Circulating Tumor Cell Analyses in Patients With Esophageal Squamous Cell Carcinoma Using Epithelial Marker-Dependent and -Independent Approaches

Abstract

In several epithelial malignancies, detection of circulating tumor cells (CTCs) in the peripheral blood has diagnostic, prognostic, and therapeutic implications. However, the clinical relevance of CTCs in esophageal squamous cell carcinoma (ESCC) has not yet been ascertained. The study was conducted with the aim of determining the clinical significance of CTCs in patients with ESCC by using 2 CTC detection systems, one epithelial marker-dependent and the other epithelial marker-independent. Paired peripheral blood samples were prospectively obtained from 61 ESCC patients before treatment and were analyzed for CTCs isolated by the CellSearch system (CS) and the method of isolation by size of epithelial tumor (ISET). Blood samples from 22 healthy volunteers were used as controls. Out of 61 study subjects, CTCs were detected in 20 patients (32.8%) by the ISET method and in only 1 patient (1.6%) by the CS method. Circulating tumor microemboli (CTM) were observed in 3 of 61 (4.9%) patients using ISET, but were undetectable in any of the patient by CS method. No CTCs/CTM were detected by either method in control groups. By ISET method, the presence of CTCs appeared to correlate with the stage of ESCC and with the baseline median platelet levels. No correlation with any other relevant clinicopathological variables was observed. Our results clearly indicate the ability of both CS and ISET methods to detect CTCs in peripheral blood samples from ESCC patients. However, the CellSearch system appears to have a poorer sensitivity as compared with the ISET method. Further studies are essential for assessing the role of such technologies in ESCC.

FIGURE 1

Within CellSearch^TM system, CTCs are defined as epithelial origin cells with round or oval morphology, cell size >4 μm in diameter, cell surface antigen EpCAM, CK, and DAPI positive, CD45 antigen negative (EpCAM+/CK+/DAPI+/CD45−).

FIGURE 2

Cytomorphological analysis of CTCs/CTM detected on filtered blood using the ISET method in patients with ESCC. (A–D) Cells showing cytological malignant features isolated by the ISET method in patients with ESCC. Romanowsky staining. (A) CTC: nuclear-cytoplasmic ratio >0.8; nucleoli is abnormally huge; (B) CTC: the diameter of the nucleus >18 μm; nuclear membranes appear thickened, sunken, wrinkled, and jagged; (C) CTC: nuclear shape is irregular; hyperchromatic nuclei with nonuniform color; presence of nuclear chromatin side shift; (D) CTM: presence of tumor cells (≥3) aggregation. The cells were analyzed under ×40 magnification. The scale bar is 20 μm.

FIGURE 3

Immunofluorescent staining for characterization of CTCs/CTM detected by ISET assay. (A–D) Immunofluorescent staining characterization of CTC/CTM detected by ISET assay. Stained with Hoechst (blue fluorescence for nucleus), CK8/18/19 (green fluorescence for epithelial cells), vimentin (purple fluorescence for EMT cells), and CD45 (red fluorescence for leukocytes). (A) Cell line control; (B) CTC: expression of CK-positive in their CTCs (CK+/Vimentin−/CD45−); (C) CTM: expression of CK-weakly positive and Vimentin-positive in cells within CTM (CK+/Vimentin+/CD45−); (D) EMT-CTM: heterogeneity was observed for CK-negative and Vimentin-positive (CK−/Vimentin+/CD45−). The cells were analyzed under ×40 magnification.