Leukocyte Membrane-Coated Filtrable Micromotors With Selective Distribution of Enzymes for Capturing and Sensing Circulating Tumor Cells.

A cancer cell-activatable aptamer-reporter system for one-step assay of circulating tumor cells.

CTC Technologies and Tools.

Simple SummaryThe present review is aimed to discuss the relevance of assaying for the presence and isolation of circulating tumor cells (CTCs) in patients with sarcoma. Just a few studies have been performed to detect and enumerate viable CTCs in sarcoma and a majority of them still represent proof-of-concept studies, while more frequently tumor cells have been detected in the circulation by using the PCR-based method. Nevertheless, recent advances in technologies allowed detection of epithelial–mesenchymal transitioned CTCs from patients with mesenchymal malignancies, despite results being mostly preliminary. The possibility to identify CTCs holds a great promise for both applications of liquid biopsy in sarcoma for precision medicine, and for research purposes to pinpoint the mechanism of the metastatic process through the characterization of tumor mesenchymal cells. Coherently, clinical trials in sarcoma have been designed accordingly to detect CTCs, for diagnosis, identification of novel therapeutic targets and resistance mechanisms of systemic therapies, and patient stratification.Bone and soft tissue sarcomas (STSs) represent a group of heterogeneous rare malignant tumors of mesenchymal origin, with a poor prognosis. Due to their low incidence, only a few studies have been reported addressing circulating tumor cells (CTCs) in sarcoma, despite the well-documented relevance for applications of liquid biopsy in precision medicine. In the present review, the most recent data relative to the detection and isolation of viable and intact CTCs in these tumors will be reviewed, and the heterogeneity in CTCs will be discussed. The relevance of epithelial–mesenchymal plasticity and stemness in defining the phenotypic and functional properties of these rare cells in sarcoma will be highlighted. Of note, the existence of dynamic epithelial–mesenchymal transition (EMT)-related processes in sarcoma tumors has only recently been related to their clinical aggressiveness. Also, the presence of epithelial cell adhesion molecule (EpCAM)-positive CTC in sarcoma has been weakly correlated with poor outcome and disease progression, thus proving the existence of both epithelial and mesenchymal CTC in sarcoma. The advancement in technologies for capturing and enumerating all diverse CTCs phenotype originating from these mesenchymal tumors are presented, and results provide a promising basis for clinical application of CTC detection in sarcoma.

Microfluidics and Circulating Tumor Cells

Abstract #4162 The detection of circulating tumor cells (CTCs) in breast cancer patients have the potential to improve prognostication and the monitoring of response to treatment. Most CTC enrichment technologies are based on binding to anti-EpCAM antibodies. The sensitivity of such assays is limited by tumors that express no or undetectable levels of EpCAM. Improvements in CTC detection coupled with the development of systems to interrogate CTCs for therapeutic target expression could lead to novel applications for patient monitoring, clinical diagnosis and treatment. In this study, we describe a sensitive and reproducible enrichment method for CTCs. We defined cells as circulating tumor cells with three criteria: Positive for cytokeratin (CK+) and DAPI (nuclear) (DAPI+) and negative staining for CD45 (CD45-). We have previously reported that this system has a higher sensitivity for circulating tumor cell detection and provides a better platform for CTC downstream analyses compare to the methods currently available in the market. Herein, we describe the use of this platform for the evaluation of breast cancer biomarkers in CTCs. Blood samples from patients with metastatic breast cancer were used for CellSearch™ assay (Veridex , LLC ) and our CTC assay (A1000 CTC enrichment and detection kit, Genetix). We performed the CTC enrichment assay using the combination of anti-CK and anti-EpCAM antibodies. CTCs were identified with brightfield and fluorescence labeled anti-CK, anti-CD45 and DAPI (nuclear stain) images. The Ariol® system (Applied Imaging Corporation) was employed for automated cell image capture and analysis of CTCs on glass slides. CTCs enriched on the glass slides were used for CTC downstream analysis. Our CTC enrichment model is designed to have the capability to enrich all the three types of CTCs including CK+ & EpCAM+, CK+ & EpCAM-/low and CK-/low & EpCAM+ cells. Compared to the enrichment methods using anti-EpCAM or anti-cytokeratin antibody alone, a higher CTC detection rate and a larger dynamic CTC detected range were obtained with our new enrichment model. Interestingly there were clear CTC number differences with enrichment methods in the higher CTC count patient samples which indicate that the different enrichment methods may enrich different types of CTCs from patient blood samples.
 Results of DNA and RNA FISH analyses on enriched CTCs indicate that the CTCs on glass slides can be used for its downstream analyses directly or indirectly. Our method may have better performance on enrichment of heterogeneous CTCs and provide a better platform for CTCs profiling for biomarker evaluations and CTC downstream analyses. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4162.

Background: Despite progressive advances in the fields of radiation and chemotherapy, metastasis remains the leading cause of death in women with recurrent breast cancer. In metastasis, cells disseminate from the primary tumor, and circulate via the vascular system to distant organs, developing tumors at these new sites. Recent studies have suggested that tumor cells disseminate early on and develop the capacity to metastasize independently from the primary tumor. These circulating tumour cells (CTCs) represent the intermediate cells between primary tumours and metastases. The presence of CTCs in blood is an established prognostic marker of shorter progression-free and overall survival. It is currently impossible to distinguish CTCs from normal epithelial cells. They are also a rare population (approximately 1 out of 109 blood cells), making genomic profiling unachievable thus far. If the whole genome of CTCs can be screened for genomic alterations, two fundamental problems can be addressed (a) identifying a gene signature describing specific genomic alterations in early breast cancer that are associated with metastasis; and (b) utilizing this signature in the development of specific markers for CTCs in blood. Materials and Methods: We have successfully designed a protocol for the isolation of CTCs from blood for subsequent whole genome amplification (WGA) and microarray analysis. Blood samples from healthy donors were spiked with MCF7 tumor cells, and then enriched by automated immunomagnetic column separation. Enriched cell smears were stained for cytokeratin, using a glucose oxidase (GO) detection system. GO is absent from mammalian cells, which abolishes false positives seen with alkaline phosphatase and horse raddish peroxidase detection. Positively stained cells were isolated by single-cell laser capture microdissection, followed by WGA. Results: Genomic amplification was observed from as few as 2 MCF7 cells; with a sufficient yield of 1.5-3 μg of DNA. Microarray analysis was carried out on the high density Affymetrix Genome Wide SNP 6.0 array. Expected regions of amplification and deletion in the MCF7 cell line were identified in WGA samples. Copy number states were found to be strictly conserved across samples with single cell starting material (P<0.0001). We achieved up to 84% SNP call concordance between amplified single cell DNA and unamplified genomic DNA (P<0.0001). Discussion: We are isolating CTCs from the peripheral blood of 60 patients with early breast cancer. We have isolated CTCs and amplified DNA from 11 of 20 patients recruited so far. This preliminary study shows regions of DNA amplification that are unique to CTCs. We hypothesize that genes within these regions have the potential of being developed into CTC specific markers, and include genes associated with epithelial-mesenchymal transition, dormancy, cancer cell-stemness, migration, and invasion of the extracellular matrix. Identification of novel genomic alterations in CTCs associated with metastasis will pave the way for the development of a robust molecular or immunohistochemical prognostic test in patients with early breast cancer, to better identify those patients whose disease will progress to metastasis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-09.

The Promise of Circulating Tumor Cells in Metastatic CRPC

Despite progressive advances in the fields of radiation and chemotherapy, metastasis remains the leading cause of death in women with recurrent breast cancer. In metastasis, a small, select group of cells disseminate from the primary breast tumor, and circulate via the vascular system to distant organs, developing tumors at these new sites. Recent studies have suggested that tumor cells disseminate early on and develop the capacity to metastasize independently from the primary tumor. Furthermore, the enumeration of circulating tumour cells (CTCs) in blood is an established prognostic marker whereby, patients with more than 5 CTCs per 7.5mL of blood have shorter progression-free and overall survival. It is currently impossible to distinguish CTCs from normal epithelial cells. If the whole genome of CTCs can be screened for genomic alterations, two fundamental problems can be addressed (a) identifying a gene signature describing specific genomic alterations in early breast cancer that are associated with metastasis; and (b) utilizing this signature in the development of specific makers for CTCs in blood. We have designed a protocol for the isolation of tumor cells from blood for subsequent whole genome amplification (WGA) and microarray analysis. Blood samples from healthy donors were spiked with MCF7 tumor cells, and then enriched by automated immunomagnetic column separation. Enriched cell smears were stained for cytokeratin, using a glucose oxidase (GO) detection system. GO is absent from mammalian cells, which abolishes false positives seen with alkaline phosphatase and horse raddish peroxidase detection. Positively stained cells were isolated by single-cell laser capture microdissection, followed by WGA. Genomic amplification was observed from as few as 2 MCF7 cells; with a sufficient yield of 1.5-3 µg. Microarray analysis was carried out on the high density Affymetrix Genome Wide SNP 6.0 array. Expected regions of amplification and deletion in the MCF7 cell line were identified in WGA samples, as well as conserved across samples with single cell starting material (p<0.0001). Concordance of 75% was achieved from WGA of 3 single cells (p<0.0001). We propose to isolate CTCs from peripheral blood of 60 patients seen at the Locally Advanced Breast Cancer clinic at Princess Margaret Hospital. We have amplified genomic DNA from 6 out of 12 patients recruited so far, each having between 3-10 CTCs. Matched normal samples were obtained from the residual WBC pellet of each patient to allow for paired analysis. Optimization of WGA concordance will be followed by analysis of CTC DNA obtained from patients. Identification of novel genomic alterations in CTCs associated with metastasis will pave the way for the development of a robust molecular or immunohistochemical prognostic test in patients with early breast cancer, to better identify those patients whose disease will progress to metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2120.

Aim and Background: Breast cancer is one of the most prevalent malignancy among women worldwide, and its early detection remains a challenge. Circulating tumour cells (CTCs) are emerging as promising biomarkers for the diagnosis, prognosis, and monitoring of cancer. This study aims to assess the diagnostic performance of CK-positive and HER2-positive CTCs in breast cancer patients and healthy controls. Methods: This prospective case-control study involved 60 breast cancer patients (cases) and 60 age-matched healthy individuals (controls). Peripheral blood samples were collected prior to any oncological intervention. CTCs were isolated using Ficoll density gradient centrifugation and identified by flow cytometry, using cytokeratin (CK), CD45, and HER2 markers. Immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) were performed to assess hormone receptor status (ER, PR, and HER2) in CTCs. All statistical analyses were performed using SPSS 14 software. Results: Circulating tumour cells (CTCs) were detected in 85% of breast cancer patients, with cytokeratin-positive (CK+) CTCs showing high specificity and sensitivity for breast cancer detection (AUC = 1.000). Mann-Whitney U test results indicated significant differences in CTC counts between breast cancer patients and controls (p < 0.001 for both CK and HER2). In HER2-positive breast cancer, the expression of the HER2 marker demonstrated a 100% negative predictive value (NPV) and positive predictive value (PPV), along with a sensitivity of 51.67% and an area under the curve (AUC) of 0.758 for differentiation. Conclusion: Our study results support the clinical utility of CTCs as non-invasive biomarkers for breast cancer detection and characterisation. Applications of liquid biopsy in precision oncology is valuable, and further large-scale validation studies are warranted to enhance the clinical implementation of CTC analysis in breast cancer management

Background. RareCyte has developed platform technology for visual identification and single cell retrieval of rare cells in blood, including circulating tumor cells (CTCs). There is increasing interest in mutational analysis of circulating tumor cells (CTCs) as a liquid biopsy application. Because of the minuscule amount of DNA present in a single cell (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as elevated error rates that can lead to false positive mutations. Amplicon-based next generation sequencing (abNGS) is a high-throughput method which enables genetic confirmation of malignancy and discovery of de novo pathogenic mutations. Here we present a method for performance of single cell abNGS on model CTCs without prior WGA using a commercially available pan-cancer hotspot panel. Methods. A549 lung cancer cells as model CTCs (mCTCs) were spiked into whole blood, which was processed by AccuCyte® separation onto slides. After formalin fixation, multi-parameter immunofluorescence and automated imaging (CyteFinder®) were used to identify CTCs - visualized as nucleated cells expressing epithelial markers (cytokeratin or EpCAM) and not expressing white blood cell markers. mCTCs were mechanically retrieved by CytePicker® into PCR tubes and either amplified by WGA (PicoPLEX®) or lysed in a PCR-compatible lysis buffer. WGA products or cell lysates were used as template for the AmpliSeq™ Cancer HotSpot Panel v2 for Illumina® library preparation; additional PCR cycles were added during target amplification to compensate for low DNA input in the non-WGA samples. Libraries were sequenced on an Illumina MiSeq and analyzed using the BaseSpace bioinformatics suite. Results. Single mCTCs that underwent amplicon-based NGS library prep direct from cell lysate (non-WGA) displayed increased uniformity of coverage with decreased target dropout when compared to WGA cells. Median read depth increased 7-fold with the non-WGA method. On average, 8 of 15 variants present in bulk A549 genomic DNA were observed in single mCTCs sequenced after WGA, while 12 out of 15 were observed with the non-WGA method. Additionally, the false positive error frequency of non-WGA samples was < 5% of the WGA samples. The non-WGA method was applied to CTCs identified in blood from a prostate cancer patient and confirmed presence of PTEN and TP53 mutations identified by cell-free DNA analysis. Conclusions. Amplicon-based targeted single-cell sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs. Citation Format: Nolan G. Ericson, Arturo B. Ramirez, Alisa C. Clein, Celestia S. Higano, Daniel E. Sabath, Eric P. Kaldjian. Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 439.

NG04: Diversity of circulating tumor cells in a mouse pancreatic cancer model identified by single cell RNA sequencing

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.

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.

Introduction: Breast cancers (BCa) in younger women are more likely to be fast-growing, higher grade and hormone receptor-negative. As such these tumors tend to be associated with a worse prognosis even after systemic treatments with surgery, chemotherapy and radiation. The mechanisms that contribute to these tumors' aggressiveness remain largely unknown. Surveillance of circulating tumor cells (CTCs) may help to understand BCa prognosis and predict treatment benefit, especially for women with metastatic disease. Herein, we report a new finding of the correlation between age and the changes of CTC after the systemic therapies. Methods: 160 whole blood samples (7.5ml/each) were collected from 100 patients with stage III/IV BCa at baseline, before and after systemic therapy. CTC enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Janssen Diagnostics) by using CELLSEARCH® CXC Kit (Cell Search). CTCs were confirmed using Anti-CK-PE, CD25, and EpCaM antibody and DAPI nuclear stain. Anti-CD45-APC antibodies were used to identify leukocytes. The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD25-. Database of CTCs was generated and linked with clinical database. Student test was used for statistics. Results: Among 100 subjects who met inclusion criteria, 54 subjects (68%) at least 1 detectable CTCs in the baseline blood drawn prior to treatment. Of those, 37 patients had ≥ 5 CTCs, 17 patients had 1-5 CTCs, and 46 patients had no CTCs. There was no significant difference on the average age between these CTCs groups, which are 54.89, 53.52 and 53.30 respectively (P>0.05). Among the initial cohort, 42 subjects had at least 2 blood draws before and after systemic therapies respectively. Among these 42 patients, 11 had more CTCs after systemic therapy [Group 1 (26%), the increased range of CTC is from 1 to 756] and 17 patients had no CTCs before or after therapy [Group 2 (40%)]. There were 14 patients who had significantly fewer CTCs after systemic therapy [Group 3 (34%), the decreased range of CTC is from -1 to -23]. There was a significant difference in the average ages of each group (48.45, 56.00 and 57.28 years respectively; P=0.02). The results indicated that younger age (less than 50 years old) is associated with persistent of higher CTCs after systemic therapy likely reflecting decreased benefit from systemic therapy. Conclusion: Our original findings suggest age-related differences in CTCs response after systemic therapies for advanced BCa. These findings may indicate that younger patients may be at higher risk of developing more distant metastases as suggested by the increased numbers of CTCs after the systemic therapies. We propose that a combination of baseline CTCs detection and age be considered as a potential new criteria for selecting the systemic therapies for BCa patients. Citation Format: Qiang Zhang, Lorenzo Gerratana, Lisa Flaum, Youbin Zhang, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased circulating tumor cell (CTC) after systemic therapy is associated with younger age in stage III/IV breast 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 1597.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly fatal disease mainly treated with standard chemotherapy, albeit with limited efficacy. New therapeutic options are greatly needed, but the use of targeted treatments is often prevented by the impossibility to obtain tissue biopsies for molecular characterization. Here, we propose the use of circulating tumor cells (CTCs) as an alternative source of tumor material to perform molecular characterization for the identification of novel therapeutic targets. MATERIALS AND METHODS: Blood samples (10 ml) from patients with advanced CCA were processed for CTC isolation as follows: -CTC enrichment with Parsortix -identification and single-cell recovery of epithelial CTCs (expressing epithelial markers) and non-conventional CTCs (lacking epithelial and leukocyte markers) using the DEPArray -whole genome amplification and quality check using Ampli1 kit and Ampli1 QC kit -mutational profiling using Ion AmpliSeq Cancer HotSpot Panel v2 and AmpliSeq somatic pipeline for variant calling -copy number alteration (CNA) analysis using Ampli1 LowPass kit, plus unsupervised clustering and frequency alteration analyses. RESULTS: We analyzed 88 single CTCs isolated from 38 blood samples longitudinally collected from 23 patients (12 with intrahepatic, 9 with extrahepatic CCA and 2 with gallbladder cancer). CNA profiles showed a high level of both inter- and intra-patient heterogeneity, with each CTC displaying a unique profile. Intra-patient heterogeneity was further confirmed by clustering analysis as, in most cases, CTCs from the same patient clustered independently. CTC clustering was also not affected by sampling time (before/during chemotherapy), nor by the anatomical location of primary tumor. Conversely, we observed an enrichment of CTCs derived from patients non-responding to therapy (showing a PD according to RECIST criteria) in 2 of the 4 identified clusters (p=0.00041). By pairwise comparison of CNAs among clusters, we identified 2 regions more frequently altered in one cluster enriched for CTCs from non-responders: 10q22.2 and 3p11.1. The latter encodes, among others, for EPHA3, a targetable gene whose involvement in chemoresistance will be investigated by in vitro studies. Mutational profiling of 19 CTCs (from 6 patients) also confirmed the high intra-patient heterogeneity with most mutations being present in only 1 CTC. This limits the applicability of this approach in patients with few CTCs. Nonetheless, in 1 patient presenting 9 CTCs, we identified 1 mutation in KIT shared by 7/9 CTCs, indicating it as a possible treatment target for this patient. CONCLUSIONS: Our results support the possibility of using CTC molecular characterization to identify both resistance mechanisms and patient-specific targets, thus opening the way for a shift in treatment management of CCA towards an innovative and personalized therapy. Citation Format: Carolina Reduzzi, Marta Vismara, Marco Silvestri, Monica Niger, Rosita Motta, Giorgia Peverelli, Patrizia Miodini, Luigi Celio, Filippo De Braud, Maria G. Daidone, Vera Cappelletti. Molecular characterization of circulating tumor cells in cholangiocarcinoma patients: A new tool for treatment management [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1390.