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中华乳腺病杂志(电子版)

中华乳腺病杂志(电子版) ›› 2022, Vol. 16 ›› Issue (02) : 84 -90. doi: 10.3877/cma.j.issn.1674-0807.2022.02.003

论著

循环肿瘤细胞分型检测在乳腺癌新辅助化疗中的应用
高娜1, 杨颖涛2,†(), 段奇2, 刘泽森2   
  1. 1. 450053　郑州人民医院乳腺外科
    2. 450052　郑州大学第五附属医院乳腺外科
  • 收稿日期:2020-08-09 出版日期:2022-04-01
  • 通信作者: 杨颖涛

Application of circulating tumor cell typing in neoadjuvant chemotherapy for breast cancer

Na Gao1, Yingtao Yang2,†(), Qi Duan2, Zesen Liu2   

  1. 1. Department of Breast Surgery, Zhengzhou People’s Hospital, Zhengzhou 450053, China
    2. Department of Breast Surgery, Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
  • Received:2020-08-09 Published:2022-04-01
  • Corresponding author: Yingtao Yang
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引用本文：

高娜, 杨颖涛, 段奇, 刘泽森. 循环肿瘤细胞分型检测在乳腺癌新辅助化疗中的应用[J/OL]. 中华乳腺病杂志(电子版), 2022, 16(02): 84-90.

Na Gao, Yingtao Yang, Qi Duan, Zesen Liu. Application of circulating tumor cell typing in neoadjuvant chemotherapy for breast cancer[J/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2022, 16(02): 84-90.

目的

探讨乳腺癌患者新辅助化疗(NAC)后外周血循环肿瘤细胞(CTCs)的变化及其与NAC疗效的关系。

方法

根据纳入、排除标准，选取2018年7月至2019年7月郑州大学第五附属乳腺外科86例行NAC的乳腺癌患者作为研究对象进行回顾性研究。分别采集患者NAC前后的外周静脉血7.5 ml，采用CanPatrol?CTC二代技术检测CTCs数目，并根据上皮-间质转化(EMT)相关标志物的表达情况将检测出的CTCs分为上皮型、间质型和混合型。CTCs数目为偏态分布数据，因此用M(P25, P75)表示。采用χ2检验分析NAC前CTCs与患者临床病理特征的关系，用Wilcoxon符号秩和检验比较各型CTCs数目化疗前后的差异，并以病理学Miller-Payne系统作为疗效评估的金标准，用Kappa一致性检验评估各型CTCs检测结果与病理学金标准评估疗效的一致性。

结果

(1)间质型CTCs与HER-2、Ki-67表达情况密切相关，即HER-2阳性及Ki-67≥15%者其间质型CTCs阳性率高于HER-2阴性及Ki-67<15%者[42.1%(16/38)比12.5%(6/48)，χ2＝9.765，P＝0.002；39.6%(19/48)比7.9%(3/38)，χ2＝11.187，P＝0.001]。(2) NAC前、后相比，CTCs总数目的差异无统计学意义[8.0 (6.0，12.0)个/7.5 ml比9.0 (6.0，13.0)个/7.5 ml，Z＝-1.214，P＝0.225]。根据术后病理检查结果将患者分为NAC治疗有效组(61例)和无效组(25例)，其中NAC有效组患者间质型和混合型CTCs数目均较NAC前明显降低[2.0(1.0，3.0)个/7.5 ml比3.0(2.0，5.0)个/7.5 ml，Z＝-4.914，P<0.001；2.0(1.0，4.5)个/7.5 ml比3.0(2.0，5.0)个/7.5 ml，Z＝-3.197，P＝0.001]，上皮型CTCs数目与NAC前相比，差异无统计学意义[3.0(1.0，5.0)个/7.5 ml比2.0(1.0，4.0)个/7.5 ml，Z＝-0.728, P＝0.467)]。无效组患者间质型CTCs数目较NAC前明显升高[3.0(2.0，6.0)个/7.5 ml比2.0(1.0，4.0)个/7.5 ml，Z＝-2.351，P＝0.019]，上皮型和混合型CTCs数目变化的差异均无统计学意义[2.0(2.0，4.0)比2.0(0.0，4.0)个/7.5 ml，Z＝-0.701，P＝0.438; 3.0(1.5，5.0)个/7.5 ml比2.0(1.0，4.0)个/7.5 ml，Z＝-1.535，P＝0.125)。(3)运用Kappa一致性检验比较上皮型、间质型、混合型CTCs检测结果与病理评估结果的一致性，结果显示Kappa值分别为0.194、0.749、0.376(P均<0.050)，间质型CTCs评估结果与病理评估结果一致性较高。

结论

CTCs总数目的变化趋势不能准确评估NAC疗效，结合CTCs的数目和型别差异才能更好地判断疗效，为调整治疗方案提供参考。

关键词: 乳腺肿瘤, 化学疗法，辅助, 循环肿瘤细胞分型
Objective

To explore the change of circulating tumor cells (CTCs) in peripheral blood after neoadjuvant chemotherapy (NAC) and its relationship with the efficacy of NAC.

Methods

According to the inclusion and exclusion criteria, this study retrospectively included 86 breast cancer patients who underwent NAC in the Department of Breast Surgery, the Fifth Affiliated Hospital of Zhengzhou University from July 2018 to July 2019. Their peripheral venous blood (7.5 ml) was collected before and after NAC. The number of CTCs was measured using CanPatrol?CTC technique. According to the expression of related markers of epithelial-mesenchymal transition (EMT), CTCs were divided into three subtypes: epithelial CTCs, mesenchymal CTCs and biphenotypic (epithelial/mesenchymal) CTCs. The number of CTCs was expressed as M(P25, P75) because of skewed distribution. χ2 test was used to analyze the relationship between CTCs before NAC and clinicopathologic characteristics of patients, and Wilcoxon signed rank sum test was used to compare the number of CTCs in different subtypes before and after chemotherapy. The Miller-Payne pathological grading system was used as the gold standard to evaluate the efficacy. The Kappa test was used to evaluate the consistency between the test results of CTCs in different subtypes and the gold standard in efficacy evaluation.

Results

(1)The mesenchymal CTCs were closely related to the expression of HER-2 and proliferative nuclear antigen Ki-67. The patients with HER-2 positive had a significantly higher positive rate of mesenchymal CTCs compared with patients with HER-2 negative[42.1%(16/38)vs 12.5%(6/48), χ2=9.765, P=0.002]; the patients with Ki-67 ≥15% had a significantly higher positive rate of mesenchymal CTCs compared with patients with Ki-67<15%[39.6%(19/48)vs 7.9%(3/38), χ2=11.187, P=0.001]. (2)The total number of CTCs showed no significant difference before and after NAC[8.0 (6.0, 12.0) cells/7.5 ml vs 9.0 (6.0, 13.0) cells/7.5 ml, Z=-1.214, P=0.225]. According to the results of postoperative pathological examination, the patients were divided into the NAC-effective group (61 cases) and the NAC-ineffective group (25 cases). The number of mesenchymal CTCs and biphenotypic CTCs in the NAC-effective group after NAC was significantly lower than that before NAC[2.0(1.0, 3.0) cells/7.5 ml vs 3.0(2.0, 5.0) cells/7.5 ml, Z=-4.914, P<0.001; 2.0(1.0, 4.5) cells/7.5 ml vs 3.0(2.0, 5.0) cells/7.5 ml, Z=-3.197, P=0.001], while the number of epithelial CTCs showed no significant difference [3.0(1.0, 5.0) cells/7.5 ml vs 2.0(1.0, 4.0) cells/7.5 ml, Z=-0.728, P=0.467]; In the NAC-ineffective group, the number of mesenchymal CTCs after NAC was significantly higher than that before NAC[3.0(2.0, 6.0) cells/7.5 ml vs 2.0(1.0, 4.0) cells/7.5 ml, Z=-2.351, P=0.019], and the number of epithelial and biphenotypic CTCs showed no significant difference [2.0(2.0, 4.0) cells/7.5 ml vs 2.0(0.0, 4.0) cells/7.5 ml, Z=-0.701, P=0.438; 3.0(1.5, 5.0) cells/7.5 ml vs 2.0(1.0, 4.0) cells/7.5 ml, Z=-1.535, P=0.125]. (3)The Kappa test was used to analyze the consistency between the test results of epithelial, mesenchymal and biphenotypic CTCs and the results of pathological evaluation (Kappa=0.194, 0.749, 0.376; all P<0.050), indicating a high consistency between test result of mesenchymal CTCs and pathological result.

Conclusions

In breast cancer patients, the change in total number of CTCs can not accurately evaluate the efficacy of NAC. Based on the number and subtype of CTCs, clinicians can make a better judgment on the efficacy of NAC and adjust the treatment plan accordingly.

Key words: Breast neoplasms, Chemotherapy, adjuvant, Circulating tumor cell typing
图1 乳腺癌患者NAC前外周血CTCs检测　a图所示上皮型CTCs；b图所示间质型CTCs；c图所示混合型CTCs注：红色荧光标记上皮型CTCs表达的EpCAM、CK8、CK18、CK19标志物；绿色荧光标记间质型CTCs表达的vimentin、twist标志物；CTCs为循环肿瘤细胞；EpCAM为上皮细胞黏附分子；CK为细胞角蛋白；NAC为新辅助化疗
表1 乳腺癌患者NAC前的临床病理特征与CTCs阳性率的关系[例(%)]
临床病理特征 例数 上皮型CTCs χ2值 P值 间质型CTCs χ2值 P值 混合型CTCs χ2值 P值
阳性 阴性 阳性 阴性 阳性 阴性
年龄                          
  ≤50岁 37 6(16.2) 31(83.8) 0.872 0.350 12(32.4) 25(67.6) 1.601 0.206 10(27.0) 27(73.0) 0.003 0.959
  >50岁 49 12(24.5) 37(75.5) 10(20.4) 39(79.6) 13(26.5) 36(73.5)
ER                          
  阳性 47 7(14.9) 40(85.1) 2.282 0.131 11(23.4) 36(76.6) 0.258 0.611 15(31.9) 32(68.1) 1.414 0.234
  阴性 39 11(28.2) 28(71.8) 11(28.2) 28(71.8) 8(20.5) 31(79.5)
PR                          
  阳性 40 10(25.0) 30(75.0) 0.748 0.387 12(30.0) 28(70.0) 0.767 0.318 11(27.5) 29(72.5) 0.022 0.883
  阴性 46 8(17.4) 38(82.6) 10(21.7) 36(78.3) 12(26.1) 34(73.9)
HER-2                          
  阳性 38 6(15.8) 32(84.2) 1.087 0.297 16(42.1) 22(57.9) 9.765 0.002 9(23.7) 29(76.3) 0.325 0.568
  阴性 48 12(25.0) 36(75.0) 6(12.5) 42(87.5) 14(29.2) 34(70.8)
Ki-67                          
  ≥15% 48 12(25.0) 36(75.0) 1.087 0.297 19(39.6) 29(60.4) 11.187 0.001 9(18.7) 39(81.3) 3.544 0.060
  <15% 38 6(15.8) 32(84.2) 3(7.9) 35(92.1) 14(36.8) 24(63.2)
肿瘤直径                          
  ≥5 cm 44 9(20.5) 35(79.5) 0.012 0.912 11(25.0) 33(75.0) 0.016 0.899 11(25.0) 33(75.0) 0.140 0.708
  <5 cm 42 9(21.4) 33(78.6) 11(26.2) 31(73.8) 12(28.6) 30(71.4)
表2 循环肿瘤细胞评估乳腺癌患者不同化疗方案的疗效[例(%)]
组别 例数 有效 无效
AC-T组 46 33(71.7) 13(28.3)
ACT组 40 31(77.5) 9(22.5)
χ2值   0.373
P值   0.541
表3 术后病理检查评为有效的61例乳腺癌患者NAC治疗前、后各型CTCs水平[个/7.5 ml，M(P25，P75)]
组别 上皮型CTCs 间质型CTCs 混合型CTCs
化疗前 2.0(1.0，4.0) 3.0(2.0，5.0) 3.0(2.0，5.0)
化疗后 3.0(1.0，5.0) 2.0(1.0，3.0) 2.0(1.0，4.5)
Z值 -0.728 -4.914 -3.197
P值 0.467 <0.001 0.001
表4 术后病理检查评为无效的25例乳腺癌患者NAC治疗前、后各型CTCs水平[个/7.5 ml, M(P25，P75)]
组别 上皮型CTCs 间质型CTCs 混合型CTCs
化疗前 2.0(0.0，4.0) 2.0(1.0，4.0) 2.0(1.0，4.0)
化疗后 2.0(2.0，4.0) 3.0(2.0，6.0) 3.0(1.5，5.0)
Z值 -0.701 -2.351 -1.535
P值 0.438 0.019 0.125
表5 乳腺癌患者NAC后各型CTCs疗效评价与病理评估的一致性分析(例)
CTCs疗效评价 病理评价 Kappa值 P值
有效 无效
上皮型CTCs        
  有效 30 6 0.194 0.032
  无效 31 19
间质型CTCs        
  有效 56 4 0.749 <0.001
  无效 5 21
混合型CTCs        
  有效 43 7 0.376 <0.001
  无效 18 18
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