Detection of Circulating Tumor Cells in the Prognostic Significance of Patients With Breast Cancer: A Retrospective Study.

X.W. and Y.F. designed this study and wrote the manuscript. X.W. collected the sample, ran the CTC assay, and performed the statistical analysis. X.W. and Y.F. interpreted the data and prepared the figures. All authors reviewed and approved the final manuscript.

All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All subjects were approved by the Hubei Cancer Hospital (Approval #:KY2018‐201).

A total of 108 BC patients with stages I–IV and 10 patients with benign nodules were involved in this cohort study. The clinical characteristics of patients are show in Tables  2 and 3 . The analysis revealed that the median age of the patients was 60 years. Cases aged ≤ 60 years accounted for 53.70% (58/108), and those aged > 60 years accounted for 46.30% (50/108). The numbers of patients with stages I–IV were 32 (29.70%), 40 (37.00%), 25 (23.10%), and 11 (10.20%), respectively. There were 65 cases with ER‐positive BC (60.20%, 65/108), 62 cases with PR‐positive BC (57.20%, 62/108), 84 cases with HER‐2‐positive BC (77.80%, 84/108), and 12 cases with TNBC (11.10%, 12/108). We speculated that there were many cases with double receptor‐positive BC (ER/PR, ER/HER2, PR/HER2) or ER/PR/HER2‐triple positive BC. Comparison of general clinical data between patients with BC and 10 patients with benign nodules. Abbreviations: ER, estrogen receptor; HER‐2, human epidermal growth factor receptor‐2; HR, hormone receptor; PR, progesterone receptor; TNBC, three negative breast cancer. Relationship between the presences of circulating tumor cells (CTCs) and the clinical features of breast cancer. Abbreviations: ER, estrogen receptor; HER‐2, human epidermal growth factor receptor‐2; HR, hormone receptor; PR, progesterone receptor; TNBC, three negative breast cancer. CTCs from the peripheral blood of 108 BC patients and 10 patients with benign nodules were verified by the CanPatrol technique combined with the tricolor RNA‐ISH method. CTCs were classified according to cellular lineage surface markers (Table  1 ). eCTCs (Figure  1A ) and MCTCs (Figure  1B ) expressed EpCAM plus CK8/18/19 and vimentin plus Twist, respectively. Mixed CTCs (Figure  1C ) expressed both epithelial and mesenchymal markers. In addition, the cellular nucleus was stained with DAPI fluorescence dye, showing blue staining. Positive CTCs, MCTCs, and mixed CTCs were identified under an immunofluorescence microscope. Among patients with benign nodules in this study, no positive CTCs were detected in peripheral blood. To catch the optimal cutoff value of CTCs for stratifying patients with BC and patients with benign nodules, ROC and AUC were used. The results showed that six CTCs/5 mL of blood was the optimal cutoff value for total CTCs, and 80.00% sensitivity and 85.20% specificity were achieved (Figure  2 and Table  4 ). We also decided the cutoff values of eCTCs, mixed CTCs, and MCTCs for stratifying clinical and pathological results using the same method (Table  4 ). The data showed that more than six CTCs/5 mL were measured as the criterion for positivity in this study. ROC curve determination of the CTC cutoff value for differentiating clinical significance. This graph shows the ROC curve of total CTCs in 108 BC patients and 10 patients with benign nodules. It determined sensitivity and specificity of CTCs at 6.0/5 mL of blood as a cutoff. AUC, area under the ROC curve; CTC, circulating tumor cells; ROC, receiver operating characteristic. ROC comparison of CTC subtypes in breast cancer patients and benign patients. Abbreviations: AUC, area under the ROC curve; CTC, circulating tumor cells; eCTCs, epithelial circulating tumor cells; MCTCs, mesenchymal circulating tumor cells; ROC, receiver operating characteristic curve. MCTCs, mixed CTCs, and positive eCTCs did not differ significantly among patients of different ages. In total, 60 patients had positive CTCs including 9 cases at stage I (28.10%, 9/32), 20 cases at stage II (50.00%, 20/40), 32 cases at stage III (80.00%, 20/25), and 11 cases at stage IV. From these data, we found that the CTC positive rate increased with advanced staging. The CTC positive rate was 100% in all 11 stage IV patients and was higher than that in stage I and II patients ( p  < 0.0001). In addition, we also calculated the CTC positive rates in patients with ER + , PR + , HER2 + as 6.10% (4/65). 19.40% (12/62), 38.10% (32/84), and 100% (12/12), respectively. These results also indicated that CTC positive rates were closely associated with hormonal receptor expression. Interestingly, all patients with TNBC had CTC positive rates, indicating that CTC positive rates are relevant to the severity of BC. We also evaluated the relationship between Ki‐67 and CTCs and found that the number of CTCs with low Ki‐67 expression (20.00%) was significantly lower than the number of CTCs with Ki‐67 expression above 20% ( p  < 0.01). In addition, the CTC positive rate of patients with invasive ductal carcinoma was higher than that of patients with other histology types ( p  < 0.01). These results showed that the number of CTCs is also associated with cancer proliferation and histological types (Table  5 ). Comparison of PFS in different CTC numbers of breast cancer patients. Abbreviations: CI, confidence interval; CTC, circulating tumor cell; eCTC, epithelial circulating tumor cell; HR, hazard ratio; M, months; MCTC, mesenchymal circulating tumor cell; PFS, progression‐free survival. The specific data of the follow‐up patients were as follows: a total of 65 CTC‐positive patients were followed up from January 2011 to December 2019, 60 patients were followed up to the endpoint, and 5 patients were lost to the endpoint, of which the number of patients who developed clinical recurrence or metastasis totaled 50, with a recurrence rate of 83.40%. Postoperative follow‐up ranged from 4 to 5 years, with a mean follow‐up of (60.72 ± 6.82) months and a median time to the endpoint event of 36.5 months. To evaluate the clinical significance of CTCs in predicting the outcomes of BC patients, we performed ROC analysis and determined the optimal cutoff value. Among 60 patients with positive CTCs, the numbers of patients with total CTCs > 6 and ≤ 6 were 24 (40%) and 36 (60%) before treatment, respectively. Kaplan–Meier analysis showed that the PFS of patients with total CTCs > 6 was significantly shorter than that of patients with total CTC ≤ 6 ( p   6 mixed CTCs (Figure  3C , p  < 0.01) and MCTCs (Figure  3D , p  = 0.00) was shorter than that of patients with ≤ 6 mixed CTCs or MCTCs. There was no statistically significant difference in PFS between eCTC‐positive or ‐negative patients (Figure  3B , p  = 0.07). Multivariate Cox regression analysis showed that the hazard ratio (HR) and 95% confidence interval (CI) for PFS in patients with a total number of CTCs > 6 were 5.70 and 3.49–9.33, respectively ( p  < 0.01), while HR = 4.10 and 95% CI = 2.59–6.50 ( p  < 0.01) for mixed CTCs and HR = 2.32 and 95% CI = 1.22–4.42, p  = 0.00 for MCTCs. These results showed that the PFS of patients with high total CTCs, mixed CTCs, and MCTCs was significant poorer than that of patients with low total CTCs, mixed CTCs and MCTCs. In contrast, positive eCTCs were not relevant to the PFS of patients (Table  5 ). Comparison of progression‐free survival (PFS) in patients with total CTCs, epithelial CTCs, Mixed CTCs, and mesenchymal CTCs (MCTCs) by Kaplan–Meier curves. (A) Total CTCs, (B) epithelial CTCs, and (C, D) mixed MCTCs and MCTCs. CTC, circulating tumor cell; MCTC, mesenchymal CTC. To further investigate the clinical significance of hormonal receptor expression levels in predicting the outcomes of BC patients, the PFS values of the patients with different hormonal receptor expressions, including ER, PR, HER‐2 positive, and TNBC, were compared. There were 65 cases with ER (60.20%, 65/108), 62 cases with PR (57.40%, 62/108), 44 cases with HER‐2 (40.70%, 44/108), and 12 cases with TNBC (11.10%, 12/108). At 60 months follow‐up, the PFS of ER‐ and PR‐positive patients was not significantly longer than that of ER‐negative ( p  = 0.66) and PR‐negative patients ( p  = 0.79) (Figure  4A,B ). However, the PFS of HER‐2‐positive patients was longer than that of HER‐2‐negative patients (Figure  4C , p  = 0.02). In contrast, the PFS of patients with TNBC+ was significant shorter than that of non‐TNBC patients (Figure  4D , p  < 0.01). Multivariable Cox regression analysis showed PFS of BC patients with different hormone levels, showing ER+ (HR = 1.11, 95% CI = 0.693–1.78, p  = 0.66), PR+ (HR = 1.065, 95% CI = 0.66–1.71, p  = 0.79), HER‐2+ (HR = 1.98, 95% CI = 1.21–3.25, p  = 0.02), and TNBC (HR = 3.97, 95% CI = 1.27–12.30, p  < 0.01), respectively (Table  6 ). This result confirmed that TNBC patients had poor survival rate than patients with positive hormone receptor expression. PFS comparison of patients with hormone receptor expression by Kaplan–Meier curves. (A) Estrogen receptor (ER), (B) progesterone receptor‐PR, (C) ER, PR, and HER2 triple positive (TR+), and (D) triple negative breast cancer (TNBC). PFS, progression‐free survival. Comparison of PFS in breast cancer with different hormonal receptor expressions. Abbreviations: CI, confidence interval; ER, estrogen receptor; HER2, human epidermal growth factor receptor‐2; HR, hazard ratio; PFS, progression‐free survival; PR, progesterone receptor; TNBC, triple negative breast cancer.

From January 2011 to December 2019, the types of CTCs and the prognosis of 108 BC patients with TNM staging in Hubei Cancer Hospital were retrospectively collected and analyzed based on clinical features, imaging data, and pathological types, while 10 patients with benign nodules were included as controls. All patients were female. Patient final diagnoses were determined by at least two clinical pathologists using tumor tissues or biopsy samples combined with magnetic resonance imaging (MRI)/CT. This study protocol was reviewed and approved by the review board and the ethics committee of Hubei Cancer Hospital (Approval #: KY2018‐201). Informed consent was obtained from all participants before our study. General patient data as well as pathological data, including gender, age, BC subtype, number of recurrences/metastases, tumor infiltration (non‐muscular vs. muscular infiltration), histological grading, and Ki‐67 level (20% was used as a cut‐off point), were collected. The characterization of CTCs followed the approach described previously [ 28 ]. Briefly, 5 mL of peripheral blood samples were taken from patients and controls and immediately transferred into ethylenediaminetetraacetic acid (ST069, Beyotime)‐coated tubes. These cells were mixed with Ficoll 400 density gradient liquid (GE healthy, USA) and centrifuged for 30 min at 1500 rpm at room temperature (RT). The enrichment of peripheral blood CTCs was accomplished using CanPatrol TMCTC‐II capture technology (SurExam, Guangzhou, China), which enables the isolation, typing, and analysis of various types of CTCs. Then, cells were centrifuged for 5 min at 1500 rpm and then incubated for 15 min with Cytofix/Cytoperm fix solution (Cat#554722, BD Biosciences, CA, USA) on ice. After filtration using a vacuum pump filter, 1 mL of formalin (ES‐GDY‐002, Eysin, Shanghai, China) was added for fixation for 1 h, and then 50%, 70%, and 100% dehydrating agents were added sequentially for dehydration. To detect three CTC subtypes, CTCs were added with target probes, incubated for 2 h at 40°C, and washed three times with 0.1 × SSC solution (GS1958, Beijing Biolab). Then, the pre‐amplification and amplification solutions were added to the hybridization mixture and incubated for 90 min at 40°C. Alexa Fluor 594 conjugated epithelial markers EpCAM and CK8/18/19, Alexa Fluor 488 conjugated mesenchymal markers vimentin and Twist, and 4′,6‐diamidino‐2‐phenylindole (DAPI; D9542, Sigma‐Aldrich) staining dye were added, respectively. After incubating for 60 min at 40°C, the cells were washed with phosphate‐buffered saline (PBS; AM9624, ThermoFisher) containing 2% serum. Then, the cells were imaged under an immunofluorescence microscope (CKX31, Olympus). Table  1 and Figure  1 show features of CTC subtypes and their typical pictures. Image features of circulating tumor cells (CTCs) subtype. Abbreviations: eCTCs, epithelial circulating tumor cells; MCTCs, mesenchymal circulating tumor cells. Images of CTCs in breast cancer by CanPatrol and the RNA in situ hybridization technique. (A) Epithelial CTCs: EpCAM and CK8/18/19 genes were labeled with AF594 immunofluroresence dye conjugation (red dots). (B) Mesenchymal CTC: Vimentin and Twist genes were labeled with AF488 immunofluroresence dye conjugation (green dots). (C) Mixed CTCs: the cellular nucleus was stained with 6‐diamidino‐2‐phenylindole (DAPI). All pictures were taken by an immunofluroscence microscope at 100× magnification. CTCs, circulating tumor cells. CTC identification: (1) The shape and texture of cells are normal and complete. (2) There are many types of cells (large cells, oval cells, irregular cells, overlapping cells, etc.). (3) The nucleus area is smaller than the cytoplasmic area, most of the nucleus should be in the cytoplasm, and the cell diameter is ≥ 4 μm. (4) The nuclear coloration can be clearly distinguished from the CK coloration. (5) CK is positive; nuclear staining is positive. The CTC positive rate was counted, which included the respective positive rates of different phenotypes. CTCs were counted under a fluorescence microscope according to the five‐point sampling method. CTC typing criteria: (A) eCTCs: only epithelial markers (EpCAM and CK8/18/19) labeled by Alexa Fluor 594 (red) are shown under the microscope. (B) Mixed (epithelial and mesenchymal) CTCs: red fluorescence and green fluorescence signals are seen under the microscope. (C) MCTCs: only mesenchymal markers (vimentin and Twist) labeled by Alexa Fluor 488 (green) are shown under the microscope. Hormonal receptors of patients with BC, including ER, PR, and HER‐2, are very critical factors for prognosis judgment therapy of patients. We routinely performed the immunohistochemistry (IHC) assay [ 29 ]. Tumors were fixed in a 10% formalin solution overnight and paraffin‐embedded. Then, slides were placed in an oven at 56°C–60°C for 15 min and transferred to xylene. Then, the slides were serially washed with fresh 100% ethanol for 3 min, 90% ethanol for 3 min and 80% ethanol for 3 min, tap water and PBS successively. ER, PR, and HER‐2 primary antibodies were incubated for 60 min at 37°C in a humidified chamber, followed by the secondary antibodies. All pictures were taken under a light microscope (DM3000, Leica). ER and PR determination criteria: ER and PR testings were updated in accordance with the 2020 edition of the ASCO/CAP Guidelines [ 30 ]. The threshold of positivity for ER and PR immunohistochemical testing remained at ≥ 1%. Of these, 1%–10% of tumor cells with nuclear staining were judged to be weakly positive, and > 10% of tumor cells with nuclear staining were judged to be positive. HER‐2 determination criteria: according to the BC HER2 Detection Guidelines (2019 edition), HER‐2 positivity is defined as the presence of strong staining of the intact cytosol membrane (3+) in more than 10% of the cells by IHC and/or the detection of amplification of the HER‐2 gene by in situ hybridization (single‐copy HER2 gene > 6 or HER2/CEP17 ratio > 2.0). Patients were followed up for up to 5 years to assess the efficacy of the treatment. Patients were followed up every 3 months during the first year of treatment and every 6 months thereafter. Progression‐free survival (PFS) was defined as the time from the start of treatment until the tumor progressed or the patient died. Among the 60 patients, the shortest follow‐up period was 4 years and the longest was 5 years. A tumor biopsy was performed at each follow‐up visit, and IHC for ER, PR, and HER‐2 was performed to assess prognosis. The study endpoint event was defined as disease progression or death, and the last follow‐up was performed in January 2024. During follow‐up, five patients were excluded due to (1) invalid, unsatisfactory, or missing specimens; (2) large amount of missing data; and (3) need to withdraw from the study. SPSS 23.0 was used for statistical analysis and GraphPad prism 9.0. software for plotting. Measurement information conforming to normal distribution was expressed as mean ± standard deviation (SD), and non‐normal distribution was expressed as median and interquartile spacing M (Q25 to Q75). Categorical information was expressed as frequencies and percentages. The optimal value of the CTC threshold for stratifying BC patients and benign controls was determined using the receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC). The relationship between CTC levels and clinic‐pathological features was assessed by the χ 2 test. ER, PR, and HER2 levels were compared using two‐tailed Student's t ‐test. Risk factors for BC recurrence or death were determined by Cox proportional risk regression model analysis. Patient survival curves were plotted using the Kaplan–Meier method, and differences between survival curves were compared using the log‐rank test. p  < 0.05 was considered as significant differences. Each result was calculated from at least three independent tests.

The prognosis of BC depends highly on its pathological subtypes. Generally, DCIS, LCIS, tubular carcinoma, and mucinous carcinoma have favorable survival outcomes, but invasive lobular carcinoma and ductal carcinoma have poor outcomes. Therefore, identifying a specific biomarker is critical for monitoring the outcomes of BC patients. Recent studies indicated that CTCs can be used to trace metastasis, relapse, and outcomes of cancer patients [ 15 , 31 , 32 , 33 ]. In BC, CTCs in advanced stages are detected with various techniques [ 26 , 34 , 35 ]. The current study indicated that the PFS of BC patients with high total CTCs, mixed CTCs, and MCTCs was significantly poorer than that for patients with low CTCs. It was also identified that hormonal receptor expression in patients with BC was also strongly associated with the prognosis of BC. CTCs are a group of cells that are shed into the blood circulation from tumor primary or metastatic foci and play an important role in the process of tumor metastasis and dissemination. One important aspect of this is the EMT of tumor cells. However, it is not necessary to form EMT for a tumor to metastasize and form metastatic foci, and it depends largely on the microenvironment of tumor cells. Generally, CTCs are divided into three subtypes based on their cellular surface markers, including eCTCs, MCTCs, and mixed CTCs. The expression of EMT markers in CTCs is involved in various cancers such as lung, breast, colorectal, gastric, and prostate cancers [ 36 , 37 , 38 , 39 ]. For malignant tumors with non‐hematogenous metastases, test results often have a high false‐negative rate. The present study utilized CanPatrol and RNA‐ISH to measure EpCAM and CK8/18/19 as epithelial markers and vimentin and Twist for mesenchymal markers. Since only a few blood cells are amplified multiple times in a short period of time, this technique has a high level of sensitivity and specificity. Current data indicated that CTCs were strongly associated with advanced stages and hormone receptor expression. These results are similar to a previous report [ 40 ]. Here, CTC positive rates are also relevant to the outcomes of BC. The PFS of patients with high total CTCs, mixed CTCs, and MCTCs was significantly poorer than that of patients with low CTCs. In contrast, there were no differences in the PFS of patients with high and low eCTCs. Ning et al. [ 41 ] performed the CTC test on 21 patients with ovarian cancer and 21 patients with the endometriosis of the ovary. As observed, 76.2% of the patients with ovarian cancer were positive for CTCs, and no CTC was found in the peripheral blood of the patients with endometriosis. This is very similar to the results of the present study. Hematogenous dissemination is the main route of BC metastasis, and the entry of tumor cells into the bloodstream is a prerequisite for metastasis. Therefore, clinically, the liquid biopsy of CTCs can realize non‐invasive real‐time detection of collective health and disease progression, which is important for the diagnosis, treatment, and prognostic monitoring of BC. Hormonal receptor expression in BC patients is strongly associated with the outcomes and therapies of BC [ 42 ]. Especially, for patients with triple negative receptor expression, how to use a sensitive and reliable biomarker is important. Here, our results indicated that the PFS of TNBC patients was significantly poorer than that of patients with positive hormonal receptors. CTCs of TNBC patients were also significantly higher than that of hormone receptor‐positive patients. Therefore, current data confirmed that CTC positivity and high HR expression detected in peripheral blood indicated a significantly increased risk of distant metastasis, both of which portend poor treatment outcomes. This study also has some limitations. PFS and OS are undoubtedly two important efficacy endpoints in clinical trials. In this study, we prioritized PFS. PFS, as a shorter‐term indicator, can show the treatment effect earlier. Meanwhile, PFS is recognized as the primary endpoint of clinical trials. In contrast, OS tends to require longer follow‐up times and larger sample sizes to obtain accurate data. Nevertheless, OS is still the most direct and reliable indicator for assessing tumor treatment effects. Therefore, we will further explore OS analysis by increasing the sample size and extending the follow‐up time in future ground studies. Secondly, this study only detected CTCs in peripheral venous blood, which could only reflect a localized moment in the systemic circulation of CTCs, could not answer the systemic circulatory path and distribution of CTCs, and could not completely reveal the panoramic view of the involvement of CTCs in recurrent metastasis. This will be a problem we need to solve in the future.

The authors have nothing to report.

Breast cancer (BC) is the leading cause of cancer‐related deaths among women worldwide. The latest statistics estimate that 2.09 million people worldwide are diagnosed with BC each year [ 1 ]. BC is classified into non‐invasive and invasive according to their pathological features, which further is divided into ductal carcinoma in situ (DCIS), invasive lobular carcinoma (ILC), lobular carcinoma in situ (LCIS), tubular/cribriform carcinoma, mucinous carcinoma, papillary carcinoma, metaplastic carcinoma, and invasive ductal carcinoma (IDC) [ 2 ]. DCIS, LCIS, tubular/cribriform carcinoma, and mucinous carcinoma have good prognosis with more than 95% 5‐year overall survival (OS) [ 3 ]. In contrast, ILC and IDC only have 66%–68% 10‐year OS [ 4 ]. The 5‐year survival rate is 55%–60% for papillary carcinoma. Tumor‐node‐metastasis (TNM) staging in BC is classified as stage 0 (including DCIS and LCIS), stages I–III (breast and regional lymph nodes), and stage IV (outside of breast and regional lymph nodes). BC is also classified according to the expression of receptors on the cell surface, in the cytoplasm, and in the nucleus as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor‐2 (HER2) positivity [ 5 , 6 ]. If none of these three receptors are expressed, it is named triple negative BC (TNBC). The management of BC includes surgery [ 7 ], chemotherapy [ 8 ], radiation therapy [ 9 ], hormonal therapy [ 10 ], and immunotherapy [ 11 ]. Surgery of these therapeutic methods no doubt is the first choice for local cancer. However, only about 10%–20% of BC tumors are resectable because most BC patients are diagnosed with cancer at an advanced stage. It is therefore inevitable that most patients will eventually experience recurrences or metastases. Moreover, the prognosis of BC is dependent on the staging and hormonal receptor expressions of patients at their initial diagnosis. In general, stage I cancers, DCIS and LCIS, have a good prognosis with surgical treatment. In contrast, patients with stage > 2 tend to have a poorer prognosis. Therefore, a sensitive and reliable technique for monitoring BC at advanced stages is a critical issue. To date, only a few biomarkers have been used to monitor BC. Currently, BC diagnosis is based on tumor biopsy and positron emission tomography/computed tomography (PET/CT) [ 12 ]. However, non‐invasive and easily accessible materials such as blood samples are more convenient for detecting the progression of BC. Circulating tumor cells (CTCs) are rare cells from primary tumors that are released into the circulation and seeded in distant organs [ 13 ]. These cells are the main resource for cancer relapse and metastasis. The epithelial‐to‐mesenchymal transition (EMT) process contributes to BC migration from the primary tumor site to blood circulation [ 14 , 15 , 16 ]. As CTCs travel from the blood circulation to distant organs, they undergo the mesenchymal‐to‐epithelial transition (MET), producing a new tumor site in distant organs [ 17 ]. Total CTCs are classified into epithelial CTCs (eCTCs), mesenchymal CTCs (MCTCs), and mixed CTCs according to lineage biomarkers such as EpCAM, CK8/18/19, vimentin, and Twist [ 18 ]. CTCs can be used to monitor the progression and prognosis of cancers such as hepatocellular carcinoma [ 19 ], ovarian cancer [ 20 ], and esophageal carcinoma [ 21 ]. Depending on the goal of the study, blood samples from cancer patients can easily be collected multiple times. These advantages of CTC testing are superior to other invasive methods and are becoming increasingly popular in monitoring the progression and prognosis of cancer patients. There are limited reports on biomarkers for monitoring BC prognosis and progress. Studies have revealed that exosomes, non‐coding RNAs, and circulating tumor DNA in BC patients may be very useful biomarkers in predicting BC outcomes [ 22 , 23 , 24 ]. Moreover, CTC detection of BC patients has a promising result in monitoring the progress of BC [ 25 , 26 , 27 ]. However, the clinical significance of CTC detection remains defined. Here, we present the results about the prognostic significance of CTC detection in BC patients.

The authors declare no conflicts of interest.