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We wanted to demonstrate the value of multiparameter flow cytometry in detecting human tumor cells of breast cancer (BC) (SKBR-3) in normal peripheral blood. In addition, we investigated a cluster of patients to compare the overall survival (OS) between advanced BC patients [circulating tumor cells (CTCs) ≥5 group] and limited BC patients (CTCs <5 group). SKBR-3 human BC cells were serially diluted in normal whole blood to demonstrate the sensitivity of multiparameter flow cytometry for detecting CTCs, and we also compared the specificity with reverse transcriptase polymerase chain reaction (RT-PCR) method. On the other hand, we detected CTCs among 45 patients by multiparameter flow cytometry. OS was calculated by the Kaplan-Meier product limit method, and compared it between CTCs <5 and CTCs ≥5 groups with the log-rank test. Cox regression models were fitted to determine the associated factors on survival. Human BC cells (SKBR-3) could be differentiated from normal blood based on the multiple light scatter and cell surface marker expression by multiparameter flow cytometry. The method was found to have a sensitivity limit of 10−5 and was effective for detecting human BC cells in vivo. It also found that this method had a higher specificity compared with RT-PCR. For the retrospective study, the median OS was 95 weeks and 65.5 weeks (P < 0.05, 2-tailed) for patients with CTCs <5 and CTCs ≥5, respectively. Kaplan-Meier was used to analyze the patients' survival with Log Rank P = 0.004 and Breslow P = 0.003, which showed that these two groups had statistically significant difference. Cox regression analysis was performed, and we found CTCslevels, metastasis and age (P < 0.05) were three relative factors for patients' survival. Multiparameter flow cytometry can detect CTCs effectively and has the potential to be a valuable tool for prognosis assessment among BC patients in clinical situations in China. © 2010 International Society for Advancement of Cytometry
Breast Cancer (BC) is the most common cancer in women in developed countries. In developing countries, such as China, the incidence of BC is currently increasing, particularly in the larger cities (1). In many patients with solid tumors of epithelial origin, circulating cells with the characteristics of tumor cells can be identified in the peripheral blood that is known as circulating tumor cells (CTCs). These cells are present not only in patients with metastatic disease but also in those whose tumors are apparently localized (2). There may be intermittent shedding of tumor cells into the circulation corresponding with micro-invasive events within the tumor. The first phase of the metastatic cascade consists of loss of tumor cell adhesion, induction of cell motility, and local tumor cell invasion (3). These steps are followed by either dissemination to regional lymph nodes or circulation through the blood, and homing to secondary organs, where the tumor cells may reside as viable cells in a “dormant” state (4). Some of these cells eventually become precursors of metastases that can arise many years after curative resection of the primary tumor (5). Although apoptosis contributed to a high rate of circulating cells, only a small part of the cells can adhere in organs through blood vessels that were named as circulating tumor stem cells (CTSCs) (6).
CTCs cells can be selected with the fluorescent antibody that linked to a monoclonal antibody directed against CD45 for negative selection of leukocytes (7–9). From these cluster cells, Ep-CAM (epithelial-cell adhesion molecule) and Cytokeratin 8,18,19 (Cytokeratin 8,18,19-phycoerythrin staining) positive cells are the target cells as these two monoclonal antibodies can be adhered in epithelial cells. Given the multistep nature of the metastatic cascade, there should be several opportunities for early identification and therapeutic targeting of metastatic cells.
In the past, various immunologic procedures, including immunocytology- and immunohistochemistry-based methods, and reverse transcriptase polymerase chain reaction (RT-PCR), have been used to detect systemic tumor cell contamination (10–14). Most current methods do not seem to be sensitive or specific enough to detect circulating cells in significant numbers of patients with carcinomas (15–17). Recently, a major advance in this area occurred with the advent of flow cytometry, which makes possible the detection of CTCs in a good balance of sensitivity and specificity. In addition, the simple procedure and lower cost have made it possible to apply in clinical situations.
The aim of this study was twofold. First, we wish to evaluate our new multiparameter flow cytometric methodology and compare it with conventional RT-PCR. Second, we wish to further evaluate the overall survival (OS) between advanced breast cancer (ABC) patients, who had CTCs ≥5, and limited breast cancer (LBC) patients, which was CTCs <5 in Chinese patients with multiparameter flow cytometry.
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The study of detecting disseminated BC cells in human blood using multiparameter flow cytometric approaches rely on the expression of epithelial-specific markers, such as cytokeratins, which are expressed on epithelial cells but not on leukocytes (2, 5, 20). In particular, cells of higher metastatic potential may lose expression of epithelial-specific markers during the course of metastatic progression (21–24). To avoid possible false-negative, we also chose another epithelial-specific marker: Ep-CAM (25). To detect the tumor cells that come from epithelium tissue, a monoclonal antibody directed against CD45 for negative selection of leukocytes was used (7–9). Therefore, the detection of dual-positive cells (CD45-Ep-CAM+CK+) is expected (such as in the peripheral blood) and has been proposed as a surrogate marker for epithelial tumor cells. BC cells in epithelial blood could be represented. The sensitivity of the assay can be assessed by analysis of serial dilutions of a breast-cancer cell line (SKBR-3) in blood from a healthy volunteer. Because the target population in prospective test samples is anticipated to be in the range of no more than 0.5%, by using all the information generated by the three fluorescence parameters and light scattering, it was possible to decrease the likelihood of a nonspecific event fulfilling the criteria of a target event to very low levels.
From the results of the multiparameter flow cytometric analysis and statistics of three separate serial dilutions experiments, it is easy to see that flow cytometric method had high sensitivity. When compared with RT-PCR method, it has more specificity although RT-PCR method had a higher sensitivity which is the detection of single epithelial cells in up to 107 peripheral blood mononuclear cells (8, 9, 26, 27). However, for nucleic acid techniques, in vitro sensitivity expressed in this way may overestimate the in vivo sensitivity of such assays because the tumor cells may crack and membrane fragments or nucleic acid in blood generally expressing markers remain. Furthermore, inhibitors of the PCR reaction present in tissues and body fluids could limit the in vivo sensitivity of nucleic acid-based techniques. CTCs of the patients that cannot be detected through multiparameter flow cytometric methods had Ep-CAM expression with RT-PCR, and there was no obvious difference of representation between CTCs ≥5 group and CTCs <5 group through RT-PCR. Therefore, in comparative studies, RT-PCR has higher rates of positive than flow cytometric assays. To make sure of the specificity of multiparameter flow cytomety technique, it is important to discard the first few milliliters of sampled blood.
Most researchers have used immunomagnetic combining flow cytometry technique to detect CTCs. However, it always loses an amount of target cells that would have a higher false negative and it is more expensive and multiple process that could not be applied in clinical reality. Additionally, although false-positive are often problematic with imaging or the use of tumor markers, the data from our study showed that patients in stage I, there were no more than 2 CTCs. Budd et al. (28) found that CTCs levels detected disease progression more accurately than imaging. Bone marrow biopsy is traumatic, whereas multiparameter flow cytomety which is atraumatic can be accepted by patients. On account of the above, we chose multiparameter flow cytometry as the appropriate technique.
The primary goal of this retrospective study was to confirm the prognostic value of CTCs in a cluster of patients by multiparameter flow cytometry method in China. This study demonstrated significantly reduced survival for patients with a baseline of ≥5 CTCs. The result suggests that the OS was independent of clinical pathology and diameter of tumor but correlated with age, CTCs level, and metastasis. For the variable of age, four patients who were more than 70 died during the follow-up including three of them who had CTCs ≥5 and two of them also had distinct metastasis. For the variable of CTCs level, in our study, the patients with ≥5 CTCs/7.5 mL of blood had a poorer median OS (65.5 weeks vs. 95 weeks; P < 0.05) compared with patients with <5 CTCs. Although Cristofanilli et al. (29) also observed that patients with ≥5 CTCs/7.5 mL of blood had poorer median OS (10.9 months vs. 21.9 months; P < 0.0001) compared with patients with <5 CTCs. The results of the current study strengthen the validity of previous finding. This outcome implied that measuring CTCs may provide significant value for clinical evaluation and for determining treatment options for patients.
From the outcome of our trial, CTCs can be considered an effective marker for prognosis, and multiparameter flow cytometry test could have great value for patients in China. By multiparameter flow cytometry test, patients who had CTCs <5 can be considered as micro-metastasis that could be treated by synthetic therapy to contain the development of disease, whereas, for the patients who do not have detected CTCs, there is no need to intensify treatments, avoiding the side effect of chemotherapy. However, larger studies are required to definitely establish the clinical usefulness of the technique. Moreover, we wish to isolate the CTSCs which have the marker of CD44+CD24-ESA-expressing and inject CTSCs into the peripheral blood of mice to create a model imitating the progression of BC in human. In conclusion, we have established a good foundation for following research.