乳腺癌患者外周血循环肿瘤细胞分子学检测方法的对比研究

Comparison of three different molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer

目的采用3种不同分子学方法检测乳腺癌患者外周血循环肿瘤细胞（CTC），并进行对比研究。方法回顾性研究。选自2011年1月至2014年6月四川省人民医院门诊、体检和住院部的30名健康女性体检者、71例良性乳腺疾病患者和167例乳腺癌患者，其中83例早期原发性乳腺癌、84例转移性乳腺癌。采用3种分子学方法检测CTC，单一逆转录荧光定量聚合酶链反应（RT．qPCR）检测B细胞淋巴瘤／白血病-2（BCL-2）；多重RT-qPCR检测BCL．2、原癌基因人类表皮生长因子受体2（HER-2）和人乳腺珠蛋白（HMAM）；AdnaTest试剂盒检测上皮细胞黏附分子（GA733-2）、黏蛋白1（MUC1）和HER-2。采用卡方检验比较各组问基因标志物表达阳性率，Kappa检验评估方法之间的一致性。结果（1）单一RT-qPCR、AdnaTest试剂盒和多重RT-qPCR检测早期乳腺癌阳性率分别为13．3％、16．9％和18．1％，检测转移性乳腺癌分别为31．0％、42．9％和35．7％。3种分子学方法检测健康体检者CTC阳性率分别与良性乳腺疾病和早期原发性乳腺癌比较，差异有统计学意义（检验值分别为4．235和4．301；5．367和5．474；5．894和6．023，均P〈0．05）；良性乳腺疾病的CTC阳性率和早期原发性乳腺癌比较，差异无统计学意义（检验值分别为0．891、0．748和0．701，均P〉0．05）；转移性乳腺癌的CTC阳性率与健康体检者、良性乳腺疾病及早期原发性乳腺癌比较，差异均有统计学意义（检验值8．429、7．553和7．061；10．24、9．025和8．745；9．658、8．417和8．201；均P〈0．05）。（2）在早期原发性乳腺癌中，AdnaTest与单一RT-qPCR、多重RT-qPCR的一致性分别为79．5％、77．1％，均不存在相关性（检验值分别为1．065和1．871，P分别为0．371和0．258）；单一RT-qPCR和多重RT-qPCR的一致性为80．7％，不存在相关性（检验值2．814，P=0．156）。（3）在转移性乳腺癌中，AdnaTest与单一RT-qPCR、多重RT-qPCR的一致性分别为78．6％、80．9％，均存在相关性（检验值分别为10．986和9．251，P分别为0．002和0．005）；单一RT-qPCR和多重RT-qPCR的一致性为88．1％，存在相关性（检验值12．364，P=0．001）。结论不同分子学方法对早期原发性乳腺癌CTC检出率不具有相关性，而在转移性乳腺癌中具有相关性，提示临床采用分子学方法检测的乳腺癌CTC结果辅助诊断和治疗乳腺癌时，应考虑CTC数量和其本身的异质性造成CTC检出率的差异。

Objective Comparison of three different molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer. Methods The retrospective study compared three different molecular assays to detect CTC in the peripheral blood of 30 healthy individuals and 71 benign breast disease patients and 83 early and 84 metastatic breast cancer patients. All samples were collected at the outpatient, inpatient and physical examination department of Sichuan Provincial People＇sHospital from January 2011 to June 2014. The same cDNAs were analyzed by： singleplex RT-qPCR assay for BCL-2, multiplex RT-qPCR for BCL-2, HER-2, HMAM, and a commercially available molecular assay （AdnaTest BreastCancer） for GA733-2, MUC-1, HER-2. The positive of CTC were compared among healthy individuals and benign breast disease patients and breast cancer patients. Chi square test was used to compare the expression of gene markers among the three groups, and the agreement of Kappa test was used to evaluate the method. Results （ 1 ） Detection rates of early breast cancer by single RT-qPCR, Adna kits and multiple RT-qPCR were 13.3% , 16. 9% and 18. 1% , respectively, and the detection of metastatic breast cancer were 31.0%, 42. 9% and 35.7%, respectively. There were significant differences in the positive of CTC by three molecular assays between healthy individuals and benign breast disease patients and early breast cancer patients （ The test values were 4. 235 and 4. 301, 5. 367 and 5. 474, 5. 894 and 6. 023 respectively, P 〈 0.05 ）. There were no differences between benign breast disease patients and early breast cancer patients （ The test values were 0. 891,0. 748 and 0. 701 respectively, P 〉 0. 05 ） . There were significant differences between metastasis breast cancer patients and healthy individuals and benign breast disease patients and early breast cancer patients （The test values were 8. 429,7. 553 and 7. 061 ; 10. 24, 9.025 and 8.745; 9.658, 8.417 and 8.201 respectively,P 〈0.05）. （2） In early breast cancer： The concordance between AdnaTest and single RT-qPCR was 79. 5% while between AdnaTest and multiplex RT- qPCR was 77. 1%. No agreement was found among them （The test values were 1. 065 and 1. 871, P were 0. 371 and 0. 258）. The concordance between single RT-qPCR and multiplex RT-qPCR was 80. 7%. No agreement was found between them （The test values was 2. 814, P was 0. 156）. （3） In patients with overt metastasis： The concordance between AdnaTest and single RT-qPCR was 78.6% （The test values was 10. 986）. While between AdnaTest and multiplex RT-qPCR was 80. 9% （The test values was 9. 251 ）. Agreements were found among them （P was 0. 002 and 0. 005 respectively）. The concordance between single and multiplex RT-qPCR was 88. 1% （The test values was 12. 364）. Agreement was found between them （P was 0. 001 ）. Conclusions No correlations were found among different molecular methods to detect CTC in the early primary breast cancer, but correlations were found in the metastatic breast cancer, suggesting that different rate of CTC caused by the number of CTC and its heterogeneity should be considered to the clinical diagnosis and treatment of breast cancer while molecular method is used.