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Circulating tumor cells are important markers of tumor progression and can reflect tumor behavior in metastatic colorectal cancer (mCRC). Identification of proteins that confer resistance to treatment is an important step to predict response and better selection of treatment for patients. Multidrug resistance-associated protein 1 (MRP1) and Multidrug resistance-associated protein 4 (MRP4) play a role in irinotecan-resistance, and Excision Repair Cross-Complementation group 1 (ERCC1) expression can confer resistance to platinum compounds. Here, we included 34 patients with mCRC and most of them received FOLFIRI or FOLFOX chemotherapy (91.1%). CTCs were isolated by ISET(®) Technology and identified in 30 patients (88.2%), with a median of 2.0 CTCs/mL (0-31.0). We analyzed the immunocytochemical expression of MRP1, MRP4 and ERCC1 only in patients who had previously detectable CTCs, accordingly to treatment received (n = 19, 15 and 13 patients, respectively). Among patients treated with irinotecan-based chemotherapy, 4 out of 19 cases with MRP1 positive CTCs showed a worse progression free survival (PFS) in comparison to those with MRP1 negative CTCs (2.1 months vs. 9.1 months; p = 0.003). None of the other proteins studied in CTCs had significant association with PFS. We analyzed also histological sections of primary tumors and metastases by immunohistochemistry, and found no association with clinicopathological characteristics or with PFS. Our results show MRP1 as a potential biomarker of resistance to treatment with irinotecan when found in CTCs from mCRC patients. This is a small proof-of-principle study and these early findings need to be validated in a larger cohort of patients.

Circulating tumor cells are important markers of tumor progression and can reflect tumor behavior in metastatic colorectal cancer (mCRC). Identification of proteins that confer resistance to treatment is an important step to predict response and better selection of treatment for patients. Multidrug resistance-associated protein 1 (MRP1) and Multidrug resistance-associated protein 4 (MRP4) play a role in irinotecan-resistance, and Excision Repair Cross-Complementation group 1 (ERCC1) expression can confer resistance to platinum compounds. Here, we included 34 patients with mCRC and most of them received FOLFIRI or FOLFOX chemotherapy (91.1%). CTCs were isolated by ISET(®) Technology and identified in 30 patients (88.2%), with a median of 2.0 CTCs/mL (0-31.0). We analyzed the immunocytochemical expression of MRP1, MRP4 and ERCC1 only in patients who had previously detectable CTCs, accordingly to treatment received (n = 19, 15 and 13 patients, respectively). Among patients treated with irinotecan-based chemotherapy, 4 out of 19 cases with MRP1 positive CTCs showed a worse progression free survival (PFS) in comparison to those with MRP1 negative CTCs (2.1 months vs. 9.1 months; p = 0.003). None of the other proteins studied in CTCs had significant association with PFS. We analyzed also histological sections of primary tumors and metastases by immunohistochemistry, and found no association with clinicopathological characteristics or with PFS. Our results show MRP1 as a potential biomarker of resistance to treatment with irinotecan when found in CTCs from mCRC patients. This is a small proof-of-principle study and these early findings need to be validated in a larger cohort of patients.

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., What's new? Currently, the common treatment strategy for metastatic colorectal cancer patients is 5‐Fluorouracil‐based chemotherapy, which shows high efficacy in a subset of patients. Even those patients, however, can experience disease progression due to 5‐FU resistance. There are indications that the DNA replication and repair enzyme thymidylate synthase (TYMS) may be involved. Here, the authors set to measure circulating tumor cells levels and search for TYMS staining to correlate these findings with clinical outcome. The results suggest that circulating tumor cells represent a powerful tool to follow up 5‐FU resistance in metastatic colorectal cancer patients in real time, by TYMS expression analysis.