Circulating Tumor Cells And, In The Research Of Western Medicine Treatment Curative Effect And Prognosis Of Patients With Lung Cancer

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.