EMT is believed to play an important role in intravasation and the release of CTCs, and the expression of EMT-inducing TF gene transcripts in breast cancer has been associated with poor prognosis 16
. In this study, 8 of the 52 (15.4%) PBC patients had overexpression of at least one EMT-inducing TF gene in the PBMC fractions depleted of both epithelial cells and leukocytes. We observed that SLUG gene transcript was the most commonly overexpressed EMT-inducing TF gene transcript (6 out of the 8 cases), while SNAIL1 and ZEB1 gene transcripts were not overexpressed in any of the samples. SLUG is believed to be required for the invasion and bone marrow homing of cancer cells of a different origin 17
. Hence, the long term follow up of patients is needed to determine if overexpression of SLUG is a significant prognostic factor for metastasis. Moreover, TWIST1 gene expression was more often detected in patients than in healthy donors (57.7% vs. 23.3%; P
= 0.003); however, only one patient overexpressed the TWIST1 gene transcript.
EMT has been previously linked with cancer stem cell properties 13
, which have been associated with increased therapeutic resistance 18–20
. We report that patients who received NAT were more likely to exhibit overexpression of EMT-inducing TF gene transcripts in their PBMC fractions depleted of both epithelial cells and leukocytes in comparison to identical cell fractions of patients who had not received NAT. This finding suggests either the intrinsic nature of CTCs undergoing EMT to resist NAT or an association between therapeutic stress and the surviving CTCs undergoing EMT. Furthermore, our data are consistent with another study that reported chemotherapy and hormonal treatment induced not only apoptosis, but also EMT in an experimental model 21
. Consistent with these findings, we also observed an increase in disseminated tumor cells with ALDH activity (Aldeflour+
) in bone marrow of PBC patients who had received NAT 22
. Unfortunately, the current study cannot definitely exclude the expression of EMT-inducing TF gene transcripts by other cells such as hematopoietic progenitor cells mobilized from the bone marrow or other EpCAM−
cells such as endothelial cells.
Paradoxically, we observed that some poor prognostic subgroups like patients with HER2 amplified, ER and PR negative, and/or triple-receptor negative tumors did not overexpress EMT-inducing TF gene transcripts. Alternatively, our observations could be a consequence of the small sample size and under representation of these patient subgroups in our study. Nevetheless, other mechanism beyond EMT could also be responsible for the outcome of these patients.
A recent study reported that there was a substantial variation in the detection rates of CTCs from breast cancer patients, 36% of patients with MBC were positive by CellSearch®
and 22% by the AdnaTest™ 23
. Consistent with these findings, we found no association between the presence of CTCs measured by CellSearch®
and the overexpression of EMT-inducing TF gene transcripts. Alternatively, the discordance between CTC and EMT-inducing TF gene transcripts in some patients suggests that the conventional CTC detection assays are incapable of detecting both epithelial and mesenchymal phenotypes. We found that HMEC-TWIST1 cells with complete EMT phenotype cannot be detected using conventional EpCAM based CTCs detection methods such as CellSearch®
Breast Cancer Select/Detect. However, HMEC-TWIST1 cells can be detected using a qRT-PCR based method in blood samples depleted of CD45+
leukocytes. Furthermore, Aktas et al
showed that more than 60% of CTCs detected by the AdnaTest-EMT™
kit expressed genes associated with EMT and stem cell phenotype (TWIST1, PI3K, Akt, ALDH) 12
. Taken together, these data suggest that there is a continuum of development of CTCs from one end of the spectrum (epithelial phenotype) to the other end of the spectrum (mesenchymal phenotype), and some CTCs with a partial EMT phenotype may co-express both epithelial and mesenchymal related genes (). Since CTCs with the EMT phenotype play a significant role in the progression of epithelial cancers, we suggest that the detection of CTCs undergoing EMT could have prognostic value in a broad range of epithelial tumors.
Despite the small sample size, this is a first study aimed to detect CTCs based on the overexpression of EMT-inducing TF gene transcripts in peripheral blood of PBC patients. Nevertheless we are prepared to concede that our study has a few limitations starting with the recognition that the qRT-PCR does not allow visualization of CTCs and therefore the detection of CTCs with EMT phenotype by this method is, at best, an indirect assessment. Secondarily, the limited sample size is an under representation of high risk subgroups such as those with HER2 amplified or triple-receptor negative primary tumors. Finally, the lack of paired samples in the current study prohibits definitive conclusions regarding the effect of NAT. On the other hand, our data are consistent with an in vitro
study and translational findings linking EMT induction and cancer stem cell characteristics with treatment resistance 21, 22
In summary, CTC expressing EMT-inducing gene transcripts are likely to be enriched in PBC patients who have received neoadjuvant chemotherapy, while there were no differences in expression levels of EMT-inducing TF gene transcripts according tumor size, tumor grade or tumor type. The loss of epithelial antigen on CTCs as a result of EMT, likely triggered by high expression of EMT-inducing TFs, may be responsible for the underestimation of CTCs by conventional methods such as CellSearch® or AdnaTest™ that rely on the expression of EpCAM/CD326 by CTCs for optimal detection. A future prospective study is warranted to characterize CTCs in partial or complete EMT that could lead to identification of additional tumor markers that might serve as potential novel therapeutic targets. In addition, we believe our methodology is a reliable assay that detects CTCs undergoing EMT in peripheral blood and may facilitate the monitoring of therapeutic agents capable of targeting CTC in epithelial-mesenchymal transition.