循环肿瘤细胞与中、西医治疗肺癌患者疗效及预后的相关性研究
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摘要
绝大多数死于恶性肿瘤的患者,死因为远处转移的出现和进展。恶性肿瘤转移的理论有多种,现在仍然没有定论,但是不可否认的是,远处转移必然经过的是肿瘤细胞通过循环系统的播散。播散进入循环系统中的肿瘤细胞,我们称之为循环肿瘤细胞(CTCs, circulating tumor cells),正在吸引越来越多肿瘤研究者的兴趣。近十几年,随着循环肿瘤细胞检测技术的不断发展,多项研究表明循环肿瘤细胞的检测在实体瘤的诊断、分期、预后、个体化治疗、肿瘤细胞特征研究等方面具有极大的潜力。
肺癌是临床常见的恶性肿瘤,发病率和死亡率在世界范围内已经逐渐升至首位,给人们的健康和国家经济发展都带来了很大的威胁。肺癌治疗发展到今天,传统的治疗手段得到了新的发展,新的治疗手段也纷纷涌现,在综合治疗和个体化治疗日益深入人心的今天,肿瘤本身的异质性和患者个体状况的复杂性考验着肿瘤医生对治疗手段和策略的选择。作为肺癌治疗的重要手段之一,多项研究证实中医药参与治疗能有效延长患者生存时间、减少或延缓术后复发、改善生活质量以及减轻临床症状。为了探索循环肿瘤细胞检测在中医药参与治疗晚期非小细胞肺癌患者中的地位和作用,本研究应用一种新发展的循环肿瘤细胞检测技术,进行了初步的实验室和临床的研究。
目的：①检测免疫磁珠阴性分选及免疫荧光染色技术对肺癌细胞系A549细胞富集的效率、稳定性和特异性,并进一步应用该技术进行临床实验以研究以下几个问题。②探讨晚期非小细胞肺癌患者循环肿瘤细胞阳性率和数量与各因素的关系。③探讨治疗前和第一周期治疗后循环肿瘤细胞数量的变化与两周期中医或中西医结合治疗后影像学评价之间的关系。④探讨循环肿瘤细胞数量对接受中医或中西医结合治疗的晚期非小细胞肺癌患者总生存时间(OS)和疾病无进展生存时间(PFS)的预测价值。
方法：①实验室研究：抽取20份健康志愿者提供的外周静脉血,每份7ml,随机等分为2组,每组10人份,两组各加入10个以内、10-100个之间的荧光标记的肺癌细胞系A549细胞。经免疫磁珠阴性分选后计数相应回收的肿瘤细胞数目,计算该技术对A549细胞的富集效率及稳定性。另抽取10位非肿瘤患者外周静脉血,经免疫磁珠阴性分选及免疫荧光染色技术计数,检验该技术对非肿瘤患者检测的特异性。②临床研究：中国中医科学院广安门医院肿瘤科住院的晚期非小细胞肺癌患者,在接受中医治疗或中西医结合治疗前、一周期治疗后进行两次循环肿瘤细胞检测,同期记录患者的肿瘤标记物、功能状态评分、中医辨证分型等信息；两周期后影像学评价疗效；随后进行随访,判定患者的总生存时间和疾病无进展生存时间,最后统计相关数据得出结论。
结果：①10个以内(101个)细胞组,平均富集效率为83.00%±8.23%,10-100个(102个)细胞组,平均富集效率为84.80%±4.44%,P=0.55；加入细胞数量和富集细胞数量之间线性相关Pearson相关系数为0.998,P<0.01；线性回归分析R2=0.996,线性回归方程：Y=-0.545+0.873X,P=0.000；10例进行特异性检验的非肿瘤患者,均未检测出循环肿瘤细胞的存在。②63例入组的患者,中医组33人,中西医结合组30人。从基线循环肿瘤细胞检测结果来看,共50例检测结果为阳性,阳性率为79.4%；最多检测出18个肿瘤细胞,总体数量均值为5.75±4.71个。其中,ⅢB期和Ⅳ期患者循环肿瘤细胞阳性率分别为59.1%(13人)和90.2%(37人),P=0.007；均值分别为2.73±3.58个和7.37±4.48个,P=0.000。原发灶直径≥3cm和<3cm的患者,阳性率分别为85.4%(35人)和68.2%(15人),P=0.190；均值分别为6.71±4.72个和3.95±4.25个,P=0.026。病理分化程度为高分化、中分化、低分化和未分化的患者,均值分别为3.25±4.17个、4.24±3.65个、6.24±4.34个和8.29±5.46个,P=0.019；LSD法两两比较,高分化和未分化(P=0.010)、中分化和未分化(P=0.007)组间均值差别有统计学意义。功能状态评分(Kps)为60分、70分、80分和90分的患者,均值分别为8.54±5.11个、6.50±4.50个、5.32±4.46个和2.54±3.07个,P=0.008；LSD法两两比较,60分的患者与80分(P=0.045)和90分(P=0.001)的患者比较,基线循环肿瘤细胞均值差异有统计学意义；70分的患者与90分(P=0.016)的患者,循环肿瘤细胞均值差异有统计学意义。③第一周期治疗后与基线比较,循环肿瘤细胞数量变化和肿瘤标记物积分变化与金标准影像学评价之间的一致性检验采用ROC曲线检验。53例循环肿瘤细胞变化可评价的患者,ROC曲线下面积为0.755,P=0.002；46例肿瘤标记物积分可评价者,ROC曲线下面积为0.683,P=0.035；40例肿瘤标记物积分和循环肿瘤细胞均可评价的患者,循环肿瘤细胞ROC曲线下面积为0.734,P=0.012；肿瘤标记物ROC曲线下面积为0.730,P=0.014；16例初治且循环肿瘤细胞可评价患者,ROC曲线下面积为0.865,P=0.015；14例初治且肿瘤标记物和循环肿瘤细胞均可评价患者,循环肿瘤细胞ROC曲线下面积为0.908,P=0.011；肿瘤标记物ROC曲线下面积为0.837,P=0.035；中医治疗组中,循环肿瘤细胞可评价患者,ROC曲线下面积为0.679,P=0.135；中西医结合治疗组中,循环肿瘤细胞可评价的患者,ROC曲线下面积为0.822,P=0.005。以不同的循环肿瘤细胞变化值为截点,发现以2为截点,即增加3个及以上判定为进展时,其进展预测值可达到86.67%；以-3为截点,即减少3个及以上判定为有效时,有效预测值达77.78%。④多因素Cox回归分析,基线循环肿瘤细胞数量、临床分期、影像学疗效评价、Kps评分为患者总生存的影响因素,P<0.05。Kaplan-Meier法单因素生存曲线比较：循环肿瘤细胞数量>5个和≤5个的两组患者,平均生存天数分别为228.10天和339.70天,P=0.000；对ⅢB期和Ⅳ期两组患者,平均生存天数分别为317.92天和270.91天,P=0.000；影像学评价为进展和有效的两组患者,平均生存天数分别为253.42天和328.50天,P=0.000；Kps评分为60分、70分、80分和90分的患者,平均生存天数分别为202.58天、280.78天、307.15天和339.85天,P=0.000。循环肿瘤细胞数量大于5个的患者,中医组和中西医结合组平均生存天数分别为202.93天和253.46天,P=0.125；循环肿瘤细胞数量在5个及以下时,两组平均生存天数分别是334.73天和344.07天,P=0.572；对评价为有效的患者,按照第一周期后循环肿瘤细胞数量分为大于5个和小于等于5个两组,疾病无进展时间均值分别为81.63天和167.42天；中位疾病无进展生存时间分别为为64.00天和172.00天,P=0.000。
结论：1.免疫磁珠阴性分选及免疫荧光染色技术对肺癌细胞系A549细胞在每7m1外周血含102以内个细胞时有较高的富集效率、稳定性和特异性。2.该技术检测晚期非小细胞肺癌患者总体循环肿瘤细胞阳性率达79.4%。阳性率只与临床分期相关,而与病理类型、既往治疗、原发灶直径、转移个数、性别、吸烟指数、病理分化程度、辨证分型、功能状态评分均没有明确关系；循环肿瘤细胞数量与临床分期、原发灶大小、病理分化程度及功能状态评分相关,而与病理类型、既往治疗、转移个数、性别、吸烟指数和辨证分型不相关。3.循环肿瘤细胞数量的变化对影像学疗效评价有中等程度的提前预测价值,但是并没有明显优于肿瘤标记物积分的预测价值；在初治、接受中西结合治疗患者中,循环肿瘤细胞数量变化表现出更好的提前预测影像学评价的作用；以循环肿瘤细胞不同的改变量为截点,可相应提高对影像学评价的预测。4.基线循环肿瘤细胞数量、临床分期、影像学疗效评价、Kps评分均为患者总生存的影响因素；循环肿瘤细胞数量>5个、Ⅳ期、影像学评价为进展、Kps评分更低者有更短的总生存时间；基线循环肿瘤细胞数量>5个的患者,接受中西医结合治疗有获得更长生存的趋势,但并无统计学意义；≤5个的患者,中医治疗和中西医结合治疗总生存相似；影像学评价为有效的患者,第一周期后循环肿瘤细胞数量>5个的患者与≤5个的患者相比,有更短的疾病无进展生存时间。
The vast majority of patients who died of malignant tumors, died because of the emergence and progress of distant metastasis. There are several theorys of metastasis, still not conclusive. But it is undeniable that distant metastasis rises up inevitablely through disseminated tumor cells disseminating into the circulatory system. Tumor cells disseminated into the circulatory system, which we call circulating tumor cells(CTCs), have been attracting more and more interest of tumor researchers. Over the last decade, with the circulating tumor cell detection technology continues to evolve, a number of studies have shown that the detection of circulating tumor cells has great potential value in solid tumor diagnosis, staging, prognosis, individualized treatment, tumor cell characteristics, and so on.
Lung cancer which is a common clinical malignancy, has gradually rose to the top of morbidity and mortality worldwide, and it has brought a great threat to people's health and the country's economic development. Nowdays, the traditional treatment has been a new development, and new treatments have emerged. Although comprehensive treatment and individual treatment is growing widely accepted today, tumor heterogeneity and the complexity of the individual patient situation make great challenge in choice of treatment and strategies to oncologists. As an important means for the treatment of lung cancer, number of studies confirm that treatment involved of traditional Chinese medicine can effectively extend the survival time of patients、reduce or delay the recurrence and improve the quality of life and relieve symptoms. In order to explore the role of circulating tumor cells detecting in Chinese medicine involved treatment of patients with advanced non-small cell lung cancer, this study, applying a new development of circulating tumor cells detection technology, carrys out the preliminary laboratory and clinical research.
Objective：①Observing the efficiency、stability and specificity of the lung cancer cell line A549cells enriched by the technology of negative immunomagnetic beads sorting and immunofluorescence staining. And further we conduct clinical trials to study the following questions applying the technology.②Exploring the relationship of positive rate and the number of circulating tumor cells with various factors in advanced non-small cell lung cancer patients.③Exploring the relationship between the change of number of circulating tumor cells before and after the first cycle of treatment and imaging evaluation after two cycles of traditional Chinese medicine or Integrative treatment.④Investigating the predictive value of the number of circulating tumor cells on overall survival (OS) and progression free time(PFS)in patients with non-small cell lung cancer accepting Chinese medicine or Integrative Medicine treatment.
Method:①Laboratory studies:20peripheral venous blood of healthy volunteers, each 7m1, were randomly divided into two groups (n =10). Two groups were putted into1-10,10-100fluorescently labeled lung cancer cell line A549cells respectively. Count the number of tumor cells in the corresponding recovery calculation efficiency and stability of the A549cells enriched immunomagnetic beads negative sorting. Counting the corresponding number of tumor cells after enriched by immunomagnetic beads negative sorting to calculate recovery efficiency and stability of the A549cells. Another10peripheral venous blood of non-tumor patients, afrer negative immunomagnetic beads sorting and immunofluorescence staining, test the specificity of the detection of non-tumor patients.②Clinical research: Patients of advanced non-small cell lung cancer in Oncology department, Guang An Men Hospital of China Academy of Traditional Chinese Medicine, circulating tumor cells were detected before and after the first cycle treatment of Chinese medicine or combination therapy, contemporaneous taking the records of tumor markers, functional status score, TCM typing information; carrying out Imaging evaluation of efficacy after two cycles of treatment. Subsequently, the patients were follow-up to determine the overall survival and progression free time, and finally statistical data to conclude.
Results:①1-10(101) cells group, the average enrichment efficiency is83.00%±8.23%;10-100(102) cells group, the average enrichment efficiency is84.80%±4.44%,P=0.55. Linear correlation Pearson correlation coefficient of the cell number between added to and enriched was0.998, P<0.01. Linear Regression Analysis, R2=0.996; Linear regression equations:Y=-0.545+0.873X, P=0.000. No circulating tumor cell was detected in the10cases of healthy volunteers for specific test.②63patients was enrolled,33cases in Chinese medicin group,30cases in the integrated Chinese and Western Medicine group. In the detetion before treatment, circulating tumor cells are positive in50cases, positive rate is79.4%. The mean number of overall is5.75±4.71, ranging0-18. In the stage of ⅢB and Ⅳ, the positive rate is59.1%(13) and90.2%(37), respectively, P=0.007; the mean number is2.73±3.58and7.37±4.48, respectively, P=0.000. In the patients with primary tumor diameter≥3cm and <3cm, the positive rate is85.4%(35) and68.2%(15) ，respectively, P=0.190; the mean number is6.71±4.72and3.95±4.25, respectively, P=0.026. In patients with tumor differentiation as well-differentiated、moderately、 differentiated、poorly differentiated and undifferentiated, the mean number is3.25±4.17、4.24±3.65、6.24±4.34and8.29±5.46, respectively, P=0.019. Compared by LSD method, there is statistically significant between well-differentiated and undifferentiated (P=0.010)、moderately differentiated and undifferentiated (P=0.007). In patients with performance status score as60、70、80and90, the mean number is8.54±5.11、6.50±4.50、5.32±4.46and2.54±3.07, respectively, P=0.008; Compared by LSD method, there is statistically significant between60 and80(P=0.045)、90(P=0.001), and between70and90(P=0.016).③The consistency test between the change of the number of circulating tumor cells and tumor markers PTS before and after the first cycle of treatment compared to gold standard imaging evaluation by ROC curves test.53cases that changes in circulating tumor cells of patients are evaluable, area under ROC curve is0.755, P=0.002.46cases that tumor markers Points are evaluators, area under ROC curve is0.683, P=0.035.40cases of tumor markers and circulating tumor cells evaluable, area under ROC curve of circulating tumor cells is0.734, P=0.012; area under ROC curve of tumor markers is0.730, P=0.014.16patients untreated with circulating tumor cells evaluated, area under ROC curve is0.865, P=0.015.14patients untreated with tumor markers and circulating tumor cells evaluable, area under ROC curve of circulating tumor cells is0.908, P=0.011; area under ROC curve of tumor markers is0.837, P=0.035. In the group treated by Chinese medicine, and with circulating tumor cells evaluable, area under ROC curve is0.679, P=0.135. In the group treated by integrative medicine, and with circulating tumor cells evaluable, area under ROC curve is0.822, P=0.005. Valued by different changes in circulating tumor cells as cut-off point. If2as cut-off point, that is an increase of more than three means progress, the predicted value of progress can be achieved86.67%; If-3as cut-off point, that is an increase of less than three means response, the predicted value of progress can be achieved77.78%.④Multivariate Cox regression analysis, number of baseline circulating tumor cells, clinical stage, the evaluation of the imaging, KPS score is the influencing factors of overall survival in patients, P<0.05. Kaplan-Meier method univariate survival curve comparison:The patients of number of circulating tumor cells>5and≤5, the mean survival days were228.10days and339.70days, respectively, P=0.000. Patients of stage IIIB and IV, the mean survival days were317.92days and270.91days, respectively, P=0.000. Patients evaluated as progress and effective by imaging, the mean survival days were253.42days and328.50days, P=0.000. Patients with KPS score as60points,70points,80points and90points, the mean survival days were202.58days、280.78days、307.15days and339.85days, P=0.000. Patients with the number of circulating tumor cells more than5, the mean survival days of the group of Chinese medicine and integrative medicine were202.93days and253.46days, P=0.125; With the number of circulating tumor cells less than5, the mean survival days were334.73days and344.07days, P=0.572. Patients evaluated as response, the mean progress free survival days of group with circulating tumor cells>5and ≤5afrer the first cycle of treatment were81.63days and167.42days; the middle progress free survival days were64.00days and172.00days, P=0.000.
Conclusions:1. Immunomagnetic beads negative sorting and immunofluorescence staining techniques has high enrichment efficiency、 stability and specificity for lung cancer cell line A549cells in the the7ml with≤102cells in peripheral blood.2. The overall circulating tumor cells positive rate reaches to79.4% in advanced non-small cell lung cancer patients. Positive rate is only related with the clinical stage, but has no obviouse relationship with pathological type、 prior treatment、 primary tumor diameter、 number of metastatic、 gender、smoking index、 tumor differentiation、 syndrome differentiation、 functional status scores. The number of CTCs is only related with the clinical stage、 primary tumor diameter、 tumor differentiation、functional status scores, but has no obviouse relationship with pathological type、 prior treatment 、 number of metastati、 gender、 smoking index、 syndrome differentiation.3. The changes of number of circulating tumor cells shows moderate predictive value in advance to imaging evaluation, but not significantly better than the predictive value of tumor markers integral. Patients untreated and then received combined treatment, the changes of number of circulating tumor cells shows better predictive value. According to the different change in the amount of circulating tumor cells as the cut-off point, there is a corresponding increase in the predictive value to the imaging evaluation.4. Number of baseline circulating tumor cells, clinical stage, imaging efficacy evaluation, KPS score are the influencing factors of overall survival in patients. Patients with the number of circulating tumor cells>5、stage IV、imaging evaluation of progress and lower KPS score have shorter overall survival time. Patients with baseline circulating tumor cells>5, when received Integrative Medicine treatment, trend to have longer survival time, but no statistically significant; patients ≤5, have the same overall survival time when received Chinese medicine treatment or Integrative Medicine treatment. The patients evaluated as response by imaging, patients with the number of circulating tumor cells>5after the first cycle of treatment have shorter progression-free time than ≤5.
肺癌是临床常见的恶性肿瘤,发病率和死亡率在世界范围内已经逐渐升至首位,给人们的健康和国家经济发展都带来了很大的威胁。肺癌治疗发展到今天,传统的治疗手段得到了新的发展,新的治疗手段也纷纷涌现,在综合治疗和个体化治疗日益深入人心的今天,肿瘤本身的异质性和患者个体状况的复杂性考验着肿瘤医生对治疗手段和策略的选择。作为肺癌治疗的重要手段之一,多项研究证实中医药参与治疗能有效延长患者生存时间、减少或延缓术后复发、改善生活质量以及减轻临床症状。为了探索循环肿瘤细胞检测在中医药参与治疗晚期非小细胞肺癌患者中的地位和作用,本研究应用一种新发展的循环肿瘤细胞检测技术,进行了初步的实验室和临床的研究。
目的：①检测免疫磁珠阴性分选及免疫荧光染色技术对肺癌细胞系A549细胞富集的效率、稳定性和特异性,并进一步应用该技术进行临床实验以研究以下几个问题。②探讨晚期非小细胞肺癌患者循环肿瘤细胞阳性率和数量与各因素的关系。③探讨治疗前和第一周期治疗后循环肿瘤细胞数量的变化与两周期中医或中西医结合治疗后影像学评价之间的关系。④探讨循环肿瘤细胞数量对接受中医或中西医结合治疗的晚期非小细胞肺癌患者总生存时间(OS)和疾病无进展生存时间(PFS)的预测价值。
方法：①实验室研究：抽取20份健康志愿者提供的外周静脉血,每份7ml,随机等分为2组,每组10人份,两组各加入10个以内、10-100个之间的荧光标记的肺癌细胞系A549细胞。经免疫磁珠阴性分选后计数相应回收的肿瘤细胞数目,计算该技术对A549细胞的富集效率及稳定性。另抽取10位非肿瘤患者外周静脉血,经免疫磁珠阴性分选及免疫荧光染色技术计数,检验该技术对非肿瘤患者检测的特异性。②临床研究：中国中医科学院广安门医院肿瘤科住院的晚期非小细胞肺癌患者,在接受中医治疗或中西医结合治疗前、一周期治疗后进行两次循环肿瘤细胞检测,同期记录患者的肿瘤标记物、功能状态评分、中医辨证分型等信息；两周期后影像学评价疗效；随后进行随访,判定患者的总生存时间和疾病无进展生存时间,最后统计相关数据得出结论。
结果：①10个以内(101个)细胞组,平均富集效率为83.00%±8.23%,10-100个(102个)细胞组,平均富集效率为84.80%±4.44%,P=0.55；加入细胞数量和富集细胞数量之间线性相关Pearson相关系数为0.998,P<0.01；线性回归分析R2=0.996,线性回归方程：Y=-0.545+0.873X,P=0.000；10例进行特异性检验的非肿瘤患者,均未检测出循环肿瘤细胞的存在。②63例入组的患者,中医组33人,中西医结合组30人。从基线循环肿瘤细胞检测结果来看,共50例检测结果为阳性,阳性率为79.4%；最多检测出18个肿瘤细胞,总体数量均值为5.75±4.71个。其中,ⅢB期和Ⅳ期患者循环肿瘤细胞阳性率分别为59.1%(13人)和90.2%(37人),P=0.007；均值分别为2.73±3.58个和7.37±4.48个,P=0.000。原发灶直径≥3cm和<3cm的患者,阳性率分别为85.4%(35人)和68.2%(15人),P=0.190；均值分别为6.71±4.72个和3.95±4.25个,P=0.026。病理分化程度为高分化、中分化、低分化和未分化的患者,均值分别为3.25±4.17个、4.24±3.65个、6.24±4.34个和8.29±5.46个,P=0.019；LSD法两两比较,高分化和未分化(P=0.010)、中分化和未分化(P=0.007)组间均值差别有统计学意义。功能状态评分(Kps)为60分、70分、80分和90分的患者,均值分别为8.54±5.11个、6.50±4.50个、5.32±4.46个和2.54±3.07个,P=0.008；LSD法两两比较,60分的患者与80分(P=0.045)和90分(P=0.001)的患者比较,基线循环肿瘤细胞均值差异有统计学意义；70分的患者与90分(P=0.016)的患者,循环肿瘤细胞均值差异有统计学意义。③第一周期治疗后与基线比较,循环肿瘤细胞数量变化和肿瘤标记物积分变化与金标准影像学评价之间的一致性检验采用ROC曲线检验。53例循环肿瘤细胞变化可评价的患者,ROC曲线下面积为0.755,P=0.002；46例肿瘤标记物积分可评价者,ROC曲线下面积为0.683,P=0.035；40例肿瘤标记物积分和循环肿瘤细胞均可评价的患者,循环肿瘤细胞ROC曲线下面积为0.734,P=0.012；肿瘤标记物ROC曲线下面积为0.730,P=0.014；16例初治且循环肿瘤细胞可评价患者,ROC曲线下面积为0.865,P=0.015；14例初治且肿瘤标记物和循环肿瘤细胞均可评价患者,循环肿瘤细胞ROC曲线下面积为0.908,P=0.011；肿瘤标记物ROC曲线下面积为0.837,P=0.035；中医治疗组中,循环肿瘤细胞可评价患者,ROC曲线下面积为0.679,P=0.135；中西医结合治疗组中,循环肿瘤细胞可评价的患者,ROC曲线下面积为0.822,P=0.005。以不同的循环肿瘤细胞变化值为截点,发现以2为截点,即增加3个及以上判定为进展时,其进展预测值可达到86.67%；以-3为截点,即减少3个及以上判定为有效时,有效预测值达77.78%。④多因素Cox回归分析,基线循环肿瘤细胞数量、临床分期、影像学疗效评价、Kps评分为患者总生存的影响因素,P<0.05。Kaplan-Meier法单因素生存曲线比较：循环肿瘤细胞数量>5个和≤5个的两组患者,平均生存天数分别为228.10天和339.70天,P=0.000；对ⅢB期和Ⅳ期两组患者,平均生存天数分别为317.92天和270.91天,P=0.000；影像学评价为进展和有效的两组患者,平均生存天数分别为253.42天和328.50天,P=0.000；Kps评分为60分、70分、80分和90分的患者,平均生存天数分别为202.58天、280.78天、307.15天和339.85天,P=0.000。循环肿瘤细胞数量大于5个的患者,中医组和中西医结合组平均生存天数分别为202.93天和253.46天,P=0.125；循环肿瘤细胞数量在5个及以下时,两组平均生存天数分别是334.73天和344.07天,P=0.572；对评价为有效的患者,按照第一周期后循环肿瘤细胞数量分为大于5个和小于等于5个两组,疾病无进展时间均值分别为81.63天和167.42天；中位疾病无进展生存时间分别为为64.00天和172.00天,P=0.000。
结论：1.免疫磁珠阴性分选及免疫荧光染色技术对肺癌细胞系A549细胞在每7m1外周血含102以内个细胞时有较高的富集效率、稳定性和特异性。2.该技术检测晚期非小细胞肺癌患者总体循环肿瘤细胞阳性率达79.4%。阳性率只与临床分期相关,而与病理类型、既往治疗、原发灶直径、转移个数、性别、吸烟指数、病理分化程度、辨证分型、功能状态评分均没有明确关系；循环肿瘤细胞数量与临床分期、原发灶大小、病理分化程度及功能状态评分相关,而与病理类型、既往治疗、转移个数、性别、吸烟指数和辨证分型不相关。3.循环肿瘤细胞数量的变化对影像学疗效评价有中等程度的提前预测价值,但是并没有明显优于肿瘤标记物积分的预测价值；在初治、接受中西结合治疗患者中,循环肿瘤细胞数量变化表现出更好的提前预测影像学评价的作用；以循环肿瘤细胞不同的改变量为截点,可相应提高对影像学评价的预测。4.基线循环肿瘤细胞数量、临床分期、影像学疗效评价、Kps评分均为患者总生存的影响因素；循环肿瘤细胞数量>5个、Ⅳ期、影像学评价为进展、Kps评分更低者有更短的总生存时间；基线循环肿瘤细胞数量>5个的患者,接受中西医结合治疗有获得更长生存的趋势,但并无统计学意义；≤5个的患者,中医治疗和中西医结合治疗总生存相似；影像学评价为有效的患者,第一周期后循环肿瘤细胞数量>5个的患者与≤5个的患者相比,有更短的疾病无进展生存时间。
The vast majority of patients who died of malignant tumors, died because of the emergence and progress of distant metastasis. There are several theorys of metastasis, still not conclusive. But it is undeniable that distant metastasis rises up inevitablely through disseminated tumor cells disseminating into the circulatory system. Tumor cells disseminated into the circulatory system, which we call circulating tumor cells(CTCs), have been attracting more and more interest of tumor researchers. Over the last decade, with the circulating tumor cell detection technology continues to evolve, a number of studies have shown that the detection of circulating tumor cells has great potential value in solid tumor diagnosis, staging, prognosis, individualized treatment, tumor cell characteristics, and so on.
Lung cancer which is a common clinical malignancy, has gradually rose to the top of morbidity and mortality worldwide, and it has brought a great threat to people's health and the country's economic development. Nowdays, the traditional treatment has been a new development, and new treatments have emerged. Although comprehensive treatment and individual treatment is growing widely accepted today, tumor heterogeneity and the complexity of the individual patient situation make great challenge in choice of treatment and strategies to oncologists. As an important means for the treatment of lung cancer, number of studies confirm that treatment involved of traditional Chinese medicine can effectively extend the survival time of patients、reduce or delay the recurrence and improve the quality of life and relieve symptoms. In order to explore the role of circulating tumor cells detecting in Chinese medicine involved treatment of patients with advanced non-small cell lung cancer, this study, applying a new development of circulating tumor cells detection technology, carrys out the preliminary laboratory and clinical research.
Objective：①Observing the efficiency、stability and specificity of the lung cancer cell line A549cells enriched by the technology of negative immunomagnetic beads sorting and immunofluorescence staining. And further we conduct clinical trials to study the following questions applying the technology.②Exploring the relationship of positive rate and the number of circulating tumor cells with various factors in advanced non-small cell lung cancer patients.③Exploring the relationship between the change of number of circulating tumor cells before and after the first cycle of treatment and imaging evaluation after two cycles of traditional Chinese medicine or Integrative treatment.④Investigating the predictive value of the number of circulating tumor cells on overall survival (OS) and progression free time(PFS)in patients with non-small cell lung cancer accepting Chinese medicine or Integrative Medicine treatment.
Method:①Laboratory studies:20peripheral venous blood of healthy volunteers, each 7m1, were randomly divided into two groups (n =10). Two groups were putted into1-10,10-100fluorescently labeled lung cancer cell line A549cells respectively. Count the number of tumor cells in the corresponding recovery calculation efficiency and stability of the A549cells enriched immunomagnetic beads negative sorting. Counting the corresponding number of tumor cells after enriched by immunomagnetic beads negative sorting to calculate recovery efficiency and stability of the A549cells. Another10peripheral venous blood of non-tumor patients, afrer negative immunomagnetic beads sorting and immunofluorescence staining, test the specificity of the detection of non-tumor patients.②Clinical research: Patients of advanced non-small cell lung cancer in Oncology department, Guang An Men Hospital of China Academy of Traditional Chinese Medicine, circulating tumor cells were detected before and after the first cycle treatment of Chinese medicine or combination therapy, contemporaneous taking the records of tumor markers, functional status score, TCM typing information; carrying out Imaging evaluation of efficacy after two cycles of treatment. Subsequently, the patients were follow-up to determine the overall survival and progression free time, and finally statistical data to conclude.
Results:①1-10(101) cells group, the average enrichment efficiency is83.00%±8.23%;10-100(102) cells group, the average enrichment efficiency is84.80%±4.44%,P=0.55. Linear correlation Pearson correlation coefficient of the cell number between added to and enriched was0.998, P<0.01. Linear Regression Analysis, R2=0.996; Linear regression equations:Y=-0.545+0.873X, P=0.000. No circulating tumor cell was detected in the10cases of healthy volunteers for specific test.②63patients was enrolled,33cases in Chinese medicin group,30cases in the integrated Chinese and Western Medicine group. In the detetion before treatment, circulating tumor cells are positive in50cases, positive rate is79.4%. The mean number of overall is5.75±4.71, ranging0-18. In the stage of ⅢB and Ⅳ, the positive rate is59.1%(13) and90.2%(37), respectively, P=0.007; the mean number is2.73±3.58and7.37±4.48, respectively, P=0.000. In the patients with primary tumor diameter≥3cm and <3cm, the positive rate is85.4%(35) and68.2%(15) ，respectively, P=0.190; the mean number is6.71±4.72and3.95±4.25, respectively, P=0.026. In patients with tumor differentiation as well-differentiated、moderately、 differentiated、poorly differentiated and undifferentiated, the mean number is3.25±4.17、4.24±3.65、6.24±4.34and8.29±5.46, respectively, P=0.019. Compared by LSD method, there is statistically significant between well-differentiated and undifferentiated (P=0.010)、moderately differentiated and undifferentiated (P=0.007). In patients with performance status score as60、70、80and90, the mean number is8.54±5.11、6.50±4.50、5.32±4.46and2.54±3.07, respectively, P=0.008; Compared by LSD method, there is statistically significant between60 and80(P=0.045)、90(P=0.001), and between70and90(P=0.016).③The consistency test between the change of the number of circulating tumor cells and tumor markers PTS before and after the first cycle of treatment compared to gold standard imaging evaluation by ROC curves test.53cases that changes in circulating tumor cells of patients are evaluable, area under ROC curve is0.755, P=0.002.46cases that tumor markers Points are evaluators, area under ROC curve is0.683, P=0.035.40cases of tumor markers and circulating tumor cells evaluable, area under ROC curve of circulating tumor cells is0.734, P=0.012; area under ROC curve of tumor markers is0.730, P=0.014.16patients untreated with circulating tumor cells evaluated, area under ROC curve is0.865, P=0.015.14patients untreated with tumor markers and circulating tumor cells evaluable, area under ROC curve of circulating tumor cells is0.908, P=0.011; area under ROC curve of tumor markers is0.837, P=0.035. In the group treated by Chinese medicine, and with circulating tumor cells evaluable, area under ROC curve is0.679, P=0.135. In the group treated by integrative medicine, and with circulating tumor cells evaluable, area under ROC curve is0.822, P=0.005. Valued by different changes in circulating tumor cells as cut-off point. If2as cut-off point, that is an increase of more than three means progress, the predicted value of progress can be achieved86.67%; If-3as cut-off point, that is an increase of less than three means response, the predicted value of progress can be achieved77.78%.④Multivariate Cox regression analysis, number of baseline circulating tumor cells, clinical stage, the evaluation of the imaging, KPS score is the influencing factors of overall survival in patients, P<0.05. Kaplan-Meier method univariate survival curve comparison:The patients of number of circulating tumor cells>5and≤5, the mean survival days were228.10days and339.70days, respectively, P=0.000. Patients of stage IIIB and IV, the mean survival days were317.92days and270.91days, respectively, P=0.000. Patients evaluated as progress and effective by imaging, the mean survival days were253.42days and328.50days, P=0.000. Patients with KPS score as60points,70points,80points and90points, the mean survival days were202.58days、280.78days、307.15days and339.85days, P=0.000. Patients with the number of circulating tumor cells more than5, the mean survival days of the group of Chinese medicine and integrative medicine were202.93days and253.46days, P=0.125; With the number of circulating tumor cells less than5, the mean survival days were334.73days and344.07days, P=0.572. Patients evaluated as response, the mean progress free survival days of group with circulating tumor cells>5and ≤5afrer the first cycle of treatment were81.63days and167.42days; the middle progress free survival days were64.00days and172.00days, P=0.000.
Conclusions:1. Immunomagnetic beads negative sorting and immunofluorescence staining techniques has high enrichment efficiency、 stability and specificity for lung cancer cell line A549cells in the the7ml with≤102cells in peripheral blood.2. The overall circulating tumor cells positive rate reaches to79.4% in advanced non-small cell lung cancer patients. Positive rate is only related with the clinical stage, but has no obviouse relationship with pathological type、 prior treatment、 primary tumor diameter、 number of metastatic、 gender、smoking index、 tumor differentiation、 syndrome differentiation、 functional status scores. The number of CTCs is only related with the clinical stage、 primary tumor diameter、 tumor differentiation、functional status scores, but has no obviouse relationship with pathological type、 prior treatment 、 number of metastati、 gender、 smoking index、 syndrome differentiation.3. The changes of number of circulating tumor cells shows moderate predictive value in advance to imaging evaluation, but not significantly better than the predictive value of tumor markers integral. Patients untreated and then received combined treatment, the changes of number of circulating tumor cells shows better predictive value. According to the different change in the amount of circulating tumor cells as the cut-off point, there is a corresponding increase in the predictive value to the imaging evaluation.4. Number of baseline circulating tumor cells, clinical stage, imaging efficacy evaluation, KPS score are the influencing factors of overall survival in patients. Patients with the number of circulating tumor cells>5、stage IV、imaging evaluation of progress and lower KPS score have shorter overall survival time. Patients with baseline circulating tumor cells>5, when received Integrative Medicine treatment, trend to have longer survival time, but no statistically significant; patients ≤5, have the same overall survival time when received Chinese medicine treatment or Integrative Medicine treatment. The patients evaluated as response by imaging, patients with the number of circulating tumor cells>5after the first cycle of treatment have shorter progression-free time than ≤5.
引文
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