Molecular Characterization Of Circulating Tumor Cells Research Articles

Abstract 3385: A pipeline for mRNA analysis on circulating tumor cells (CTCs) collected and stored in preservative tubes

Abstract Introduction: Circulating tumor cells (CTCs) are associated with prognosis in breast cancer. In addition to enumeration, there is great interest in the molecular characterization of CTCs. Nonetheless, this is technically challenging, especially with regards to RNA analysis, which requires immediate CTC processing to avoid RNA degradation. The purpose of this study was to evaluate the performance of the CellRescueTM preservative tubes in preserving RNA, and to optimize a method for mRNA expression analysis on CTCs up to 4 days after blood collection. Methods: Ten blood sample from one healthy donor (10 mL each) were collected into CellRescueTM tubes and spiked-in with 5 (n=4) or 500 (n=4) MCF7; two tubes were used for white blood cell (WBC) collection on day (d) 1, 2, 4, and 8 (4 mL used for each timepoint). The spiked-in samples were processed for CTC enrichment on d2 and d4 using CellSearch® (cs5d2, cs500d2, cs5d4, cs500d4) or Parsortix® (p5d2, p500d2, p5d4, p500d4). RNA was extracted, quantified, and subjected to reverse transcription to cDNA. Half cDNA from each sample was analyzed through TaqMan real-time PCR (in technical duplicates) for the detection of 3 transcripts of interest: ACTB (housekeeping), CD45 (WBC marker), and EPCAM (tumor cell marker). Considering the low amount of RNA, the second half of cDNA for each sample underwent preamplification of the 3 target transcripts, followed by real-time PCR (with technical triplicates) to evaluate whether this could increase sensitivity. Results: RNA quantification by Qubit was 11.6, 23, 9.9 and 4.8 ng/uL in WBC samples collected on d1, d2, d4 and d8, respectively. Thus, using CellRescueTM tubes, RNA was preserved up to 8 days, with a slight reduction in RNA quantity on d8. For spiked-in samples, RNA was below detection at any timepoint. Real-time PCR analysis of CD45 and ACTB in WBC samples confirmed a similar quantity of transcripts across all timepoints. For spiked-in samples processed with CellSearch®, CTCs were detected only after preamplification and when 500 MCF7 were present. For spiked-in samples processed with Parsortix®, 500 CTCs were detected both at d2 and d4, regardless of preamplification. Preamplification allowed for the detection of 5 CTCs as well, but only at d2. The spiked-in samples confirmed that by using CellRescueTM tubes, RNA is preserved up to 4 days, with slight transcript signal decrease between D2 and D4. Conclusions: With the developed pipeline, it is possible to detect 500 CTCs after blood storage for up to 4 days, and as low as 5 CTCs after 2-days storage. The enrichment methods can however affect the detection. More tests need to be performed to define the pipeline’s sensitivity using different enrichment methods and with different numbers of cells. Using this pipeline, up to a total of 100 gene expression assays of interest can be evaluated in a single sample. Citation Format: Eleonora Nicolo, Marwa Manai, Youbin Zhang, Laura Munoz Arcos, Strickland Kaylan, Paolo D'Amico, Huiping Liu, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. A pipeline for mRNA analysis on circulating tumor cells (CTCs) collected and stored in preservative tubes [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 3385.

The Plasticity of Circulating Tumor Cells in Ovarian Cancer During Platinum-containing Chemotherapy.

Circulating Tumor Cells (CTCs) are a potential source of metastases and relapses. The data on molecular characteristics of Ovarian Cancer (OC) CTCs are limited. This study aims to assess the TGFβ, CXCL2, VEGFA and ERCC1 expressions in two OC CTC subpopulations before and during chemotherapy (CT), and their relation to clinical characteristics. Two CTCs subpopulations (EpCAM+CK18+E-cadherin+; EpCAM+CK18+Vimentin+) were enriched using immunomagnetic separation before treatment and after 3 cycles of platinumcontaining CT. The expression of mRNA was assessed using RT-qPCR. The study included 31 I-IV stage OC patients. During CT, TGFβ levels increased in both fractions (p=0.054) compared with the initial levels. ERCC1 expression in E-cadherin+ CTCs was higher during neoadjuvant than adjuvant CT (p=0.004). CXCL2 level in E-cadherin+ CTCs increased (p=0.038) during neoadjuvant CT compared with the initial. TGF-β expression in vimentin+ CTCs during CT was negatively correlated to disease stage (p=0.003). Principal component analysis before CT revealed a component combining VEGFA, TGFβ, CXCL2, and a component with ERCC1 and VEGFA; during CT, component 1 contained ERCC1 and VEGFA, and component 2 - TGFβ and CXCL2 in both fractions. Increased ERCC1 expression in E-cadherin+ CTCs during CT was associated with decreased Progression-Free Survival (PFS) (HR 1.11 (95% CI 1.03-1.21, p=0.009) in multivariate analysis. EpCAM+ OC CTCs are phenotypically heterogeneous, which may reflect variability in their metastatic potential. CT changes the molecular characteristics of CTCs. Expression of TGFβ in EpCAM+ CTCs increases during CT. High ERCC1 expression in EpCAM+CK18+E-cadherin+ CTCs during CT is associated with decreased PFS in OC.

Systemic immune responses are associated with molecular characteristics of circulating tumor cells in head and neck squamous cell carcinoma.

Systemic immunity mediated by circulating immune cells may affect clinical features, as well as the characteristics of circulating tumor cells (CTCs) in patients with head and neck squamous cell carcinoma (HNSCC). The present study aimed to analyze the influence of circulating immune cells, using their markers, on clinical features to investigate the association between systemic immunity and the molecular characteristics of CTCs. Circulating immune-cell markers were associated with disease progression and clinical outcomes in patients with HNSCC. Meanwhile, there was no significant association between the presence of CTCs and systemic immune-related markers. Moreover, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit a expression in CTCs was significantly associated with higher lymphocyte counts (P=0.035) and an increased prognostic nutrition index (P=0.0157). Patients with CTCs expressing CD47 exhibited significantly higher neutrophil (P=0.0031) and monocyte (P=0.0016) counts. Patients with CTCs expressing programmed cell death 1 ligand 2 exhibited lower C-reactive protein (CRP) levels (P=0.0271) and a decreased CRP/albumin ratio (P=0.0207). The current results suggested that the interaction between CTCs and circulating immune cells may provide survival advantages via molecular alterations to CTCs.

Abstract PS2-11: Ctc-her2+ a novel subset in stage IVaggressive: Molecular correlations, outcome and clinical characteristics in metastatic breast cancer

Abstract Background The enumeration of Circulating Tumor Cells (CTCs) in the peripheral blood of patients (pts) with metastatic breast cancer (MBC) showed prognostic value independent of clinic-pathological characteristics and proposed as a new tool for Stage IV stratification. Molecular characterization of CTCs with analysis of standard HER2 and estrogen-receptor (ER) status provides additional predictive value. The presence of HER2 positive CTCs (CTCs-HER2+) has been documented in pts with HER2 negative MBC and suggestive of tumor heterogeneity. The molecular features associated with the detection of these cells and the clinical significance of CTCs-HER2+ remains of unclear clinical significance. The lack of validated prospective trials limits the use of this immunophenotypic characterization as a prognostic and predictive factor. We conducted an analysis of a large cohort of patients with MBC evaluated for CTCs enumeration (CTCs+), molecular analysis of CTCs (CTCs-HER2+) and ctDNA. Methods The IRB-approved study prospectively analyzed blood samples of pts with MBC enrolled before starting a new line of therapy. Samples were collected from pts treated at Northwestern University (Chicago, IL) between 2016 and 2020. ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health) with a specific focus on the detection of ERBB2 mutations. CTCs were enumerated through CellSearch™ (Menarini Silicon Biosystems, Bologna, Italy), and characterized for HER2 expression using the CellSearch CXC Kit. HER2+ CTCs were divided in 4 different categories (0,1+,2+,3+), leaning on fluorescence intensity, as previously reported by Riethdorf et al. Pts with <5 CTCs (stage IV indolent ) were excluded from the analysis. The cHER2 ratio, defined as the sum of 2+ CTCs and 3+ CTCs divided by the total number of HER2+ CTCs was used to split the remaining pts in 2 different subgroups: cHER2 ratio> 0.75 (cHER2high), cHER2 ratio<0.75 (cHER2low). Pts' characteristics and genomic alterations were compared between these two subgroups and a long-rank test was performed to analyze the outcome (progression-free survival, PFS). Results A total of 247 pts with MBC about to start new therapy were enrolled. 136 patients (55%) demonstrated CTCs count ≥ 5 (stage IV aggressive). Of these, 111 pts had a blood sample bearing HER2+ CTCs. 88 pts were classified as cHER2low, while 32 were allocated in the second subgroup (cHER2high). The clinic subtype distribution of the two groups was as follows: 65% ER+, 19% HER2+ and 16% TNBC among cHER2low, compared to 55% ER+, 38% HER2+ and 7% TNBC among cHER2high pts. The HER2+ subtype was significantly more represented in the cHER2high group (p<0.05), along with ERBB2 mutations (p<0.05). We observed a total of 89 events (progression to treatment), 67 and 22 respectively. The median PFS was 87 days in the cHER2low and 209 days among cHER2high pts (log Rank test, p<0.001). Conclusion This study shows a significant association between the cHER2 ratio and outcome in pts with Stage IV aggressive disease, suggesting a potential predictive/prognostic role of this biomarker among MBC pts bearing CTC-HER2+. cHER2high pts, in which blood was collected a prevalent number of CTC-HER2+ 2+ and 3+, were more likely to have a HER2 FISH amplification by tissue biopsy and ERBB2 gene alteration by ctDNA. These findings, taken together, suggest a potential role of these subsets of CTC-HER2+ in the characterization of the HER2+ metastatic disease. The cHER2low is intrinsically characterized by a higher percentage of CTC-HER2+ 1+. This CTC-HER2+ subset of cells is commonly detected among all subtypes but, more frequently in ER+ disease and their significance remains unclear. Exploring the underlying biological nature of the CTC-HER2+ 1+ could allow to better understand the role these cells in the natural evolution of the MBC. Citation Format: Paolo D'Amico, Qiang Zhang, Lorenzo Gerratana, Youbin Zhang, Ami Shah, Carolina Reduzzi, Andrew Davis, Saya Jacob, Firas Wehbe, Amir Behdad, Giuseppe Curigliano, Massimo Cristofanilli. Ctc-her2+ a novel subset in stage IVaggressive: Molecular correlations, outcome and clinical characteristics in metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-11.

MiR-146a Regulates Migration and Invasion by Targeting NRP2 in Circulating-Tumor Cell Mimicking Suspension Cells

Cancer metastasis is the primary cause of cancer-related death and metastatic cancer has circulating-tumor cells (CTCs), which circulate in the bloodstream before invading other organs. Thus, understanding the precise role of CTCs may provide new insights into the metastasis process and reduce cancer mortality. However, the molecular characteristics of CTCs are not well understood due to a lack of number of CTCs. Therefore, suspension cells were generated from MDA-MB-468 cells to mimic CTCs, and we investigate the microRNA (miRNA)-dependent molecular networks and their role in suspension cells. Here, we present an integrated analysis of mRNA and miRNA sequencing data for suspension cell lines, through comparison with adherent cells. Among the differentially regulated miRNA–mRNAs axes, we focus on the miR-146a-Neuropilin2 (NRP2) axis, which is known to influence tumor aggressiveness. We show that miR-146a directly regulates NRP2 expression and inhibits Semaphorin3C (SEMA3C) signaling. Functional studies reveal that miR-146a represses SEMA3C-induced invasion and proliferation by targeting NRP2. Finally, high-NRP2 is shown to be associated with poor outcomes in breast cancer patients. This study identifies the key role of the miR-146a–NRP2 signaling axis that is critical for the regulation of migration and invasion in CTC-mimicking cells.

Clinical utility of circulating tumor cells: an update.

The prognostic role of circulating tumor cells (CTCs) has been clearly demonstrated in many types of cancer. However, their roles in diagnostic and treatment strategies remain to be defined. In this review, we present an overview of the current clinical validity of CTCs in nonmetastatic and metastatic cancer, and the main studies or concepts investigating the clinical utility of CTCs. In particular, we focus on breast, lung, colorectal, and prostate cancer. Two major topics concerning the clinical utility of CTC are discussed: treatment based on CTC count or CTC variations, and treatment based on the molecular characteristics of CTCs. Although some of these studies are inconclusive, many are still ongoing, and their results could help to define the role of CTCs in the management of cancers. A summary of published or ongoing phase II‐III trials is also presented.

Development of High Throughput Single-Cell Analysis System for Circulating Tumor Cells Based on Digital Micromirror Device

Circulating tumor cells (CTCs) are widely known as useful biomarkers in the liquid biopsies of cancer patients. Recently, in addition to counting the number of CTCs, genetic analysis of CTCs provides critical insights into cancer metastases and add detailed clinical information that enhances patient care. It is widely known that CTCs are genetically heterogeneous, the molecular characterization of CTCs should be performed at the single-cell level. In fact, several studies have identified inter- and intra-patient heterogeneity in the mutational status of CTCs in metastatic breast and lung cancers. These studies also reported that CTCs related to drug resistance are novel therapeutic targets. Thus, genetic analysis of single CTCs has the potential to be widely applicable in personalized medicine and drug discovery. However, CTCs are extremely rare cells that only 1-100 cells are contained in 1 ml of blood, so the recovery of such extremely rare CTCs from the peripheral blood is technically challenging. So far, we have developed a CTC recovery system using a microcavity array (MCA), which demonstrates highly efficient recovery of cancer cells based on differences in cell size and deformability. Furthermore, we have proposed a novel cell manipulation method through the visualization of single cells through hydrogel encapsulation for subsequent genetic analysis of single CTCs. Subsequently, recovered CTCs can be subjected to a hydrogel-encapsulation for the following genetic analysis of single cells. Clinical studies indicated that the genetic analysis of CTCs from lung, pancreatic and gastric cancer patients was successfully achieved. However, this hydrogel encapsulation has limitations in throughput. In this study, we demonstrate a high-throughput single-cell analysis for CTCs using a multiple single-cell encapsulation system with a digital micromirror device (DMD). The novel multiple single-cell encapsulation systemwas composed of two optical systems: the wide-field fluorescence imaging system and the photopolymerization system equipped with DMD. The wide-field fluorescence imaging system using a CMOS sensor was designed to visualize 2D fluorescence imaging of the entire MCA (6.0 × 6.0 mm2). We evaluated the sensitivity of fluorescence detection of the wide-field fluorescence imaging system using the calibration slide. The limit of detection of the wide-field fluorescence imaging system was 2.1 × 102 molecules-Cy3/μm2, which is comparable to conventional fluorescence microscopy (1.0 × 102 molecules-Cy3/μm2). Generally, the expression of cellular marker protein, such as cytokeratin, was estimated to be 1.3 × 103 molecules/μm2. Therefore, our proposed system had enough sensitivity for the detection of antibody-stained CTCs. Furthermore, this wide-field fluorescence imaging system for CTC detection enables the rapid visualization of all stained cells within the field of the MCA via one-shot imaging. This rapid detection method also allows for the rapid completion of single-CTC manipulation. The photopolymerization system for single-cell encapsulation was controlled by the DMD. The curing light (λmax = 365 nm) modulated by the DMD was projected onto an MCA for hydrogel generation, and single-cell isolation by hydrogel-photopolymerization was examined at the optimized conditions. After recovery of cancer cells from whole blood onto MCA and staining by the fluorescent-labeled antibodies, cancer cells were identified by the wide-field imaging system. Then light irradiation (3 sec) was performed by the photopolymerization system. To minimize the area of the hydrogel reserved for a single cell, we newly designed the convex-type hydrogel. In the spike-in experiment, single-cell isolation rates were 97.6% (41/42) for NCI-H1975 cells (non-small cell lung cancer cell) and 94.4% (51/54) for NCI-N87 (gastric cancer cell) that were comparable with our previous study using fluorescence microscopy. With this proposed system, more than 50 single cells were encapsulated simultaneously within 1 min and isolated with an efficiency. Furthermore, single cells were successfully isolated from adjacent cells on the same microcavity without any contamination. Single cancer cell was partially encapsulated on the hydrogel, and the partial encapsulation allows us to subject them to whole genome amplification (WGA). Light irradiation and photopolymerizaion did not affect the quality of the amplified WGA products for single-cell genomic analyses. We are currently investigating single cell genetic analysis of metastatic cancer patients, and this developed multiple single-cell encapsulation system will improve the high throughput single-cell genetic analysis of CTCs in clinical samples. Our system provides an attractive application for other targets such as adherent cells, tissue samples, and microorganisms, in addition to widespread use in the isolation of CTCs for liquid biopsy.

Pancreatic circulating tumor cell profiling identifies LIN28B as a metastasis driver and drug target

Pancreatic ductal adenocarcinoma (PDAC) lethality is due to metastatic dissemination. Characterization of rare, heterogeneous circulating tumor cells (CTCs) can provide insight into metastasis and guide development of novel therapies. Using the CTC-iChip to purify CTCs from PDAC patients for RNA-seq characterization, we identify three major correlated gene sets, with stemness genes LIN28B/KLF4, WNT5A, and LGALS3 enriched in each correlated gene set; only LIN28B CTC expression was prognostic. CRISPR knockout of LIN28B—an oncofetal RNA-binding protein exerting diverse effects via negative regulation of let-7 miRNAs and other RNA targets—in cell and animal models confers a less aggressive/metastatic phenotype. This correlates with de-repression of let-7 miRNAs and is mimicked by silencing of downstream let-7 target HMGA2 or chemical inhibition of LIN28B/let-7 binding. Molecular characterization of CTCs provides a unique opportunity to correlated gene set metastatic profiles, identify drivers of dissemination, and develop therapies targeting the “seeds” of metastasis.

Abstract IA09: Beyond counting: Imaging flow cytometry-based detection of therapeutic molecular targets in circulating tumor cells

Abstract Detection of circulating tumor cells (CTCs) has been established as a blood-based biomarker of poor prognosis in most types of epithelial cancer. The observation of increased CTC detection rates in patients with metastatic disease supported the hypothesis that CTCs might represent the precursor cells of distant metastases. Experimental proof for this hypothesis was provided by studies in experimental mouse models in which single metastasis-initiating cells within the total pool of CTCs could be causally linked to the formation of distant metastases. In addition, a potential of CTCs of reseeding at the primary site has been reported, suggesting that CTCs could also play a role in local tumor recurrence. Enumeration and molecular characterization of CTCs might thus be clinically exploited for improving the clinical management of epithelial cancers both in the locally advanced and recurrent/metastatic disease setting. Numerous studies reported on the presence of CTCs in peripheral blood samples of head and neck squamous cell carcinoma (HNSCC) patients. Significant associations with clinical high-risk parameters such as advanced stage, high tumor burden, nodal disease, and distant metastasis were reported. Accordingly, CTC detection was correlated with increased risk of local and/or distant recurrence, progression-free and overall survival. Current data in HNSCC are not yet robust enough for the implementation of CTC detection in clinical routine. In addition, the mere enumeration of CTCs has proven insufficiently informative to prompt widespread clinical adoption. There is accumulating evidence that more extended phenotyping of CTCs might be necessary for improving their diagnostic value. Indeed, despite the estimated daily shedding of millions of tumor cells from the bulk tumor tissue, only a small subpopulation of CTCs is able to reach and survive in the blood circulation and to contribute to the formation of distant metastasis. Identification of the true metastasis-inducing subclones within the bulk CTC population remains a challenge but is imperative in order to improve the diagnostic potential of CTCs. In addition, improved knowledge of the relevant biologic mechanisms endowing CTCs with the potential to emigrate from the primary site to blood circulation, to survive their journey, and to reseed at distant organs will support the development of novel antimetastatic therapies. Several technologies are currently available that allow multiparametric CTC phenotyping at the single-cell level. These platforms include imaging flow cytometry (IFC), by which multispectral images from a large number of cells acquired in suspension can be analyzed. By combining the features of flow cytometry and fluorescent microscopy, information on the cell morphology together with the extent of target protein expression and its subcellular localization can be collected. This provides a unique opportunity for statistical analysis of large populations, but also helps in avoiding artifacts. In the talk, the evidence of a prognostic role of CTCs in HNSCc will be summarized. Results from recent studies reporting on multiparametric CTC phenotyping at the single-cell level will be presented. Examples of CTC-based biomarkers that could be exploited for the optimization of current molecular treatment strategies in HNSCC will be discussed. Citation Format: Ingeborg Tinhofer. Beyond counting: Imaging flow cytometry-based detection of therapeutic molecular targets in circulating tumor cells [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA09.

Circulating tumor cells in breast cancer.

Breast cancer is the most recurrent cancer in female population, its mortality is related to the presence of distant metastases. Distant metastasis begins as a small group of cells that spread regionally and remotely from the site of primary origin. Circulating tumor cells (CTC) are present in the blood of cancer patients and are therefore considered disease markers and precursors of metastasis. It is trascendental the review of the molecular aspects of the metastatic process, the available techniques for the enrichment, identification and molecular characterization of CTC, as well as some examples of its usefulness and clinical application in patients with breast cancer, including aspects related to perioperative management during breast cancer surgery.

Direct comparison of size-dependent versus EpCAM-dependent CTC enrichment at the gene expression and DNA methylation level in head and neck squamous cell carcinoma

We directly compared two different approaches used for Circulating Tumor Cell (CTC) isolation, a size-dependent microfluidic system versus an EpCAM-dependent positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in Head and Neck Squamous Cell Carcinoma (HNSCC). A size-dependent microfluidic device (Parsortix, ANGLE) and an EpCAM-dependent positive immune-magnetic isolation procedure were applied in parallel, using 10 mL PB from 50 HNSCC patients and 18 healthy donors. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M (reference gene). Real time methylation specific PCR (MSP) was used to study the methylation status of RASSF1A and MLL3 genes. In identical blood draws, the label-free size-dependent CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-dependent CTC enrichment, since a significantly higher percentage of identical PB samples was found positive at the gene expression and DNA methylation level, while the specificity was not affected. Our results indicate that future studies focused on the evaluation of clinical utility of CTC molecular characterization in HNSCC should be based on size-dependent enrichment approaches.

Abstract OT3-18-01: Detect III/IV study trial - The multicenter study program in patients with HER2-negative MBC and circulating tumor cells

Abstract Background: The collaborative DETECT study program represents a large program on metastatic breast cancer (MBC) worldwide. The main objective of the DETECT trial is to evaluate the efficacy of individualized breast cancer treatment based on the presence and phenotype setting of circulating tumor cells (CTCs). Thus, the DETECT study program is one of the first clinical trials translating the role of CTC enumeration/phenotyping directly into treatment intervention within different breast cancer subtypes. Trial design: The DETECT-III trial is a multicenter, randomized, phase III study comparing standard therapy alone versus standard therapy plus lapatinib in patients with initially HER2-negative MBC and HER2-positive CTCs. Patients with HER2-negative MBC and only HER2-negative CTCs can be included within the DETECT-IV trial, a prospective, multicenter, open-label, phase II study including patients with HER2-negative MBC. Within the DETECT-IV study setting postmenopausal patients with hormone-receptor positive MBC are treated with the CDK 4/6 inhibitor ribociclib and endocrine therapy, while women with triple negative MBC or a hormone-receptor positive tumor and indication for chemotherapy will receive eribulin. Specific aims: The DETECT study program offers various up-to-date treatment options, generating a wealth of clinical data including long-term follow-up data, evaluated in the controlled setting of a single large clinical trial program. The primary endpoint of the DETECT III trial is the comparison of efficacy as assessed using CTC clearance rate between patients receiving standard anticancer therapy with lapatinib and patients receiving standard anticancer therapy alone. The secondary objective of this trial is to assess the level of compliance to study procedures comparing the efficacy of lapatinib between given treatment groups. The primary objectives of the DETECT- IV trial are to evaluate CTC clearance rate for the ribociclib cohort and progression-free survival (defined as time interval from date of recruitment until progressive disease) for the eribulin cohort. The main focal point of the extensive collaborative translational oncology research projects is to apply innovative biomarkers and assays focusing on molecular characteristics of CTCs. This “biological status” of CTCs may provide new information about their potential function in terms of a repeatedly performed liquid biopsy to assess the patients’ response to therapy. Citation Format: Sabrina Krause, Thomas WP Friedl, Peter A Fasching, Andreas Schneeweiss, Volkmar Müller, Sabine Riethdorf, Klaus Pantel, Florin-Andrei Taran, Arkadius Polasik, Marie Tzschaschel, Amelie de Gregorio, Franziska Meier-Stiegen, Jens Huober, Wolfgang Janni, Tanja Fehm. Detect III/IV study trial - The multicenter study program in patients with HER2-negative MBC and circulating tumor cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT3-18-01.

Looking for a Better Characterization of Triple-Negative Breast Cancer by Means of Circulating Tumor Cells.

Traditionally, studies to address the characterization of mechanisms promoting tumor aggressiveness and progression have been focused only on primary tumor analyses, which could provide relevant information but have limitations to really characterize the more aggressive tumor population. To overcome these limitations, circulating tumor cells (CTCs) represent a noninvasive and valuable tool for real-time profiling of disseminated tumor cells. Therefore, the aim of the present study was to explore the value of CTC enumeration and characterization to identify markers associated with the outcome and the aggressiveness of triple-negative breast cancer (TNBC). For that aim, the CTC population from 32 patients diagnosed with TNBC was isolated and characterized. This population showed important cell plasticity in terms of expression of epithelia/mesenchymal and stemness markers, suggesting the relevance of epithelial to mesenchymal transition (EMT) intermediate phenotypes for efficient tumor dissemination. Importantly, the CTC signature demonstrated prognostic value to predict the patients’ outcome and pointed to a relevant role of tissue inhibitor of metalloproteinases 1 (TIMP1) and androgen receptor (AR) for TNBC biology. Furthermore, we also analyzed the usefulness of the AR and TIMP1 blockade to target TNBC proliferation and dissemination using in vitro and in vivo zebra fish and mouse models. Overall, the molecular characterization of CTCs from advanced TNBC patients identifies highly specific biomarkers with potential applicability as noninvasive prognostic markers and reinforced the value of TIMP1 and AR as potential therapeutic targets to tackle the most aggressive breast cancer.

Antibody-Free Hydrogel with the Synergistic Effect of Cell Imprinting and Boronate Affinity: Toward the Selective Capture and Release of Undamaged Circulating Tumor Cells.

The selective and highly efficient capture of circulating tumor cells (CTCs) from blood and their subsequent release without damage are very important for the early diagnosis of tumors and for understanding the mechanism of metastasis. Herein, a universal strategy is proposed for the fabrication of an antibody-free hydrogel that has a synergistic effect by featuring microinterfaces obtained by cell imprinting and molecular recognition conferred by boronate affinity. With this artificial antibody, highly efficient capture of human hepatocarcinoma SMMC-7721 cells is achieved: as many as 90.3 ± 1.4% (n = 3) cells are captured when 1 × 105 SMMC-7721 cells are incubated on a 4.5 cm2 hydrogel, and 99% of these captured cells are subsequently released without any loss of proliferation ability. In the presence of 1000 times as many nontarget cells, namely, leukaemia Jurkat cells, the SMMC-7721 cells can be captured with an enrichment factor as high as 13.5 ± 3.2 (n = 3), demonstrating the superior selectivity of the artificial antibody for the capture of the targeted CTCs. Most importantly, the SMMC-7721 cells can be successfully captured even when spiked into whole blood, indicating the great promise of this approach for the further molecular characterization of CTCs.

Epigenetics of Circulating Tumor Cells in Breast Cancer.

Liquid biopsy based on the analysis of circulating tumor cells (CTCs) has emerged as an important field of research. Molecular characterization of CTCs can provide insights into cancer biology and biomarkers for the clinic, representing a non-invasive powerful tool for monitoring breast cancer metastasis and predict the therapeutic response. Epigenetic mechanisms play a key role in the control of gene expression and their alteration contributes to cancer development and progression. These epigenetic modifications in CTCs have been described mainly related to modifications of the DNA methylation pattern and changes in the expression profile of noncoding RNAs. Here we summarize the recent findings on the epigenetic characterization of CTCs in breast cancer and their clinical value as tumor biomarkers, and discuss challenges and opportunities in this field.

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 2241: Molecular characterization of circulating tumor cells in head and neck squamous cell carcinoma: Direct comparison of a label-independent size-based microfluidic device with EpCAM-based CTC enrichment

Abstract Background: Circulating tumor cells (CTCs) heterogeneity is highly affecting the efficiency of their isolation and thus the reliability of downstream analysis. Especially in Head and Neck Squamous Cell Carcinoma (HNSCC) epithelial mesenchymal transition (EMT) is highly affecting CTC isolation and downstream analysis. We directly compared two different approaches used for CTC isolation, a label-independent size-based microfluidic-based system versus an EpCAM-based positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in HNSCC. Methods: Peripheral blood (PB) in EDTA (20mL) was collected from 50 HNSCC patients and 18 healthy donors (HD). A size-based microfluidic device (Parsortix, ANGLE) and an EpCAM-based positive immune-magnetic isolation procedure were applied in parallel, using 10mL PB in each case. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M. Real time methylation specific PCR (MSP) was used to study the methylation status of SOX17, RASSF1A and MLL3 genes in DNAs isolated from the same enriched CTCs. Results: In identical blood draws, the label-free size-based CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-based CTC enrichment, since a significantly higher percentage of identical PB samples was found positive for all genes tested both at the gene expression and DNA methylation level, while the specificity was not affected. Conclusions: In HNSCC CTC molecular characterization at the gene expression and DNA methylation level should be based on a label-free size-based isolation system. Citation Format: Martha Zavridou, Sophia Mastoraki, Areti D. Strati, George Koutsodontis, Apostolos Klinakis, Amanda Psyrri, Evi S. Lianidou. Molecular characterization of circulating tumor cells in head and neck squamous cell carcinoma: Direct comparison of a label-independent size-based microfluidic device with EpCAM-based CTC enrichment [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 2241.

Abstract 427: Detection and molecular characterization of circulating tumor cells in advanced head and neck squamous cell carcinoma

Abstract Malignant properties of tumor cells are genetically and epigenetically altered during tumor development and progression. Moreover, tumor cells rapidly change to treatment resistant phenotype by various therapeutic pressures, and not only become more heterogeneous but also possess metastatic potential. Detection and characterization of circulating tumor cells (CTCs) in the bloodstream of cancer patients have been currently remarked as a valuable tool for real-time evaluation of tumor tissue with phenotypic heterogeneity in the diagnosis and treatment of various cancers, however these have been still challenging. In the present study, we investigated the presence of CTCs and their molecular characterization by the combination of microfiltration system and RT-qPCR method. Peripheral blood samples were collected from 30 patients with advanced head and neck squamous cell carcinoma (HNSCC) and passed through two CellSieveTM microfilters using a low-pressure filtration system. The existence of CTCs collected from the first filter was determined by the expression of epithelial-related genes (KRT19, EPCAM, EGFR, or MET), then the expression of cell proliferation (PIK3CA and CCND1)-, EMT (SNAI1 and VIM)-, cancer stemness (CD44, NANOG, and ALDH1A1)-, and immune regulation (CD47, CD274, and PDCD1LG2)-related genes was analyzed for molecular characterization of CTCs. The gene expression in cells collected from the second filter was used for normalization of target genes. Sixteen (53.3%) of 30 patients were positive for at least one epithelial-related gene. Interestingly, the number of patients with locoregional recurrence or distant metastasis in 16 CTC-positive patients was 7 (43.8%) or 5 (31.3%); whereas that in 14 CTC-negative patients was 2 (14.3%) or 1 (7.1%), respectively. The gene expression profile in CTCs collected from 16 CTC-positive patients has revealed remarkable heterogeneity between HNSCC patients. Of 16 CTC-positive patients, PIK3CA was overexpressed in 56.3%, CCND1 in 43.8%, SNAI1 in 62.5%, VIM in 43.8%, CD44, in 50.0%, NANOG in 31.3%, ALDH1A1 in 56.3%, CD47 in 62.5%, CD274 in 37.5%, and PDCD1LG2 in 31.3%. Our results suggest that CTCs may play an important role in locoregional recurrence as well as distant metastasis in HNSCC. Further molecular profiling at the single-cell level of CTCs is needed to clarify the clinical significance of molecular phenotype of CTCs. Citation Format: Hiroe Tada, Hideyuki Takahashi, Hiroki Ishii, Yuki Kuwabara-Yokobori, Masato Shino, Kazuaki Chikamatsu. Detection and molecular characterization of circulating tumor cells in advanced head and neck squamous cell carcinoma [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 427.

Abstract OT1-10-01: DETECT III/IV study trial – The multicenter study program in patients with HER2-negative metastatic breast cancer and circulating tumor cells

Abstract Background: The collaborative DETECT study program represents one of the largest study program on metastatic breast cancer worldwide. The main objective of the DETECT trial is to evaluate the efficacy of individualized breast cancer treatment based on the presence and phenotype setting of circulating tumor cells (CTCs). Thus, the DETECT study program is one of the first clinical trial translating the role of CTC enumeration/phenotyping directly into treatment intervention within different breast cancer subtypes. Trial design: The DETECT III trial is a multicenter, randomized, phase III study to compare standard therapy alone versus standard therapy plus lapatinib in patients with initially HER2-negative metastatic breast cancer and HER2-positive circulating tumor cells. Patients with persisting HER2-negative circulating tumor cells (CTCs) can be included within the DETECT IV trial, a prospective, multicenter, open-label, phase II study including patients with HER2-negative metastatic breast cancer. Within the DETECT IV study setting postmenopausal patients with hormone-receptor positive metastatic breast cancer are treated with everolimus or ribociclib and endocrine therapy, while women with triple negative metastatic breast cancer or a hormone-receptor positive tumor and indication for chemotherapy receive eribulin. Specific aims: The DETECT study program comprises all breast cancer subtypes and therefore offers various up-to-date treatment options, generating a wealth of clinical data including long-term follow-up data, evaluated in a controlled setting of a single large clinical trial. The primary endpoint of the DETECT III trial is the comparison of patients receiving standard anticancer therapy with lapatinib and patients receiving standard anticancer therapy alone, with regard to the CTC clearance rate. The secondary objective of this trial is to assess the level of compliance to study procedures comparing the efficacy of lapatinib between given treatment groups (Progression free survival, overall response rate and dynamic of CTCs). Primary objective of the DETECT IV trial is to evaluate CTC clearance rate within the everolimus/ribociclib cohort and additionally assess progression-free survival defined as time interval from date of recruitment until progressive disease within the eribulin cohort. The main focal point of the extensive collaborative translational oncology research projects is to apply innovative biomarkers and assays focusing on molecular characteristics of CTCs. This “biological status” may give new information about CTCs potential function as liquid biopsy in order to determine their relevance for therapy prediction. Citation Format: Krause S, Friedl T, Romashova T, Fasching PA, Schneeweiss A, Müller V, Taran F-A, Arkadius P, Marie T, De Gregorio A, Meier-Stiegen F, Huober J, Janni W, Fehm T. DETECT III/IV study trial – The multicenter study program in patients with HER2-negative metastatic breast cancer and 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 OT1-10-01.

The Isolation and Characterization of Circulating Tumor Cells from Head and Neck Cancer Patient Blood Samples Using Spiral Microfluidic Technology.

Metastasis is responsible for 90% of cancer-related deaths. The study of circulating tumor cells (CTCs) enables the study of the units of disease responsible for the process of metastasis. While the biology of the primary tissue is relatively known, little is understood about the cells en route to distant sites. Here we describe the isolation of CTCs using the spiral microfluidic technology for the efficient sorting of CTCs from head and neck cancer (HNC) patient blood samples. Furthermore, the molecular characterization of CTCs can aid in stratifying patients for targeted therapy such as immunotherapy, which is having a profound impact in the treatment of metastatic HNC.