| Backgroung and Objective:Circulating Tumor Cells (CTCs) are tumor cells which are detectable in blood circulation system of patients with tumor, and may result in overt metastatic recurrence. The number of circulating tumor cells is correlate with prognosis in patients with recurrence/metastatic breast cancer(MBC). The more the number of CTCs in blood, the worse the prognosis. Meanwhile, in the treatment of MBC, CTCs may reflect the changes in tumor bueden and assess treatment efficacy in advance.Recent studies have found that circulating DNA fragments carrying tumor specific sequence alterations (circulating tumor DNA, ctDNA) which is present in the peripheral blood of patients with tumor is a highly sensitive biomarker than CTCs. In the treatment of patients with tumor, CTCs and ctDNA act as new liquid biopsy, providing the possibility of real-time monitoring of tumor therapeutic efficacy and predicting the prognosis.This study mainly observed CTCs in patients with operable breast cancer and analysed the correlation between CTCs and clinicopathologic features to assesse the prognosis role of CTCs in patients with operable breast cancer. With dynamic testing ctDNA and CTCs changes in the blood in patients with Advanced Breast Cancer (ABC), we compared the result of Magnetic Resonance Imaging (MRI) to explore the role of treatment efficacy of ctDNA/CTCs in preoperative chemotherapy for patients with advanced breast cancer.Method:From January to September in 2014,73 operable breast cancer patients in department of center for breast diseases of PLA general hospital enrolled in this study. 7.5 ml peripheral blood were extracted from each patient in the first day of postoperative, then stores in CellSave tubes, CTCs were analyzed using CellSearch system. Their primary tumor size, axillary lymph node status. pTNM stage, molecular subtyping, ER, PR. Her-2, Ki-67 were analyzed; the relationship between CTCs and clinicopathologic features of primary tumor were investgated; the relationship between CTCs and the prognosis of operable breast cancer was analyzed with the disease-free survival time (DFS) as the observed indicators.From January to November in 2014, six patients with advanced breast cancer from department of center for breast diseases of PLA general hospital enrolled in the study. The tumor-associated gene loci of patients'biopsy specimens from the first preoperative chemotherapy and residual tumor tissue after chemotherapy were deteced using the next generation sequencing. Breast tumor tissue-specific gene mutation were analysed by gene sequence alignment. Using digital PCR technology, the study detected the change of mutation gene loci in the blood samples (10ml blood samples from each cylcle of the first day of chemotherapy and preoperative stored in the BCT) of the patients with consecutive blood samples. Their CTCs from consecutive blood samples (7.5ml blood samples from each cylcle of the first day of chemotherapy and preoperative stored in the CellSave) were analyed using CelleSearch system. Compared the imaging results of tumors with ctDNA and CTCs, we evaluated the treatment efficacy of prechemotheraoy in patients with advanced breast cancer.Results:Relationship between CTCs and clinicopathological features, prognosis in patients with operable breast cancer:(1) The detection rate of CTCs in patients with operable breast cancer was 16.4%.(2) The detection rate of patients with tumor diameter greater than 5cm in CTCs positive and negative group were 25% and 3.3% with statistically significant difference (P=0.036).(3) In the multivariate analysis, tumor size is an independent factor in CTCs positive(P=0.024, OR=2.024)(4) The 2-year disease free survival in CTCs positive and negative patient were 82.0% and 92.9% with statistically significant difference (P=0.045).Efficacy evaluation of ctDNA in preoperative chemotherapy for advanced breast cancer:(1) The total clinical response was 100% in 6 patients, clinical complete response was 83.3%(5/6), clinical partial response was 16.7%(1/6).(2) 8 tumor-associated mutations was successfully deteced in the tissue of 5 patients:PIK3CA?GRIN2A?TP53?EWSR1?PDGFRA?FYN?FANCF?KMT2C; and significant changes before and after treatment. The coverage of the target area reached more than 99.7%, and the average sequencing depth was more than 200X;(3) Through the analysis of gene database, only PIK.3CA is the hot spot mutation gene of breat cancer in 8 mutant genes,2 patients were detected with PIK3CA in 5 patients, the detection rate was 40%. The frequency of PIK3CA mutation in 2 patients showed a decreasing trend in the course of treatment.(4) The detection of ctDNA in serially collected plasma specimens showed a wide dynamic range, and it had a correlation with CTCs(P=0.013).(5) Compared with CTCs, the result of imaging examination and ctDNA in the evaluation of the preoperative chemotherapy, ctDNA and CTCs provided the earliest measure of treatment response in 2 patients than the result of imaging examination.Conclusion:These results preliminarily showed that:(1) The overall detection rate of CTCs in patients with operable breat cancer is low, and the detection rate is correlated with tumor size. The larger the tumor, the more the detection rate is. In addition, the presence of CTCs in patients with operable breast cancer prompt more likely to have tumor relapse and metastasis.(2) PIK3CA mutant gene is a hot spot mutation associated with breast cancer. The change of ctDNA in the blood is more advanced than that of CTCs and MRI in reflecting the treatment effecy. It suggests that ctDNA in the blood of PIK3CA mutation may be a new therapeutic evaluation indicator, which can be applied to the preoperative chemotherapy of advanced breast cancer. |
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