Thymidylate synthase expression in circulating tumor cells: a new tool to predict 5-fluorouracil resistance in metastatic colorectal cancer patients

Emne Ali Abdallah, Marcello Ferretti Fanelli, Marcilei Eliza Cavicchioli Buim, Marcelo Calil Machado Netto, José Luiz Gasparini Junior, Virgílio Souza E Silva, Aldo Lourenço Abbade Dettino, Natalia Breve Mingues, Juliana Valim Romero, Luciana Menezes Mendonça Ocea, Bruna Maria Malagoli Rocha, Vanessa Silva Alves, Daniel Vilarim Araújo, Ludmilla Thomé Domingos Chinen, Emne Ali Abdallah, Marcello Ferretti Fanelli, Marcilei Eliza Cavicchioli Buim, Marcelo Calil Machado Netto, José Luiz Gasparini Junior, Virgílio Souza E Silva, Aldo Lourenço Abbade Dettino, Natalia Breve Mingues, Juliana Valim Romero, Luciana Menezes Mendonça Ocea, Bruna Maria Malagoli Rocha, Vanessa Silva Alves, Daniel Vilarim Araújo, Ludmilla Thomé Domingos Chinen

Abstract

Thymidylate synthase (TYMS) is an important enzyme for 5-fluorouracil (5-FU) metabolism in metastatic colorectal cancer (mCRC) patients. The search for this enzyme in circulating tumor cells (CTCs) can be a powerful tool to follow-up cancer patients. mCRC patients were enrolled before the beginning of 5-FU-based chemotherapy. The blood was filtered on Isolation by Size of Epithelial Tumor Cells (ISET), and the analysis of TYMS expression in CTCs was made by immunocytochemistry. Additionally, we verified TYMS staining in primary tumors and metastases from the same patients. There were included 54 mCRC patients and 47 of them received 5-FU-based chemotherapy. The median CTCs number was 2 per mL. We were not able to analyze immunocytochemistry in 13 samples (9 patients with absence of CTCs and 4 samples due to technical reasons). Therefore, TYMS expression on CTCs was analyzed in 34 samples and was found positive in 9 (26.5%). Six of these patients had tumor progression after treatment with 5-FU. We found an association between CTC TYMS staining and disease progression (DP), although without statistical significance (P = 0.07). TYMS staining in primary tumors and metastases tissues did not have any correlation with disease progression (P = 0.67 and P = 0.42 respectively). Patients who had CTC count above the median (2 CTCs/mL) showed more TYMS expression (P = 0.02) correlating with worse prognosis. Our results searching for TYMS staining in CTCs, primary tumors and metastases suggest that the analysis of TYMS can be useful tool as a 5-FU resistance predictor biomarker if analyzed in CTCs from mCRC patients.

Keywords: chemotherapy resistance; circulating tumor cells; isolation by size of epithelial tumor cells; metastatic colorectal cancer; thymidylate synthase.

© 2015 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

Figures

Figure 1
Figure 1
Immunostaining of Thymidylate synthase (TYMS). (a) Primary tumor tissue positive for TYMS. (b) Positive control, a normal palatine tonsil tissue. (c) Negative control, tumor tissue without antibody. (d) CTC TYMS positive. (e) Positive control, a white blood cell. (f) Negative control, a CTC without antibody. Thin arrows represent pores of ISET membrane, thick arrows show leukocytes and asterisks indicate CTCs. Images were taken at ×600 magnification using a light microscope (Research System Microscope BX61—Olympus, Tokyo, Japan) coupled to a digital camera (SC100—Olympus, Tokyo, Japan). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 2
Figure 2
Immunostaining showing heterogeneity of TYMS expression in the same sample. Figures (a), (b) and (c) present primary tumor from the same patient with no expression, moderate and strong expression of TYMS respectively. Figures (d), (e) and (f) present metastasis tissue from another patient with different grades of staining (weak, moderate and strong expression of TYMS respectively). Images were taken at ×200 magnification using a light microscope (Research System Microscope BX61—Olympus, Tokyo, Japan) coupled to a digital camera (SC100—Olympus, Tokyo, Japan). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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