M.M. and S.A.M. share first authorship
Cancer Cell Biology
Expression of epithelial–mesenchymal transition-inducing transcription factors in primary breast cancer: The effect of neoadjuvant therapy†
Version of Record online: 27 APR 2011
Copyright © 2011 UICC
International Journal of Cancer
Volume 130, Issue 4, pages 808–816, 15 February 2012
How to Cite
Mego, M., Mani, S. A., Lee, B.-N., Li, C., Evans, K. W., Cohen, E. N., Gao, H., Jackson, S. A., Giordano, A., Hortobagyi, G. N., Cristofanilli, M., Lucci, A. and Reuben, J. M. (2012), Expression of epithelial–mesenchymal transition-inducing transcription factors in primary breast cancer: The effect of neoadjuvant therapy. Int. J. Cancer, 130: 808–816. doi: 10.1002/ijc.26037
The methods described in this article are representative of a patent application by Michal Mego, Sendurai Mani, Massimo Cristofanilli and James M. Reuben.
- Issue online: 16 DEC 2011
- Version of Record online: 27 APR 2011
- Accepted manuscript online: 8 MAR 2011 10:43AM EST
- Manuscript Accepted: 1 FEB 2011
- Manuscript Revised: 18 JAN 2011
- Manuscript Received: 21 DEC 2010
- UICC American Cancer Society International Fellowship for Beginning Investigators, ACSBI Award. Grant Number: ACS/08/006
- National Cancer Institute. Grant Numbers: R01 CA138239-02, 1 P50 CA116199
- CDMRP Department of Defense. Grant Number: BC087443
- State of Texas Rare and Aggressive Breast Cancer Research Program
- Society of Surgical Oncology Clinical Investigator Award
- circulating tumor cells;
- epithelial–mesenchymal transition;
- primary breast cancer;
- neoadjuvant therapy
Epithelial cancer cells are likely to undergo epithelial–mesenchymal transition (EMT) prior to entering the peripheral circulation. By undergoing EMT, circulating tumor cells (CTCs) lose epithelial markers and may escape detection by conventional methods. Therefore, we conducted a pilot study to investigate mRNA transcripts of EMT-inducing transcription factors (TFs) in tumor cells from the peripheral blood (PB) of patients with primary breast cancer (PBC). PB mononuclear cells were isolated from 52 patients with stages I–III PBC and 30 healthy donors (HDs) and were sequentially depleted of EpCAM+ cells and CD45+ leukocytes, henceforth referred to as CD45−. The expression levels of EMT-inducing TFs (TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2) in the CD45− cells were determined using quantitative real-time polymerase chain reaction. The highest level of expression by the CD45− cell fraction of HD was used as “cutoff” to determine if samples from patients with PBC overexpressed any EMT-inducing TFs. In total, 15.4% of patients with PBC overexpressed at least one of the EMT-inducing TF transcripts. Overexpression of any EMT-inducing TF transcripts was more likely to be detected in patients with PBC who received neoadjuvant therapies (NAT) than patients who received no NAT (p = 0.003). Concurrently, CTCs were detected in 7 of 38 (18.4%) patients by CellSearch® and in 15 of 42 (35.7%) patients by AdnaTest™. There was no association between the presence of CTCs measured by CellSearch® or AdnaTest™. In summary, our results demonstrate that CTCs with EMT phenotype may occur in the peripheral circulation of patients with PBC and that NAT is unable to eliminate CTCs undergoing EMT.
Recent studies have demonstrated that circulating tumor cells (CTCs) in the peripheral blood (PB) is an independent prognostic factor for progression-free survival and overall survival in patients with metastatic breast,1 colon2 or prostate3 cancers. Commercially available CTCs detection kits, including the U.S. Food and Drug Administration-cleared CellSearch® system (Veridex LLC, Warren, NJ) and the real-time polymerase chain reaction (RT-PCR)-based AdnaTest Breast Cancer Select/Detect kit or AdnaTest™ (AdnaGen AG, Langenhagen, Germany), exploit the expression of epithelial cell adhesion molecule (EpCAM; also known as epithelial surface antigen/CD326) and epithelial cytokeratins (CKs) to detect CTCs.1, 4–6 In addition to EpCAM, AdnaTest™ recognizes a CTC as expressing MUC1 and/or HER2.
A common cause of death for patients diagnosed with epithelial tumors, including breast tumors, is the development of distant metastatic disease, which is difficult to predict and diagnose in the early stages. To metastasize to distant organs, epithelial tumor cells must detach from the primary tumor, traverse the peripheral circulation, extravasate into the parenchyma and establish a new tumor.7 A number of studies have shown that carcinoma cells often activate a transdifferentiation program, termed epithelial–mesenchymal transition (EMT), to acquire the traits needed to execute the multiple steps of metastasis.8 Through the EMT process, epithelial cells lose cell–cell contacts and cell polarity, downregulate epithelial-associated genes, acquire mesenchymal gene expression and undergo major changes in their cytoskeleton. This cellular process culminates in a mesenchymal appearance and increased motility and invasiveness.8
Cancer cells can be induced to undergo EMT by several signaling pathways, most notably those involving the cooperation between TGF-β1 signaling and oncogenic Ras or other receptor tyrosine kinases, as well as Wnt, Notch and the signaling activated by Hedgehog.9 In addition, certain transcription factors (TF), including TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2, can induce EMT in mammary epithelial cells and/or breast cancer cells.10 Moreover, blocking the expression of Twist in the highly metastatic 4T1 murine mammary cell line reduced both metastatic burden and the number of CTCs in mice bearing xenograft mammary tumors,11 thus linking EMT, metastasis and the presence of CTCs. These findings suggest that the expression of epithelial cell surface markers, such as EpCAM, may not be optimal for detecting a heterogeneous population of CTCs including those with a mesenchymal phenotype. Currently, a CTC detection kit is available to detect CTCs expressing EMT-associated genes by semiquantitative RT-PCR (AdnaTest EMT-1/Stem Cell Select/Detect kit or AdnaTest-EMT™). However, this kit includes an initial step to enrich EpCAM-expressing cells.12
Recently, we found that induction of EMT in immortalized human mammary epithelial cells (HMECs) results in de novo expression of stem cell markers and acquisition of functional stem cell properties, including mammosphere-forming potential. These findings illustrated a link between the EMT process and tumor-initiating cells (TICs)13 and suggest that EMT contributes to the heterogeneity of tumor-initiating potential observed amongst breast tumor cells. Because of the aggressive nature of TICs, these findings support the need to detect tumor cells with mesenchymal features in patients with breast cancer.
EMT plays a pivotal role during breast cancer progression14 and results in the loss of epithelial markers, and current CTCs detection kits may not detect CTCs with EMT features. Thus, we determined the feasibility of detecting CTCs based on the mRNA expression of EMT-inducing TF in PB of patients with primary breast cancer (PBC) and to correlate these findings with clinical parameters as well as the presence of CTCs determined by EpCAM-based CTCs detection methods.
Patients and Methods
This is a translational study (Protocol 04-0657; Chair: A. Lucci) approved by the Institutional Review Board (IRB) of the University of Texas MD Anderson Cancer Center and was conducted between November 2008 and May 2009. The study included 52 patients with stages I–III PBC who were undergoing definitive surgery. We assessed the presence of CTCs measured by the CellSearch® system and the AdnaTest Breast Cancer Select/Detect test (AdnaTest™) at the time of surgery. Each patient was given a complete diagnostic evaluation to exclude the presence of distant metastasis. Healthy donors (HDs) were age-matched women without breast cancer who were recruited and consented according to the IRB-approved protocol. All samples were processed as described in Figure 1.
Detection of CTCs in PB using the CellSearch® system
The CellSearch® system (Veridex LLC) was used to detect CTCs in 7.5 mL of PB, as previously described.4 Briefly, PB samples from patients with breast cancer were subjected to enrichment of EpCAM+ cells with anti-EpCAM-coated ferrous particles. CTCs were defined as nucleated cells lacking surface expression of the leukocyte antigen CD45 but expressing cytoplasmic CK-8, −18 or −19.1 Samples were considered to be positive if they had ≥1 CTC per 7.5 mL PB.
Detection of CTC using AdnaTest Breast Cancer Select and AdnaTest Breast Cancer Detect kits (AdnaTest™)
Five milliliters of PB was collected in AdnaCollect™ tubes (AdnaGen AG), transported to the laboratory on ice and processed within 4 hr of collection according to the manufacturer's instructions. Briefly, samples were enriched for epithelial cells with anti-EpCAM-coated magnetic beads (AdnaTest Breast Cancer Select kit). Thereafter, mRNA was isolated from EpCAM-enriched cells and followed by reverse transcription to cDNA and PCR to detect expression of tumor-associated antigens (EpCAM, MUC1 and HER-2) and the housekeeping gene, β-actin (AdnaTest Breast Cancer Detect kit). Samples were considered positive if the PCR product expressed transcripts of at least one of the tumor-associated genes.
Depletion of CD45+ leukocytes prior to detection of EMT-inducing TF genes by a quantitative RT-PCR assay
The AdnaTest Breast Cancer Select kit was used to deplete the PB of EpCAM+ cells. Next, the EpCAM-depleted PB was subjected to a ficoll-hypaque density gradient to isolate PB mononuclear cells (PBMCs). The PBMCs were collected, washed twice with sterile phosphate-buffered saline and adjusted to a concentration of 107 PBMCs per milliliter and incubated with 40 μL of magnetic beads coated with anti-CD45 antibody (Miltenyi-Biotec, Auburn, CA) for 15 min on 4°C. Thereafter, the PBMCs were passed through a magnet-filled column on an AutoMACSPro Cell Separator (Miltenyi-Biotec) using the negative selection protocol (DEPLETE protocol) to enrich for CD45-depleted CTCs. The CD45-depleted (CD45−) sample was recycled through the magnet-filled column to deplete the sample of any residual CD45+ cells using the MACS DEPLETES protocol. Using this approach, a median percentage of 97.4% CD45+ cells (range: 90.1–99.8%) were depleted.
RNA extraction and cDNA synthesis
CD45-depleted cells were mixed with TRIzol® LS Reagent (Invitrogen Corporation, Carlsbad, CA) and stored at −80°C until it was necessary to extract RNA according to the manufacturer's instructions. The isolated RNA was treated by DNAse (Ambion, Austin, TX) to minimize contamination by genomic DNA and stored at −80°C. All RNA preparation and handling steps took place in a laminar flow hood, under RNase-free conditions. RNA concentration was determined by absorbance readings at 260 nm. RNA extracted from HMEC transduced with TWIST1 (HMEC-TWIST1)13 and SUM149 cells8 were used as positive controls. Reverse transcription of RNA was carried out with the cDNA archive kit (Applied Biosystems, Foster City, CA).
Identification of gene transcripts in CD45−-enriched subsets
Synthesized cDNA was subjected to quantitative RT-PCR (qRT-PCR) to detect EMT-inducing TF gene transcripts (TWIST, SNAIL1, SLUG, ZEB1 and FOXC2) and EpCAM. In brief, 2.5 μL of cDNA were placed in 25 μL of reaction volume containing 12.5 μL of TaqMan Universal PCR Master Mix, No AmpErase UNG, 8.75 μL water and 1.25 μL of primers. The following TaqMan assays were purchased from ABI: TWIST1: Hs00361186_m1; SNAIL1: Hs00195591_m1; SLUG: Hs00161904_m1; ZEB1: Hs01566408_m1; FOXC2: Hs00270951_s1 and EpCAM: Hs00158980_m1. Amplicons spanned intron–exon boundaries, with the exception of FOXC2 because it is a single exon.
Amplification was performed using an ABI Fast 7500 Real-Time PCR system (ABI) using the cycling program: 95°C for 10 min; 40 cycles of 95°C for 15 sec and 60°C for 60 sec. All samples were analyzed in triplicate. Calibrator samples were run with every plate to ensure consistency of the PCR. DNA contamination was assessed by performing PCR on the nonreverse transcribed portion of each sample. All samples demonstrated sufficient elimination of genomic DNA. For all fluorescence-based RT-PCR, fluorescence was detected between 0 and 40 cycles for the control and marker genes in single-plex reactions, which allow for the deduction of the cycles at threshold (CT) value for each product. Expression of the genes of interest was calibrated against expression of the housekeeping gene, GAPDH. Target cDNA was quantified using the delta-CT method with the formula: ½Ct(target-GAPDH).
Detection of CTC in normal PB spiked with HMEC-TWIST1 cells
The immortalized HMECs were transduced with TWIST1 as previously described.13 Four 7.5 mL samples of peripheral blood from HDs were collected. Two of the PB samples were spiked with either 200 HMEC-Control or 200 HMEC-TWIST1 and were analyzed by CellSearch® to determine its ability to detect the spiked epithelial cells. Each of the remaining two 7.5 mL PB samples was spiked with HMEC-TWIST1 cells and were analyzed by AdnaTest™ to determine its ability to detect the spiked cells. Thereafter, the four PB samples containing the spiked epithelial cells were sequentially depleted of EpCAM+/CD326+ epithelial cells and CD45+ leukocytes before being assessed for the presence of TWIST1 gene transcripts by RT-PCR (Fig. 2).
The SUM149 cell line was developed from pleural effusions of a patient with inflammatory breast cancer.8 SUM149 cells display a partial EMT phenotype as evidenced by relatively high expression of fibronectin, N-cadherin and vimentin along with expression of EpCAM.15 SUM149 cells were suspended in F-12 Hams medium (Gibco™, Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (Tissue Culture Biologicals, Seal Beach, CA), 5 μg/mL of insulin and 1 μg/mL hydrocortisone and were cultured in a humidified incubator at 37°C with 5% CO2. As with the HMEC-Control and HMEC-TWIST1 cells, 200 SUM149 cells were spiked into 7.5 mL of PB sample of a HD and were analyzed with AdnaTest™ and CellSearch® assays to detect the spiked cells. Additionally, the SUM149-spiked PB sample was sequentially depleted of EpCAM/CD326+ epithelial cells and CD45+ leukocytes before being assessed for TWIST1 gene transcripts by RT-PCR (Fig. 3).
The patients' characteristics were summarized using the median (range) for continuous variables and frequency (percentage) for categorical variables. Fisher's exact test was used to assess the association between overexpression of EMT-inducing TF transcripts and other patients' characteristics. A p-value of <0.05 was deemed to detect statistically significant differences between samples. All statistical analyses were conducted using SAS 9.1 (SAS Institute, Cary, NC).
Expression of EMT-inducing TF gene transcripts TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2 in CD45-depleted PBMC from HDs
To analyze for overexpression of EMT-inducing TF transcripts in CD45-depleted PBMC from patients, we first established the range of expression of these genes in HDs. By using TaqMan-based qRT-PCR, we were unable to detect SLUG transcripts in any of the 30 CD45-depleted PBMC fractions from HD. Conversely, TWIST1, SNAIL1, ZEB1 and FOXC2 gene transcripts were detected in 23.3, 86.7, 93.3 and 93.3% of HD samples, respectively. The levels of TWIST1, SNAIL1, ZEB1 and FOXC2 transcripts in the HD samples were calculated relative to that of the housekeeping gene, GAPDH. The highest expression levels of the EMT-inducing TF gene transcripts relative to that of GAPDH were 2.0 × 10−4, 1 × 10−2, 2.2 × 10−2 and 2.1 × 10−2 for TWIST1, SNAIL1, ZEB1 and FOXC2, respectively. These values were used as “cutoff” to determine if breast cancer patients have overexpression of EMT-inducing TF gene transcripts in the PBMC samples depleted of both EpCAM/CD326+ epithelial cells and CD45+ leukocytes.
Relative quantification of TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2 gene transcripts in CD45-depleted PBMC of patients with primary breast cancer depleted of epithelial cells and leukocytes
The study population consisted of 52 PBC patients with median age of 54 years (range: 34–72 years). There were 42 (80.8%) patients with estrogen receptor positive (ER+) and/or progesterone receptor positive (PR+) tumors, 6 (11.2%) with HER-2/neu amplified and 7 (13.5%) with triple receptor negative (TN) tumors (Table 1). Twenty-three (44.2%) patients had positive lymph nodes and 14 (26.9%) patients received neoadjuvant therapy (NAT), including five patients who achieved pathological complete responses (pCRs). Patients treated with NAT were more likely to have lymph node metastasis than non-NAT patients (100% vs. 23.7%; p < 0.001), whereas the differences in stage of disease (stage ≥ T2: 78.6% vs. 44.7%; p = 0.06), grade of disease (grade ≥ 3: 35,7% vs. 24.3%; p = 0.49) and tumor type (infiltrative ductal carcinoma: 78.6% vs. 78.9%; p = 0.99) were not statistically significant between NAT and non-NAT patients.
Among the 52 PBC patient samples analyzed, the expression of EMT-inducing TF transcripts were detected as follows: 30 (57.7%) TWIST1, 47 (90.4%) SNAIL1, 6 (11.5%) SLUG and 52 (100%) ZEB1. TWIST1 transcript was more commonly expressed in patients than in HDs (57.7% vs. 23.3%; p = 0.003). The FOXC2 transcript was detected in all of the 31 patient samples tested for the presence of this TF.
To determine overexpression of the EMT-inducing TF gene transcripts in PBC patients, we compared the expression levels in patient samples with those of HDs. Relative to the highest levels of SNAIL and ZEB1 transcripts detected in HD samples, none of the patient samples overexpressed these gene transcripts. Among the patient samples, TWIST1, SLUG and FOXC2 transcripts were overexpressed in 1 of 52 (1.9%), 6 of 52 (11.5%) and 1 of 31 (3.2%) samples, respectively. Overexpression of at least one of the EMT-inducing TF gene transcripts was detected in 8 (15.4%) of the 52 patients. There was no overlap between overexpression of EMT-inducing TF gene transcripts in patient samples.
Overexpression of EMT-inducing TF gene transcripts in relation to clinicopathological parameters
Overexpression of TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2 gene transcripts in relation to various clinicopathological characteristics is shown in Table 2. There was no association between overexpression of these EMT-inducing TF transcripts and tumor size, ER/PR status, HER2/neu amplification or TN tumors. Patients who received NAT were more likely to have an overexpression of EMT-inducing TF gene transcripts in their PBMC fractions depleted of both epithelial cells and leukocytes compared to those of patients who did not receive NAT (42.9% vs. 5.3%; p = 0.003). Of the 14 patients who received NAT, five achieved a pCR. Although only one of five (20%) patients with pCR had overexpression of EMT-inducing TF gene transcripts, five of nine patients (55%) without pCR had overexpression of EMT-inducing TF gene transcripts (p = 0.30).
Overexpression of EMT-inducing TF gene transcripts relative to CTCs measured by CellSearch® and AdnaTest™
CTC enumeration by CellSearch® was available for 38 of the 52 patients. Seven of the 38 (18.4%) patients had a detectable CTC count by CellSearch® (range: 1–17 per 7.5 mL of PB; Table 1). Of the 42 patients who were tested for CTCs by the RT-PCR-based AdnaTest™, at least one of the tumor-associated gene transcripts was detected in 15 (35.7%) patients. We found that 20 of the patient samples tested positive for CTCs by either the CellSearch® or AdnaTest™ assays or both. Patients who tested positive for CTCs, by either CellSearch® or AdnaTest™, had a higher tendency to overexpress EMT-inducing TF gene transcripts compared to patients with a negative CTC test (25.0% vs. 7.7%; Table 2). However, there was no association between the overexpression of any EMT-inducing TF gene transcripts and the presence of CTCs detected by either CellSearch® (p = 0.30) or AdnaTest™ (p = 0.23; Table 3).
Detection of spiked cells with EMT phenotype in PB samples of HDs
To test if cells that had undergone EMT can be detected by EpCAM-based detection methods, we first used HMECs that ectopically expressed TWIST1 (HMEC-TWIST1) and the respective control vector-infected HMECs (HMEC-Control). A previous study showed that HMEC-TWIST1 cells exhibited an EMT phenotype as evidenced by spindle-shaped morphology, inability to form epithelial-like colonies in monolayer culture, the complete loss of E-cadherin protien and de novo expression of N-cadherin, vimentin and fibronectin.10 These traits suggest that HMEC-TWIST1 cells had fully acquired a mesenchymal phenotype or, in other words, had undergone a “complete EMT.” To test whether the commercially available CTC detection assays could detect HMECs that had undergone EMT, we analyzed PB samples in HDs that were spiked with either HMEC-TWIST1 or HMEC-Control cells using both CellSearch® and AdnaTest™ kits. The CellSearch® and AdnaTest™ kits were able to detect the HMEC-Control cells, whereas the mesenchymal-like HMEC-TWIST cells were not detected by either CellSearch® or the AdnaTest™ (Figs. 2a and 2b). On the other hand, PB samples of HDs that were spiked with SUM149 cells with a partial EMT phenotype were detected by the PCR-based AdnaTest™ and the AdnaTest-EMT™ kits (Fig. 3a) as well as the CellSearch® system (Fig. 3b). These results suggest that CellSearch®, AdnaTest™ and AdnaTest-EMT™ kits can detect cancer cells that have undergone a partial EMT.
As the CellSearch® and AdnaTest™ kits were unable to detect CTCs with a complete mesenchymal phenotype, we next spiked HMEC-TWIST1 and SUM149 cells into PB samples of HDs and then depleted the samples of CD45+ leukocytes before determining the expression of EMT-inducing TF gene transcripts using qRT-PCR. Indeed, we were able to detect TWIST1 gene transcripts in the CD45− fraction of HMEC-TWIST1 (Fig. 2c) and SUM149 cells (Fig. 3c) spiked blood samples of HDs. In addition, we detected SNAIL1 and SLUG gene transcripts in CD45-depleted PBMC isolated from blood samples of HDs spiked with SUM149 (data not shown).
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%) patients with PBC 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 (six of eight cases), whereas 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 HDs (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 aldehyde dehydrogenase (ALDH) activity (Aldeflour+/CD45−/CD326+) in bone marrow of patients with PBC 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−/CD45− 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. Nevertheless, other mechanisms 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 patients with breast cancer, 36% of patients with metastatic breast cancer 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® or AdnaTest™ 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® or AdnaTest™ 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.12 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 and ALDH). 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 coexpress both epithelial- and mesenchymal-related genes (Fig. 3). As 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 the first study that aimed to detect CTCs based on the overexpression of EMT-inducing TF gene transcripts in PB of patients with PBC. 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 our 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 patients with PBC who have received neoadjuvant chemotherapy, while there were no differences in expression levels of EMT-inducing TF gene transcripts according to 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 that our methodology is a reliable assay that detects CTCs undergoing EMT in PB and may facilitate the monitoring of therapeutic agents capable of targeting CTC in EMT.
The authors thank the patients treated at the Nellie B. Connally Breast Center, the University of Texas MD Anderson Cancer Center, for their willingness to participate in translational research studies. Michal Mego was supported by a UICC American Cancer Society International Fellowship for Beginning Investigators, ACSBI Award. Evan Cohen is the recipient of CDMRP Department of Defense Predoctoral Fellowship Award. Massimo Cristofanilli, James M. Reuben and Sendurai Mani are the recipients of a grant from the National Cancer Institute to study human breast cancer stem cell surrogates. Massimo Cristofanilli is also the recipient of a grant from the State of Texas Rare and Aggressive Breast Cancer Research Program. We also acknowledge the support of American Airlines Susan G. Komen Promise Grant for Novel Targets for Treatment and Detection of Inflammatory Breast Cancer, KGO71287. Anthony Lucci is the recipient of the Society of Surgical Oncology Clinical Investigator Award and a grant from the CDMRP Department of Defense. Gabriel N. Hortobagyi is the principal investigator of a Breast Cancer SPORE and has received a grant from the National Cancer Institute.