Cancer Res Treat.  2016 Oct;48(4):1229-1242. 10.4143/crt.2015.500.

A New Cell Block Method for Multiple Immunohistochemical Analysis of Circulating Tumor Cells in Patients with Liver Cancer

• Nam SJ
• Yeo HY
• Chang HJ
• Kim BH
• Hong EK
• Park JW

• KMID: 2356225
• DOI: http://doi.org/10.4143/crt.2015.500

Abstract

PURPOSE
We developed a new method of detecting circulating tumor cells (CTCs) in liver cancer patients by constructing cell blocks from peripheral blood cells, including CTCs, followed by multiple immunohistochemical analysis.
MATERIALS AND METHODS
Cell blockswere constructed from the nucleated cell pellets of peripheral blood afterremoval of red blood cells. The blood cell blocks were obtained from 29 patients with liver cancer, and from healthy donor blood spikedwith seven cell lines. The cell blocks and corresponding tumor tissues were immunostained with antibodies to seven markers: cytokeratin (CK), epithelial cell adhesion molecule (EpCAM), epithelial membrane antigen (EMA), CK18, α-fetoprotein (AFP), Glypican 3, and HepPar1.
RESULTS
The average recovery rate of spiked SW620 cells from blood cell blocks was 91%. CTCs were detected in 14 out of 29 patients (48.3%); 11/23 hepatocellular carcinomas (HCC), 1/2 cholangiocarcinomas (CC), 1/1 combined HCC-CC, and 1/3 metastatic cancers. CTCs from 14 patients were positive for EpCAM (57.1%), EMA (42.9%), AFP (21.4%), CK18 (14.3%), Gypican3 and CK (7.1%, each), and HepPar1 (0%). Patients with HCC expressed EpCAM, EMA, CK18, and AFP in tissue and/or CTCs, whereas CK, HepPar1, and Glypican3 were expressed only in tissue. Only EMA was significantly associated with the expressions in CTC and tissue. CTC detection was associated with higher T stage and portal vein invasion in HCC patients.
CONCLUSION
This cell block method allows cytologic detection and multiple immunohistochemical analysis of CTCs. Our results show that tissue biomarkers of HCC may not be useful for the detection of CTC. EpCAM could be a candidate marker for CTCs in patients with HCC.

Keyword

Circulating neoplastic cells; Hepatocellular carcinoma; Immunohistochemistry

MeSH Terms

Antibodies
Biomarkers
Blood Cells
Carcinoma, Hepatocellular
Cell Line
Cholangiocarcinoma
Epithelial Cells
Erythrocytes
Glypicans
Humans
Immunohistochemistry
Keratins
Liver Neoplasms*
Liver*
Methods*
Mucin-1
Neoplastic Cells, Circulating*
Portal Vein
Tissue Donors
Antibodies
Biomarkers
Glypicans
Keratins
Mucin-1

Figure

• 

  Fig. 1. Western blot analysis of expression of E-cadherin, vimentin, SLUG, and SNAIL proteins in human liver cancer cell lines. β-Actin was used as a loading control. HepG2, Huh7, and SNU-182 showed epithelial characteristics. SNAIL, SNU-387, and SNU-449 showed mesenchymal characteristics. PLC/RPF5 showed semi-epithelial characteristics.

• 

  Fig. 2. Histologic and immunohistochemical images (×400) of peripheral blood cell block containing spiked hepatocellular carcinoma cell line HepG2. The HepG2 cells show strong expression of cytokeratin (CK), Glypican3, and α-fetoprotein (AFP), and weak expression of epithelial cell adhesion molecule (EpCAM), but the cells are negative for HepPar1.

• 

  Fig. 3. Representative images (×400) of marker expression in circulating tumor cell (CTC) and hepatocellular carcinoma (HCC) tissue samples from three patients. All three CTCs showed strong cytoplasmic expression of epithelial cell adhesion molecule (EpCAM), but their corresponding HCC tissues were negative for EpCAM. The pairs of samples showed identical expression patterns for cytokeratin 18 (CK18), α-fetoprotein (AFP), epithelial membrane antigen (EMA), and Glypican3.

• 

  Fig. 4. Association of tumor marker expression in circulating tumor cells (CTCs) and tumor tissue of the 14 hepatocellular carcinoma (HCC) patients positive for CTCs. Three patients with HCC showed aberrant epithelial cell adhesion molecule (EpCAM) expression in CTC. Two patients with HCC showed aberrant epithelial membrane antigen (EMA) expression in CTC. AFP, α-fetoprotein; CK, cytokeratin.

• 

  Fig. 5. Survival analysis according to presence of circulating tumor cell (CTC). Kaplan-Meier analyses of survival of patients with liver cancer (A-D) and patients with hepatocellular carcinoma (HCC) (E-H). p-values were determined using log-rank tests. The presence of CTCs and CTCs positive for epithelial cell adhesion molecule (EpCAM) and α-fetoprotein (AFP) resulted in poorer prognosis, but the differences were not statistically significant. EMA, epithelial membrane antigen.

Reference

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