EpCAM-positive Circulating Tumor Cells Research Articles

EpCAM-positive circulating tumor cells and serum AFP levels predict outcome after curative resection of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has high recurrence rates exceeding 50% despite curative resection. The serum biomarker alpha-fetoprotein (AFP) is a well-known prognostic marker for HCC. EpCAM-positive circulating tumor cells (CTC) have a high predictive value for early HCC recurrence after curatively intended resection, most likely indicating micro-metastases at the time of resection. However, sensitivity remains low. The objective of this study was to evaluate a composite test comprising both CTC and AFP to identify patients at high risk for early HCC recurrence. We prospectively enrolled 58 patients undergoing curative intended resection for HCC at a tertiary referral center. Blood specimens were obtained prior to resection and analyzed for EpCAM-positive CTC and serum AFP levels. A positive result was defined as either detection of CTC or AFP levels ≥ 400 ng/ml. Eight patients tested positive for CTC, seven for AFP, and two for both markers. A positive composite test was significantly associated with shorter early recurrence-free survival (5 vs. 16 months, p = 0.005), time to recurrence (5 vs. 16 months, p = 0.011), and overall survival (37 vs. not reached, p = 0.034). Combining CTC and AFP identified patients with poor outcome after surgical resection, for whom adjuvant or neoadjuvant therapies may be particularly desirable.

Abstract 2311: Extensive molecular heterogeneity in circulating tumor cells: A key player in oral squamous cell carcinoma prognosis

Abstract Background: Oral Squamous cell carcinoma (OSCC) often results in unfavorable outcomes due to locoregional relapse. While circulating tumor cells (CTCs) may be attributed to these locoregional metastases, detecting CTCs and their molecular profiling remains challenging. Here we report a novel strategy for isolating CTCs in OSCC. Secondly, we characterized the CTCs to prognosticate the disease outcome using their transcriptomic signature. Methods: Keeping in view the inherent disadvantages of currently available CTC-isolation techniques, like the use of single marker or size-based isolation, we developed a novel FACS–based strategy, combining pre-enrichment by immunomagnetic depletion of CD45+ cells, followed by positive selection of CTCs using multiple FITC-conjugated epithelial markers (EpCAM, EGFR, CK) and EMT marker (Vimentin), with a novel gating strategy for isolating pure CTCs. Isolated CTCs were reconfirmed using various methods. CTCs and Primary tumors were isolated in 63 OSCC patients, and their transcriptomic signature was correlated with disease outcome. Results: Seventy-one percent (45/63) of patients were positive for CTCs, and associated with poor OS and DFS, while there is no difference between the proportion of CTC-positivity between early (23/33;69%) and late (22/30;73%) TNM stage. CTC-specific markers (EpCAM, EGFR, CK, and Vim) are expressed in various combinations, indicating extensive phenotypic heterogeneity. Importantly, EpCAM-positive CTCs were detected only in 46% (29/63) patients, and patients with EpCAM-negative CTCs had worse OS (p=0.044*), indicating the importance of a multi-marker approach. On unsupervised hierarchical clustering of RNAseq data of isolated CTCs and primary tumors, we found that CTCs and primary tumors clustered separately, indicating that the CTCs are transcriptionally different from the primary tumors. Moreover, CTCs were clustered into two distinct sub-clusters with significantly different OS (p=0.0004***) and DFS (p=0.0097***), suggesting that the inter-patient transcriptomic heterogeneity of CTCs is an important prognostic factor. We found coagulation, IL-6 JAK STAT3 signaling, allograft rejection, complement, EMT, inflammatory response, heme metabolism, UV response, and ECM receptor interaction pathways significantly enriched in CTCs compared to the primary tumor. TNF-alpha signaling via NF-kB and KRAS signaling pathways were also positively enriched in CTCs with poor prognostic traits. Interestingly, for the first time, we found various Cancer/Testis (CT) antigens upregulated in CTCs, while not detected in primary tumors. Conclusion: Here we report a novel, technologically less difficult, cost-effective CTC isolation technique suitable for developing countries. We explored the prognostic implications of the extensive heterogeneity in CTCs isolated from OSCC patients for the first time. Citation Format: Anshika Chauhan, Geeta S. Boora, Sahana Ghosh, Monil Parsana, Subrata K. Patra, Roshan K. Verma, Jaimanti Bakshi, Debajyoti Chatterjee, Radhika Srinivasan, Sushmita Ghoshal, Arindam Maitra, Arnab Pal. Extensive molecular heterogeneity in circulating tumor cells: A key player in oral squamous cell carcinoma prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2311.

Nondestructive separation/enrichment and rolling circle amplification-powered sensitive SERS enumeration of circulating tumor cells via aptamer recognition

Nondestructive separation/enrichment and reliable detection of extremely rare circulating tumor cells (CTCs) in peripheral blood are of considerable importance in tumor precision diagnosis and treatment, yet this remains a big challenge. Herein, a novel strategy for nondestructive separation/enrichment and ultra-sensitive surface-enhanced Raman scattering (SERS)-based enumeration of CTCs is proposed via aptamer recognition and rolling circle amplification (RCA). In this work the magnetic beads modified with "Aptamer (Apt)-Primer" (AP) probes were utilized to specifically capture CTCs, and then after magnetic separation/enrichment, the RCA-powered SERS counting and benzonase nuclease cleavage-assisted nondestructive release of CTCs were realized, respectively. The AP was assembled by hybridizing the EpCAM-specific aptamer with a primer, and the optimal AP contains 4 mismatched bases. The RCA enhanced SERS signal nearly 4.5-fold, and the SERS strategy has good specificity, uniformity and reproducibility. The proposed SERS detection possesses a good linear relationship with the concentration of MCF-7 cells spiked in PBS with the limit of detection (LOD) of 2 cells/mL, which shows good potential practicality for detecting CTCs in blood with recoveries ranging from 100.56% to 116.78%. Besides, the released CTCs remained good cellular activity with the normal proliferation after re-culture for 48 h and normal growth for at least three generations. The proposed strategy of nondestructive separation/enrichment and SERS-based sensitive enumeration is promising for reliable analysis of EpCAM-positive CTCs in blood, which is expected to provide a powerful tool for analysis of extremely rare circulating tumor cells in complex peripheral blood for liquid biopsy.

Detection and Isolation of Circulating Tumor Cells from Breast Cancer Patients Using CUB Domain-Containing Protein 1.

In cancer metastasis, single circulating tumor cells (CTCs) in the blood and disseminated tumor cells (DTCs) in the bone marrow mediate cancer metastasis. Because suitable biomarker proteins are lacking, CTCs and DTCs with mesenchymal attributes are difficult to isolate from the bulk of normal blood cells. To establish a procedure allowing the isolation of such cells, we analyzed the cell line BC-M1 established from DTCs in the bone marrow of a breast cancer patient by stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry. We found high levels of the transmembrane protein CUB domain-containing protein 1 (CDCP1) in breast cancer cell lines with mesenchymal attributes. Peripheral blood mononuclear cells were virtually negative for CDCP1. Confirmation in vivo by CellSearch revealed CDCP1-positive CTCs in 8 of 30 analyzed breast cancer patients. Only EpCam-positive CTCs were enriched by CellSearch. Using the extracellular domain of CDCP1, we established a magnetic-activated cell sorting (MACS) approach enabling also the enrichment of EpCam-negative CTCs. Thus, our approach is particularly suited for the isolation of mesenchymal CTCs with downregulated epithelial cancer that occur, for example, in triple-negative breast cancer patients who are prone to therapy failure.

Association of emergent neuroendocrine prostate cancer detected by liquid biopsies with survival and treatment resistance.

247 Background: The mainstay of therapy in metastatic prostate cancer is androgen receptor (AR) signaling inhibition. However, the emergence of early castration resistance or neuroendocrine transformation is associated with poor prognosis. Reliable biomarkers are needed to identify these patients and guide selection of clinical therapy. Methods: mRNA was isolated from EpCAM-positive circulating tumor cells (CTCs) isolated from patients with CSPC, CRPC, or NEPC to measure expression of KLK2, KLK3 (PSA), TMPRSS2, FOLH1 (PSMA), synaptophysin ( SYP), and chromogranin ( CHGA). Post-hoc retrospective analysis of an institutional review board–approved prospective cohort (N = 98) was performed to identify patterns of gene expression. Samples were considered AR+ if 3 of 4 AR pathway genes ( TMPRSS2, KLK2, KLK3, and FOLH1) were positive, and were considered NE+ if either or both SYP or CHGA were positive. Blood samples from two prospective clinical trials of men with mCRPC treated with abiraterone and enzalutamide, respectively, were analyzed to confirm results. Longitudinal samples were collected from 17 patients (6 NEPC and 11 Adenocarcinoma) and cell free DNA was isolated and sequenced using a novel targeted exon panel. Results: AR and/or NE positive patients were found to have a median overall survival (OS) of 8.58 months as compared to a median OS of 29.6 months in the AR and NE negative population (p<0.0001; HR=2.75 [1.60-4.56]). In the subset of castrate resistant prostate cancer (CRPC) patients, AR+ and/or NE+ patients (n=31) were found to have a median OS of 6.74 months vs 18.79 months in the AR- and NE- group (n=39) (p= 0.0009; HR 2.38 [1.36-4.18]). We also tested samples from a phase II ARSI trials with abiraterone and enzalutamide, respectively. None of the baseline samples from these two trials met the above criteria for NEPC (AR- NE+) on their CTCs or histologically. Three of 48 total patients were identified with expression in their baseline blood samples without loss of AR target gene expression (AR+/NE+). Preliminary analysis of this small patient cohort in comparison to patients who were NE- shows that NE+ patients had worse OS (HR= 5.5906 [1.143-27.36), as would be expected by patients with emerging neuroendocrine differentiation. Integrated ctDNA sequencing identified mutations in genes associated with NEPC. Conclusions: The expression of NE genes in liquid biopsies while retaining AR target gene expression is associated with worse OS and may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype. Early identification of these patients may improve therapeutic decisions and improved patient outcomes. Pairing genomic alteration with changes in gene expression may additionally offer the basis for a new mechanism to assess efficacy of novel therapeutics in future clinical trials.

Development and Analytical Validation of a 6-Plex Reverse Transcription Droplet Digital PCR Assay for the Absolute Quantification of Prostate Cancer Biomarkers in Circulating Tumor Cells of Patients with Metastatic Castration-Resistant Prostate Cancer.

Gene expression in circulating tumor cells (CTCs) can be used as a predictive liquid biopsy test in metastatic castration-resistant prostate cancer (mCRPC). We developed a novel 6-plex reverse transcription droplet digital PCR (RT-ddPCR) assay for the absolute quantification of 4 prostate cancer biomarkers, a reference gene, and a synthetic DNA external control (DNA-EC) in CTCs isolated from mCRPC patients. A novel 6-plex RT-ddPCR assay was developed for the simultaneous absolute quantification of AR-FL, AR-V7, PSA, and PSMA, HPRT (used as a reference gene), and a synthetic DNA-EC that was included for quality control. The assay was optimized and analytically validated using DNA synthetic standards for each transcript as positive controls. Epithelial cellular adhesion molecule (EpCAM)-positive CTC fractions isolated from 90 mCRPC patients and 11 healthy male donors were analyzed, and results were directly compared with reverse transcription quantitative PCR (RT-qPCR) for all markers in all samples. Linear dynamic range, limit of detection, limit of quantification, intra- and interassay precision, and analytical specificity were determined for each marker. Application of the assay in EpCAM-positive CTC showed positivity for AR-FL (71/90; 78.9%), AR-V7 (28/90; 31.1%), PSA (41/90; 45.6%), PSMA (38/90; 42.2%), and HPRT (90/90; 100%); DNA-EC concentration was constant across all samples. Direct comparison with RT-qPCR for the same markers in the same samples revealed RT-ddPCR to have superior diagnostic sensitivity. Our 6-plex RT-ddPCR assay was highly sensitive, specific, and reproducible, and enabled simultaneous and absolute quantification of 5 gene transcripts in minute amounts of CTC-derived cDNA. Application of this assay in clinical samples gave diagnostic sensitivity and specificity comparable to, or better than, RT-qPCR.

Large-scale whole-genome sequencing identifies genetic alterations in circulating hepatocellular carcinoma cells.

e15019 Background:Hepatocellular carcinoma (HCC) is one of the most lethal malignancies. Most of the patients have large tumor burden, vascular invasion, or metastasis at initial diagnosis. Liver biopsy is usually inaccessible in HCC patients due to the high risk of hemorrhage and tumor dissemination. Circulating tumor cells (CTCs) are malignant cells shed from the tumor tissue into the blood or lymphatic vessel. The fact that CTCs are live tumor cells makes them distinct from any of the existing cancer biomarkers. The biophysical property-based enrichment methods make downstream process easier as the isolated CTCs are not tagged with antibodies and the cell viability can be largely remained. Our objective is to isolate CTCs using a novel inertial focusing-base microfluidic device (CTC-100) and determine both their clinical value and the genomic features. Methods: We enriched and isolated CTCs in 155 HCC patients, 51 cirrhosis patients and 10 healthy donors. These patients were enrolled from Sep, 2020 to Feb, 2022 in the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China). This method enriches CTCs according to the cell size and is label-free. The enriched CTCs are then validated via immunostaining. The CD45-EpCAM+DAPI+ cells with size larger than 15μm are considered as epithelial CTCs, and CD45-EpCAM-DAPI+ cells are considered mesenchymal. The CTCs were then micromanipulated from the slide and the pooled CTCs from each patient were then subjected to MALBAC whole genome amplification and whole genome sequencing, which has been finished in CTCs from 12 patients receiving surgery and 4 without tumor biopsy. The other samples are under processing. Results: Our data revealed that the number of total CTCs or mesenchymal CTCs were significantly higher in HCC patients than that in cirrhosis or healthy donors. The detection rate of CTC in HCC patients was 100%. The EpCAMpositive CTCs accounted only for 16.6% (298/1798) of the total CTCs. Moreover, the number of CTCs was positively associated with macrovessel invasion and tumor size (P < 0.01). Most of the mutated genes in CTC samples were accordant with corresponding cancer tissues. We have detected 24 CTC-specific mutated genes, including FBXL22, USP3, MIR1827, S100A1. We then figure out the frequently mutated genes in CTC samples. Finally, we cluster the SNVs in CTCs according to clinicopathological parameters including macrovessel invasion, distant metastasis, tumor size. The results showed that part of the mutated genes might be associated with tumor invasion or metastasis. Conclusions: The novel microfluidic platform can efficiently detect CTCs in HCC patients. CTC number is associated with macrovessel invasion and tumor size. Finally, the SNV but not CNV in CTC samples is tightly consistent with that in tumor tissue. We also find out some CTC-specific mutated genes that might be involved in HCC metastasis. Clinical trial information: ChiCTR2100051584.

DNA walker-powered ratiometric SERS cytosensor of circulating tumor cells with single-cell sensitivity

Identification and detection of extreme rare circulating tumor cells (CTCs) in peripheral blood can precisely monitor cancer recurrence and metastasis, however, how to ultra-sensitively and reliably detect CTCs is a big challenge. In this work, a ratiometric surface-enhanced Raman spectroscopy (SERS)-based strategy for ultra-sensitively and nondestructively detecting CTCs was proposed via CTCs-triggered DNA walker-assisted assembly of plasmonic nanostructure networks consisting of Walker probes and SERS tags. The Walker probes were prepared by modifying Fe3O4@SiO2@Au nanoparticles (GMNPs) with ROX-labeled EpCAM aptamer-blocked Zn2+-specific DNAzyme and hairpin-structured single-stranded DNAs H1, and the SERS tags were constructed by co-labelling hairpin-structured single-stranded DNAs H2 and Raman molecules (DTNB) on Au NPs. The aptamers can recognize EpCAM-positive CTCs via the specific binding to EpCAM, so that the activity of DNAzymes is activated with the assistance of Zn2+ to launch the DNA walker to move around for the cleavage of H1 on GMNPs. The residual fragments of H1 on GMNPs can hybridize with H2 on SERS tags and result in the formation of Walker probe-SERS tag network nanostructures (Nw NSs) with rich SERS hot spots. The reliable SERS detection of CTCs is achieved by the stable ratiometric SERS signals of DTNB and ROX generated from the Nw NSs, and a good linear relation between ratiometric SERS signal and MCF-7 cells concentration was obtained with the detection limit low to 1 cell/mL. The recovery rate of MCF-7 cells in peripheral blood is in the range of 94.0%–104.5%, which indicates a good application prospect of the novel ratiometric SERS cytosensor in the clinic detection of EpCAM-positive CTCs.

In situ monitoring of circulating tumor cell adhered on three-dimensional graphene/ZnO macroporous structure by resistance change and electrochemical impedance spectroscopy

A three-dimensional (3D) graphene macroporous foam substrate with ZnO nanorod array was developed for specific and non-invasive detection of circulating tumor cells (CTCs). With the help of the epithelial cell adhesion molecule antibody (anti-EpCAM) and the array structure, the EpCAM-positive CTCs can adhere on the detection substrate (rGO-ZnO-antiEpCAM foam). Moreover, the resistance and the electrochemical impedance spectroscopy (EIS) of the rGO-ZnO-antiEpCAM foam was changing during the CTCs adhering process with high sensitivity. Compared to the EpCAM-negative cancer cell, the resistance of the substrate is rising with the amount of the EpCAM-positive CTCs. The possible reason is that the negative charges of CTCs affected the conductivity of the p-type reduced graphene oxide (rGO), and when CTCs were adhering to the rGO-ZnO-antiEpCAM foam, the resistance of the rGO-ZnO-antiEpCAM foam increased. In addition, the EIS measurement also shows the regular changes during the adhering process. From the equivalent circuit modeling of EIS, the charge transfer resistance (Rct) on the surface of the rGO-ZnO-antiEpCAM foam electrode had a linear growth. This may be due to the fixation of negatively charged Breast cancer cells (MCF-7) on the substrate, the charge transfer process was hindered, which caused the semicircle domain of the EIS curve to expand significantly with time. These results reveal that CTCs can not only be specifically adhered on the rGO-ZnO-antiEpCAM foam, but also the electrical microenvironment of the detection substrate was changed during CTCs adhering process. The construction of the detection substrate, the measurement and analysis methods of the electrical signals are of great significance in CTCs acquisition and sensing, which will provide an effective strategy for the real-time monitoring of CTCs at the clinic.

Modification of Hemodialysis Membranes for Efficient Circulating Tumor Cell Capture for Cancer Therapy.

Background: It is well known that more than 90% of cancer deaths are due to metastases. However, the entire tumorigenesis process is not fully understood, and it is evident that cells spreading from the primary tumor play a key role in initiating the metastatic process. Tumor proliferation and invasion also elevate the concentration of regular and irregular metabolites in the serum, which may alter the normal function of the entire human homeostasis and possibly causes cancer metabolism syndrome, also referred to as cachexia. Methods: We report on the modification of commercially available hemodialysis membranes to selectively capture circulating tumor cells from the blood stream by means of immobilized human anti-EpCAM antibodies on the inner surface of the fibers. All critical steps are described that required in situ addition of the immuno-affinity feature to hemodialyzer cartridges in order to capture EpCAM positive circulating tumor cells, which represents ~80% of cancer cell types. Results: The cell capture efficiency of the suggested technology was demonstrated by spiking HCT116 cancer cells both into buffer solution and whole blood and run through on the modified cartridge. Flow cytometry was used to quantitatively evaluate the cell clearance performance of the approach. Conclusions: The suggested modification has no significant effect on the porous structure of the hemodialysis membranes; it keeps its cytokine removal capability, addressing cachexia simultaneously with CTC removal.

A Comprehensive Molecular Analysis of in Vivo Isolated EpCAM-Positive Circulating Tumor Cells in Breast Cancer.

Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa). In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining. All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection. In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs.

Prognostic Significance of Gene Expression and DNA Methylation Markers in Circulating Tumor Cells and Paired Plasma Derived Exosomes in Metastatic Castration Resistant Prostate Cancer

Simple Summary“Liquid biopsy”, based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides non-invasive real-time monitoring of tumor evolution and therapeutic efficacy. We performed for the first time a direct comparison study on gene expression and DNA methylation markers in CTCs and paired plasma-derived exosomes and evaluated their prognostic significance in metastatic castration resistant prostate cancer. Our results revealed for the first time a significantly higher positivity of all markers in EpCAM-positive CTCs compared to plasma-derived exosomes. We report that in EpCAM-positive CTCs, CK-19, PSMA, TWIST1 expression and GSTP1 methylation are significantly correlated with worse overall survival (OS), while in exosomes, CK-8 expression and GSTP1 and RASSF1A methylation status were significantly correlated with a lower OS. We also enumerated CTC and tumor-derived extracellular vesicles (tdEVs) using CellSearch (CS) and found a correlation between the CTC and tumor-derived extracellular vesicles (tdEVs) enumeration values.Liquid biopsy, based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides non-invasive real-time monitoring of tumor evolution and therapeutic efficacy. We performed for the first time a direct comparison study on gene expression and DNA methylation markers in CTCs and paired plasma-derived exosomes and evaluated their prognostic significance in metastatic castration resistant prostate cancer. This prospective liquid biopsy (LB) study was based on a group of 62 metastatic castration resistant prostate cancer (mCRPC) patients and 10 healthy donors (HD) as controls. Identical blood draws were used to: (a) enumerate CTC and tumor-derived extracellular vesicles (tdEVs) using CellSearch (CS) and (b) analyze CTCs and paired plasma-derived exosomes at the gene expression and DNA methylation level. CTCs were enumerated using CellSearch in 57/62 patients, with values ranging from 5 to 854 cells/7.5 mL PB. Our results revealed for the first time a significantly higher positivity of gene expression markers (CK-8, CK-18, TWIST1, PSMA, AR-FL, AR-V7, AR-567 and PD-L1 mRNA) in EpCAM-positive CTCs compared to plasma-derived exosomes. GSTP1, RASSF1A and SCHLAFEN were methylated both in CTC and exosomes. In CTCs, Kaplan–Meier analysis revealed that CK-19 (p = 0.009), PSMA (p = 0.001), TWIST1 (p = 0.001) expression and GSTP1 (p = 0.001) methylation were correlated with OS, while in exosomes GSTP1 (p = 0.007) and RASSF1A (p = 0.001) methylation was correlated with OS. Our direct comparison study of CTCs and exosomes at gene expression and DNA methylation level, revealed for the first time a significantly higher positivity in EpCAM-positive CTCs compared to plasma-derived exosomes. Future perspective of this study should be the evaluation of clinical utility of molecular biomarkers in CTCs and exosomes on independent multicentric cohorts with mCRPC patients.

Radioiodine therapy reduces the frequency of circulating tumour cells in patients with differentiated thyroid cancer.

The aim of the study was the quantification of circulating tumour cells (CTCs) in differentiated thyroid cancer (DTC) patients before and 6weeks after radioiodine therapy (RIT). Circulating tumour cells (CTCs) were described more recently in cancer patients, mostly correlating with poor outcome and advanced metastases. Peripheral blood for identification and quantification of CTC before RIT or/and 6weeks after RIT was provided by 55 DTC patients that received RIT for remnant tissue ablation. 13 follicular thyroid cancer (FTC) patients, 31 papillary thyroid cancer (PTC) patients and 11 patients having the follicular variant PTC (FV-PTC) were included. Peripheral blood mononuclear cells (PBMCs) were isolated and EpCAM-positive CTCs were counted by immune fluorescent staining. A CTC positivity of 31.8% before RIT could be observed. Six weeks after RIT, the CTC positivity was reduced to 13.6%. Paired data at both time points of blood sampling could be gathered for n=33 DTC patients. These patients had significantly higher CTC numbers before RIT than 6weeks afterwards (0.27±0.47 vs 0.05±0.15, P=.0215). Additionally, significantly reduced CTC numbers were also demonstrated in pre-RIT CTC-positive patients (0.88±0.43 vs 0.05±0.16, P=.0039). Our results indicate a reducing effect on the number of CTCs by RIT. Therefore, CTC enumeration should be considered as efficient tool for treatment monitoring during RIT.

THU481 - The combination of EpCAM-positive circulating tumor cells and serum AFP/AFP-L3/DCP predicts outcome after curative resection of hepatocellular carcinoma

THU481 - The combination of EpCAM-positive circulating tumor cells and serum AFP/AFP-L3/DCP predicts outcome after curative resection of hepatocellular carcinoma

1923P - Prognostic value of CTCs in advanced NSCLC patients treated with platinum-based chemotherapy

BackgroundLung cancer is the leading cause of cancer-related mortality worldwide. At diagnosis, 70% of patients present advanced disease being chemotherapy the standard of care. Although, specific tumour biomarkers that allow a better treatment selection and monitoring is absent at the moment. Enumeration and characterization of circulating tumor cells (CTCs) have the potential to perform as a prognostic biomarker for a precision medicine approach to lung cancer care. The present study was conducted to validate the characterization CTCs from patients with advanced Non-small cell lung cancer (NSCLC) as a valuable tool for anticipating the disease evolution and the therapy response. Methods78 patients with advanced NSCLC were enrolled in the study at HGUV and CHUS. EpCAM positive CTCs from these patients were isolated and analysed using both CellSearch technology and a qRT-PCR based approach at different times: at baseline and before the 2nd and 5th cycle treatment. A panel of genes with a relevant role for NSCLC aggressiveness and the resistance to platinum-based treatments were analysed. ResultsFrom all patients included in the study 46% were positive for CTCs using CellSearch system at baseline, showing only 12% of patients ≥5 CTCs. Additionally, patients with ≥5 CTCs showed poor PFS (4.66 vs 7.12 months, p=0.040) and OS (3,9 vs 13.43 months, p<0.001) rates compared with those patients with <5CTCs. In addition, patients with high CTCs levels during treatment also had a more aggressive disease evolution in terms of PFS and OS (p=0.007 and p=0.001, respectively). From the analyzed gene panel, patients with lower CHOKα expression at baseline had increased PFS (7.03 vs. 3.9 months, p=0.009) and OS (11.4 vs. 4.07 months, p=0.001). Furthermore, we found low CHOK α levels as a powerful marker to discriminate patients with a good response to chemotherapy from those that progressed during treatment administration (14.35 vs 5.07 months, respectively, p=0.011). ConclusionsWe demonstrated that quantification and gene expression characterization of CTCs represents an adequate strategy to identify prognostic biomarkers in NSCLC patients Supported by RTC-2014-1532-1 from MINECO and CB16/12/00350-CB16/12/00328 from CIBERONC. Legal entity responsible for the studyFIHGUV. FundingSupported from RTC-2014-1532-1 (Spanish Ministry of Economy, Industry and Competitiveness) and CB16/12/00350 and CB16/12/00328 from CIBERONC. DisclosureAll authors have declared no conflicts of interest.

Detection of circulating tumor cells (CTCs) in cerebrospinal fluid of a patient with HER2-overexpressing gastric cancer and single cell analysis of intra-patient heterogeneity of CTCs.

BackgroundThe purpose of this study was to identify circulating tumor cells (CTCs) through single cell sorting using DEPArray technology in cerebrospinal fluid (CSF) samples from an advanced gastric cancer (AGC) patient with leptomeningeal seeding. We also demonstrated intra-patient heterogeneity of CTCs isolated from CSF samples at the single cell level.MethodsWe used the DEPArray system to identify and align single enabled CSF and CTCs based on a multi-parallel fluorescence analysis. The CSF samples were stained with an antibody cocktail recognizing DAPI, cytokeratin, EpCAM, and HER2. The stained cells were sorted and identified into a single, variable cell based on a multiparametric fluorescence analysis. The CSF and CTC subpopulations were quantified for absolute cell counts and relative frequency. Based on the stained morphology of each single CSF and CTC, five groups were identified.ResultsWe stratified the CTCs to evaluate the character of the CTCs isolated from the CSF, according to CK, EpCAM and HER2. In total, 78.2% of the CTCs isolated from the CSF showed HER2 overexpression, which was consistent with the status of HER2 in the patient’s primary gastric tumor. The most common types were HER2 positive, CK positive, and EpCAM positive CTCs (56.6%). On the other hand, 21.8% of the CTCs isolated from the CSF did not show HER2 overexpression, which was inconsistent with the primary tumor. Her2 negative, CK negative, and EpCAM positive CTCs were the predominant types among the CTCs without HER2 overexpression (19.7%).ConclusionsWe enriched the CTCs through a single cell sorting process with DEPArray technology in CSF samples of an AGC patient with leptomeningeal seeding. We also demonstrated intra-patient heterogeneity of the CTCs at the single cell level.

Biology and clinical relevance of EpCAM.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein primarily known to mediate homotypic cell contacts in epithelia tissues. Because EpCAM expression is limited to normal and malignant epithelia, it has been used as diagnostic marker for the detection of carcinoma cells in mesenchymal organs such as blood, bone marrow or lymph nodes. In particular, the detection and molecular characterization of EpCAM-positive circulating tumor cells (CTCs) in the blood of carcinoma patients has gained considerable interest over the past ten years. EpCAM is primarily considered as an adhesion molecule, but recent studies have shown diverse biological functions including regulation of cell proliferation and cancer stemness. In this review, we summarize the current knowledge on the biological properties of EpCAM with emphasis on mechanisms involved in cancer progression and discuss the clinical implications of these findings for the clinical use of EpCAM as a diagnostic marker.

Mycoplasma infection promotes tumor progression via interaction of the mycoplasmal protein p37 and epithelial cell adhesion molecule in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer death worldwide. To study how mycoplasma infection affects HCC progression, we investigated the characteristics of mycoplasma-infected tumor tissues and circulating tumor cells (CTCs) in HCC patients. The mycoplasmal membrane protein p37 showed significant correlations with higher histologic stages and vascular invasion and predicted poor disease-free survival of HCC patients. p37-positive CTCs were detected in 42 out of 47 HCC patients (89%). p37-positive circulating cells were also detected in 4 out of 10 healthy donors (40%), and all were epithelial cell adhesion molecule (EpCAM)-positive. In HCC patients, most of p37-negative CTCs (95%) showed intermediate phenotype with neither EpCAM nor vimentin expression, but p37-positive CTCs were EpCAM-positive (44%), vimentin-positive (32%), and both negative (24%), suggesting that EpCAM-positive CTCs are enriched with mycoplasma infection. Mycoplasma infection promoted migratory capacity of HCC cells with increased expression of EpCAM. Immunoprecipitation analysis revealed that p37 associates with EpCAM. The results suggest that mycoplasma infection promotes tumor progression in HCC patients via interaction of the mycoplasmal p37 and EpCAM.

Abstract 3604: Centrosome amplification in epcam-captured circulating tumor cells from metastatic cancer patients

Abstract Background: Genomic instability and evolution of cancers is linked to chemotherapy sensitivity and resistance in patients with metastatic cancer. Taxane-based chemotherapies operate by promoting chromosomal instability (CIN) by increasing multipolar mitoses. Breast tumors with CIN are more sensitive to paclitaxel compared to tumors without CIN. There is a critical need for new biomarkers based on biological evaluation of mechanisms of response and resistance in metastatic cancer. Although CIN is difficult to detect directly, centrosome amplification (CA) is a common mechanism of CIN. In this study, we develop an assay to detect CA in circulating tumor cells (CTCs) as a minimally invasive assay to evaluate sensitivity to taxane-based chemotherapies. Methods: Blood samples from patients with metastatic breast and prostate cancer were collected and processed for nucleated cells using a Ficoll gradient and magnetic depletion of CD45-positive cells. EpCAM-positive CTCs were captured using an exclusion-based sample preparation technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. CTCs were defined as cells positive for Hoechst, cytokeratin and negative for multiple immune and endothelial cell markers (CD45/CD11b/CD14 and CD34). EpCAM-captured CTCs were also stained with pericentrin and centrin to identify CA and evaluated for pericentrin (whole centrosome marker) and centrin (centriole marker) protein-staining intensity, number and size. Results: CA was detected in a subset of CTCs from patients with metastatic breast and prostate cancer patients. As a control, centrosomes were also analyzed in matched peripheral blood mononuclear cells from the same patients. CA was heterogeneous with 100% of patients demonstrating some degree of CA (&amp;gt;4 centrioles), ranging from 15-48% of CTCs. Conclusion: In conclusion, we report the presence of CA in EpCAM-captured CTCs from metastatic cancer patients. Evaluation of CA in CTCs using pericentrin and centrin staining may serve as a predictive biomarker of taxane based chemotherapy response and resistance. We are recruiting taxane-naïve metastatic cancer patients to test the hypothesis that CA and/or CIN predict taxane sensitivity. Citation Format: Ashok Singh, Ryan A. Denu, Jamie M. Sperger, Beth A. Weaver, Mark E. Burkard, Joshua M. Lang. Centrosome amplification in epcam-captured circulating tumor cells from metastatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3604.

Expanded Genomic Profiling of Circulating Tumor Cells in Metastatic Breast Cancer Patients to Assess Biomarker Status and Biology Over Time (CALGB 40502 and CALGB 40503, Alliance).

Purpose: We profiled circulating tumor cells (CTCs) to study the biology of blood-borne metastasis and to monitor biomarker status in metastatic breast cancer (MBC).Methods: CTCs were isolated from 105 patients with MBC using EPCAM-based immunomagnetic enrichment and fluorescence-activated cells sorting (IE/FACS), 28 of whom had serial CTC analysis (74 samples, 2-5 time points). CTCs were subjected to microfluidic-based multiplex QPCR array of 64 cancer-related genes (n = 151) and genome-wide copy-number analysis by array comparative genomic hybridization (aCGH; n = 49).Results: Combined transcriptional and genomic profiling showed that CTCs were 26% ESR1-ERBB2-, 48% ESR1+ERBB2-, and 27% ERBB2+ Serial testing showed that ERBB2 status was more stable over time compared with ESR1 and proliferation (MKI67) status. While cell-to-cell heterogeneity was observed at the single-cell level, with increasingly stable expression in larger pools, patient-specific CTC expression "fingerprints" were also observed. CTC copy-number profiles clustered into three groups based on the extent of genomic aberrations and the presence of large chromosomal imbalances. Comparative analysis showed discordance in ESR1/ER (27%) and ERBB2/HER2 (23%) status between CTCs and matched primary tumors. CTCs in 65% of the patients were considered to have low proliferation potential. Patients who harbored CTCs with high proliferation (MKI67) status had significantly reduced progression-free survival (P = 0.0011) and overall survival (P = 0.0095) compared with patients with low proliferative CTCs.Conclusions: We demonstrate an approach for complete isolation of EPCAM-positive CTCs and downstream comprehensive transcriptional/genomic characterization to examine the biology and assess breast cancer biomarkers in these cells over time. Clin Cancer Res; 24(6); 1486-99. ©2018 AACR.