breast cancer patients with isolated tumor cells in the sentinel node:implications for completion axillary node disseetion . Am J Surg 2005 : 190:588-591.
[24] Lyman G H, Giuliano A E, SomergieldM R, et al. American Society of Clinical Oncology guideline recommendations for sentinel lymph node biop sy in early stage breast cancer. J Clin Oncol, 2005, 23: 7703 - 7720.
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