Identification Of Circulating Tumor Cells Research Articles

PIC&RUN: An integrated assay for the detection and retrieval of single viable circulating tumor cells

Circulating tumor cells (CTCs) shed from solid tumors can serve as a minimally invasive liquid biopsy for monitoring disease progression. Because CTCs are rare and heterogeneous, their biological properties need to be investigated at the single cell level, which requires efficient ways to isolate and analyze live single CTCs. Current methods for CTC isolation and identification are either performed on fixed and stained cells or need multiple procedures to isolate pure live CTCs. Here, we used the AccuCyte-RareCyte system to develop a Protocol for Integrated Capture and Retrieval of Ultra-pure single live CTCs using Negative and positive selection (PIC&RUN). The positive selection module of PIC&RUN identifies CTCs based on detection of cancer surface markers and exclusion of immune markers. Combined with a two-step cell picking protocol to retrieve ultrapure single CTCs, the positive selection module is compatible for downstream single cell transcriptomic analysis. The negative selection module of PIC&RUN identifies CTCs based on a live cell dye and the absence of immune markers, allowing retrieval of viable CTCs that are suitable for ex vivo culture. This new assay combines the CTC capture and retrieval in one integrated platform, providing a valuable tool for downstream live CTC analyses.

Expression Profiling of Circulating Tumor Cells in Pancreatic Ductal Adenocarcinoma Patients: Biomarkers Predicting Overall Survival.

The interest in liquid biopsy is growing because it could represent a non-invasive prognostic or predictive tool for clinical outcome in patients with pancreatic ductal adenocarcinoma (PDAC), an aggressive and lethal disease. In this pilot study, circulating tumor cells (CTCs), CD16 positive atypical CTCs, and CTC clusters were captured and characterized in the blood of patients with PDAC before and after palliative first line chemotherapy by ScreenCell device, immunohistochemistry, and confocal microscopy analysis. Gene profiles were performed by digital droplet PCR in isolated CTCs, five primary PDAC tissues, and three different batches of RNA from normal human pancreatic tissue. Welsh's t-test, Kaplan-Meier survival, and Univariate Cox regression analyses have been performed. Statistical analysis revealed that the presence of high CTC number in blood is a prognostic factor for poor overall survival and progression free survival in advanced PDAC patients, before and after first line chemotherapy. Furthermore, untreated PDAC patients with CTCs, characterized by high ALCAM, POU5F1B, and SMO mRNAs expression, have shorter progression free survival and overall survival compared with patients expressing the same biomarkers at low levels. Finally, high SHH mRNA levels are negatively associated to progression free survival, whereas high vimentin mRNA levels are correlated with the most favorable prognosis. By hierarchical clustering and correlation index analysis, two cluster gene signatures were identified in CTCs: the first, with high expression of VEGFA, NOTCH1, EPCAM, IHH, is the signature of PDAC patients before chemotherapy, whereas the second, with an enrichment in the expression of CD44, ALCAM, and POU5F1B stemness and pluripotency genes, is reported after palliative chemotherapy. Overall our data support the clinic value of the identification of CTC's specific biomarkers to improve the prognosis and the therapy in advanced PDAC patients.

Abstract 2285: Comparison of the nCyte Dx and CellSearch systems for CTC enumeration

Abstract Introduction: Circulating Tumor Cells (CTCs) have clinical utility for cancer prognosis and monitoring disease. The goal of this study was to compare CTC enumeration by the FDA-approved CellSearch technology to a new technology, the nCyte Dx system by Axon Dx, LLC. Methods: Duplicate peripheral blood samples from 24 patients with advanced prostate cancer were collected in CellSave tubes as standard of care and submitted for CTC quantification using the CellSearch system at ARUP Laboratories. Following CellSearch testing, a paired sample was sent from ARUP to Axon Dx for nCyte Dx analyses. For every pair of samples, the blood was collected at the same time, but the sample tested by Axon Dx was aged 24 to 72 hours beyond the sample tested with CellSearch. CellSearch identifies CTCs by immunomagnetic capture of EpCAM-postive cells, while nCyte Dx does not incorporate specific CTC enrichment. With nCyte Dx, all PBMCs are isolated via density-based separation in a CPT tube and stained with Axon’s immunofluorescent cocktail (nPac). The cells are drawn down onto semi-porous membranes and placed onto microscope slides. Slides are imaged with the nCyte, a rapid fluorescent scanning instrument, and Axon’s advanced cell finding and analysis software (nAble) facilitates automated CTC detection. Due to the high-quality optical properties of the nCyte, the detected material can be characterized and categorized based on morphology and cellular structure. Results: CellSearch-derived CTC counts were compared with the enumeration results from the nCyte Dx system. Results were reported per 7.5mL blood for both methods. Bland-Altman analysis demonstrates that CellSearch and nCyte Dx results agreed in 22 of the 24 patient sample sets tested within a 95% confidence interval (CI). For both discrepant samples, the CellSearch count was found to be higher. The nCyte Dx method generally detected more CTCs than CellSearch in the lower enumeration range (<10 CTCs). Because previous data from Axon Dx shows no CTCs detected in healthy patients, this finding may be attributable to increased sensitivity rather than decreased specificity. Differences between the results may also be attributable to the natural variation between duplicate blood draws, best characterized by the Poisson distribution model. Summary and Conclusions: A comparison of CTC enumeration results using the CellSearch and nCyte Dx platforms was performed using duplicate blood samples from 24 prostate cancer patients. The results reveal high similarity between the two technologies, with 22 of 24 patients (92%) having enumeration results that agreed within a 95% CI. While both technologies deliver similar results, there are notable advantages of the nCyte Dx system over CellSearch, including a lack of bias toward EpCAM positive material. Additionally, the superior image quality of nCyte Dx may limit subjectivity in CTC identification and offer the ability to characterize CTC heterogeneity. Citation Format: Josefa Andrade, Ramsey Mohl, Stephanie Thomas, John Kearney, Phillip Bernard, Hillary S. Sloane. Comparison of the nCyte Dx and CellSearch systems for CTC enumeration [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 2285.

Abstract 454: Transferrin receptor identifies a novel circulating tumor cell population in patients with pancreatic cancer with a unique metastasis-associated molecular signature

Abstract Pancreatic Ductal Adenocarcinomas (PDAC) is the fourth most common cancer, expected to be the second leading cause of cancer mortality by 2030. Most PDAC patients are diagnosed with metastatic disease, with dismal prognosis of less than 1% 5-year survival. A major challenge in PDAC treatment is the absence of actionable molecular targets and the scarcity of available tumor tissue impeding molecular understanding of PDAC progression. Circulating Tumor Cells (CTCs) provide an easily accessible source for tumor tissue. CellSearchTM is the only FDA-cleared platform for CTC isolation based on positive selection of EpCAM+ cells. However, this method performs poorly in PDAC failing to show any prognostic relevance, likely due to EpCAM down-regulation during epithelial-to-mesenchymal transition (EMT), which precedes metastasis. To overcome this limitation, we tested Transferrin Receptor 1 (TfR) in the identification and isolation of CTCs from PDAC patients. TfR is a cell surface protein that mediates iron uptake, is overexpressed in multiple tumors with sustained expression throughout EMT, making it a suitable candidate for CTC isolation. To test this hypothesis, we first assessed TfR expression in a panel of 9 human PDAC cell lines, and we observed that TfR was expressed in 9/9 (100%) in contrast to 6/9 (66.7%) being EpCAM positive. No TfR expression was detected in healthy donor and PDAC patients’ PBMCs. To assess the performance of TfR in CTC identification and isolation from the peripheral blood of PDAC patients, we collected samples from 37 PDAC patients with stage 4 disease, enriched CTCs by negative CD45 depletion (RossetteSepTM) and subsequently stained live CTCs with TfR, EpCAM, and CD45. CTCs were defined as CD45- cells, positive for TfR+ or EpCAM+. We observed that the number of TfR+ CTCs (median: 148, range 2-4182) was significantly higher than EpCAM+ CTCs (median: 68, range 0-1552) within the same patient and across different patients (P-value 0.007). In addition, serial sampling at baseline (chemotherapy-naïve) and at disease progression from 5 PDAC patients revealed that TfR+CTC enumeration correlated with disease progression radiologically and/or CA19-9 (PDAC tumor marker) levels. In contrast, there was no correlation between the number of EpCAM+CTCs and clinical outcomes. RNA-Sequencing of isolated pools of TfR+ or EpCAM+ CTCs, followed by gene set enrichment analysis, revealed significant enrichment in oncogenic pathways, such as EMT, G2M, MYC and KRAS in TfR+ CTCs as compared with EpCAM+ CTCs. Taken together, our results reveal that TfR identifies a clinically relevant CTC subpopulation which correlates with disease progression and is molecularly distinct. Ongoing studies are focused on the function of TfR in disease progression and the potential use of TfR+ CTC molecular profiles in personalized PDAC treatment. Citation Format: Ahmed Halima, Jiaren Zhang, Giuseppe Galletti, Allyson J. Ocean, Paraskevi Giannakakou. Transferrin receptor identifies a novel circulating tumor cell population in patients with pancreatic cancer with a unique metastasis-associated molecular signature [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 454.

Abstract 1390: Molecular characterization of circulating tumor cells in cholangiocarcinoma patients: A new tool for treatment management

Abstract 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.

Abstract 1336: An assay for the detection and isolation of single live circulating tumor cells using Accucyte/Rarecyte platform

Abstract Introduction: Circulating tumor cells (CTCs) are critical metastatic drivers for the interface between the primary tumor and the target secondary organs. CTCs are heterogeneous, therefore, investigating their phenotypic, molecular and biological properties on a single cell level warrant a better understanding to metastasis and patient’s response to therapy. AccuCyte® - Rarecyte® system is a density based CTC isolation platform which allows for automated visual identification and retrieval of fixed rare cells from blood. Aims: To establish an assay for the detection and isolation of live single CTCs valid for subsequent molecular and functional characterization. Methods: Normal human whole blood samples were spiked with cells from breast cancer patient-derived CTC lines and were processed using the AccuCyte system. Buffy coats containing spiked CTCs were stained live with a cocktail of PE-594-conjugated antibodies for immune cell markers (CD14, CD16 and CD45) and either a live cell dye or a cocktail of Alexa Fluor 488-conjugated antibodies for cancer cell surface markers (EpCAM, Her2, EGFR and CDH11). Stained cells were then plated on chamber slides in the presence of serum free RPMI media and investigated using RareCyte fluorescence platform. CTCs were identified based on the lack of immune cell marker expression and the presence of the live cell dye (negative selection) or the expression of cancer cell surface markers and the lack of immune cell markers (positive selection). Single CTCs detected by both approaches were retrieved using our optimized interactive picking module. Cells detected by negative selection were tested for their ability to proliferate ex-vivo, whereas those isolated by positive selection were processed for single cell RNA sequencing analyses. Results: Detection rates of our protocols reached up to 85% (12 out of 14 CTCs). Single CTCs isolated by negative selection showed a slower proliferation rate during the first 6 days of culture (1.1 Fold increase relative to Day 0) compared to control cells (2.5 Fold increase). However their proliferation rates were comparable to control cells at day 13 (2.7 Vs. 3.2) and day 21 (2.2 Vs. 2.5). Currently, other live cell dyes are being tested to improve the proliferation ability of CTCs isolated by negative selection. Moreover, single cells which were retrieved by positive selection are currently being validated for RNA sequencing analyses. Conclusion: We have developed a novel assay which allows for the detection and isolation of single viable CTCs. Our assay has two modules; one uses a positive detection approach combined with a two-step cell picking protocol to provide ultrapure single CTCs valid for gene expression analyses. The other module provides an unbiased identification of CTCs using the negative selection approach and a single step of cell picking that provides viable single CTCs able to proliferate in culture. Citation Format: Mohamed Kamal, Shahin Saremi, Remi V. Klotz, Oihana Iriondo, Min Yu. An assay for the detection and isolation of single live circulating tumor cells using Accucyte/Rarecyte platform [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 1336.

Abstract 451: Transferrin Receptor identifies a distinct pool of circulating tumor cells from metastatic prostate cancer patients with unique molecular profiles enriched in AR variants

Abstract Androgen receptor (AR) drives prostate cancer (PC) progression, even in castrate-conditions due to expression of AR variants (AR-V), which are constitutively active in the nucleus. Castration-resistant prostate cancer (CRPC) is currently lethal, due to the development of drug resistance. The molecular basis of clinical drug resistance remains poorly elucidated, due to tumor heterogeneity and limited availability of serial tumor sampling as disease progresses. Circulating tumor cells (CTCs), isolated from patient peripheral blood, have emerged as a reliable, easy to obtain source of tumor cells which can be and used to interrogate the molecular disease evolution. To date, EpCAM-based CTC enrichment is commonly used but has important shortcomings as EpCAM is often lost during EMT, thus, missing the more aggressive mesenchymal CTCs. On the other hand, Transferrin Receptor (TfR) which is overexpressed in cancer cells, is a cell surface protein required for iron uptake and is critical for several biological processes such as cell proliferation and metabolism. Mining large gene expression databases in PC we observed that TfR expression increased with disease stage, from adenocarcinoma (TCGA) to CRPC (SU2C) to the more aggressive neuroendocrine PC (NEPC), while EpCAM expression remained unchanged. We also found that TfR expression was increased in EMT cell models, while EpCAM expression was lost, suggesting that TfR may perform well in CTC isolation across the EMT spectrum. We then tested TfR- and EpCAM-based CTC enrichment in a cohort of 31 CRPC patients, where we observed a significantly higher number of TfR+ CTCs (median, 304) than EpCAM+-CTCs (median, 21) (p<0.01). RNA-sequencing of TfR+ CTCs in comparison to EpCAM+ CTCs, followed by gene-set enrichment analyses revealed that the TfR+ subpopulation was significantly enriched in prostate specific oncogenic pathways including EMT, suggesting distinct molecular profiles between the two subsets. To investigate the clinical relevance of the two CTC subsets, we quantitated AR-FL and splice variants AR-V7 and AR-v567es expression by digital droplet PCR (ddPCR) in pools of TfR+-CTCs vs EpCAM+-CTCs. Our results from 16 CRPC patients showed that while expression of AR-FL was similar in the two CTC subsets, both AR variants were enriched in TfR+-CTCs. To better map the tumor heterogeneity of AR-V expression we isolated 102 single TfR+- and 63 single EpCAM+-CTCs from 3 CRPC patients and quantified AR-V expression by ddPCR. The single CTC analysis revealed that AR-V7 and AR-v567es were expressed in 21% and 18% of TfR+-CTCs, respectively, vs 0% in EpCAM+-CTCs. These data identify TfR as a promising marker for CTC identification across the EMT gradient, and provide a unique insight into tumor cell heterogeneity, which was not appreciated before, and which may have significant clinical impact. [A. Gjyrezi and G. Galletti contributed equally to this work.] Citation Format: Ada Gjyrezi, Giuseppe Galletti, Jiaren Zhang, Rohan Bareja, Ahmed Halima, Daniel Worroll, David Nanus, Scott Tagawa, Himisha Beltran, Paraskevi Giannakakou. Transferrin Receptor identifies a distinct pool of circulating tumor cells from metastatic prostate cancer patients with unique molecular profiles enriched in AR variants [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 451.

Abstract LB-236: Genomic characterization of circulating tumor cells in de novo metastatic prostate cancer

Abstract Background: The absence of biomarkers that identify clinically meaningful biological subsets of prostate cancer results in the homogeneous application of systemic therapies to a heterogeneous disease and hampers therapy development. This heterogeneity may become more evident at the molecular level under the pressure of androgen withdrawal. Here, we longitudinally characterized peripheral blood circulating tumor cells (CTCs) from patients with de novo metastatic prostate cancer to examine the phenotypic and genomic changes induced by androgen suppression. Methods: Peripheral blood samples were collected at MD Anderson Cancer Center from a cohort of DNM1PCa patients under clinical trial NCT01751438. Timepoints of collection includes baseline (BSL, before treatment) and 6 months (6mo, after 6 months of systemic therapy). We used the High-Definition Single Cell Assay (HD-SCA) workflow for CTC enumeration, morphology (nuclear size, DAPI intensity, cytokeratin (CK), androgen receptor (AR) intensity and genomics (whole-genome copy number aberration analysis) evaluation. This workflow utilizes an enrichment-free fluorescence based (DAPI/ CK/ CD45/ AR) CTC identification system and single-cell genomic sequencing. Results: Twenty-nine of the 58 available blood samples contained CTCs: 15 of the 28 (53.6%) collected at BSL (median 2.00 CTCs/mL, range 0.84-74.15) and 14 of the 30 (46.7%) collected at 6mo (median 2.43 CTCs/mL, range 0.96-13.85). However, at the 6mo timepoint, the CTCs had undergone a significant morphological shift in nuclear area local ratio (BSL vs 6mo: 1.036 vs 1.165, p=0.039), DAPI intensity (0.199 vs 1.030, p=0.036), CK intensity (18.978 vs 15.651, p=0.006), AR intensity (0.681 vs -0.468, p=0.037) and AR+ percentage (36.25% vs 15.38%, p=0.020). Further, genomic analysis of two patients at BSL indicated copy number aberrations including PTEN, TP53, RB1 and BRCA2 loss and MYC gain. Discussion: This study aims at characterizing changes induced by systemic therapies in CTC potential biomarkers that identify distinct subsets of disease biology. The changes observed in patient subsets may serve as clinically relevant biomarkers to optimize treatment and improve prognosis. Citation Format: Shoujie Chai, Paymaneh D. Malihi, Ana M. Apariciop, Brian F. Chapin, Matthew Lin, Ryan Storgard, Carmen Ruiz Velasco, Anand Kolatkar, James Hicks, Peter Kuhn. Genomic characterization of circulating tumor cells in de novo metastatic prostate cancer [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 LB-236.

Pathologic complete response and serial circulating tumor cell monitoring during neoadjuvant therapy in nonmetastatic triple-negative breast cancer patients.

e12111 Background: ARTEMIS (A Randomized, TNBC Enrolling trial to confirm Molecular profiling Improves Survival) is a randomized trial to determine if precision guided neoadjuvant chemotherapy (NAC) influences rates of pathologic complete response in the breast and axillary nodes (pCR). We hypothesized that blood circulating tumor cell (CTC) identification before and/or after NAC (just before surgery) would be associated with pCR in non-metastatic TNBC patients. Methods: Blood was assessed for CTCs at baseline (pre-NAC) and after NAC (post-NAC), as part of an IRB approved study, ARTEMIS (2014 – 0185/PA15-1050). CTCs (per 7.5 ml blood) were identified using the Cell Search System (Menarini Silicon Biosystems). Samples > one cell having morphologic criteria for malignancy were deemed CTC+. Fisher exact test was used to evaluate associations between CTC detection and clinicopathologic characteristics, and Chi2 test was used to analyze associations between CTC detection pre- and post- NAC, and pCR. Results: Fifty-nine patients had pre-NAC and 43 patients had post-NAC CTC assessments. One or more CTC was detected in 13/59 (22%) patients pre-NAC and in 16/43 (37%) of post NAC samples. CTC detection was not associated with patient stage, tumor androgen receptor expression, vimentin expression, Ki-67 staining, or tumor-infiltrating lymphocyte levels. Pre-NAC CTC detection was not (chi2 P = 0.98), but post-NAC (chi2 P = 0.0017) CTC identification was negatively associated with pCR. Conclusions: CTC detection post-NAC was negatively associated with pCR in non-metastatic TNBC patients. These findings warrant larger studies studying serial CTC detection throughout treatment to assess response to NAC.

Prognostic impact of baseline circulating tumor cells (CTCs) detected by the isolation by size of epithelial tumor cells (ISET) in locally advanced head and neck squamous cell carcinoma (LAHNSCC): Results of a prospective study.

6061 Background: The prognostic impact of CTCs in LAHNSCC is yet to be determined, with conflicting results in previous trials, the majority utilizing cytokeratin dependent techniques for identification and counting of CTCs. The primary objective of this study is to determine the detection rates using the ISET method, and the prognostic role of CTCs in LAHNSCC patients (pts) treated with a curative intent. Methods: In this prospective study, peripheral blood samples of pts with non-metastatic LAHNSCC, stages III/IV, were analyzed for CTCs using the ISET method, in two scenarios: curative surgical resection and adjuvant radiotherapy (CTCs before RT) and candidates for a non-surgical strategy (unresectable or organ preservation) either with upfront RT concurrent with chemotherapy (CT) or cetuximab (CTCs before RT), or preceded by induction CT (CTCs before ICT). Results: Eighty-three pts were included, the majority males (83%), with oropharynx primary (50%) and submitted to ICT (40%). The detection rate of baseline CTCs was 94% (78/83), and CTCs counts were significantly correlated with survival. For each increase of 1 CTC at baseline there was a relative increase of 18% in the risk of death (HR = 1.18; 95%CI: 1.06-1.31; p < 0.001), 16% in the risk of progression (HR = 1.16; 95%CI: 1.04-1.28; p = 0.004) and a reduction of 26% in the odds of complete response to treatment (non-surgical group only - OR = 0.74; 95%CI: 0.58-0.95; p = 0.022). Using the maximum of the standardized log-rank statistic proposed by Lausen and Schumacher we establish cut off points for overall survival (OS) and progression-free survival (PFS). Pts with CTCs < 6.5/ml had an estimated 2y OS of 85.6% versus 22.9% for CTCs ≥ 6.5/ml (HR = 0.18; 95%CI: 0.06-0.49; P < 0.0001) and pts with CTCs ≤ 3.8/ml had an estimated 2y PFS of 71.8% versus 37% for CTCs > 3.8/ml (HR = 0.32; 95%CI:0.15-0.67; p = 0.001). Conclusions: The detection rate of baseline CTCs using the ISET method was very high in LAHNSCC and the counts of CTCs were strongly correlated with survival and response to treatment.

Rapid and efficient isolation and detection of circulating tumor cells based on ZnS:Mn2+ quantum dots and magnetic nanocomposites

Rapid and non-destructive detection of circulating tumor cells (CTCs) with no disruption of their functions is of great significance for clinical tumor therapy. However, many existing methods for CTC detection commonly rely on conventional three-color immunofluorescence identification, which damages CTCs and easily causes loss of cells. Here, we employed a method to simultaneously capture and authenticate CTCs based on immunonanocomposites (ZnS:Mn2+ QDs and Fe3O4/SiO2) equipped with permanent fluorescent and magnetic properties. A multifunctional nanocomposite was synthesized by encapsulating ZnS:Mn2+ quantum dots (QDs) and Fe3O4 nanoparticles into SiO2 nanospheres and bio-conjugating tumor-specific anti-EpCAM antibodies onto the surface. The resulting nanocomposite had a high tumor cell binding ability, and the Fe3O4 nanoparticles had a rapid magnetic response that enabled capture of circulating tumor cells from patients' blood within minutes. In addition, the cell-immunonanocomposites complexes could be directly recognized by the yellow-orange light emitted by the ZnS:Mn2+ quantum dots, thus labeling cells without utilizing the complicated and destructive procedures involved in traditional CTCs identification. We successfully achieved a high capture efficiency of up to 90.8%, and the specific fluorescence labeling of CTCs was realized in 9 clinical breast cancer patients’ samples. Furthermore, this simple, convenient and cell-friendly approach is significant for solving the problems of cell viability and enables non-destructive CTC detection, which marks an advance in cancer treatment and clinical applications.

21O - EPAC-Lung: Pooled analysis of circulating tumor cells in advanced non-small cell lung cancer

Background: We assessed the clinical validity of circulating tumor cell (CTC) quantification for prognostication of patients with advanced non-small cell lung cancer (NSCLC) by undertaking a European pooled analysis of individual patient data. This is the largest study of its kind and the first to examine between-centre heterogeneity of CTC identification in NSCLC. Methods: Nine European NSCLC CTC centers were asked to provide reported/unreported anonymised data for patients with advanced NSCLC who participated in CellSearch CTC studies from January 2003 - March 2017. We used Cox regression models, stratified by centre, to establish the association between CTC count and survival. We assessed the added value of CTCs to prognostic clinico-pathological models using likelihood ratio (LR) statistics and c-indices. Results: Seven out of nine eligible centers provided data for 550 eligible patients, including 209 patients whose prognostic information was previously unpublished. CTC counts of ≥ 2 and ≥5 per 7·5 mL were associated with reduced progression-free survival (≥2 CTCs: HR 1.72, p < 0·001; ≥5 CTCs: HR 2.21, p < 0·001) and overall survival (≥2 CTCs: HR 2·18, p < 0·001; ≥5 CTCs: HR 2·75, p < 0·001), respectively. Survival prediction was significantly improved by addition of baseline CTC count to LR clinico-pathological models (log-transformed CTCs p < 0·0001; ≥2 CTCs p < 0·0001; ≥5 CTCs p < 0·0001), while more moderate improvements were observed with the use of c-index models. There was minor evidence of between-center heterogeneity in the effect on PFS, but not OS.No difference in CTC profile was observed between key NSCLC molecular subsets such as EGFR, ALK, and KRAS. Conclusions: These data confirm CTCs as an independent prognostic indicator of progression-free survival and overall survival in advanced NSCLC. CTC count improves prognostication when added to full clinico-pathological predictive models. ≥2 CTCs is an appropriate cutoff to move towards establishing clinical utility. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: C.R. Lindsay: Institutional funding for an ongoing phase II trial for which I am PI; Supported by Roche as part of an ESMO translational fellowship awarded in 2014-2016. F.H. Blackhall: Grants: AstraZeneca, Novartis, Pfizer, Amgen, BMS; Consultancy fees: Cell Medica, MSD; Speaker bureau: BI; Advisory board work: Regeneron, Medivation, AbbVie, Takeda, Roche, Ibsen. M.G. Krebs: Advisory board: J&J. L. Terstappen: Inventor on a number of US patents related to CellSearch, rights of which assigned to Johnson&Johnson, CellSearch kits obtained from Johnson&Johnson through a collaborative agreement with the MCBP. J-C. Soria: Consultancy fees: AZ, Astex, Clovis, GSK, GamaMabs, Lilly, MSD, Mission Therapeutics, Merus, Pfizer, PharmaMar, Pierre Fabre, Roche/Genentech, Sanofi, Servier, Symphogen, Takeda; Full time employee: MedImmune; Shareholder: AZ, Gritstone. All other authors have declared no conflicts of interest.

Androgen receptor nuclear localization correlates with AR-V7 mRNA expression in circulating tumor cells (CTCs) from metastatic castration resistance prostate cancer patients

Androgen receptor (AR) signaling drives prostate cancer (PC) progression and remains active upon transition to castration resistant prostate cancer (CRPC). Active AR signaling is achieved through the nuclear accumulation of AR following ligand binding and through expression of ligand-independent, constitutively active AR splice variants, such as AR-V7, which is the most commonly expressed variant in metastatic CRPC (mCRPC) patients. Most currently approved PC therapies aim to abrogate AR signaling and activity by inhibiting this ligand-mediated nuclear translocation. In a prospective multi-institutional clinical study, we recently showed that taxane based chemotherapy is also capable of impairing AR nuclear localization (ARNL) in circulating tumor cells (CTCs) from CRPC patients, whereas taxane induced decreases in ARNL were associated with response. Thus, quantitative assessment of ARNL in CTCs can be used to monitor therapeutic response in patients and help guide clinical decisions.Here, we describe the development and implementation of quantitative high throughput (QHT) image analysis algorithms to aid in CTC identification and quantitative assessment of percent ARNL (%ARNL). We applied this algorithm to fifteen CRPC patients at the start of taxane chemotherapy, quantified %ARNL in CTCs, and correlated with expression of AR-V7 mRNA (from CTCs enriched via negative, CD45+ depletion of peripheral blood) and with biochemical (prostate specific antigen; PSA) response to taxane chemotherapy.We found that CTCs from AR-V7 positive patients had higher baseline %ARNL compared to CTCs from AR-V7 negative patients, consistent with the constitutive nuclear localization of AR-V7. In addition, lower %ARNL in CTCs at baseline was associated with biochemical response to taxane chemotherapy. High inter- and intra-patient heterogeneity was also observed. As ARNL is required for active AR signaling, the QHT algorithms described herein can provide prognostic and/or predictive value in future clinical studies.

Development and Preliminary Clinical Application of Circulating Tumor Cell Detection System for Prostate Cancer.

Real-time detection of circulating tumor cell (CTC) markers that are constantly changing and renewing during disease progression is of great significance for the timely regimen switch or individualized target therapy. The abnormally expressed special AT-rich sequence binding protein 1 (SATB1), a nuclear matrix attachment region binding protein, in various tumors, promotes the growth and metastasis of tumor cells by regulating gene expression. In this paper, a CTC detection system for prostate cancer (PCa) was developed on the basis of epithelial cell adhesion molecule (EpCAM)-targeted immunomagnetic separation and CK-FITC and SATB-1-APC immunofluorescence assay, and the recovery rate of tumor cells in PBS and simulated whole blood by this system was detected. Subsequently, we isolated, identified, and counted SATB-1 ositive CTCs in the peripheral blood and urine samples of 60 tumor-bearing nude mice, 5 healthy volunteers and 13 PCa patients. Combined with the clinicopathological factors, the clinical value of the system was analyzed, and the possibility of SATB-1-positive CTCs in the diagnosis of PCa was evaluated. The results showed that the CTC sorting and identification system for prostate cancer constructed in this study had a recovery rate of more than 85% for CTC in PBS, urine and blood simulation samples. The expression level of SATB-1 was different in different PCa cell lines, which was relatively high in the highly invasive PCa DU-145 cell line. The expression of SATB-1 in CTCs in the blood samples of PCa patients with different clinical characteristics and in the urine samples of a few PCa patients with bone metastases were different, and the detection sensitivity of peripheral blood was higher than that of urine. This study has important clinical reference value for the early diagnosis of PCa and the evaluation of bone metastasis based on the CTC counting and the SATB-1 expression in CTCs.

Abstract P3-01-01: Circulating tumor cells (CTCs) after neoadjuvant chemotherapy for triple negative breast cancer (TNBC)

Abstract Background: ARTEMIS (A Randomized, TNBC Enrolling trial to confirm Molecular profiling Improves Survival) is a randomized trial to determine if precision guided neoadjuvant chemotherapy (NAC) impacts rates of pathologic complete response in the breast and axillary nodes (pCR). We hypothesized that CTCs in peripheral blood after completion of NAC would provide prognostic information beyond pCR alone in TNBC patients. Methods: Blood was assessed for CTCs after NAC as part of two IRB approved studies, ARTEMIS (2014 – 0185/PA15-1050), and LAB04-0698. CTCs were identified using the Cell Search® System (Menarini Silicon Biosystems). Samples with one or more cells, also having morphologic criteria for malignancy, were deemed CTC positive. Log-rank test and Cox regression analysis were applied to evaluate associations between CTC positive, pCR, and overall survival. Results: pCR was achieved in 24/68 (35%) patients with TNBC. Twenty four patients (35%) were CTC positive. Three year overall survival was evaluated in 4 groups of patients: pCR and no CTCs (n=20), pCR and CTC positive (n=4), non-pCR and no CTCs (n=24) and non-pCR and CTC positive (n=20). Three year overall survival was higher in the pCR and no CTCs cohort (100%), compared to pCR and CTC positive (50%), non-pCR and no CTCs (83%), non-pCR and CTC positive (19%); log rank p&amp;lt;0.0001. In the non-pCR and CTC positive patient cohorts, the presence of CTCs was associated with significant risk of death at 3 years [hazard ratio of 12.3 (95% CI 3.4-454, p=0.00002)], whereas a favorable, but non-significant trend was noted for pCR [hazard ratio of 0.2 (95% CI 0.0, 1.4, p=0.11)]. Conclusion: The identification of CTCs after NAC has prognostic significance beyond that of pCR, and should be considered in evaluation of patients for clinical trials of adjuvant therapies. Citation Format: Lucci A, Hall C, Hess K, Ravenberg E, Clayborn A, Mittendorf E, Rauch G, Candelaria R, Moulder S, Thompson A. Circulating tumor cells (CTCs) after neoadjuvant chemotherapy for triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-01.

Abstract P3-01-11: A novel six-parameter assay for comprehensive phenotyping of circulating tumor cells

Abstract Background. The presence and number of circulating tumor cells (CTCs) are prognostic for breast cancer treatment outcome. Direct imaging assays traditionally employ four markers to identify canonical epithelial CTCs: nucleus, exclusion (CD45), and inclusion (EpCAM and cytokeratin). There is intense interest in the ability to phenotype CTCs in order to provide a noninvasive means by which to predict treatment benefit from endocrine therapy and/or HER2-directed therapy in breast cancer. To address this, a 6-parameter assay for detection of ER and HER2 expression on CTCs was developed. We applied this assay to four well characterized breast cancer cell lines representative of various ER and HER2 phenotypes. Methods. BT474, MCF-7, SKBR3, or MDA-MB-231 cells were spiked into peripheral blood from healthy donors and processed using the AccuCyte® sample preparation system; nucleated cells, including CTCs, are captured onto glass slides (8 slides per 7.5 mL blood sample) for subsequent immunofluorescent staining. Slides were stained using the combined epithelial marker and ER/HER2 CTC assay and then analyzed with the CyteFinder® imaging system. CTCs were identified as nucleated cells with positive EpCAM and/or cytokeratin staining, and negative CD45 staining. ER and HER2 expression were assessed as present or absent. Results. All cell lines expressed both cytokeratin and EpCAM, except for MDA-MB-231 which was EpCAM-negative. The ER / HER2 expression patterns observed were consistent with reported phenotype: BT474 (+/+), MCF-7 (+/–), SKBR3 (–/+), and MDA-MB-231 (–/–). Conclusions. Identification of epithelial CTCs and phenotypic characterization of ER and HER2 status are feasible in a combined assay applied to a single blood sample. This approach has implications for efficiency and cost effectiveness, which are of particular importance given the interest in longitudinal testing. Assay evaluation is currently underway using blood samples from breast cancer patients with known receptor status, treatment history, and clinical outcomes. Results will be available for presentation at the meeting. Citation Format: Liu MC, Sun Y, Ramirez A, Campton D, George T, Haselkorn KE, Clein A, Gadi V, Sabath D, Kaldjian E. A novel six-parameter assay for comprehensive phenotyping of circulating tumor cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-11.

Immunomagnetic isolation of circulating melanoma cells and detection of PD-L1 status

Personalised medicine targeted to specific biomarkers such as BRAF and c-Kit has radically improved the success of melanoma therapy. More recently, further advances have been made using therapies targeting the immune response. In particular, therapies targeting the PD-1/PD-L1 or CTLA-4 axes alone or in combination have shown more sustained responses in 30–60% of patients. However, these therapies are associated with considerable toxicities and useful biomarkers to predict responders and non-responders are slow to emerge. Here we developed a reliable melanoma circulating tumor cell (CTC) detection method with PD-L1 evaluation on CTCs. A set of melanoma cell surface markers was tested as candidates for targeted melanoma CTC isolation and a melanoma specific immunostaining-based CTC identification protocol combined with PD-L1 detection was established. In vitro testing of the effect of exposure to blood cells on melanoma cell PD-L1 expression was undertaken. Immunomagnetic targeting isolated melanoma CTCs in up to 87.5% of stage IV melanoma patient blood samples and 3 8.6% of these had some PD-L1 expressing CTCs. Our in vitro data demonstrate PD-L1 induction on melanoma cells in the blood.This study established a robust, reliable method to isolate melanoma CTCs and detect expression of PD-L1 on these cells.

Colorectal Cancer CTCs Detection Using Two FCM Protocol Approaches.

The presence and quantification of circulating tumor cells (CTCs) could minimize the mortality among cancer patients by tailored, personalized, and targeted therapy and could also help in the field of investigation about new therapeutic targets. Identification of CTCs has been performed using molecular techniques and more advanced automated techniques such as CellSearch® and Amnis®. Our aim was to test the possibility of identifying CTCs in colorectal cancer patients using a lower cost and less complex flow cytometry-based method. Besides the CellSearch® system for CTCs enumeration and Amnis® Imaging Flow Cytometers, which are both commercially available, we tested and developed two FCM protocol approaches after immunomagnetic enrichment for CTCs enumeration in the blood of colorectal cancer patients. The CTCs numbers were assessed at baseline before anesthesia and the curative surgery and day one after the curative surgery. Blood from healthy donors was used as negative control. The research was performed by the Cyflow® Space cytometer (Partec, Münster, Germany). In the patient group, the enumeration using the direct protocol with a threshold 3 cells/mL, showed a mean of CTCs before surgery of 32, while after surgery it was 20, with sensitivity (SN) of 49.2% and specificity (SP) of 58.3%. On the other hand, using the intracellular protocol with a threshold of 1 cell/mL in patients' group, the mean of CTCs before surgery was 65 and after surgery it was 60, with a sensitivity (SN) of 62.7% and specificity (SP) of 70%. In the intracellular protocol the most significant correlation identified was for the expression of CKs with adenocarcinomas (r = 0.256, p = 0.044), with the existence of lymph node infiltration (r = 0.380, p = 0.008), and with the stage of the disease (r = 0.391, p = 0.003). For the direct protocol, considerable correlation was found with the samples of the right colon (r = 0.369, p = 0.002). Our findings demonstrate that we established two FCM low cost protocol approaches to detect and enumerate CTCs in colorectal cancer patients. In both FCM protocols (direct and intracellular) we observed a statistically significant increase (p ˂ 0.05) of CTCs number in the patient group. No statistical significance (p ˂ 0.05) of CTCs before and after surgery in patient group in both protocols was observed.

Heterogeneous expression of EPCAM in human circulating tumour cells from patient-derived xenografts

BackgroundWe aim to characterize the heterogeneous circulating tumour cells (CTCs) in peripheral blood, independently of physical or immunological purification, by using patient-derived xenografts (PDXs) models. CTC studies from blood generally rely on enrichment or purification. Conversely, we devised a method for the inclusive study of human cells from blood of PDX models, without pre-selection or enrichment.MethodsA qRT-PCR assay was developed to detect human and cancer-related transcripts from CTCs in PDXs. We quantified the EPCAM and keratins CTC markers, in a PDX cohort of breast cancer. The murine beta actin gene was used for normalization. Spearman’s rho coefficients were calculated for correlation.ResultsWe demonstrated, for the first time, that we can quantify the content of CTCs and the expression of human CTC markers in PDX blood using human-specific qRT-PCR. Our method holds strong potential for the study of CTC heterogeneity and for the identification of novel CTC markers.ConclusionsThe identification and the relative quantification of the diverse spectrum of CTCs in patients, irrespective of EPCAM or other currently used markers, will have a great impact on personalized medicine: unrestricted CTCs characterization will allow the early detection of metastases in cancer patients and the assessment of personalized therapies.

Detection of Circulating Tumor Cells in Fluorescence Microscopy Images Based on ANN Classifier

Circulating tumor cells (CTCs) is a clinical biomarker for cancer metastasis. CTCs are cells circulating in the body of patients by being separated from primary cancer and entering into blood vessel. CTCs spread every positions in the body, and this is one of the cause of cancer metastasis. To analyze them, pathologists get information about metastasis without invasive test. CTCs test is conducted by analyzing the blood sample from patient. The fluorescence microscope generates a large number of images per each sample, and images contain a lot of cells. There are only a few CTCs in images and cells often have blurry boundaries. So CTCs identification is not an easy work for pathologists. In this paper, we develop an automatic CTCs identification method in fluorescence microscopy images. This proposed method has three section. In the first approach, we conduct the cell segmentation in images by using filtering methods. Next, we compute feature values from each CTC candidate region. Finally, we identify CTCs using artificial neural network algorithm. We apply the proposed method to 5895 microscopy images (7 samplesas), and evaluate the effectiveness of our proposed method by using leave-one-out cross validation. We achieve the result of performance tests, a true positive rate is 92.57% and false positive rate is 9.156%.