Molecular Detection of EMT Markers in Circulating Tumor Cells from Metastatic Non-Small Cell Lung Cancer Patients: Potential Role in Clinical Practice

Abstract

Background Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related mortality; nevertheless, there are few data regarding detection of circulating tumor cells (CTCs) in NSCLC, compared to other kinds of cancers in which their prognostic roles have already been defined. This difference is likely due to detection methods based on the epithelial marker expression which ignore CTCs undergoing epithelial-mesenchymal transition (CTCsEMT). Methods After optimization of the test with spiking experiments of A549 cells undergoing TGF-β1-induced EMT (A549EMT), the CTCsEMT were enriched by immunomagnetic depletion of leukocytes and then characterized by a RT-PCR assay based on the retrieval of epithelial and EMT-related genes. Blood samples from ten metastatic NSCLC patients before starting treatment and during chemotherapy were used to test this approach by longitudinal monitoring. Ten age- and sex-matched healthy subjects were also enrolled as controls. Results Recovery experiments of spiked A549EMT cells showed that the RT-PCR assay is a reliable method for detection of CTCsEMT. CTCsEMT were detected in three patients at baseline and in six patients after four cycles of cysplatin-based chemotherapy. Longitudinal monitoring of three patients showed that the CTCsEMT detection is related to poor therapeutic response. Conclusions The RT-PCR-based approach for the evaluation of CTCsEMT phenotype could be a promising and inexpensive tool to predict the prognosis and the therapeutic response in NSCLC patients.

Epithelial‑mesenchymal transition phenotype of circulating tumor cells is associated with distant metastasis in patients with NSCLC

“…During the dynamic testing, the expression of the mesenchymal phenotype appeared to be associated with a more unfavorable prognostic factor. However, the clinical significance of the EMT phenotypic CTC needs to be further validated considering the limited patient number in the study by Milano et al (36). In the present study, 110 patients (85 patients with NSCLC from different clinical stages and 25 patients with benign diseases) were recruited.…”

“…Thus, the expression of epithelial cell markers including EpCAM and members of CKs are downregulated and that of mesenchymal cell markers is upregulated (14)(15)(16)35). Milano et al (36) designed a study to detect the EMT of CTCs in patients with metastatic NSCLC and analyzed the expression of epithelial markers (carcinoembryonic antigen-CK19), EMT-associated genes (vimentin) and EMT transcription factors (zinc finger protein SNAI2, zinc finger E-box-binding homeobox 1-2 and TWIST1-2) using a reverse transcription-polymerase chain reaction assay in order to calculate the cell numbers. However, in the present study, RNA in situ hybridization with a combination of epithelial (EpCAM and CK8/18/19) and mesenchymal (vimentin and TWIST1) markers were used to detect the epithelial and mesenchymal phenotype of CTCs, and an automated imaging fluorescent microscope was used to directly count the cells with different phenotypes.…”

“…However, in the present study, RNA in situ hybridization with a combination of epithelial (EpCAM and CK8/18/19) and mesenchymal (vimentin and TWIST1) markers were used to detect the epithelial and mesenchymal phenotype of CTCs, and an automated imaging fluorescent microscope was used to directly count the cells with different phenotypes. Furthermore, in the study by Milano et al (36), only 10 metastatic patients with NSCLC and 10 healthy volunteers were recruited to test the feasibility of CTC testing in the clinic. This previous study revealed that 3 of 10 samples were positive for CTCs at the baseline and the positive percentage of the patients increased subsequent to treatment with the platinum-based chemotherapy.…”

Intravenous 5-fluoro-2′-deoxycytidine administered with tetrahydrouridine increases the proportion of p16-expressing circulating tumor cells in patients with advanced solid tumors

“…While CTC analyses have typically relied upon selection of epithelial-phenotype CTCs via EpCAM, CK, or other epithelial markers, mesenchymal-and mixed epithelial/ mesenchymal-phenotype CTCs are, a priori, more consistent with the characteristics of metastatic tumor cells, including enhanced motility, invasiveness, and self-renewal [42]; indeed, data from several recent studies across multiple tumor types have suggested that mesenchymal-or mixed E/M-phenotype CTCs may be of greater prognostic and/or predictive value than their epithelial-phenotype counterparts [24][25][26][27]. Following the emergence of this information regarding the potential predictive value of mesenchymal-or mixed E/M-phenotype CTCs, we developed and validated a novel custom CellSearch ® assay to assess p16 expression in the putative mixed E/M-phenotype subpopulation.…”

“…Initially, we focused this analysis on epithelial-phenotype (cytokeratin-positive, putatively EpCAM-expressing) CTCs. However, during the course of the trial, new knowledge came to light regarding the biological relevance and potential prognostic value of mesenchymal-and mixed epithelial/mesenchymal (E/M)-phenotype CTCs in patients with metastatic disease [24][25][26][27]. Therefore, we developed and validated a novel 5-channel CellSearch ® assay to assess treatment-induced changes in p16 expression in the putative mixed E/M-phenotype (vimentin-positive, putatively EpCAM-expressing) CTC subpopulation for patients who enrolled during the final years of the study, after validation of this assay for E/M-phenotype CTCs had been completed.…”