**4.2. The significances of CTSCs in breast cancer**

**Patient characteristics CD45-**

\* *P* < 0.05. a

b

c

in ER<sup>+</sup>

ANOVA test.

206 Tumor Metastasis

Student's *t*‐test.

Kruskal‐Wallis test.

**4. Discussion**

**Table 5.** Percentage of CD45-

**/MNC (%) CTC/CD45-**


1+ 2.93 ± 5.20 0.33 ± 0.47 0.03 ± 0.05 3.21 ± 7.63 2+ 2.45 ± 2.32 0.36 ± 0.35 0.09 ± 0.14 1.68 ± 2.80 3+ 1.57 ± 0.99 0.61 ± 0.83 0.11 ± 0.15 5.51 ± 7.96

cells, CTC and CTSC, and their clinical relevance.

stage and RLNM status according to the percentage of CTSC on CD45‐C.

CK+

we found that the percentage of CTCs on CD45‐C in ER-

with breast cancer and assess the progression of disease.

**4.1. The significances of CTCs in breast cancer**

dual‐positive cells (CD45‐EpCAM<sup>+</sup>

The percentages of CTCs on CD45‐C with TNM and histology stage increasing. In addition,

Therefore, multiparameter flow cytometry technique is capable enough to identify patients

The detection of CTCs by using multiparameter flow cytometry relied on the epithelial‐specific marker, which was expressed on epithelial cells but not on leukocytes [2, 27, 28]. Some of the CTCs that had higher metastatic potential may lose the expression of epithelial‐specific markers during the migration process [29–31]. We also target another epithelial‐specific marker—Ep‐CAM (epithelial‐cell adhesion molecule) to avoid possible false negative [32]. In order to detect the tumor cells that come from epithelium tissue, a monoclonal antibody directed against CD45 for negative selection of leukocytes [9–11]. Therefore, we targeted the

test was demonstrated to confirm the sensitivity of the assay by adding SKBR‐3 into healthy sample. At the same time, we also verified the higher specificity of multiparameter flow cytometry by comparing with RT‐PCR, although RT‐PCR had a higher sensitivity [10, 11, 33, 34]. However, the detection of CTCs by nucleic acid techniques may overestimate the sensi‐ tivity, which resulted from the membrane fragments or nucleic acid of markers because of the crack of tumor cells in circle. CTC detection should be performed on cell level. On the other

 and PR<sup>+</sup> groups. And so was on CTSCs. Above all, we found the relationship between the percentage of CTC on CD45‐C and clinical pathology. Then, it was good for evaluate TNM

**TNM stage <sup>a</sup> <sup>a</sup> \*c <sup>c</sup>**

Her‐2 <sup>a</sup> <sup>a</sup> <sup>c</sup> <sup>a</sup>

 **(%) CTSC/CD45-**

 **(%) CTSC/CTC (%)**

and PR- groups was higher than that

) as a surrogate marker for CTCs. And the serial dilution

We chose CD44<sup>+</sup> CD24 as an excellent marker in CTSCs identify. CD44<sup>+</sup> CD24 had been considered as the marker of CSC [17, 37]. While CD133 was much more restricted in expression compared with CD44 that was the reason we did not chose CD133 as the CTSCs marker [38– 40]. ALDH1 was another maker to identify CSC from BC. Ginestier et al. reported that the expression of ALDH1 in normal and breast cancer was 3–10%, while expression of CD44<sup>+</sup> CD24 was 31% in contrast [41, 42]. In summary, CD44<sup>+</sup> CD24 was an excellent maker in CSC identify.

CTCs may exist after mastectomy and chemotherapy; even there was no clinical manifestation of breast cancer. It was explained as the theory of "dormancy" in tumor cells [43]. And it was a part of tumor stem cells. Once the balance between proliferation and apoptosis was destroyed by some inducement, the disease progressed. The immune system and angiogenesis was reported, which were correlated closely with tumor cell dormancy [43, 44]. When the tumor stem cells fell off the primary tumor, they came into the peripheral circle and became the circulating tumor stem cells (CTSCs).

We successfully confirmed that the existence of CTSCs and also reveal the relationship between CTSCs level and different TNM stages. The correlation of CTSCs and clinical pathologic features remains unclear before. Previous study had reported that the CD44+ CD24 cancer stem cells was not correlated with clinical features such as lymph node status, tumor size, histology grade, ER, and PR, or HER‐2 [45, 46].

It was reported that the patients who had high expression of CD44+ CD24- tumor stem cells were related with distant metastasis, particularly osseous [45]. And we found the expression of CTSCs was quite related with RLNM status. It was a novel way for treatment targeting at CTSCs to prevent metastasis and to evaluate the prognosis.

Cancer stem cells (CSCs) had been confirmed existed in many kinds of epithelial malignancy [47]. And the CSCs were considered as a subpopulation of tumor cells [48]. The mutation of normal stem cells resulted in the genesis of CSCs, which had been demonstrated by Cariati and Purushotham [49]. Therefore, CTSCs were suggested that it was generated not only from CTCs but also from normal stem cells. Further researches needed to be performed to confirm it.

Detection of CTCs and CTSCs by using multiparameter flow cytometry was considered as an effective technique on monitoring disease development and evaluating prognosis [50, 51]. Using multiparameter flow cytometry to detect CTC and CTSC in peripheral blood may provide new opportunities for the early diagnosis of invasive breast cancer, guide us to select optimal therapeutic regimens and help us to predict the prognosis. Moreover, we wish to isolate the CTSCs that have the marker of CD44+ CD24- ESA- expressing. Further researches on CTSCs needed to be demonstrated and we have established a firm basis for following research.
