摘要
乳腺癌是女性发病率和死亡率最高的恶性肿瘤，肿瘤复发和转移仍是导致患者死亡的首位原因。存在于患者血液、骨髓及淋巴结中不被常规手段检出的微转移正是导致患者复发、转移的重要原因。外周血标本的检测具有创伤小、便于重复采集等优势，血液中的微转移即循环肿瘤细胞(circulating tumor cell，CTC)是乳腺癌发生远处转移的重要中间环节，检测CTC并探讨其意义已成为当前临床研究的热点。既往研究显示，检测并定期监测外周血CTC水平及变化趋势有助于判断患者预后、评估治疗方案，为临床决策提供更确切的参考信息。已有研究表明，手术切除原发肿瘤会增加肿瘤细胞脱落入血风险，而乳腺癌不同手术方式致血液微转移风险是否存在差异尚未见报道。同时，有关CTC能否有效预测、评估乳腺癌辅助治疗近期疗效的研究结论尚存在较大争议。为此，本课题拟对上述问题进行初步探讨。
实验方法和主要结果
1.循环肿瘤细胞评估乳腺癌手术致微转移风险差异的临床初步研究
方法：110例(常规开放手术组57例；腔镜手术组53例)可手术女性乳腺癌患者分别于术前和术后12小时采集静脉血5ml，利用密度梯度离心法富集单个核细胞，通过荧光定量PCR技术检测所获单个核细胞中细胞角蛋白(cytokeratin, CK)19mRNA表达阳性的CTC。对比分析两组手术前后CTC检出率、检出数目及变化趋势是否存在差异。
结果：两组基线信息具有可比性。常规开放手术组手术前后CTC检出率分别为22.81%(13/57)、33.33%(19/57)(P=0.211)，CTC检测数目分别为0.21(0-15.16)MCF-7细胞/2μg RNA、0.43(0-9)MCF-7细胞/2μg RNA(P=0.123)，血液微转移风险增加者占29.8%(17/57)；腔镜手术组手术前后CTC检出率分别为24.53%(13/53)、28.3%(15/53)(P=0.659)，CTC检测数目分别为0.27(0-26.49)MCF-7细胞/2μg RNA、0.36(0-10.56)MCF-7细胞/2μg RNA (P=0.717)，血液微转移风险增加者占13.2%(7/53)。两组术后CTC检出率及检出数目均较术前有增高趋势，但差异尚不具统计学意义；组间比较，常规开放手术致CTC检出率及检出数目增加趋势高于腔镜手术，但差异也不具统计学意义；常规开放手术较腔镜手术增加血液微转移的风险更高(χ2=4.446，P=0.035；OR=2.79，95%CI1.05-7.42)；不同手术方式致血液微转移风险差异与患者绝经状态、区域淋巴结状态、肿瘤分期、HER-2表达状态等临床病理因素均不相关，仅与患者激素受体状态有关。
2.循环肿瘤细胞评估乳腺癌辅助治疗近期疗效的Meta分析
方法：计算机检索PubMed、Web of Science、EMBASE、中国生物医学文献数据库、中国期刊全文数据库等医学文献数据库截止至2012年4月公开发表的文献，同时辅助手工检索参考文献列表中的遗漏文献及圣安东尼奥乳腺癌大会、美国临床肿瘤协会、美国癌症研究学会2000年~2011年年会的会议论文，筛选有关CTC预测、评估乳腺癌辅助治疗近期疗效的临床研究。对符合筛选条件的研究进行资料提取及质量评价后，采用Review Manager5.0软件进行Meta分析，并采用GRADE系统对证据质量进行评价。
结果：共纳入24项研究，实际纳入分析乳腺癌患者共1551人，其中，评价辅助治疗前基线CTC水平与辅助治疗总有效率、临床获益率关系的文献分别为18篇、10篇，纳入分析患者984人、537人；评价辅助治疗期间/治疗结束后CTC水平与辅助治疗TRR、CBR关系的文献分别为13篇、7篇，纳入分析患者795人、462人。Meta分析结果显示：辅助治疗前以及治疗期间/治疗结束检测CTC阳性乳腺癌患者的近期总有效率分别是CTC阴性患者的75%(RR=0.75，95%CI0.63-0.89，P=0.001)和31%(RR=0.31，95%CI0.22-0.45， P<0.00001)，临床获益率分别是CTC阴性患者的73%(RR=0.73，95%CI0.59-0.92，P<0.00001)和53%(RR=0.53，95%CI0.42-0.67，P<0.00001)，提示乳腺癌辅助治疗前后外周血CTC检测结果阴性的乳腺癌患者，其近期总有效率和临床获益率均优于阳性患者，显示出更好的临床获益，且结果比较，差异具有统计学意义。GRADE系统评价结果显示，辅助治疗前以及治疗期间/治疗结束患者CTC水平与近期总有效率关系的证据评价水平均为B，辅助治疗前、治疗期间/治疗结束患者CTC水平与近期临床获益率关系的证据评价水平分别为D和C，提示乳腺癌辅助治疗前外周血CTC的检测结果对预测近期疗效的参考价值更高。
结论
1.乳腺癌手术有增加患者外周血CTC水平的趋势，常规开放手术较腔镜手术增加血液微转移的风险更高。
2.乳腺癌患者辅助治疗前后外周血CTC检测结果能够预测、评估乳腺癌辅助治疗近期疗效，辅助治疗前CTC检测结果对预测近期疗效的参考价值更高。
Breast cancer is by far the most common malignancy in women and it is ranked as theleading cause of cancer death. However,tumor relapse and metastasis are still the primaryreason for mortality, which mainly result from micrometastasis undetected by conventionalapproaches in blood, bone marrow or lymph nodes. Recent clinical research has focused ondetection and exploration of micrometastasis in the form of circulating tumor cells (CTC) inperipheral blood, which significantly contributes to distant metastasis. Results from studieshave showed a clinical value of detecting and monitoring levels of CTC for evaluation ofprognosis and therapeutic regime, as well as providing reference information for clinicaldecision-making. Previous studies have demonstrated surgery can contribute to the seeding ofcancer cells, while there has been no research on difference in different surgical approaches.Meanwhile, there still have been controversies on the role of CTC detection in predicting theeffect of treatment. Accordingly, the aim of this pilot study was to evaluate the effect of thesurgical approach on differentiation of micrometastatic risk correlated to surgery and thepredictive and prognostic value of CTC for assessing response to breast cancer relatedadjuvant therapies.
Methods&Results
Part I. Decreased risk of circulating tumor cells after endoscopic breast surgerythan open surgery for breast cancer
Methods:. Pre-and postoperative (12h after surgery) peripheral blood samples (5ml)obtained from110female patients with operable breast cancer (53patients underwentendoscopic breast surgery and57patients underwent open radical mastectomy) were enrichedby density gradient centrifugation for mononuclear cells and then detected by quantitativereal-time reverse transcription-PCR (qPCR) for cytokeratin19mRNA-positive CTC. The difference in detection rate, median levels expressed as MCF-7cell equivalents/2μg RNA ofCK19mRNA-positive CTC, micrometastatic risk on surgery between endoscopic breastsurgery and open surgery were compared and analyzed.
Results: There was no significant difference in clinicopathologic characteristics betweenthese two groups. In the open-group, the positive rate of CTC before and after surgery were22.81%(13/57) and33.33%(19/57)(P=0.211), the median levels of CK19mRNA-positiveCTC expressed as MCF-7cell equivalents/2μg RNA before and after surgery were0.21(0-15.16) and0.43(0-9)(P=0.123) and17patients (29.8%) had increased micrometastaticrisk related to surgery; In the endoscopic-group, the positive rate of CTC before and aftersurgery were24.53%(13/53) and28.3%(15/53)(P=0.659), the median levels of CK19mRNA-positive CTC expressed as MCF-7cell equivalents/2μg RNA before and after surgerywere0.27(0-26.49) and0.36(0-10.56)(P=0.717), respectively, and7patients (13.2%) hadincreased micrometastatic risk related to surgery.The results showed a higher clinical but notstatistical elevation in postoperative CTC detection rate and median levels in the open-groupcompared with the endoscopic-group.An increased micrometastatic risk caused by operationwas observed in the open-group as compared to the endoscopic approach (OR=2.79,95%CI1.05-7.42), and the difference of micrometastatic risk related to surgery between these twogroups correlated with the status of hormone receptors, not the status of menopause, lymphnode, tumor stage or HER-2.
Part II. Predictive and prognostic value of circulating tumor cells for assessingresponse to breast cancer related adjuvant therapies: a meta-analysis
Methods: All eligible studies assessing the predictive and prognostic value of CTC forevaluating tumor response to breast cancer related adjuvant therapies were identified bysearch of medical databases including PubMed, Web of Science, EMBASE, and ChineseBiomedical Literature database and China Academic Literature Full-text Database for theperiod up to April2012, and the reference lists of identified studies, conference proceedingsfrom San Antonio Breast Cancer Symposium, annual meetings of American Society ofClinical Oncology and American Association for Cancer Research from2000to2011werereviewed as a augmented searching. The quality of all included studies were assessed and thedata were extracted. A meta-analysis was performed with RevMan5.0software, and then theGRADE System was used to evaluate the level of evidence.
Results: Among the24studies involving1551cases included, there were984cases of18studies involved in the analysis of total response rate (TRR) and537cases of10studies inclinical benefit response (CBR) correlated with the detection of CTC prior to adjuvant therapy,795cases of13studies involved in the analysis of TRR and462cases of7studies in CBRrelated to the detection of CTC during/at the end of adjuvant therapy.The results of thismeta-analysis showed a higher TRR in the group of patients with CTC-negative result thanCTC-positive both prior to treatment (RR=0.75，95%CI0.63-0.89，P=0.001) and during/at theend of therapy (RR=0.31，95%CI0.22-0.45，P<0.00001), there was also a higher CBR in thegroup of patients with CTC-negative result than CTC-positive both prior to treatment(RR=0.73，95%CI0.59-0.92，P<0.00001) and during/at the end of therapy (RR=0.53，95%CI0.42-0.67，P<0.00001), which indicated a better TRR and CBR could be obtained in patientswith a negative result of CTC detection before and after therapy than positive ones.Accordingto GRADE system, the levels of evidence in the relationship between TRR and CTC detectionresult before and during/at the end of therapy were all Grade B, which were Grade D and Cfor the evidence in the relationship between CBR and CTC detection result before andduring/at the end of therapy, respectively. suggesting a more predictive and prognostic valueof CTC detection result for assessing response to breast cancer related adjuvant therapies.
Conclusions
1. CTC tends to be detected in patients with operative breast cancer after surgery, andthere is an increased micrometastatic risk on surgery in the open-group as compared to theendoscopic breast surgery.
2. CTC might have a clinically valuable predictive and prognostic power in patients withbreast cancer to evaluate tumor response to adjuvant therapies, and the presence of CTC priorto therapy or not might provide more effective reference information.
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