Epithelial Circulating Tumor Cells Research Articles

Relationship between circulating tumor cells, blood coagulation, and urokinase-plasminogen-activator system in early breast cancer patients.

Cancer is a risk factor for venous thromboembolism (VTE) and plasma d-dimer (DD) and tissue factor (TF) are established VTE associated markers. Circulating tumor cells (CTCs) are associated with the risk of VTE in metastatic breast cancer. This study aimed to correlate CTCs, blood coagulation and the urokinase plasminogen activator (uPA) system in primary breast cancer (PBC) patients. This prospective study included 116 PBC patients treated by primary surgery. CTCs were detected by quantitative RT-PCR assay for expression of epithelial (CK19) or epithelial-mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1, FOXC2). Plasma DD, TF, uPA system proteins were detected by enzyme-linked immunosorbent assays, while expressions of uPA system in surgical specimens were evaluated by immunohistochemistry. CTCs were detected in 27.6% patients. Patients with CTCs had a significantly higher mean plasma DD (ng/mL) than those of patients without CTCs (632.4 versus 365.4, p = 0.000004). There was no association between plasma TF and CTCs. Epithelial CTCs exhibit higher expression of uPA system genes compared to EMT_CTCs. Patients with CTCs had higher plasma uPA proteins than those of patients without CTCs; there was no correlation between tissue expression of uPA system, CTCs, DD or TF levels. In multivariate analysis CTCs and patients age were independent factors associated with plasma DD. We found association between plasma DD and CTCs indicating a potential role for activation of the coagulation cascade in the early metastatic process. CTCs could be directly involved in coagulation activation or increased CTCs could be marker of aggressive disease and increased VTE risk.

Circulating tumor cell enumeration with a combination of epithelial cell adhesion molecule- and cell-surface vimentin-based methods for monitoring breast cancer therapeutic response.

Detection, isolation, and enumeration of circulating tumor cells (CTCs) from cancer patients has become an important modality in clinical management of patients with breast cancer. Although CellSearch, an epithelial cell adhesion molecule (EpCAM)-based method that is used to isolate epithelial CTCs, has gained prominence, its inability to detect mesenchymal CTCs from breast cancer patients raises concerns regarding its utility in clinical management. To address this gap in technology, we recently discovered the utility of cell-surface vimentin (CSV) as a marker for detecting mesenchymal CTCs from sarcoma tumors. In the present study, we tested the sensitivity and specificity of detecting CTCs from blood collected at a random time during therapy from each of 58 patients with metastatic breast cancer by use of 84-1 (a monoclonal antibody against CSV to detect epithelial/mesenchymal-transition CTCs) and CellSearch methods. Additionally, we tested the possibility of improving the sensitivity and specificity of detection by use of additional parameters including nuclear EpCAM localization and epithelial mesenchymal ratios. CTC counts with CSV were significant (P = 0.0053) in differentiating populations responsive and nonresponsive to treatment compared with CTC counts with CellSearch (P = 0.0564). The specificity of CTC detection was found to be highest when the sum of CTC counts from the 2 methods was above a threshold of 8 CTCs/7.5 mL. The sum of CTC counts from the CellSearch and CSV methods appears to provide new insights for assessment of therapeutic response and thus provides a new approach to personalized medicine in breast cancer patients.

Overall Survival in Small Cell Lung Cancer Detected with Epithelial, Mesenchymal and Stem Cell Biomarkers

ABSTRACT Aim: Markers of epithelial to mesenchymal transition (EMT) and markers associated with cancer stem cells (CSC) have been associated with increased chemoresistance in small cell lung cancer (SCLC). We and others previously demonstrated that elevated circulating tumour cells (CTC) are associated with worse survival. In the current exploratory study we hypothesized that markers of EMT and CSC in tumour biopsies obtained at diagnosis were associated with overall survival in patients with SCLC. Methods: Biopsies selected from small cell lung cancer pts study published previously by our group [ Ann Oncol 2012;23:2937-42]. Epithelial markers [Cytokeratin 8,18,19 (CK; ab41825, Abcam), EPCAM (VU1D9, cellsignaling), E-cadherin (24E10, cellsignaling)] and mesenchymal markers [Vimentin (V9, Santa Cruz) and c-MET (EP1454Y, Abcam)] were used as biomarkers for EMT. SOX2 (L1D6A2, cellsignaling) and CD44 (IM7, Biolegend) were used as CSC markers. Scoring by two independent observers having no information about clinical outcome. Immuno-Reactive Score (IRS) was made by multiplying percentage (0-6) and intensity (0-3). IRS score>=5 was high, =2 low Results: From 38 patients (mean age 65 yrs [49-84], 18 female pts, ECOG performance score 1 [0-3], extensive disease stage 26 pts, CTC at baseline 19 [0-14040]) adequate material was available to cut at least 10 tumour containing slides for immunohistochemical staining. Staining intensity high/low: CK 10/27; Vimentin 23/15; CD44 19/19; SOX-2 17/21; cMET 19/19; IRS high/low EPCAM 20/18, E-cadherin 14/24. Combining primary tumour low c-MET intensity with high CK intensity and high E-Cadherin IRS resulted in a significantly worse survival vs the c-METhigh/CKlow/E-cadherinlow group, OS 10 vs 24 month, p=0.040. A trend towards a positive correlation between CTC and primary tumor c-METlow was seen, p=0.062. Conclusions: SCLC with an epithelial phenotype (E-Cadhigh, CKhigh, cMETlow) may give rise to more epithelial CTCs in circulation than those with an mesenchymal phenotype and have a worse prognosis. Disclosure: All authors have declared no conflicts of interest.

IGFBP1 in epithelial circulating tumor cells as a potential response marker to selective internal radiation therapy in hepatocellular carcinoma.

Local ablative techniques such as selective internal radiation therapy (SIRT) have become the mainstay of treating hepatocellular carcinoma (HCC) in the bridging-to-transplant and palliative setting. We recently demonstrated that epithelial circulating tumor cells (CTCs) correlate to an unfavorable outcome. We wanted to scrutinize whether molecular markers detected in this specific CTC subgroup may also have clinical implications. Mononuclear cells and CTCs were isolated from peripheral blood samples using density gradient centrifugation followed by depletion of hematopoietic and enrichment of epithelial (EpCAM(+)) cells employing immunomagnetic beads. The mRNA expression of candidate markers was correlated with response to SIRT in 25 patients using quantitative real-time reverse-transcription PCR. IGFBP1 mRNA expression levels were significantly correlated with time to progression in a Kaplan-Meier log rank test (p = 0.04; 0 vs 4 months) and receiver operating characteristic analysis demonstrated a potential use to predict patients with shortened time to progression (area under the curve: 0.8; 95% CI: 0.44-0.98; p = 0.03). The EpCAM fraction of CTCs may be useful to detect novel molecular markers to individualize treatment decision in patients with HCC.

Mesenchymal Phenotype of CTC-Enriched Blood Fraction and Lymph Node Metastasis Formation Potential

IntroductionCirculating tumor cells (CTCs) that present mesenchymal phenotypes can escape standard methods of isolation, thus limiting possibilities for their characterization. Whereas mesenchymal CTCs are considered to be more malignant than epithelial CTCs, factors responsible for this aggressiveness have not been thoroughly defined. This study analyzed the molecular profile related to metastasis formation potential of CTC-enriched blood fractions obtained by marker unbiased isolation from breast cancer patients without (N−) and with lymph nodes metastases (N+).Materials and MethodsBlood samples drawn from 117 patients with early-stage breast cancer were enriched for CTCs using density gradient centrifugation and negative selection with anti-CD45 covered magnetic particles. In the resulting CTC-enriched blood fractions, expression of CK19, MGB1, VIM, TWIST1, SNAIL, SLUG, HER2, CXCR4 and uPAR was analyzed with qPCR. Results were correlated with patients' clinicopathological data.ResultsCTCs (defined as expression of either CK19, MGB1 or HER2) were detected in 41% (20/49) of N− and 69% (34/49) of N+ patients (P = 0.004). CTC-enriched blood fractions of N+ patients were more frequently VIM (P = 0.02), SNAIL (P = 0.059) and uPAR-positive (P = 0.03). Positive VIM, CXCR4 and uPAR status correlated with >3 lymph nodes involved (P = 0.003, P = 0.01 and P = 0.045, respectively). In the multivariate logistic regression MGB1 and VIM-positivity were independently related to lymph node involvement with corresponding overall risk of 3.2 and 4.2. Moreover, mesenchymal CTC-enriched blood fractions (CK19−/VIM+ and MGB1+ or HER2+) had 4.88 and 7.85-times elevated expression of CXCR4 and uPAR, respectively, compared with epithelial CTC-enriched blood fractions (CK19+/VIM− and MGB1+ or HER2+).ConclusionsTumors of N+ patients have superior CTC-seeding and metastatic potential compared with N- patients. These differences can be attributed to VIM, uPAR and CXCR4 expression, which endow tumor cells with particularly malignant phenotypes.

Abstract P1-04-02: Correlation between blood markers of hemostasis and circulating tumor cells (CTCs) in early breast cancer patients

Abstract Background: Cancer is a risk factor for venous thromboembolism (VTE) and plasma D-dimer (DD) and tissue factor (TF) are established blood markers associated with VTE. CTCs are an independent predictor of survival in early and metastatic breast cancer (BC) patients (pts) and CTCs are associated with the risk of VTE in metastatic BC pts. In this study, we hypothesized correlation between plasma DD and TF with presence of CTCs. Moreover, we hypothesized, that activation of urokinase plasminogen activator system (uPA) could be involved in CTCs released into peripheral blood (PB) as well as coagulation activation. Methods: This prospective study included 119 early BC patients treated by primary surgery followed by systemic therapies when indicated, at the NCI in Slovakia from March to December 2012. Isolated peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells (CD45+) using RossetteSepTM negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT-inducing transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by qRT-PCR. Expressions of gene transcripts in CD45- PBMC of pts and healthy donors (HD) were compared. Patient samples with higher epithelial and/or mesenchymal gene transcripts than those of HD (n = 60) were considered as CTC positive. Plasma DD, TF, uPA (urokinase-type plasminogen activator) and PAI-1 (plasminogen activator inhibitor) levels were detected by ELISA, while expressions of TF and uPA system in surgical specimens were evaluated by immunohistochemistry. Results: CTCs were detected in 27.7% pts. The majority of CTCs exhibited either epithelial differentiation (13.5% pts) or mesenchymal phenotype (EMT markers in 15.1% of pts). A small fraction of CTCs showed co-expression of both markers (0.8%). Pts with any CTCs in PB had significantly higher mean ± SEM plasma DD levels (ng/mL) than those of pts without CTCs (632.4 ± 75.9 vs. 365.4 ± 47.0, p = 0.00002). This association was observed for both, epithelial CTCs (688.4 ± 110.4 vs. 400.7 ± 43.5 (p = 0.026) and mesenchymal CTCs (573.1 ± 105.8 vs. 415.6 ± 44.7, p = 0.0007). There was no association between plasma TF levels or breast tumor TF expression and CTCs. Patients with any CTCs had higher mean ± SEM plasma uPA (ng/mL) (374.6 ± 32.6 vs. 307.1 ± 20.3, p = 0.02) and PAI-1 (pg/mL) levels (5.3 ± 0.7 vs. 4.4 ± 0.4, p = 0.28), than those of patients without CTCs, however, there was no correlation between plasma uPA system activation and DD or TF levels. In multivariate analysis CTCs, patients age and tumor grade were independent factors associated with plasma DD levels. Conclusion: This prospective study showed for the first time a positive association between plasma D-dimer levels and CTCs indicating a potential role for activation of the coagulation cascade in the early metastatic process. We hypothesize that CTCs could be directly involved in coagulation activation in breast cancer patients, or alternatively an increased CTCs count could be a marker of more aggressive disease and increased risk of VTE. Future studies will need to address the prognostic implications of these observations with the potential for therapeutic interventions for the metastatic process. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-02.

Circulating tumor cells (CTCs) and epithelial mesenchymal transition (EMT) in primary breast cancer.

e22023 Background: CTCs are prognostic in breast cancer (BC) patients and represent a heterogeneous population of cells with different phenotypes and biological value. EMT gives rise to cells with stem cell-like properties with increased chemotherapy resistance and may be responsible for under detection of CTCs by currently approved assays. The aim of this study was to characterize CTCs based on the expression of epithelial and mesenchymal markers in non-metastatic BC patients. Methods: This prospective ongoing study included 124 patients (pts) with non-metastatic BC enrolled from March 2012 to December 2012. CTCs were detected before surgery (124 pts), before 1st cycle (28 pts) and before 2nd cycle (23 pts) of adjuvant therapy. Isolated peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSep kit negative selection with anti-CD45 antibody. RNA extracted from CD45-depleted (CD45-) PBMC was interrogated for expression of EMT-inducing transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by qRT-PCR. Expressions of gene transcripts in CD45- PBMC of patients were compared to those of CD45- PBMC of 60 healthy donors. Results: CTCs were detected in 26.6% pts before surgery, in 21.4% pts before 1st cycle and in 43.5% of pts before 2nd cycle of adjuvant therapy. Before surgery, CTCs exhibiting only epithelial makers were present in 12.9% pts, whereas CTCs with only EMT markers were observed in 12.1% of pts and CTCs coepressing both markers were detected in 1.6% pts. There was a trend for higher CTC positivity in high grade, triple negative tumors and in the presence of lymphovascular invasion. We detected CTCs with only an EMT phenotype more often in pts with N+ than in N0 disease (20.4% vs. 10.2%; p = 0.03) and in pts after 1stcycle of adjuvant chemotherapy compared to before surgery (30.4% vs. 12.1%; p = 0.03). There was no association between epithelial CTCs and analyzed clinico-pathological variables. Conclusions: Our data suggest that CTCs with EMT phenotype are involved in tumor dissemination and treatment resistance, and support the role of EMT in cancer pathogenesis. Moreover, these results underlay the concept of CTCs heterogeneity.

The prognostic value of circulating tumor cells lacking cytokeratins in metastatic breast cancer patients

In this study, we detected epithelial circulating tumor cells (CTCs) that expressed cytokeratins and potential circulating tumor cells (pCTCs) that had lost expression of cytokeratins in metastatic breast cancer (MBC) patients. The aim of the study was to evaluate the prognostic significance of these two kinds of CTCs in MBC patients. We detected CTCs and pCTCs from 66 MBC patients using MACS immunomagnetic enrichment technology combined with immunocytochemistry (ICC). A cutoff score of 5 CTCs (or pCTCs) was set as a benchmark for prognosis in patients. Progression free survival (PFS) was calculated and analyzed during the following 24 months. We evaluated the sensitivity of this method and recovery rates of the CTCs by spiking experiments. The loss number of tumor cells by our method was 0-15. The population with fewer than 5 CTCs showed significantly higher PFS than the group with 5 or more CTCs. The difference in PFS between the patients with 5 or more pCTCs and those with fewer than 5 pCTCs was statistically significant. The presence of these pCTCs more accurately predicted poor prognosis than the CTCs that express cytokeratins. There is a subset of CTCs that lose epithelial markers such as pCTCs. Due to the heterogeneity of the expression of epithelial antigens in CTCs, different subtypes of CTCs exist. Independently of CTCs, the groups of patients with pCTCs had poorer prognoses.

Abstract P2-01-14: circulating tumor cells in breast cancer exhibit dynamic changes in epithelial and mesenchymal cell composition

Abstract Epithelial to mesenchymal transition (EMT) has been postulated to contribute to the migration and dissemination of cancer cells, but supporting histopathological evidence is limited. We used a microfluidic device to isolate circulating tumor cells (CTCs), combined with multiplex fluorescent RNA-in-situ hybridization (ISH) and RNA sequencing, to quantify and characterize EMT in breast cancer cells within the bloodstream. Whereas only rare (0.1–10%) cells in the primary tumor expressed both mesenchymal and epithelial markers, such biphenotypic as well as purely mesenchymal cells were enriched among CTCs, across all histological subtypes of breast cancer. Analysis of the therapy response in 8 patients suggest an association of mesenchymal CTCs with disease progression. In an index patient followed longitudinally, fluctuation in epithelial and mesenchymal states was observed as a function of initial response and subsequent resistance to therapy. Mesenchymal markers were predominant in clusters of tumor cells, many of which had adherent platelets. Finally, RNA sequencing of mesenchymal CTC clusters identified TGF-B and other EMT-related signatures, which were absent from more epithelial CTCs. FOXC1, a known regulator of EMT, was abundantly expressed in mesenchymal CTCs and was detectable within localized regions of the primary breast tumor. Together, these data support a role for EMT in the blood-borne dissemination of breast cancer and point to the dynamic nature of this cell fate change. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-14.

Analysis of Circulating Tumor Cells in Patients with Non-small Cell Lung Cancer Using Epithelial Marker-Dependent and -Independent Approaches

Epithelial circulating tumor cells (CTCs) are detectable in patients with non-small cell lung cancer (NSCLC). However, epithelial to mesenchymal transition, a widely reported prerequisite for metastasis, may lead to an underestimation of CTC number. We compared directly an epithelial marker-dependent (CellSearch) and a marker-independent (isolation by size of epithelial tumor cells [ISET]) technology platform for the ability to identify CTCs. Molecular characteristics of CTCs were also explored. Paired peripheral blood samples were collected from 40 chemonäive, stages IIIA to IV NSCLC patients. CTCs were enumerated by Epithelial Cell Adhesion Molecule-based immunomagnetic capture (CellSearch, Veridex) and by filtration (ISET, RareCell Diagnostics). CTCs isolated by filtration were assessed by immunohistochemistry for epithelial marker expression (cytokeratins, Epithelial Cell Adhesion Molecule, epidermal growth factor receptor) and for proliferation status (Ki67). CTCs were detected using ISET in 32 of 40 (80%) patients compared with 9 of 40 (23%) patients using CellSearch. A subpopulation of CTCs isolated by ISET did not express epithelial markers. Circulating tumor microemboli (CTM, clusters of ≥ 3 CTCs) were observed in 43% patients using ISET but were undetectable by CellSearch. Up to 62% of single CTCs were positive for the proliferation marker Ki67, whereas cells within CTM were nonproliferative. Both technology platforms detected NSCLC CTCs. ISET detected higher numbers of CTCs including epithelial marker negative tumor cells. ISET also isolated CTM and permitted molecular characterization. Combined with our previous CellSearch data confirming CTC number as an independent prognostic biomarker for NSCLC, we propose that this complementary dual technology approach to CTC analysis allows more complete exploration of CTCs in patients with NSCLC.

A Novel Platform for Detection of CK+ and CK− CTCs

Metastasis is a complex, multistep process that begins with the epithelial-mesenchymal transition (EMT). Circulating tumor cells (CTC) are believed to have undergone EMT and thus lack or express low levels of epithelial markers commonly used for enrichment and/or detection of such cells. However, most current CTC detection methods target only EpCAM and/or cytokeratin (CK) to enrich epithelial CTCs, resulting in failure to recognize other, perhaps more important, CTC phenotypes that lack expression of these markers. Here, we describe a population of complex aneuploid CTCs that do not express CK or CD45 antigen in patients with breast, ovarian, or colorectal cancer. These cells were not observed in healthy subjects. We show that the primary epithelial tumors were characterized by similar complex aneuploidy, indicating conversion to an EMT phenotype in the captured cells. Collectively, our study provides a new method for highly efficient capture of previously unrecognized populations of CTCs. Current assays for CTC capture likely miss populations of cells that have undergone EMT. Capture and study of CTCs that have undergone EMT would allow a better understanding of the mechanisms driving metastasis.

Circulating Tumor Cells in Head and Neck Squamous Cell Carcinoma: Evaluation of a Novel Method

Objective: The primary objective of this project is to determine if circulating tumor cells (CTCs) are identifiable in peripheral blood from patients with advanced head and neck squamous cell carcinoma using a commercially available semi-automated technique. Method: Prior to treatment, adult patients with advanced Head and Neck Squmous Cell Carcinoma had whole peripheral blood drawn and analyzed using the CellSearch Epithelial CTC kit. The target molecules of the system include EpCAM and cytokeratins 8, 18, and 19. Results: There was a total of 18 patients with additional positive and negative controls. The average age was 65 years and 61% of the population was female. Primary tumor sites were varied with 39% laryngeal, 33% pharyngeal, 22% oral cavity, and 6% cutaneous. The majority of patients, 78%, had stage IV disease at the time of evaluation. The positive control, SCC-1483, identified over 305 CTCs. There were no CTCs identified in clinical samples. Conclusion: HNSCC cells are detectable using the CellSeach system, as evidenced by the positive control. Despite testing a population with advanced disease, the system failed to detect a CTC in a clinical sample. Therefore, the system is not a sensitive technique for identifying CTCs in HNSCC.

Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients

Recently, miRNA-expression profiling in primary tumors has yielded promising results. However, establishing miRNA expression in the circulation probably has advantages over determination in primary tumor tissue, further augmenting the potential applications of miRNA determination in oncology. Circulating tumor cells (CTCs) have rapidly developed as important prognostic and therapy-monitoring biomarkers in metastatic breast, colorectal and prostate cancer when enumerated, and their isolation enables subsequent analysis using various molecular applications, including miRNA-expression analysis. In addition to CTC-associated miRNAs, free circulating miRNAs have been identified in whole blood, plasma and serum. Determination of miRNAs in peripheral blood, either cell-free or CTC-associated, is expected to become important in oncology, especially when linked to and interpreted together with epithelial CTCs. In this article, we will discuss miRNA-expression profiling in primary tumors, depict the potential applications of measuring miRNA in the circulation and review the literature on cell-free circulating miRNAs, as well as offering some methodological and technical considerations on the measurement of circulating miRNAs.

Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells

This paper presents development of a parylene membrane microfilter device for single stage capture and electrolysis of circulating tumor cells (CTCs) in human blood, and the potential of this device to allow genomic analysis. The presence and number of CTCs in blood has recently been demonstrated to provide significant prognostic information for patients with metastatic breast cancer. While finding as few as five CTCs in about 7.5 mL of blood (i.e., 10 10 blood cells in) is clinically significant, detection of CTCs is currently difficult and time consuming. CTC enrichment is performed by either gradient centrifugation of CTC based on their buoyant density or magnetic separation of epithelial CTC, both of which are laborious procedures with variable efficiency, and CTC identification is typically done by trained pathologists through visual observation of stained cytokeratin-positive epithelial CTC. These processes may take hours, if not days. Work presented here provides a micro-electro-mechanical system (MEMS)-based option to make this process simpler, faster, better and cheaper. We exploited the size difference between CTCs and human blood cells to achieve the CTC capture on filter with ∼90% recovery within 10 min, which is superior to current approaches. Following capture, we facilitated polymerase chain reaction (PCR)-based genomic analysis by performing on-membrane electrolysis with embedded electrodes reaching each of the individual 16,000 filtering pores. The biggest advantage for this on-membrane in situ cell lysis is the high efficiency since cells are immobilized, allowing their direct contact with electrodes. As a proof-of-principle, we show beta actin gene PCR, the same technology can be easily extended to real time PCR for CTC-specific transcript to allow molecular identification of CTC and their further characterization.