Epithelial Circulating Tumor Cells Research Articles

Characteristics of circulating tumor cells in organ metastases, prognosis, and T lymphocyte mediated immune response.

Circulating tumor cells (CTCs) possess profound influence on tumor metastases and disease progression. This study aimed to investigate the correlation of CTCs with clinical characteristics and T-cell immunity, and to explore whether CTCs and the subpopulations can serve as an independent prognostic factor in advanced non-small cell lung cancer (NSCLC). A prospective study was conducted in late stages of NSCLC patients. The levels of overall CTCs and the three subpopulation CTCs were enumerated using the CanPatrol™ CTC enrichment system. The information about the patients which included the clinical characteristics, survival status at the 200th day postdiagnosis, and the levels of T cells was collected. Mann–Whitney U test, Kruskal–Wallis H test, Cox regression, and Spearman’s rank correlation coefficient were the statistical methods used in this study. We detected CTCs in 27 of the 31 eligible patients; the level of epithelial–mesenchymal circulating tumor cells (EMCTCs) was higher than that of epithelial circulating tumor cells and that of mesenchymal circulating tumor cells (MCTCs) in the majority of NSCLC patients. Organ metastases were positively associated with the levels of overall CTCs, EMCTCs, and MCTCs (P<0.05). EMCTCs and MCTCs were associated with worse clinical outcomes. Additionally, the levels of EMCTCs were negatively associated with the levels of CD3+ T cells (P=0.01) and CD8+ T cells (P=0.04). In conclusion, the levels of CTCs were positively associated with organ metastases, particularly bone metastases, but were negatively associated with T-cell levels. The levels of EMCTCs and MCTCs had negative prognostic value.

Detection of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer

PURPOSE: Gastric cancer studies indicated a potential correlation between circulating tumor cells (CTCs) in peripheral blood and tumor relapse/metastasis. The prevalence and significance of circulating tumor microemboli (CTM) in gastric cancer remain unknown. We investigated the prevalence and prognostic value of CTCs and CTM for progression-free survival (PFS) and overall survival (OS) in gastric cancer patients. METHODS:Eighty-one gastric cancer patients consented to provide 5ml of peripheral blood before systematic therapy. CTCs and CTM were isolated using isolation by size of epithelial tumor cells and characterized by cytopathologists. For 41 stage IV gastric cancer patients, CTM was investigated as a potential biomarker to predict prognosis. RESULTS:CTCs were detected in 51 patients; the average count was 1.81. In clinical stage I, II, III, and IV patients, the average CTC counts were 1.40, 0.67, 1.24, and 2.71, respectively. CTM were detected in 3 of 33 clinical stage I to IIIb patients, at an average of 0.12 (0-2). CTM were detected in 13 of 53 clinical stage IIIc to IV patients, at an average of 1.26 (0-22). In stage IV patients, CTM positivity correlated with the CA125 level. PFS and OS in CTM-positive patients were significantly lower than in CTM-negative patients (P<.001). CTM positivity was an independent factor for determining the PFS (P=.016) and OS (P=.003) of stage IV patients in multivariate analysis. Using markers of the epithelial-mesenchymal transition, single CTCs were divided into three phenotypes including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. For CTM, CK−/Vimentin+/CD45− and CK+/Vimentin+/CD45− phenotypes were observed, but the CK+/Vimentin−/CD45− CTM phenotype was not. CA125 was detected in gastric cancer cell lines BGC823 and MGC803. CONCLUSIONS: In stage IV patients, CTM positivity was correlated with serum CA125 level. CTM were an independent predictor of shorter PFS and OS in stage IV patients. Thus, CTM detection may be a useful tool to predict prognosis in stage IV patients.

EMT-like circulating tumor cells in ovarian cancer patients are enriched by platinum-based chemotherapy.

BackgroundAssuming that tumor cell dissemination requires a shift to a mesenchymal phenotype, we analyzed the incidence of epithelial-to-mesenchymal-transition (EMT)-like circulating tumor cells (CTCs) in ovarian cancer patients and inquired, how their molecular phenotypes respond to platinum-based chemotherapy and influence outcome.ResultsBefore surgery, overall detection rate for epithelial CTCs was 18%. EMT-like CTCs were more frequently observed (30%) and were mutually exclusive to epithelial CTCs in the majority of patients (82%). After chemotherapy, EMT-like CTCs increased up to 52%, accompanied by the “de novo” emergence of PI3Kα+/Twist+ EMT-like CTCs. Before surgery, PI3K+ EMT-like CTCs in combination with epithelial CTCs indicated decreased OS (p = 0.02) and FIGO I-III patients with residual tumor burden after surgery were more likely to be positive for EMT-like CTCs after chemotherapy (p = 0.02). In the latter group, epithelial CTCs alone significantly correlated with decreased PFS and OS (p = 0.02, p = 0.002), supported by an additional inclusion of PI3K+ CTCs (OS, p = 0.001).Materials and MethodsBlood samples of 91 ovarian cancer patients before surgery and 31 matched samples after adjuvant chemotherapy were evaluated for CTCs with the AdnaTest ovarian cancer and EMT-1, analyzing the epithelial-associated transcripts EpCAM, Muc-1 and CA125 and the EMT-associated transcripts PI3Kα, Akt-2 and Twist.ConclusionsPlatinum-based chemotherapy seems to select for EMT-like CTCs in ovarian cancer patients and provokes a shift towards PI3Kα and Twist expressing CTCs, which may reflect clonal tumor evolution towards therapy resistance. It has to be determined, whether this CTC subgroup may serve as a biomarker to identify patients at high risk.

FAM172A expression in circulating tumor cells for prediction of high-risk subgroups of colorectal cancer.

ObjectivesPrevious studies used enumerated circulating tumor cells (CTCs) to predict prognosis and therapeutic effect in several types of cancers. However, increasing evidence showed that only enumerated CTCs were not enough to reflect the heterogeneity of tumors. Therefore, we classified different metastasis potentials of CTCs from colorectal cancer (CRC) patients to improve the accuracy of prognosis by CTCs.MethodsBlood samples were collected from 45 primary CRC patients. CTCs were enriched by blood filtration, and the RNA in situ hybridization method was used to identify and discriminate subgroups of CTCs. Later, FAM172A expression in individual CTCs was measured.ResultsThree CTC subgroups (epithelial/biophenotypic/mesenchymal CTCs) were identified using epithelial–mesenchymal transition markers. In our research, mesenchymal CTCs significantly increased along with tumor progression, including developing distant metastasis and vascular invasion. Furthermore, FAM172A expression rate in mesenchymal CTCs was significantly higher than that in epithelial CTCs, which suggested that FAM172A may correlate with tumor malignancy. This hypothesis was further verified by FAM172A expression in mesenchymal CTCs strictly related to tumor aggressiveness factors. Finally, we revealed that mesenchymal CTCs and FAM172A expression may predict high-risk subgroups in stage II CRC.ConclusionOur research proved that CTCs could serve as feasible surrogate samples to detect gene expression as a predictive biomarker for tumor evaluation.

Whole-exome sequencing of single circulating tumor cells is a useful tool for studying the intrapatient genetic heterogeneity in metastatic prostate cancer.

148 Background: Molecular characterization of metastatic castration resistant prostate cancer (mCRPC) is limited by tumor tissue availability. The analysis of circulating tumor cells (CTC) offers an attractive noninvasive surrogate option to analyze molecular alterations. We report whole exome sequencing (WES) of CTCs at the single cell level in mCRPC patients. Methods: Blood samples were drawn from 11 enzalutamide or abiraterone pre-treated mCRPC patients enrolled in the clinical program MOSCATO (NCT02613962). CTC enrichment, immunofluorescent detection and single cell isolation were performed using three methods (ISET filtration, CellSearch and the VyCap puncher system and RosetteSep enrichment) to obtain pools of 1-10 CTCs with distinct epithelial or mesenchymal phenotypes. After Whole Genome Amplification (WGA), WES was performed on the Illumina HiSeq 2000 platform. GATK Haplotype Caller enabled identification of germline polymorphisms from each patient in normal DNA, metastatic sample and CTCs in order to consider WGA induced bias. The detection of sSNV in tumor biopsies and CTCs was assessed with Mutect and IndelGenotyper respectively. Results: 189 WGA of CTC pools were performed. 34 pools of phenotypically different CTCs from 7 patients were selected and sequenced. Mean coverage of 51% was obtained at a sequencing depth of 10X. Allelic drop out was lower for CTC pools containing 5-10 cells. 17/34 (50%) CTC samples had shared sSNV with the paired tumor sample (range 0.35%-68%) Epithelial CTCs had more shared sSNV with metastatic biopsies than CTCs of other phenotypes but shared sSNV were also detected in large non epithelial CTC pointing out a high level of genetic heterogeneity between CTC. Overall, 89 deleterious protein-coding mutations were found only in pools of CTC, including mutations affecting oncogenic drivers such as MAPK1, HSP90AB1 or KDM5B. Conclusions: We present single cell WES of CTCs harboring distinct phenotypes. The detection of shared sSNV between CTC pools and corresponding biopsy could validate the use of CTCs as a liquid biopsy. The finding of sSNV specific to CTCs could offer additional data on tumor heterogeneity.

Association between circulating tumor cells in peripheral blood of patients with esophageal squamous cell carcinoma and tumor relapse and metastasis

Objective To explore the association between the number and classification of circulating tumor cells (CTCs) in peripheral blood with clinicopathological characteristics, relapse and metastasis of esophageal squamous cell carcinoma. Methods Fifty-one patients who accepted surgical resection of esophageal squamous cell carcinoma and CTCs detection between 2013-2015 were retrospectively included in the study. The association between the number and classification of CTCs and age, sex, differentiation degree, T stage, N stage, location, tumor relapse and metastasis was analyzed. Results CTCs in peripheral blood were detected in 68.6% of patients (35/51). The detection rate of epithelial CTCs, biophenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs was 56.9% (29/51), 66.7% (34/51) and 49.0% (25/51) respectively. CTCs (+ ) patients had shorter disease-free survival (10.8 months) than CTCs (-) patients (14.9 months, χ2=6.981, P=0.000). In 35 CTCs (+ ) patients, the 25 mesenchymal CTCs (+ ) patients had shorter disease-free survival (9.5 months) than other patients (12.0 months, χ2=2.127, P=0.007). Conclusion The number of CTCs in the peropheral blood of patients with esophageal squamous cell carcinoma is an important potential marker of disease-free survival, and the CTCs (+ ) patients are at higher risk of replapse and metastasis. Key words: Esophageal squamous cell carcinoma; Circulating tumor cells; Relapse; Metastasis

Expression and clinical relevance of epithelial and mesenchymal markers in circulating tumor cells from colorectal cancer.

Circulating tumor cells (CTCs) with phenotypic hallmarks of epithelial-mesenchymal transition (EMT) reportedly contribute to tumor metastasis in different cancer types. We therefore evaluated the expression of EMT markers in CTCs obtained from a large cohort of Chinese patients with colorectal cancer (CRC) and investigated their clinical relevance. The CanPatrolTM CTC enrichment technique was used to isolate and classify CTCs. CTCs were detected in 1046 of 1203 patients (86.9%), and three phenotypes were identified based on the expression of epithelial and mesenchymal markers: epithelial CTCs, biophenotypic (epithelial/mesenchymal) CTCs, and mesenchymal CTCs. Total CTC numbers positively correlated with both clinical stage and lymph node metastasis and distant metastasis. Furthermore, both biophenotypic and mesenchymal, but not epithelial, CTCs, correlated with the above parameters, suggesting CTCs displaying a mesenchymal phenotype denote more aggressive disease and metastatic potential. This is the first study to demonstrate a significant correlation between CTCs displaying a mesenchymal phenotype and both clinical stage and metastasis in a large cohort of patients with CRC. Our findings suggest that assessment of not only epithelial, but also mesenchymal markers in CTC analyses may offer valuable assistance for tumor staging and metastasis evaluation in patients with CRC.

Abstract 4953: Whole-exome sequencing of single circulating tumor cells according to epithelial-mesenchymal marker expression in metastatic prostate cancer

Abstract Molecular characterization of metastatic castration-resistant prostate cancer (mCRPC) is limited by tumor tissue availability. The analysis of circulating tumor cells (CTCs) offers an attractive non invasive surrogate option to analyze molecular alterations. We report whole exome sequencing (WES) of CTCs at the single cell level in 11 mCRPC patients. We examined single somatic nucleotide variant (sSNV) shared between matched metastatic tumor sample and CTCs and sSNV specific to CTCs. Blood samples were drawn from 11 patients enrolled in the clinical program MOSCATO (2011-A00841-40). CTC enrichment, detection and single cell isolation were performed using three methods to obtain pools of 1-10 CTCs. The first method used ISET filtration, immunofluorescent staining (CD45, pan-cytokeratin, EpCAM, Vimentin and Hoechst 33342) on filters and laser microdissection of single CTCs; the second combined CellSearch and the VyCap puncher system; the third used RosetteSep enrichment, immunofluorescent staining and isolation by cell sorting. Whole Genome Amplification (WGA) was performed using the Ampli1 kit. WGA quality was assessed by qPCR of 7 genes located on different regions of the genome. WES was performed by preparation of a genomic DNA bank, Agilent capture and sequencing on the Illumina HiSeq 2000 platform. Data were aligned to the human genome reference hg19. GATK Haplotype Caller enabled identification of germline polymorphisms from each patient in normal DNA, metastatic sample and CTCs in order to consider WGA induced bias. The detection of sSNV in tumor biopsies and CTCs was assessed with Mutect and IndelGenotyper respectively. 189 WGAs of CTC pools were performed. A first round of WES showed that at least 3 well amplified genes were required to obtain a coverage of at least 50% at 10X depth sequencing. 34 pools of phenotypically different CTCs from 7 patients were selected and sequenced. Mean coverage of 51% was obtained at a sequencing depth of 10X. Allelic drop out was lower for CTC pools containing 5 to 10 cells. 17/34 (50%) CTC samples (4 patients) had shared sSNV with the paired tumor sample (range 0.35%-68%). Epithelial CTCs had more shared sSNV with metastatic biopsies than CTCs of other phenotypes but shared sSNV were also detected in large Cytokeratin-Vimentin- CTC. Shared sSNV in cancer genes between epithelial CTC pools, but not in the paired biopsy, were present in 2 patients. We report WES of CTC pools harboring distinct EMT marker phenotypes is possible with the use of 3 different approaches to enrich, detect and isolate CTCs. The detection of shared sSNV between CTC pools and corresponding biopsy could validate the use of CTCs as a liquid biopsy. The finding of sSNV specific to CTCs could offer additional data on tumor heterogeneity. Ongoing work examining if sSNV detected in phenotypically different CTCs converge to similar signaling pathways will be presented. Citation Format: Vincent Faugeroux, Céline Lefebvre, Emma Pailler, Valérie Pierron, Fanny Billiot, Charles Marcaillou, Philippe Vielh, Semih Dogan, Philippe Rameau, Maud Ngocamus, Jean Charles Soria, Karim Fizazi, Yohann Loriot, Sylvia Julien, Françoise Farace. Whole-exome sequencing of single circulating tumor cells according to epithelial-mesenchymal marker expression in metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4953.

Abstract 1388: Multi-template analysis in metastatic breast cancer blood samples

Abstract Introduction The presence of Circulating Tumor Cells (CTC) has been observed in advanced cancer patients and studies have indicated that these cells contribute to the process of metastasis. Historically CTCs with metastatic potential have been characterized as cells that are EpCAM positive, Cytokeratin positive (CK+), and CD45 negative. We report the results of a prospective study challenging these historical definitions of circulating tumor cells. Description In a prospective study, patients with metastatic breast cancer there were about to start a new systemic therapy were enrolled The samples and representative tissue were collected at baseline. The patients were predominantly female (97%) and all had stage IV disease. From these whole blood samples a LiquidBiopsy® was performed using the “MultiTemplate” format which directly compares CTC, circulating cell free (CCF), and WBC patient-matched DNA in a NGS based resequencing test. Of 22 patients, 18 biopsy samples were successfully evaluated; the balance had insufficient tissue or DNA for analysis. Summary The LiquidBiopsy compared two phenotypic definitions for CTC capture. Tumor cell populations were enriched using epithelial targeted capture and compared to a novel cocktail of antibodies. The cocktail of reagents gave 77% average recovery of engineered samples when a target receptor was present on cell lines representing the spectrum of breast cancer subtypes. This enrichment protocol was applied to serial patient samples. Epithelial based enrichment served as a control and showed an average recovered purity of 10.7% CK+ cells. By comparison, cocktail selection enriched populations with on average 8.8% CK+ cell populations but a larger range of cells than epithelial selection. Importantly, the median number of CK+ cells recovered from cocktail capture was almost twice that of epithelial capture alone. (median of 35.8 vs 22.1 for cocktail and epithelial respectively). The median CD45+ non-target cells background was 80 and 155 cells respectively. This background supports sequencing detection of mutations present at &amp;gt;1%. ccfDNA sample was purified from the same tube of blood. The average concentration of patient matched ccfDNA recovered was 7.3 ng/mL. The combined results of ctcDNA and ccfDNA template sequencing gave productive samples showed similar detection frequency (55% vs 58%), were temporally flexible, and were complementary both to each other and the “gold standard” (FFPE). Conclusions The data from this prospective clinical study reveals subsets of CTC, including an important cohort that is not detected using the standard definition for epithelial CTCs. Furthermore, we present evidence for the successful use of a “Multi-Template” approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and justifies the redefinition of CTC phenotype based upon cell surface biomarkers and mutation content. Citation Format: William M. Strauss, Chris Carter, Erich Klem, Jill Simmons, Keerthi Gogineni, Ruth O’Regan, Laura Austin, Paul W. Dempsey, Massimo Cristofanilli. Multi-template analysis in metastatic breast cancer blood samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1388.

Phenotypic and genetic heterogeneity of tumor tissue and circulating tumor cells in patients with metastatic castration-resistant prostate cancer: A report from the PETRUS prospective study.

Molecular characterization of cancer samples is hampered by tumor tissue availability in metastatic castration-resistant prostate cancer (mCRPC) patients. We reported the results of prospective PETRUS study of biomarker assessment in paired primary prostatic tumors, metastatic biopsies and circulating tumor cells (CTCs). Among 54 mCRPC patients enrolled, 38 (70%) had biopsies containing more than 50% tumour cells. 28 (52%) patients were analyzed for both tissue samples and CTCs. FISH for AR-amplification and TMPRSS2-ERG translocation were successful in 54% and 32% in metastatic biopsies and primary tumors, respectively. By comparing CellSearch and filtration (ISET)-enrichment combined to four color immunofluorescent staining, we showed that CellSearch and ISET isolated distinct subpopulations of CTCs: CTCs undergoing epithelial-to-mesenchymal transition, CTC clusters and large CTCs with cytomorphological characteristics but no detectable markers were isolated using ISET. Epithelial CTCs detected by the CellSearch were mostly lost during the ISET-filtration. AR-amplification was detected in CellSearch-captured CTCs, but not in ISET-enriched CTCs which harbor exclusively AR gain of copies. Eighty-eight percent concordance for ERG-rearrangement was observed between metastatic biopsies and CTCs even if additional ERG-alteration patterns were detected in ISET-enriched CTCs indicating a higher heterogeneity in CTCs.Molecular screening of metastatic biopsies is achievable in a multicenter context. Our data indicate that CTCs detected by the CellSearch and the ISET-filtration systems are not only phenotypically but also genetically different. Close attention must be paid to CTC characterization since neither approach tested here fully reflects the tremendous phenotypic and genetic heterogeneity present in CTCs from mCRPC patients.

Distribution and clinical significance of circulating tumor cells in nasopharyngeal carcinoma.

Nasopharyngeal carcinomais one of the leading malignancies with obscure etiology. Circulating tumor cellshave been showed to intimately correlate with characteristics in different kinds of cancer. But links between circulating tumor cells andnasopharyngeal carcinoma were still lacking. Therefore, we exploredcirculating tumor cells' distribution innasopharyngeal carcinoma and their possible associations withnasopharyngeal carcinoma. Firstly, we found that the positive ratio ofcirculating tumor cells is extremely high in four stages ofnasopharyngeal carcinoma. Meanwhile, positive ratios of mesenchymalcirculating tumor cells were higher in early stages ofnasopharyngeal carcinoma. Apart from epithelialcirculating tumor cells, total, hybrid and mesenchymalcirculating tumor cells were correlated withnasopharyngeal carcinoma clinical stage. Our results showed that hybrid and mesenchymalcirculating tumor cells were associated withnasopharyngeal carcinoma metastasis (both distant and lymph node) and smoking. Meanwhile, hybridcirculating tumor cells expressed the highest Epstein-Barr virus proteins and deoxyribonucleic acid in three types ofcirculating tumor cells. Moreover, we found that Epstein-Barr virusproteins viral-caspid antigen-immunoglobulin A (VCA/IgA) and early antigen-immunoglobulin A (EA/IgA), but not Epstein-Barr virus-deoxyribonucleic acid, had a closed association withnasopharyngeal carcinoma metastasis. However,Epstein-Barr virus hallmarks failed to associate with othernasopharyngeal carcinoma characteristics. Furthermore, we confirmed that matrix metalloproteinase 9existed incirculating tumor cells and expressed most in mesenchymalcirculating tumor cells. In addition, matrix metalloproteinase 9-expressed extent in hybridcirculating tumor cells is somewhat different from epithelial and mesenchymalcirculating tumor cells in matrix metalloproteinase 9-positivecirculating tumor cells. Nevertheless, matrix metalloproteinase 9 had no relationship with othernasopharyngeal carcinoma characteristics. Finally, our results showed thatcirculating tumor cells were decreased in patients after therapies. Taken together,circulating tumor cells were tightly correlated withnasopharyngeal carcinoma characteristics. In addition, Epstein-Barr virus was associated withnasopharyngeal carcinoma metastasis. Of note, decreasedcirculating tumor cells indicated a favorable curative effect in nasopharyngeal carcinoma patients.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study

Over the past decade, numerous reports describe the generation and increasing utility of non-small-cell lung cancer (NSCLC) patient-derived xenografts (PDX) from tissue biopsies. While PDX have proven useful for genetic profiling and preclinical drug testing, the requirement of a tissue biopsy limits the available patient population, particularly those with advanced oligometastatic disease. Conversely, 'liquid biopsies' such as circulating tumour cells (CTCs) are minimally invasive and easier to obtain. Here, we present a clinical case study of a NSCLC patient with advanced metastatic disease, a never smoker whose primary tumour was EGFR and ALK wild-type. We demonstrate for the first time, tumorigenicity of their CTCs to generate a patient CTC-derived eXplant (CDX). CTCs were enriched at diagnosis and again 2 months later during disease progression from 10 ml blood from a 48-year-old NSCLC patient and implanted into immunocompromised mice. Resultant tumours were morphologically, immunohistochemically, and genetically compared with the donor patient's diagnostic specimen. Mice were treated with cisplatin and pemetrexed to assess preclinical efficacy of the chemotherapy regimen given to the donor patient. The NSCLC CDX expressed lung lineage markers TTF1 and CK7 and was unresponsive to cisplatin and pemetrexed. Examination of blood samples matched to that used for CDX generation revealed absence of CTCs using the CellSearch EpCAM-dependent platform, whereas size-based CTC enrichment revealed abundant heterogeneous CTCs of which ∼80% were mesenchymal marker vimentin positive. Molecular analysis of the CDX, mesenchymal and epithelial CTCs revealed a common somatic mutation confirming tumour origin and showed CDX RNA and protein profiles consistent with the predominantly mesenchymal phenotype. This study shows that the absence of NSCLC CTCs detected by CellSearch (EpCAM(+)) does not preclude CDX generation, highlighting epithelial to mesenchymal transition and the functional importance of mesenchymal CTCs in dissemination of this disease.

Establishment of a multimarker qPCR panel for the molecular characterization of circulating tumor cells in blood samples of metastatic breast cancer patients during the course of palliative treatment.

BackgroundCirculating tumor cells (CTC) are discussed to be an ideal surrogate marker for individualized treatment in metastatic breast cancer (MBC) since metastatic tissue is often difficult to obtain for repeated analysis. We established a nine gene qPCR panel to characterize the heterogeneous CTC population in MBC patients including epithelial CTC, their receptors (EPCAM, ERBB2, ERBB3, EGFR) CTC in Epithelial-Mesenchymal-Transition [(EMT); PIK3CA, AKT2), stem cell-like CTC (ALDH1) as well as resistant CTC (ERCC1, AURKA] to identify individual therapeutic targets.ResultsAt TP0, at least one marker was detected in 84%, at TP1 in 74% and at TP2 in 79% of the patients, respectively. The expression of ERBB2, ERBB3 and ERCC1 alone or in combination with AURKA was significantly associated with therapy failure. ERBB2 + CTC were only detected in patients not receiving ERBB2 targeted therapies which correlated with no response. Furthermore, patients responding at TP2 had a significantly prolonged overall-survival than patients never responding (p = 0.0090).Patients and Methods2 × 5 ml blood of 62 MBC patients was collected at the time of disease progression (TP0) and at two clinical staging time points (TP1 and TP2) after 8–12 weeks of chemo-, hormone or antibody therapy for the detection of CTC (AdnaTest EMT-2/StemCell Select™, QIAGEN Hannover GmbH, Germany). After pre-amplification, multiplex qPCR was performed. Establishment was performed using various cancer cell lines. PTPRC (Protein tyrosine phosphatase receptor type C) and GAPDH served as controls.ConclusionsMonitoring MBC patients using a multimarker qPCR panel for the characterization of CTC might help to treat patients accordingly in the future.

Circulating Tumor Cell Phenotype Predicts Recurrence and Survival in Pancreatic Adenocarcinoma.

We assessed circulating tumor cells (CTCs) with epithelial and mesenchymal phenotypes as a potential prognostic biomarker for patients with pancreatic adenocarcinoma (PDAC). PDAC is the fourth leading cause of cancer death in the United States. There is an urgent need to develop biomarkers that predict patient prognosis and allow for better treatment stratification. Peripheral and portal blood samples were obtained from 50 patients with PDAC before surgical resection and filtered using the Isolation by Size of Epithelial Tumor cells method. CTCs were identified by immunofluorescence using commercially available antibodies to cytokeratin, vimentin, and CD45. Thirty-nine patients (78%) had epithelial CTCs that expressed cytokeratin but not CD45. Twenty-six (67%) of the 39 patients had CTCs which also expressed vimentin, a mesenchymal marker. No patients had cytokeratin-negative and vimentin-positive CTCs. The presence of cytokeratin-positive CTCs (P < 0.01), but not mesenchymal-like CTCs (P = 0.39), was associated with poorer survival. The presence of cytokeratin-positive CTCs remained a significant independent predictor of survival by multivariable analysis after accounting for other prognostic factors (P < 0.01). The detection of CTCs expressing both vimentin and cytokeratin was predictive of recurrence (P = 0.01). Among patients with cancer recurrence, those with vimentin-positive and cytokeratin-expressing CTCs had decreased median time to recurrence compared with patients without CTCs (P = 0.02). CTCs are an exciting potential strategy for understanding the biology of metastases, and provide prognostic utility for PDAC patients. CTCs exist as heterogeneous populations, and assessment should include phenotypic identification tailored to characterize cells based on epithelial and mesenchymal markers.

CXCR4-SDF-1 interaction potentially mediates trafficking of circulating tumor cells in primary breast cancer.

BackgroundCytokines are involved in cancer invasion and metastasis. Circulating tumor cells (CTCs) play key role in tumor dissemination and are an independent survival predictor in breast cancer patients. The aim of this study was to assess correlation between CTCs and plasma cytokines in primary breast cancer (PBC) patients.MethodsThis study included 147 chemotherapy naïve PBC patients. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoetic cells using RossetteSep™ negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (Twist1, Snail1, Slug, Zeb1) and epithelial (Ck19) gene transcripts by qRT-PCR. The concentrations of 51 plasma cytokines were measured using multiplex bead arrays.ResultsCTCs were detected in 25.2 % patients. CTCs exhibiting only epithelial markers (CTC_EP) and only EMT markers (CTC_EMT) were present evenly in 11.6 % patients, while CTCs co-expressing both markers were detected in 2.0 % patients. Patients with presence of CTC_EP in peripheral blood had significantly elevated levels of plasma IFN-α2, IL-3, MCP-3, β-NGF, SCF, SCGF-β, TNF-β and SDF-1 compared to patients without CTC_EP. CTC_EP exhibited overexpression of SDF-1 receptor and CXCR4, but not other corresponding cytokine receptor, and in multivariate analysis SDF-1 was independently associated with CTC_EP. There was an inverse correlation between CTC_EMT and plasma cytokines CTACK, β-NGF and TRAIL, while presence of either subtype of CTCs was associated with increased level of TGF-β2.ConclusionUsing cytokine profiling, we identified cytokines associated with CTCs subpopulations in peripheral blood of PBC. Our data suggest that CXCR4-SDF-1 axis is involved in mobilization and trafficking of epithelial CTCs.

Abstract P2-02-04: Distinct clinical and biological values of subpopulations of circulating tumor cells (CTCs) in primary breast cancer

Abstract Background: CTCs represent a heterogeneous population of cells with different phenotypes and biological values. Epithelial to mesenchymal transition (EMT) gives rise to cells with stem cell-like properties with increased resistance to chemotherapy that may be under detected by currently approved assays. The aim of this study was to characterize CTCs based on the expression of epithelial and mesenchymal markers in primary breast cancer (BC) and to correlate them with patients'/tumor characteristics. Methods: This prospective translational study included 422 patients with primary BC enrolled from March 2012 to February 2015. Blood for CTC detection was drawn before surgery (422 patients), before 1st cycle (95 patients) and before 2nd cycle (53 patients) of adjuvant therapy. Isolated peripheral blood mononuclear cells (PBMC) were depleted of cells of hematopoietic origin (CD45+) using RossetteSep kit (StemCell Technologies) 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 quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC from patients were compared to those of CD45- PBMC of 60 healthy donors. Results: Totally, CTCs were detected in 116/422 (27.5%) patients before surgery, in 21/95 (22.1%) patients after surgery and before 1st cycle and in 19/53 (35.8%) of patients before 2nd cycle of adjuvant therapy. Before surgery, CTCs exhibited only epithelial markers in 38 (9.0%) patients, only EMT markers in 68 (16.1%) of patients, while in 10 (2.4%) patients CTCs with both epithelial and EMT markers were detected. Epithelial CTCs were more often detected before surgery compared to after surgery (11.4% vs. 2.1%; p = 0.003), while mesenchymal CTCs were more often detected after the 1st cycle of chemotherapy as opposed to detection before surgery (30.2% vs. 18.2%; p = 0.05). Patients with N2-3 disease had more often detectable CTCs compared to patients with N0-1 disease (41.4% vs. 24.9%, p = 0.01) and this was mainly driven by mesenchymal CTCs (31.0% for N2-3 vs. 16.0% for N0-1; p = 0.007). Similarly, patients that lacked p53 expression (wild type TP53) in primary tumor had more often CTCs with EMT phenotype opposite to patients with p53 expression (p = 0.02). Presence of epithelial CTCs was significantly associated with lower absolute lymphocyte (p = 0.02) and neutrophil (p = 0.02) counts in peripheral blood. Conclusions: Our results support the concept of CTCs phenotypic heterogeneity in breast cancer patients. These results support the role of EMT in cancer pathogenesis and suggest that CTCs with EMT phenotype are involved in tumor dissemination while their increase after chemotherapy might be a mechanism of treatment resistance. Moreover, these data suggest inverse relationship between immune cells and epithelial CTCs which stress the role of immune cells in tumor dissemination. Citation Format: Mego M, Jurisova S, Karaba M, Minarik G, Benca J, Sedlackova T, Manasova D, Malejcikova M, Sieberova G, Macuch J, Gronesova P, Sufliarsky J, Pindak D, Cristofanilli M, Reuben JN, Mardiak J. Distinct clinical and biological values of subpopulations of circulating tumor cells (CTCs) in primary breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-04.

Abstract 1595: Analysis of circulating epithelial and EMT-like CTCs in pancreatic cancer using a sensitive microfluidic CTC capture device

Abstract Background &amp; Aims: A critical factor in the poor outcomes in pancreatic cancer is due to it's silent nature until late in the disease process, so early detection has remained a major issue in this type of cancer. Detection, quantification, and molecular characterization of Epithelial circulating tumor cells (CTCs) as well as Epithelial-to-Mesenchymal Transition (EMT)-like CTCs in peripheral blood have been recognized as a means of “liquid biopsy” technique in the diagnosis of cancer and metastasis. Methods: A modified version of CTC chip, CTC Carpet chip was used to investigate the inextricable relationship between circulating epithelial and EMT-like CTCs in 40 pancreatic cancer patients at both protein and RNA levels. Results: The recovery from whole blood spiked with different cancer cell line was validated, achieving a mean efficiency of ∼100% for both HT-29 and Panc-1 cell lines compared to &amp;lt;80% for PC-3 cell line. Out of 40 patients, 38 patients had ≥5 CK+ CTCs with a mean of 21.84 per mL and all 40 samples had ≥ 15 Vim+ CTCs with mean of 89.77 per mL of whole blood. Up to 67 CK+ epithelial and 257 Vim+ EMT like CTCs were detected in these set of samples. The results indicated the significant differences between the numbers of Epithelial CTCs vs. EMT-like CTCs in both stage I-II and IV of these 40 pancreatic cancer patients’ samples. Conclusion: Although the isolation of CTCs from pancretic cancer is possible nowadays, investigation of their clinical utility has proven less successful than the other cancer types, due to poor sensitivity of many CTC assays for patients with PDAC. Herein, we present an integrated microfluidic technology- and biology-based translational approach using bioengineering tools to identify and study the biological relevance of rare CTCs including both circulating epithelial and EMT-like tumor cells simultaneously from the peripheral blood of pancreatic cancer patients by using multiple markers of interest. Citation Format: Mina Zeinali, Vasudha Murlidhar, Shamileh Fouladdel, Mathius Hafner, Shimeng Shao, Ebrahim Azizi, Max S. Wicha, Kyle Cuneo, Diane M. Simeone, Sunitha Nagrath. Analysis of circulating epithelial and EMT-like CTCs in pancreatic cancer using a sensitive microfluidic CTC capture device. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1595. doi:10.1158/1538-7445.AM2015-1595

Relationship between circulating tumor cells and epithelial to mesenchymal transition in early breast cancer.

BackgroundCirculating tumor cells (CTCs) play a crucial role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. Epithelial to mesenchymal transition (EMT) is involved in cancer invasion and metastasis. The aim of this study was to assess correlation between CTCs and expression of EMT transcription factors TWIST1 and SLUG in breast tumor tissue.MethodsThis study included 102 early BC patients treated by primary surgery. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSep™ negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, FOXC2 and ZEB1) and epithelial (KRT19) gene transcripts by qRT-PCR. Expression of TWIST1 and SLUG in surgical specimens was evaluated by immunohistochemistry and quantified by multiplicative score.ResultsCTCs were detected in 24.5 % patients. CTCs exhibiting only epithelial markers were present in 8.8 % patients, whereas CTCs with only EMT markers were observed in 12.8 % of pts and CTCs co-expressing both markers were detected in 2.9 % pts. We observed lack of correlation between CTCs and expression of TWIST1 and SLUG in breast cancer cells or cancer associated stroma. Lack of correlation was observed for epithelial CTCs as well as for CTCs with EMT.ConclusionsIn this translational study, we showed a lack of association between CTCs and expression of EMT-inducing transcription factors, TWIST1 and SLUG, in breast tumor tissue. Despite the fact that EMT is involved in cancer invasion and metastasis our results suggest, that expression of EMT proteins in unselected tumor tissue is not surrogate marker of CTCs with either mesenchymal or epithelial features.

Different subpopulations of circulating tumor cells in patients with hepatocellular carcinoma and clinical relevance.

e15130 Background: The existence of circulating tumor cells (CTCs) in peripheral blood has a correlation with prognosis and tumor metastasis in patients with hepatocellular carcinoma (HCC). Epithelial-mesenchymal transition (EMT) is regarded as pivotal process in tumor metastasis, and plays an important role in tumor local invasion and subsequent dissemination. According to the EMT status, CTCs can be classified into different subpopulations, including epithelial CTCs, mesenchymal CTCs, hybrid (epithelial/mesenchymal) CTCs etc. Furthermore, the mesenchymal CTCs hold a strong metastatic potential. Methods: We isolated CTCs from peripheral blood of 13 HCC patients. The CanPatrol CTC enrichment technique (SurExam China) was used to enumerate and characterize the phenotypes of CTCs. The first step of this technique was to isolate CTCs via a filter-based method; then, a multiplexed mRNA fluorescence in situ hybridization method was used to characterize CTCs according to the expression status of epithelial and mesenchymal biomarkers. Results: Among the 13 patients, 12 patients had detectable CTCs. All CTC-positive patients had detectable hybrid CTCs, four CTC-positive patients had detectable mesenchymal CTCs, and one CTC-positive patient had detectable three phenotypes: epithelial, hybrid and mesenchymal CTCs. Of the four mesenchymal CTC-positive patients, three of them were undergoing metastasis, and it suggested that metastasis in patients with HCC was positive associated with mesenchymal CTCs (p < 0.01). Conclusions: Although the sample size of this study was small, our findings suggested that the existence of mesenchymal CTCs might have a positive correlation with tumor metastasis in HCC.

Classification of circulating tumor cells by epithelial-mesenchymal transition markers.

In cancer, epithelial-mesenchymal transition (EMT) is associated with metastasis. Characterizing EMT phenotypes in circulating tumor cells (CTCs) has been challenging because epithelial marker-based methods have typically been used for the isolation and detection of CTCs from blood samples. The aim of this study was to use the optimized CanPatrol CTC enrichment technique to classify CTCs using EMT markers in different types of cancers. The first step of this technique was to isolate CTCs via a filter-based method; then, an RNA in situ hybridization (RNA-ISH) method based on the branched DNA signal amplification technology was used to classify the CTCs according to EMT markers. Our results indicated that the efficiency of tumor cell recovery with this technique was at least 80%. When compared with the non-optimized method, the new method was more sensitive and more CTCs were detected in the 5-ml blood samples. To further validate the new method, 164 blood samples from patients with liver, nasopharyngeal, breast, colon, gastric cancer, or non-small-cell lung cancer (NSCLC) were collected for CTC isolation and characterization. CTCs were detected in 107(65%) of 164 blood samples, and three CTC subpopulations were identified using EMT markers, including epithelial CTCs, biophenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. Compared with the earlier stages of cancer, mesenchymal CTCs were more commonly found in patients in the metastatic stages of the disease in different types of cancers. Circulating tumor microemboli (CTM) with a mesenchymal phenotype were also detected in the metastatic stages of cancer. Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulation and provides useful evidence for determining an appropriate clinical approach. This method is suitable for a broad range of carcinomas.