Detection of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer

Abstract

Purpose: Gastric cancer studies indicated a potential correlation between circulating tumor cells (CTCs) in peripheral blood and tumor relapse/metastasis. The prevalence and significance of circulating tumor microemboli (CTM) in gastric cancer remain unknown. We investigated the prevalence and prognostic value of CTCs and CTM for progression-free survival (PFS) and overall survival (OS) in gastric cancer patients.

Methods: Eighty-one gastric cancer patients consented to provide 5ml of peripheral blood before systematic therapy. CTCs and CTM were isolated using isolation by size of epithelial tumor cells and characterized by cytopathologists. For 41 stage IV gastric cancer patients, CTM was investigated as a potential biomarker to predict prognosis.

Results: CTCs were detected in 51 patients; the average count was 1.81. In clinical stage I, II, III, and IV patients, the average CTC counts were 1.40, 0.67, 1.24, and 2.71, respectively. CTM were detected in 3 of 33 clinical stage I to IIIb patients, at an average of 0.12 (0-2). CTM were detected in 13 of 53 clinical stage IIIc to IV patients, at an average of 1.26 (0-22). In stage IV patients, CTM positivity correlated with the CA125 level. PFS and OS in CTM-positive patients were significantly lower than in CTM-negative patients (P<.001). CTM positivity was an independent factor for determining the PFS (P=.016) and OS (P=.003) of stage IV patients in multivariate analysis. Using markers of the epithelial-mesenchymal transition, single CTCs were divided into three phenotypes including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. For CTM, CK-/Vimentin+/CD45- and CK+/Vimentin+/CD45- phenotypes were observed, but the CK+/Vimentin-/CD45- CTM phenotype was not. CA125 was detected in gastric cancer cell lines BGC823 and MGC803.

Conclusions: In stage IV patients, CTM positivity was correlated with serum CA125 level. CTM were an independent predictor of shorter PFS and OS in stage IV patients. Thus, CTM detection may be a useful tool to predict prognosis in stage IV patients.

Figure 1

Cells enriched by the ISET method in patients with gastric cancer. Diff-Quick staining. (A) CTC: irregular nucleus; nuclear diameter >15 μm; nuclear-cytoplasmic ratio >0.8; hyperchromatic nucleus and nonhomogeneous staining. (B) CTM: presence of tumor cell aggregation, prominent nucleoli seen in the nuclei. (C) Nucleus without plasma. (D) Normal blood cells isolated on the membrane. The scale bar represents 20 μm.

Figure 2

Immunofluorescent staining of CTCs/CTM isolated by ISET assay. (A–D) Stained with CK8/18/19 (green fluorescence for epithelial cells), Vimentin (purple fluorescence for EMT cells), CD45 (red fluorescence for leukocytes), and Hoechst (blue fluorescence for nuclei). (A) Single CTC: CK+/Vimentin−/CD45− phenotype; (B) single CTC: CK+/Vimentin+/CD45− phenotype; (C) single CTC: CK−/Vimentin+/CD45− phenotype; (D) CTM: CK−/Vimentin+/CD45− phenotype; (E) CTM: CK+/Vimentin+/CD45− phenotype. The cells were analyzed at 40× magnification.

Figure 3

(A) In 41 patients with stage IV gastric cancer, the PFS rate was significantly lower in patients with CTM than in those without CTM (P < .05). (B) In 41 patients with stage IV gastric cancer, the OS rate was significantly lower in patients with CTM than in those without CTM (P < .05). (C) In 30 patients with single CTCs or CTM, the PFS rate was significantly lower in patients with CTM than in those with only single CTCs (P < .05). (D) In 30 patients with single CTCs or CTM, the OS rate was significantly lower in patients with CTM than in those with only single CTCs (P < .05).

Figure 4

Detection of CA125 expression in human gastric epithelial cell line GES-1 and human gastric cancer cell lines. (A) Immunofluorescent staining of GES-1, BGC823, MGC803, MKN45, and SGC7901 cell lines with CA125 (red fluorescence for CA125) and DAPI (blue fluorescence for nuclei). (B) Quantitative RT-PCR was carried out to detect relative CA125 mRNA expression in GES-1, BGC823, MGC803, MKN45, and SGC7901 cell lines. The cells were analyzed at 40× magnification.