**2. Methods**

which can be sampled at any stage of the disease. Then, the detection of CTCs for monitoring therapy was highly investigated in breast cancer. Circulating cells with the characteristics of tumor cells can be identified in the peripheral blood that is known as circulating tumor cells (CTCs) in many patients with solid tumors of epithelial origin. These cells are present both in patients with metastasis and in those whose tumors are localized [3]. Tumor cells shed into the circulation intermittently which was corresponding with microinvasive events. The first phase of metastatic consists of lessens of tumor cell adhesion, induction of tumor cell motility and local tumor cell invasion [4]. These steps are followed by either spread to circulation in peripheral blood or regional lymph nodes, and locating in secondary organs [5]. Some of these cells generate metastases eventually that can arise many years after therapy of the primary tumor at earlier phase [2]. CTCs also may be related to a half‐life probably 1–2.4 h, which means it cannot always exist in circulating [6]. Some authors argued that these cells were predominantly in G0 phase and thus are not replicating [7]; however, they did not exclude the existence that the prolifera‐ tion of CTCs can occur, although it was a rare event. Considering the half‐life of CTCs, the presence of CTCs in the blood could be maintained by a balance between replication and cell death. While apoptosis contributed to a high rate of circulating tumor cells, only a small part of the cells can adhere in second organs through blood vessels that were named as circulating

CTCs can be selected with a monoclonal antibody directed against CD45 for negative selection of leukocytes [9–11]. And from this cluster cells, EpCAM (epithelial‐cell adhesion molecule) and cytokeratin‐8 (CK‐8), CK‐18, CK‐19 (CK‐8, CK‐18, CK‐19 phycoerythrin staining)‐positive cells are the target cells, which were known as the marker of epithelial cells. The characteri‐ zation of CTCs presents a very hot topic in breast cancer research nowadays [12]. It helped to identify diagnosis and provide individual therapies according to the characterization of CTCs [12–14]. The characterization of CTSCs contributed to the identification and targeted therapy in breast cancer in the near future [15]. Molecularly targeted cancer therapies contributed great help which according to the characterization of CTCs especially on patients whose tumors have a particular mutation [16]. Some of the biological properties and the molecular charac‐ teristics of CTCs were connected to CTSCs and the genomic profiles have been completed [15]. Following that, the CD44‐positive CD24‐negative cells with their tumor‐initiating ability had been considered as CTSCs [17]. Hence, from CTC, CD44‐positive cell and CD24‐negative cell are CTSCs. Molecular characterization of CTCs, which is important for the identification of diagnostically and therapeutically relevant for individual therapies, it is difficult to address since they are very rare and the amount of available sample is very limited. Given the properties of the metastatic CTCs, there should be some opportunities for early identification

Some immunologic procedures, such as immunohistochemistry‐based methods and reverse transcriptase polymerase chain reaction (RT‐PCR), have been used to detect CTCs in past time [18–22]. However, current methods of detection do not seem to be sensitive or specific enough to apply in clinical [23–25]. Nowadays, we demonstrate the advent of the flow cytometry in the detection of CTCs, which makes a good balance of sensitivity and specificity. In addition,

tumor stem cells (CTSCs) [8].

196 Tumor Metastasis

and therapeutic targeting in breast cancer.

### **2.1. Patients**

We investigated 45 patients with breast cancer in the Union Hospital in Wuhan during September 2006 and June 2008 with three normal people as negative control. Twenty‐five patients had overt metastasis and 20 patients had no sign of overt metastasis. The character‐


ALND, axillary lymph node dissection.

**Table 1.** Patient characteristics.

istics of the patients are shown in **Table 1**, including mean age, TNM phase, histopathology, lymph node status, metastasis and so on. All these patients were incipient and were treated by systemic therapy, including 12 patients cured by cytokine‐induced killer cells therapy (CIK). And all the patients were drawn blood for the detection of CTCs.
