Circulating Tumor Cells Population Research Articles

Two-color diffuse in vivo flow cytometer.

Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse in vivo flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal. The goal of this work was to develop and validate a two-color DiFC system capable of non-invasively detecting circulating cells expressing two distinct FPs. A DiFC instrument was designed and built to detect cells expressing either green FP (GFP) or tdTomato. We tested the instrument in tissue-mimicking flow phantoms in vitro and in multiple myeloma bearing mice in vivo. In phantoms, we could accurately differentiate GFP+ and tdTomato+ CTCs and CTCCs. In tumor-bearing mice, CTC numbers expressing both FPs increased during disease. Most CTCCs (86.5%) expressed single FPs with the remainder both FPs. These data were supported by whole-body hyperspectral fluorescence cryo-imaging of the mice. We showed that two-color DiFC can detect two populations of CTCs and CTCCs concurrently. This instrument could allow study of tumor development and response to therapies for different sub-populations in the same animal.

Abstract 5864: YAP1 defines a highly plastic, persister cell population in small cell lung cancer (SCLC) following relapse

Abstract SCLC is an aggressive, neuroendocrine malignancy notable for rapid, albeit transient, responses to therapy. We have previously shown that transcriptional heterogeneity among treatment-naïve SCLC tumors underlies four distinct transcriptional subtypes, each with distinct clinical vulnerabilities. Though previously hypothesized to delineate a distinct subtype, expression of YAP1 is largely absent from treatment-naïve SCLC. Less clearly defined is the transcriptional landscape of relapsed SCLC, the evolution of which may underlie SCLC’s capacity for insurmountable resistance. To better define the biology of relapsed SCLC, we performed mass cytometry analysis of patient circulating tumor cells (CTCs), immunohistochemistry for ASCL1, NEUROD1, POU2F3, MYC and YAP1, and single-cell (sc)RNAseq of core needle biopsies from SCLC patients and preclinical models following resistance to standard-of-care therapies. In contrast to treatment-naïve SCLC, YAP1 mRNA and protein are detectable in patient CTC populations and tumors from relapsed patients and PDXs, albeit at levels lower than subtype-defining transcription factors (e.g. ASCL1, NEUROD1, or POU2F3). Notably, however, scRNAseq reveals these YAP1 expressing cells to be mutually exclusive with ASCL1 and NEUROD1, though not necessarily POU2F3. YAP1-expressing cells possess unique mesenchymal, inflamed features and exhibit high levels of transcriptional plasticity. Consistent with the upregulation of YAP1 following chemotherapy, YAP1-positive cell populations express senescence associated secretory phenotype genes and cancer stem cell genes, suggesting that these populations represent drug tolerant persister cells that may serve as a source for renewal of classical neuroendocrine populations within the tumor. Targeting these resistant populations may be critical to re-establish response in relapsed SCLC, and these analyses identified high expression of several surface proteins that are currently under investigation as targets for antibody-drug conjugates in SCLC clinical trials, including B7H3, TROP2 and HER2 antibody drug conjugates. These data suggest that the acquisition of YAP1 expression delineates a particularly recalcitrant tumor cell population observed almost exclusively in relapsed SCLC that is capable of driving tumor persistence and treatment resistance. Clinically, these data underscore the importance of constraining transcriptional plasticity in the frontline management of SCLC, but also of developing therapies aimed specifically at emergent populations capable of driving resistance, as with the YAP1-high cells identified here. The emergence of YAP1 following resistance in SCLC and the observation of YAP1 as a common feature of other high-grade neuroendocrine carcinomas exclusive of SCLC, likely explains the initial mischaracterization of YAP1 as a subtype-defining feature of SCLC. Citation Format: C. Allison Stewart, Sarah M. Groves, Runsheng Wang, Kavya Ramkumar, Yuanxin Xi, Lixia Diao, Alejandra G. Serrano, Azusa Tanimoto, Ashley M. Victorian, Sayantan Bhattacharyya, Alexa J. Halliday, Neda Kalhor, Luisa Maren Soto, Pedro Filipe Da Rocha, Natalie Vokes, Loukia G. Karacosta, Jing Wang, John V. Heymach, Vito Quaranta, Bonnie Glisson, Lauren Averett Byers, Carl M. Gay. YAP1 defines a highly plastic, persister cell population in small cell lung cancer (SCLC) following relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5864.

Abstract 7504: Longitudinal multi-omics characterization of circulating tumor cells from ALK positive NSCLC patients

Abstract Introduction: Anaplastic lymphoma kinase positive non-small cell lung cancer (ALK+NSCLC) represents a distinct subtype of NSCLC characterized by oncogenic ALK gene mutations leading to ALK overexpression and activation in tumor cells. ALK+ NSCLC will frequently develop mutations during treatment, leading to nonresponse and eventually cancer relapse. Therefore, there is a critical need for sensitive technologies to monitor ALK+ NSCLC disease status in patients over time. To facilitate longitudinal monitoring, there has been a rapid evolution in diagnostic technologies leveraging circulating tumor cells (CTCs) as a liquid biopsy tool. CTCs can be collected non-invasively at various intervals throughout patient treatment. Moreover, they shed from solid tumors and disseminate into the bloodstream, which makes isolating CTCs a valuable source of genomic, transcriptomic, and proteomic insights pertaining to the primary tumor. In this study, we present a comprehensive workflow for conducting multi-omic analysis on CTCs derived from ALK+ NSCLC patients. Methods: Blood samples were collected from a cohort of ALK+ NSCLC patients, and CTCs were isolated using a high throughput, label free microfluidic device, Labyrinth. Enriched CTCs were immunofluorescently stained for cytokeratin, CD45, DAPI, epithelial cell adhesion molecule (EpCAM), and vimentin markers. Heterogeneous CTC populations with hybrid epithelial/mesenchymal (EM) phenotypes are reported. Fluorescence in situ hybridization (FISH) was performed on enriched CTC samples. DNA was extracted and sequenced using a 523 gene targeted sequencing panel. Single cell RNA-seq technology was performed in parallel on CTC samples to investigate the gene expression throughout treatment. Results: The median concentration of CTCs in the ALK+ NSCLC cohort (n=33) is 137 CTCs per ml of blood, and 64% of CTCs are in an EM hybrid state. In ALK+ NSCLC patients (n=3) with an average of 4.6 visits per sample, we observed a trend of decreasing CTC concentration for patients with partial response to the treatment. The presence of ALK fusion in ALK+ NSCLC patient’s CTC is confirmed by FISH. Oncogenic mutations (JAK1, SPEN, ERBB4, NTRK3) are observed in bulk CTC samples from two NSCLC patients. Single cell RNA sequencing was performed on three ALK+ NSCLC patients; 58, 199, and 700 potential CTCs were detected with positive cytokeratin expression and no white blood cell marker expression. Conclusions: This study examined the feasibility of using CTCs for studying and monitoring the clinical status of the ALK+ NSCLC patients as well as the genomic and transcriptomic signatures within their tumors. More patients will need to be processed in order to validate our workflow and reinforce our conclusions. Citation Format: Yuru Chen, Shamileh Fouladdel, Harrison Ball, Zhaoping Qin, Albert Liu, Xu Cheng, Liwei Bao, Habib Serhan, Bryce Vandenburg, Varun Kathawate, Larua Goo, Peter Ulintz, Nathan Merrill, Aaron Udager, Angel Qin, Sofia D. Merajver, Sunitha Nagrath. Longitudinal multi-omics characterization of circulating tumor cells from ALK positive NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7504.

The circulation stage of the metastatic cascade: A mathematical description and its clinical implications

Metastatic cascade is a multi-stage process that starts with separation of a cancer cell from the primary tumor and ends with the emergence of a detectable metastasis. In the process the initiator cancer cell enters the circulatory system (intravasates), flows with the blood, and exits the circulation (extravasates) into an organ or tissue. The time period between intravasation and extravasation constitutes the circulation stage of the metastatic cascade. This stage is unique in that it lends itself naturally to various non-invasive observations and measurements in an individual cancer patient. This creates an opportunity for gaining insight into metastasis, its mathematical modeling, and designing diagnostic/prognostic tools and new cancer therapies. Although mechanisms of intravasation, survival and extravasation of circulating tumor cells (CTCs) are very complex and largely unknown, mathematical modeling of the circulation stage of the metastatic cascade is facilitated by two inter-related factors: a relative simplicity of the circulatory network and the cyclic nature of blood flow. The article presents a single-subject stochastic model of CTC dynamics that leads to simple formulas, applicable to any homogeneous CTC population, for organ-specific extravasation probabilities, the distribution and expected value of the number, X, of circulation cycles completed by a CTC prior to extravasation, and the average circulation time. In particular, we found that the distribution of random variable X is geometric G(x), where parameter x is measurable, at least in principle, in an individual subject. We also discuss implications of our results for cancer research and treatment.

Single-cell metabolic fingerprints discover a cluster of circulating tumor cells with distinct metastatic potential

Circulating tumor cells (CTCs) are recognized as direct seeds of metastasis. However, CTC count may not be the “best” indicator of metastatic risk because their heterogeneity is generally neglected. In this study, we develop a molecular typing system to predict colorectal cancer metastasis potential based on the metabolic fingerprints of single CTCs. After identification of the metabolites potentially related to metastasis using mass spectrometry-based untargeted metabolomics, setup of a home-built single-cell quantitative mass spectrometric platform for target metabolite analysis in individual CTCs and use of a machine learning method composed of non-negative matrix factorization and logistic regression, CTCs are divided into two subgroups, C1 and C2, based on a 4-metabolite fingerprint. Both in vitro and in vivo experiments demonstrate that CTC count in C2 subgroup is closely associated with metastasis incidence. This is an interesting report on the presence of a specific population of CTCs with distinct metastatic potential at the single-cell metabolite level.

Dynamics of CXCR4 positive circulating tumor cells in prostate cancer patients during radiotherapy.

Ablative radiotherapy is a highly efficient treatment modality for patients with metastatic prostate cancer (PCa). However, a subset of patients does not respond. Currently, this subgroup with bad prognosis cannot be identified before disease progression. We hypothesize that markers indicative of radioresistance, stemness and/or bone tropism may have a prognostic potential to identify patients profiting from metastases-directed radiotherapy. Therefore, circulating tumor cells (CTCs) were analyzed in patients with metastatic PCa (n=24) during radiotherapy with CellSearch, multicolor flow cytometry and imaging cytometry. Analysis of copy-number alteration indicates a polyclonal CTC population that changes after radiotherapy. CTCs were found in 8 out of 24 patients (33.3%) and were associated with a shorter time to biochemical progression after radiotherapy. Whereas the total CTC count dropped after radiotherapy, a chemokine receptor CXCR4-expressing subpopulation representing 28.6% of the total CTC population remained stable up to 3 months. At once, we observed higher chemokine CCL2 plasma concentrations and proinflammatory monocytes. Additional functional analyses demonstrated key roles of CXCR4 and CCL2 for cellular radiosensitivity, tumorigenicity and stem-like potential in vitro and in vivo. Moreover, a high CXCR4 and CCL2 expression was found in bone metastasis biopsies of PCa patients. In summary, panCK+ CXCR4+ CTCs may have a prognostic potential in patients with metastatic PCa treated with metastasis-directed radiotherapy.

Abstract P2-26-06: Association between CK+/CD45+ circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) alterations in advanced breast cancer patients

Abstract Background: Circulating tumor cells (CTCs) in breast cancer (BC) are commonly defined as epithelial cells (EPCAM and cytokeratin (CK) positive), lacking the universal blood cell marker CD45. Nonetheless, CTCs expressing both CK and CD45 (= dual-positive, DP cells) can be observed in the blood of cancer patients. Early evidence suggests that DP cells might derive from the fusion of tumor cells and macrophages, and we have previously demonstrated that they present aberrant genomes and are associated with worse prognosis in BC [1,2]. Here, to further investigate the mechanisms/pathways underlying their presence, we analyzed the association between DP cells and circulating tumor DNA (ctDNA) alterations. Methods: Blood samples were collected from patients with advanced BC (aBC), before starting a new line of therapy. All patients were enrolled in a prospective clinical trial. For CTC and DP cells analysis, 7.5 ml of blood collected in CellSave® tubes was processed with the FDA-approved CellSearch® platform (positivity cutoffs were ≥1 cell for DPcells and ≥5 cells for CTCs). For ctDNA analysis, plasma was collected from Streck stabilizing tubes and analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs), insertions/deletions (indels), gene fusions/rearrangements and copy number variations (CNVs), which were then classified into pathways based on previously defined profiles generated on the Cancer Genome Atlas database (RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, PI3K). Associations between ctDNA-detected gene alterations and circulating cell types were analyzed through chi square test, while mutant allele frequency (MAF) and number of detected alterations (NDA) were tested by Mann Whitney test. Results: We analyzed blood samples from 169 patients with luminal-like (n=80), HER2+ (n=34) and triple-negative (n=52) aBC. DPcells were detected in 85 patients (50.3 %, range 0-53), of which 40 (47 %) were CTC-positive and 45 (53%) CTC-negative. Somatic ctDNA alterations were detected in all analyzed samples. In the overall population, the presence of ≥1 DPcell was associated with SNVs in the cell cycle pathway (p = 0.043), a numerically higher incidence was also observed for CNVs in this pathway. SNVs and CNVs in the cell cycle pathway were associated with CTCs ≥ 5 as well (p = 0.005 and p = 0.003, respectively). Moreover, associations with CTCs ≥ 5 were observed for RTK SNVs and CNVs (p = 0.041 and p = 0.046, respectively), PI3K SNVs and CNVs (p = 0.006 and p = 0.007, respectively), MYC SNVs and CNVs (p = 0.042). No associations were observed in terms of MAF and NDA. In the luminal-like subgroup an association was highlighted for CNVs in the cell cycle pathway, p = 0.038. CTCs ≥ 5 were associated with PI3K SNVs (p = 0.031). In the triple-negative subgroup DPcells were associated with SNVs in the RAF pathway (p = 0.041), whereas CTCs ≥ 5 were associated with PI3K SNVs and CNVs (p = 0.044 and p = 0.024, respectively) and RTK SNVs (p = 0.008). In the HER2 positive subgroup, a higher MAF and number of detected SNVs was observed for samples with ≥1 DPcell (p = 0.0286 and p = 0.0099, respectively). Conclusions: The study analyzed ctDNA features associated with canonical and CK+/CD45+ CTCs, showing differential gene alteration profiles. Cell cycle pathway SNVs were common in both CTC populations, while other pathways (RTK, PI3K, MYC and RAF) were significantly altered in a mutually exclusive pattern. These results suggest that DPcells might have a different biological meaning compared to canonical CTCs. More studies need to be conducted to better characterize this understudied CTC subpopulation and understand their specific contribution to cancer progression.

Settling the uncertainty about unconventional circulating tumor cells: Epithelial-to-mesenchymal transition, cell fusion and trogocytosis.

Circulating tumor cells (CTCs) were first described 150 years ago. The so-called "classical" CTC populations (EpCAM+/CK+/CD45-) have been fully characterized and proposed as the most representative CTC subset, with clinical relevance. Nonetheless, other "atypical" or "unconventional" CTCs have also been identified, and their critical role in metastasis formation was demonstrated. In this chapter we illustrate the studies that led to the discovery of unconventional CTCs, defined as CTCs that display both epithelial and mesenchymal markers, or both cancer and immune markers, also in the form of hybrid cancer-immune cells. We also present biological explanations for the origin of these unconventional CTCs: epithelial to mesenchymal transition, cell-cell fusion and trogocytosis. We believe that a deeper knowledge on the biology of CTCs is needed to fully elucidate their role in cancer progression and their use as cancer biomarkers.

Detection of circulating tumor cells using antibody-functionalized microchips to monitor tumorigenesis in a mouse model of metastatic breast cancer

Circulating tumor cell (CTC)-based liquid biopsy has been a promising and potentially informative diagnostic approach. However, its clinical application has remained limited until now, in part due to the lack of systematic research to validate the new technologies for CTC enrichment. In this work, we established a 4T1 mouse model of breast cancer recurrence after surgery to evaluate microfluidic-based CTC detection and monitor tumor recurrence. The time point detected by CTC analysis using EpCAM antibody-functionalized chip was first compared with in vivo bioimaging and pathological examination for monitoring metastasis. The CTCs were monitored after tumor resection to evaluate CTC detection for postoperative monitoring of tumor recurrence. Using an antibody-functionalized chip, CTCs can be detected 10-days earlier than being detected with other imaging technologies. The CTC detection assay also showed that EpCAM+ CTC populations increased with tumor growth, whereas EpCAM+ CSV+ CTCs increased first and then decreased, possibly indicating multistep tumorigenesis in this model. Monitoring of CTCs after tumor resection indicated that CTCs were gradually cleared from the blood during mice recovery. These results provide a basis for CTC detection to monitor tumorigenesis and metastasis, as well as a model for validating other liquid biopsy methods.

Signs of apoptosis in circulating tumor cell subpopulations with phenotypes associated with stemness and epithelial-mesenchymal transition in breast carcinoma

Introduction. Ability of circulating tumor cells (CTC) initiate metastases in distant sites is associated primarily with their resistance to apoptosis which allows them to retain viability in the blood. Knowledge of phenotypical signs associated with this ability would allow to predict the risk of metastases and optimize adjuvant therapy.Aim. To examine signs of apoptosis in CTC populations with various phenotypical characteristics.Materials and methods. The study included 58 patients with invasive breast carcinoma of unspecified type, stages T1–4N0–3M0. Cell concentrates extracted from patients’ whole blood were stained with an antibody cocktail against CK7 / 8, CD45, EpCAM, CD44, CD24, CD133, ALDH, N-cadherin which allowed to identify CTC with signs of stemness and epithelial-mesenchymal transition. Annexin V and 7‑amino-actinomycin D staining was used for evaluation of apoptosis stage in CTC populations.Results. Circulating tumor cells are characterized by heterogeneity in respect to signs of stemness and epithelial-mesenchymal transition and presence of early and late signs of apoptosis and necrosis. CTC phenotypes including co-expression of epithelial marker CK7 / 8 and stemness marker CD133 (but not CD44) are characterized by absence of signs of apoptosis. Co-expression of CK7 / 8 and CD133 in CTC with stemness markers CD44+ / C D24– is associated with development of early but not late signs of apoptosis and necrosis. Circulating tumor cells without co-expression of CK7 / 8 and CD133 could have both early and late signs of apoptosis and necrosis. Circulating tumor cells phenotypes with signs of early apoptosis expressing CD133 remain in blood after non-adjuvant chemotherapy opposed to CTC without CD133 expression.Conclusion. There are CTC phenotypical signs associated with stemness and epithelial-mesenchymal transition and linked to apoptosis resistance or sensitivity.

Marker-free characterization of full-length transcriptomes of single live circulating tumor cells.

The identification and characterization of circulating tumor cells (CTCs) are important for gaining insights into the biology of metastatic cancers, monitoring disease progression, and medical management of the disease. The limiting factor in the enrichment of purified CTC populations is their sparse availability, heterogeneity, and altered phenotypes relative to the primary tumor. Intensive research both at the technical and molecular fronts led to the development of assays that ease CTC detection and identification from peripheral blood. Most CTC detection methods based on single-cell RNA sequencing (scRNA-seq) use a mix of size selection, marker-based white blood cell (WBC) depletion, and antibodies targeting tumor-associated antigens. However, the majority of these methods either miss out on atypical CTCs or suffer from WBC contamination. We present unCTC, an R package for unbiased identification and characterization of CTCs from single-cell transcriptomic data. unCTC features many standard and novel computational and statistical modules for various analyses. These include a novel method of scRNA-seq clustering, named deep dictionary learning using k-means clustering cost (DDLK), expression-based copy number variation (CNV) inference, and combinatorial, marker-based verification of the malignant phenotypes. DDLK enables robust segregation of CTCs and WBCs in the pathway space, as opposed to the gene expression space. We validated the utility of unCTC on scRNA-seq profiles of breast CTCs from six patients, captured and profiled using an integrated ClearCell FX and Polaris workflow that works by the principles of size-based separation of CTCs and marker-based WBC depletion.

Defining A Liquid Biopsy Profile of Circulating Tumor Cells and Oncosomes in Metastatic Colorectal Cancer for Clinical Utility.

Simple SummaryMetastatic colorectal cancer (mCRC) is typified by its tumor heterogeneity and changing disease states, suggesting that personalized medicine approaches could be vital to improving clinical practice. As a minimally invasive approach, the liquid biopsy has the potential to be a powerful longitudinal prognostic tool. We investigated mCRC patients’ peripheral blood samples using an enrichment-free single-cell approach to capture the broader rare-event population beyond the conventionally detected epithelial-derived circulating tumor cell (CTC). Our analysis reveals a heterogenous profile of CTCs and oncosomes not commonly found in normal donor samples. We identified select rare cell types based on their distinct immunofluorescence expression and morphology across multiple assays. Lastly, we highlight correlations between enumerations of the blood-based analytes and progression-free survival. This study clinically validates an unbiased rare-event approach in the liquid biopsy, motivating future studies to further investigate these analytes for their prognostic potential.Metastatic colorectal cancer (mCRC) is characterized by its extensive disease heterogeneity, suggesting that individualized analysis could be vital to improving patient outcomes. As a minimally invasive approach, the liquid biopsy has the potential to longitudinally monitor heterogeneous analytes. Current platforms primarily utilize enrichment-based approaches for epithelial-derived circulating tumor cells (CTC), but this subtype is infrequent in the peripheral blood (PB) of mCRC patients, leading to the liquid biopsy’s relative disuse in this cancer type. In this study, we evaluated 18 PB samples from 10 mCRC patients using the unbiased high-definition single-cell assay (HDSCA). We first employed a rare-event (Landscape) immunofluorescence (IF) protocol, which captured a heterogenous CTC and oncosome population, the likes of which was not observed across 50 normal donor (ND) samples. Subsequent analysis was conducted using a colorectal-targeted IF protocol to assess the frequency of CDX2-expressing CTCs and oncosomes. A multi-assay clustering analysis isolated morphologically distinct subtypes across the two IF stains, demonstrating the value of applying an unbiased single-cell approach to multiple assays in tandem. Rare-event enumerations at a single timepoint and the variation of these events over time correlated with progression-free survival. This study supports the clinical utility of an unbiased approach to interrogating the liquid biopsy in mCRC, representing the heterogeneity within the CTC classification and warranting the further molecular characterization of the rare-event analytes with clinical promise.

Genome-wide in vivo screen of circulating tumor cells identifies SLIT2 as a regulator of metastasis.

Circulating tumor cells (CTCs) break free from primary tumors and travel through the circulation system to seed metastatic tumors, which are the major cause of death from cancer. The identification of the major genetic factors that enhance production and persistence of CTCs in the bloodstream at a whole genome level would enable more comprehensive molecular mechanisms of metastasis to be elucidated and the identification of novel therapeutic targets, but this remains a challenging task due to the heterogeneity and extreme rarity of CTCs. Here, we describe an in vivo genome-wide CRISPR knockout screen using CTCs directly isolated from a mouse xenograft. This screen elucidated SLIT2-a gene encoding a secreted protein acting as a cellular migration cue-as the most significantly represented gene knockout in the CTC population. SLIT2 knockout cells are highly metastatic with hypermigratory and mesenchymal phenotype, resulting in enhanced cancer progression in xenograft models.

Circulating tumour cell gene expression and chemosensitivity analyses: predictive accuracy for response to multidisciplinary treatment of patients with unresectable refractory recurrent rectal cancer or unresectable refractory colorectal cancer liver metastases

BackgroundPatients with unresectable recurrent rectal cancer (RRC) or colorectal cancer (CRC) with liver metastases, refractory to at least two lines of traditional systemic therapy, may receive third line intraarterial chemotherapy (IC) and targeted therapy (TT) using drugs selected by chemosensitivity and tumor gene expression analyses of liquid biopsy-derived circulating tumor cells (CTCs).MethodsIn this retrospective study, 36 patients with refractory unresectable RRC or refractory unresectable CRC liver metastases were submitted for IC and TT with agents selected by precision oncotherapy chemosensitivity assays performed on liquid biopsy-derived CTCs, transiently cultured in vitro, and by tumor gene expression in the same CTC population, as a ratio to tumor gene expression in peripheral mononuclear blood cells (PMBCs) from the same individual. The endpoint was to evaluate the predictive accuracy of a specific liquid biopsy precision oncotherapy CTC purification and in vitro culture methodology for a positive RECIST 1.1 response to the therapy selected.ResultsOur analyses resulted in evaluations of 94.12% (95% CI 0.71–0.99) for sensitivity, 5.26% (95% CI 0.01–0.26) for specificity, a predictive value of 47.06% (95% CI 0.29–0.65) for a positive response, a predictive value of 50% (95% CI 0.01–0.98) for a negative response, with an overall calculated predictive accuracy of 47.22% (95% CI 0.30–0.64).ConclusionsThis is the first reported estimation of predictive accuracy derived from combining chemosensitivity and tumor gene expression analyses on liquid biopsy-derived CTCs, transiently cultured in vitro which, despite limitations, represents a baseline and benchmark which we envisage will be improve upon by methodological and technological advances and future clinical trials.

Multiparametric Phenotyping of Circulating Tumor Cells for Analysis of Therapeutic Targets, Oncogenic Signaling Pathways and DNA Repair Markers.

Simple SummaryDetection of circulating tumor cells (CTCs) has been established as an independent prognostic marker in solid cancer. In order to expand the clinical utility of this blood–based minimally invasive biomarker we established a protocol allowing multiparametric phenotyping of CTCs to analyze the expression levels of therapeutic target proteins. By applying this assay, we demonstrated intratumoral heterogeneity of PD–L1 expression in CTCs from head and neck cancer patients, an observation previously reported in tumor tissue specimens. We further verified the feasibility of applying the protocol to analyze the activation status of important oncogenic pathways and the extent of DNA repair following radiation. These promising preliminary results warrant further study and may lead to the implementation of this assay in clinical routine for improved treatment selection and monitoring.Detection of circulating tumor cells (CTCs) has been established as an independent prognostic marker in solid cancer. Multiparametric phenotyping of CTCs could expand the area of application for this liquid biomarker. We evaluated the Amnis® brand ImageStream®X MkII (ISX) (Luminex, Austin, TX, USA) imaging flow cytometer for its suitability for protein expression analysis and monitoring of treatment effects in CTCs. This was carried out using blood samples from patients with head and neck squamous cell carcinoma (n = 16) and breast cancer (n = 8). A protocol for negative enrichment and staining of CTCs was established, allowing quantitative analysis of the therapeutic targets PD–L1 and phosphorylated EGFR (phospho–EGFR), and the treatment response marker γH2AX as an indicator of radiation–induced DNA damage. Spiking experiments revealed a sensitivity of 73% and a specificity of 100% at a cut–off value of ≥3 CTCs, and thus confirmed the suitability of the ISX-based protocol to detect phospho–EGFR and γH2AX foci in CTCs. Analysis of PD–L1/–L2 in both spiked and patient blood samples further showed that assessment of heterogeneity in protein expression within the CTC population was possible. Further validation of the diagnostic potential of this ISX protocol for multiparametric CTC analysis in larger clinical cohorts is warranted.

Differential Kinase Activity Across Prostate Tumor Compartments Defines Sensitivity to Target Inhibition.

Single-cell mass cytometry analyses provide insights into the differences in kinase activities across tumor compartments and cell states, which contribute to heterogeneous responses to targeted therapies.

Disease characterization in liquid biopsy from HER2-mutated, non-amplified metastatic breast cancer patients treated with neratinib

Metastatic breast cancer (mBC) patients have a high risk of progression and face poor prognosis overall, with about one third (34%) surviving five years or more. In rare instances (2–4% of cases) patients with mBC have ERBB2 (HER2) activating mutations but are ERBB2 non-amplified. Neratinib is a potent, irreversible inhibitor that binds HER2 and inhibits downstream signaling. We used the previously validated high-definition single cell assay (HDSCA) workflow to investigate the clinical significance of the liquid biopsy in ERBB2 mutant, non-amplified, post-menopausal mBC patients starting neratinib and fulvestrant combination therapy. Characterization with a comprehensive liquid biopsy methodology (HDSCA) included genomic analysis of both the cell-free DNA (cfDNA) and single circulating tumor cells (CTCs) to monitor tumor evolution and identify potential mutational variants unique to the patient’s clinical response. A limited series of five sequentially enrolled patients presented here were from the MutHER (https://www.clinicaltrials.gov, NCT01670877) or SUMMIT (https://www.clinicaltrials.gov, NCT01953926) trials. Patients had an average of 5.4 lines of therapy before enrollment, variable hormone receptor status, and ERBB2 mutations at diagnosis and during treatment. CTC enumeration alone was not sufficient to predict clinical response. Treatment pressure was shown to lead to an observable change in CTC morphology and genomic instability (GI), suggesting these parameters may inform prognosis. Single cell copy number alteration (CNA) analysis indicated that the persistence or development of a clonal population of CTCs during treatment was associated with a worse response. Hierarchical clustering analysis of the single cells across all patients and timepoints identified distinct aberrant regions shared among patients, comprised of 26 genes that are similarly affected and may be related to drug resistance. Additionally, the genomic analysis of the cfDNA, identified new mutations in ERBB2, PIK3CA, and TP53 that arose likely due to treatment pressure in a patient with poor response, further providing insights on the dynamics of the cancer genome over the course of therapy. The data presented in this small cohort study demonstrates the feasibility of real-time molecular profiling of the cellular and acellular fractions of the liquid biopsy using the HDSCA methodology. Additional studies are necessary to determine the potential use of morphometric and genomic analysis as a prognostic tool to advance personalized oncology.

Abstract P2-02-07: Unbiased liquid biopsy in breast cancer patients utilizing novel light sheet microscopy of circulating tumor cells. Proof of concept in 10 patients and 13 controls

Abstract Background It is widely expected that circulating tumor cells (CTCs) in cancer patients shall turn out to be good prognosticators for outcomes. However, enumeration and monitoring of CTCs continues to pose serious hurdles. Current methods to assess CTCs utilize various approaches to enrich for CTCs. The only FDA-approved system detects CTCs via enrichment for EpCAM. Cancer heterogeneity requires an enrichment-free characterization of all CTCs to better identify this ever-changing landscape for biologic understanding and clinical management. Methods CLINBREAC is a single site, prospective, longitudinal trial for detecting and characterizing all CTCs in breast cancer patients. We present results from 8 metastatic and 2 neoadjuvant patients followed over time. Collection of 18ml blood samples occurred at enrollment and at change of treatment, or at 3-month intervals. Using the RareScope™ Fluorescence Light Sheet Microscope (QCDx LLC, Farmington CT), an aliquot of all morphologically intact, un-enriched nucleated cells placed in immobilized suspensions were analyzed. All cells were stained with immunofluorescent markers against CD45, Vimentin (Vim), EpCAM and Cytokeratin (CK) for characterization of epithelial or mesenchymal CTC phenotypes and trophoblast surface antigen 2 (TROP-2), ER-α and HER2 for treatment-specific characterization. Results No CTCs were observed in blood samples from 13 healthy volunteers. All patient samples contained CTCs collected over all time-points from 10 patients. In all, 1261 CTCs were identified, range, 18-85 CTCs per 1.7K to 2.7K nucleated cells analyzed. Each sample had a unique pattern of epithelial and mesenchymal markers that changed with time and treatment and mirrored clinical course. Of the CTC populations seen, 239 (19%) stained EpCAM+. Of the remaining CTCs, 798 CK+ (63%) and 842 Vim+ (67%) . An example: Patient 1: progression of metastatic triple negative breast cancer on nab-paclitaxel and atezolizumab after which anti-TROP-2 directed therapy with sacituzumab govitecan was initiated. In 39 CTCs present in sample 1, EpCAM-5%, CK-36%, and Vim-92% alone and co-expressed with EpCAM and CK. In subsequent samples, CTCs decreased from 39 to 23 to 6 consistent with response to treatment. TROP2+ CTCs decreased from 14/25 cells to 1/10 with treatment. CTCs co-expressed TROP2, HER2 and ER-α. As TROP2+ CTCs decreased from Sample 1 to 3 (56 to 10%) HER2+ CTCs increased (12 to 70%). The correlation of CTCs with clinical course of disease was observed in all 8 metastatic patients. Phenotypic changes occurred in all patients’ CTCs followed over time. Conclusions RareScope technology, utilized here, is a novel CTC-detection methodology that does not rely on prior enrichment of blood for CTCs. In patients analyzed here, all samples contained CTCs in higher numbers than described previously; a minority of CTCs contain EpCAM+ CTCs and may account for lower numbers in prior studies. Continuing refinement of this technology with addition of multiplex testing on single cells with a larger panel of antibodies will enhance targeted analysis and shall be useful in detection and detailed characterization of CTCs with un-precedented resolution and in real time. Citation Format: Susan Tannenbaum, Emily Hsu, Ashwanth Reddy, Spencer Keilich, Triantafyllos Tafas. Unbiased liquid biopsy in breast cancer patients utilizing novel light sheet microscopy of circulating tumor cells. Proof of concept in 10 patients and 13 controls [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-07.

Abstract P097: Comparative single cell transcriptome profiling of primary tumors, CTCs and metastatic sites from a bladder cancer PDX model

Abstract Background: A PDX bladder cancer model, BL0293-F563, grows large subcutaneous tumors, spontaneously metastasizes to the liver and bone, and sheds high numbers of circulating tumor cells (CTCs). This PDX model provides a unique opportunity to explore the relationships between primary tumors, CTCs and metastatic cell subpopulations. Methods: BL0293-F563 tumors (available from the NCI Patient-Derived Models Repository [https://pdmr.cancer.gov/] and originally developed by Jackson Laboratories) were implanted into NSG mice and and primary tumors, metastatic nodules in the liver, and blood were collected at maximal allowable tumor burden. Tumor tissue was dissociated using Miltenyi Tumor Dissociation Kit with OctoDissociator, and Human CTCs were enriched from whole mouse blood through negative selection with anti-mouse CD45 and anti-mouse MHC-1 magnetic beads. Single cell sequencing was done using 10X Genomics 3’ gene expression assay v3.1. Sequencing libraries were prepared using 10X Genomics Chromium and 3’ gene expression kit v3.1. Data processing and analysis was done using 10X Genomics’ Cell Ranger pipeline, Seurat, and consensus non-negative matrix factorization. Results: Using Seurat FindNeighbors, cells in the aggregated dataset were classified into 17 distinct clusters. All clusters were comprised of cells from multiple sites (primary tumor, CTCs, metastases), but three clusters were enriched in CTCs and one cluster was composed of mostly primary tumor cells. All clusters exhibited an epithelial-like gene expression signature score, suggesting that CTC shedding was occurring without prominent epithelial-mesenchymal transition. Consistent with expected differences in oxygenation states, CTC-enriched clusters exhibited a lower hypoxia gene expression score than primary tumor and metastasis-enriched clusters. CTC-enriched clusters also showed higher expression of oxidative phosphorylation genes, suggesting metabolic differences between CTCs and cells from primary tumors and metastases. Based on Human Primary Cell Atlas phenotype prediction, several clusters were associated with stem cell like phenotypes. Additionally, two of three CTC-enriched clusters had elevated expression of mitosis-associated genes, suggesting that at least some populations of CTCs are not quiescent but actively cycling. Conclusions: Utilizing single cell gene expression profiling, we have linked the gene expression profile of CTCs to specific cell subpopulations in primary tumors and metastases. We show that CTC-enriched cell clusters appear to maintain an epithelial phenotype. Subpopulations of CTC cells exhibited enrichment of stemness-associated transcripts and features of active cell cycling. Citation Format: Tomas Vilimas, Brandie Fullmer, Alyssa Chapman, Li Chen, Ting-Chia Chang, Rini Pauly, Biswajit Das, Chris Karlovich, Yvonne A. Evrard, Howard Stotler, Michelle M. Gottholm-Ahalt, Tara Grinnage-Pulley, Melinda G. Hollingshead, James H. Doroshow, P. Mickey Williams. Comparative single cell transcriptome profiling of primary tumors, CTCs and metastatic sites from a bladder cancer PDX model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P097.

1820P Hybrid circulating tumor cells in breast cancer

The CTC (circulating tumor cells) population is heterogeneous, and includes canonical tumor cells (CD45-EpCAM+ and/or cytokeratins+) and cells with a hybrid phenotype (CD45+EpCAM+ and/or cytokeratins+). Hybrid CTCs are very poorly studied. Their molecular characteristics, determining invasive and metastatic properties (stemness and epithelial-mesenchymal transition (EMT)), need to be clarified. The aim of this study was to evaluate phenotypic features and gene expression profile of hybrid CTCs.