Single Circulating Tumor Cells Research Articles

Abstract B042: Molecular assessment of cell surface targets using integrated circulating tumor cell (CTC) RNAseq and single CTC phenotyping

Abstract Background: Androgen-targeted therapies are a mainstay of treatment for metastatic prostate cancer and have significantly improved patient outcomes. However, development of treatment resistance remains universal, occurring through androgen receptor (AR) alterations driving constitutive AR signaling, or lineage state transitions that bypass AR entirely and culminate in a small cell/neuroendocrine phenotype (NEPC). Cell surface targeted therapies, including antibody drug conjugates and targeted radioligand therapies, represent a new therapeutic class that has shown promise for treatment of androgen-resistant metastatic prostate cancer (mCRPC). Circulating tumor cells (CTCs) present an accessible source of tumor material to identify expression of known and novel targets for therapeutic development, as well as the unique potential for longitudinal profiling to understand the evolution of target expression and association with clinical resistance to cell surface targeted therapies. We have developed a CTC platform allowing for gene expression and protein profiling of cell surface targets on CTCs. Methods: Live CTCs were isolated with our automated microfluidic technology integrating negative and positive selection for CTC enrichment with a cohort of 273 samples. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA-seq, or protein staining on chip for single cell fluorescent quantification of prostate adenocarcinoma and NEPC cell surface targets including PSMA, TROP2, B7H3 and DLL3. Results: CTC RNA sequencing can be used to understand the expression of cell surface targets across different CTC phenotypes. In CTCs from patient with mCRPC, canonical prostate adenocarcinoma cell surface targets STEAP1, KLK2 and FOLH1 and epithelial cell surface target TACSTD2 had the highest expression, while prostate adenocarcinoma target STEAP2, pan-tumor target ERBB2, and tumor immune checkpoint cell surface protein CD276 (B7H3) had intermediate expression. Expression of DLL3 and SSTR2, targets associated with neuroendocrine differentiation, was lowest, though still detected in a subset of CRPC CTCs. Comparison of expression levels between CRPC and NEPC CTCs demonstrated lower expression of most adenocarcinoma cell surface targets in NEPC CTCs as expected, and a trend towards higher expression of DLL3 and SSTR2. Cell surface expression is being assessed for proteins including Trop-2, PSMA and DLL3. Evaluation of DLL3 at the protein level in a subset of patients including both CRPC (n=13) and NEPC (n=12) confirmed that DLL3 was detected in both adenocarcinoma and neuroendocrine CTCs, with a median of 45.8% DLL3-positive CTCs per sample. Conclusions: Biomarkers are needed to better predict response and resistance to targeted therapies. CTC RNAseq and single CTC phenotyping hold potential for predictive and on-treatment biomarkers of response to cell surface targeted therapies. Further study of mechanisms driving response and resistance can be evaluated with these biomarkers. Citation Format: Jamie M. Sperger, Marina N. Sharifi, Katherine R. Kaufmann, Matthew L. Bootsma, Shannon R. Reese, Viridiana Carreno, Alex H. Chang, Luke A. Nunamaker A. Nunamaker, Charlotte Linebarger, Amy K. Taylor, Katherine E Tippins, Kyle T. Helzer, Shuang G. Zhao, Joshua M Lang. Molecular assessment of cell surface targets using integrated circulating tumor cell (CTC) RNAseq and single CTC phenotyping [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B042.

Abstract IA005: Tumor cell-based liquid biopsy to characterize prostate cancer

Abstract Circulating Tumor Cells (CTCs) hold the promise of comprehensive yet noninvasive molecular characterization of cancer cells during the course of tumor evolution and progression. To dissect the epigenetic profile of prostate CTCs, we undertook paired DNA methylation and RNA sequencing of single CTCs isolated from blood specimens of patients with prostate cancer. Across the genome, our study identified 40 core Partial Methylation Domains (PMDs) that arise early in tumorigenesis and are shared by all individual CTCs across multiple patients. Key among these is a single genomic locus harboring multiple interferon-inducible genes along with all the CD1 gene family members, implicated in innate immunity. Based on mouse models, we propose that early biallelic silencing of multiple genes residing within a single PMD may contribute to the escape from immune surveillance in prostate and potentially other cancers. Studies of CTC heterogeneity within individual patients are limited by the very small number of cells that can be isolated from standard 10ml blood volumes. To enable such analyses, we applied a new high throughput microfluidic platform, capable of processing an entire leukapheresis product, interrogating 2-3 L of blood volume equivalent. Applying this technology to patients with metastatic prostate cancer, we successfully enriched thousands of CTCs from individual cases, enabling the characterization of intra-patient heterogeneity at the level of cell morphology and marker expression, chromosome copy number variation and exome-wide genotyping, and transcriptionally-defined tumor cell subpopulations. Such high-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies. Citation Format: Daniel A Haber, Shyamala Maheswaran, Hongshan Guo, Joanna Vuille, Shih-Bo Huang, Ben S Wittner, Lecia V Sequist, Richard J Lee, Patricia AR Brunker, David T Miyamoto, Avanish Mishra, Mehmet Toner. Tumor cell-based liquid biopsy to characterize prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA005.

A novel electrochemiluminescent cytosensor using dual-target magnetic probe recognition and nanozymes-catalyzed cascade signal amplification for precise phenotypic enumeration of CTCs.

The inability of surgical biopsy to monitor the dynamic evolution of cancer cells hampers its capacity to reflect real-time tumor heterogeneity. Circulating tumor cells (CTCs), as a crucial target in liquid biopsy, offer a novel approach for accurate monitoring of tumors. However, the rarity and complex phenotype resulting from epithelial-mesenchymal transition pose challenges for conventional methods such as CellSearch and immunohistochemistry, which have insufficient ability for simultaneous phenotyping and enumeration of CTCs. The enumeration of a single phenotype CTCs is insufficient for accurately assessing disease progression. Herein, we propose a strategy to address this issue by fabricating an electrochemiluminescence cytosensor via the integration of dual-target enrichment and nanozymes-catalyzed cascade signal amplification. The graphene oxide@hollow mesoporous Prussian blue/Pt (GO@HMPB/Pt) complex, possessing a large specific surface area and exceptional catalytic activity, is employed for loading a substantial amount of luminol as the signal probe. Dual-target magnetic PPy@Fe3O4/Au-antibody/aptamer is utilized for the magnetic capture of both epithelial and interstitial CTCs. Glutathione (GSH) can disrupt theAu-S bond on aptamer by a thiol exchange reaction and selectively releases a specific subset of phenotypic CTCs, thereby facilitating the efficient capture, accurate classification, and ultrasensitive detection of CTCs in peripheral blood. Using the epithelial MCF-7 and mesenchymal Hela cells as models, the ECL cytosensor demonstrates excellent performance in identifying cells spiked into whole blood. This study presents a novel approach for early detection of metastasis, tracking tumor recurrence, and monitoring therapeutic efficacy.

Measurable morphological features of single circulating tumor cells in selected solid tumors-A pilot study.

Liquid biopsies developed into a range of sensitive technologies aiming to analyze for example, circulating tumor cells (CTCs) in peripheral blood, which significantly deepens understanding of the metastatic process. Nevertheless, examination of CTCs is mostly limited to their enumeration and usually only 2-3 markers-based phenotyping, not offering yet sufficient insight into their biology. In contrast, quantitative analysis of their morphological details might extend our knowledge about dissemination and even improve CTC isolation or label-free identification methods dependent on their physical features such as size, and deformability. Current study was conducted to describe CTCs' and their size, shape, presence of protrusions, and micronuclei across various types of cancers (lung, n = 29; ovarian, n = 24, breast, n = 54; and prostate, n = 33). Epithelial (pan-keratins), mesenchymal (vimentin), and two exclusion markers were used to identify CTCs and classify them into four epithelial and epithelial-mesenchymal transition-related phenotypes using standardized and throughput method, imaging flow cytometry. The morphological characteristics of CTCs, including their nuclei, such as circularity, the maximum, and minimum diagonal values were determined using an open-source software QuPath. On average, detected CTCs (n = 1156) were larger, and more irregular in shape compared to leukocytes/endothelial cells (n = 400). Epithelial and mesenchymal CTCs had the largest (median = 18.2 μm) and the smallest diameter (median = 10.4 μm), respectively. In terms of cancer-specific variations, the largest CTCs were identified in lung cancer, whereas the smallest-in prostate and breast cancers. Epithelial CTCs and those negative for both epithelial and mesenchymal markers exhibited the highest degree of elongation, whereas mesenchymal CTCs were the most irregular in shape. Protrusions and micronuclei were observed extremely rarely within CTCs of breast and prostate cancer (0.6%-0.8% of CTCs). Micronuclei were observed only in epithelial and epithelial-mesenchymal CTCs. This study underscores the significant variability in the morphological features of CTCs in relation to their phenotypic classification or even the particular organ of origin, potentially influencing for example, size-dependent CTC isolation methods. It demonstrates for the first time the morphological measurements of CTCs undergoing epithelial-mesenchymal transition, and some specific morphological details (i.e., protrusions, micronuclei) within CTCs in general.

Deep Learning-Assisted Assessing of Single Circulating Tumor Cell Viability via Cellular Morphology.

Circulating tumor cells (CTCs) are closely associated with cancer metastasis and recurrence, so the assessment of CTC viability is crucial for diagnosis, prognosis evaluation, and efficacy judgment of cancer. Due to the extreme scarcity of CTCs in human blood, it is difficult to accurately evaluate the viability of a single CTC. In this study, a deep learning model based on a convolutional neural network was constructed and trained to extract the morphological features of CTCs with different viabilities defined by cell counting kit-8, achieve accurate CTC identification, and assess the viability of a single CTC. Being efficient, accurate, and noninvasive, it has a broad application prospect in biomedical directions.

Development and validation of a multi-marker liquid bead array assay for the simultaneous detection of PIK3CA and ESR1 hotspot mutations in single circulating tumor cells (CTCs)

BackgroundPIK3CA and ESR1 mutations are associated with progression and therapy resistance in metastatic breast cancer (MBC). CTCs are highly heterogeneous and their analysis at single cell level can provide unique information for mutational profiling and the existence of different sub-clones related to tumor progression. We have developed a novel multi-marker liquid bead array assay based on combination of an enzymatic mutation enrichment method, multiplex PCR-based assay, and liquid bead array technology for the simultaneous detection of PIK3CA and ESR1 hotspot mutations in liquid biopsy samples. We focus on single CTCs, however the assay can be used for bulk CTC and ctDNA analysis. Materials and methodsSingle CTCs were isolated from an ER+/HER2+ MBC patient from CellSearch® cartridges using the VyCAP Puncher System and subjected to whole genome amplification followed by nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO) enrichment. The assay was validated for analytical sensitivity and specificity for the simultaneous detection of PIK3CA (E545K, E542K, H1047R, H1047L) and ESR1 (Y537S, Y537C, Y537N, D538G, L536H) mutations in single CTCs, while its clinical performance was evaluated on 22 single CTCs and three single white blood cells (WBCs). ResultsThe developed multi-marker liquid bead array assay is novel, highly specific and sensitive for both mutation panels. The assay can reliably detect mutation-allelic-frequencies (MAFs) as low as 0.1 %. The presence of PIK3CA and ESR1 mutations was detected in 13.6 % and 72.7 % of single CTCs, respectively. The developed assay is sample-saving since it requires only 2 μL of amplified DNA to check for nine hotspot PIK3CA and ESR1 mutations in a single cell. The developed liquid bead array assay (Luminex, US), based on a 96 microwell plate format, enables the simultaneous analysis of 96 single cells. ConclusionsThe developed novel multi-marker liquid bead array assay for the simultaneous detection of PIK3CA and ESR1 hotspot mutations in single CTCs is highly specific, highly sensitive, high-throughput, and sample-, cost-, and time-saving. This multi-marker liquid bead array assay can be extended to detect up to 100 mutations in many genes at once and can be applied for bulk CTC and ctDNA analysis.

A comparative study of circulating tumor cell isolation and enumeration technologies in lung cancer.

Circulating tumor cells (CTCs) have potential as diagnostic, prognostic, and predictive biomarkers in solid tumors. Despite Food and Drug Administration (FDA) approval of CTC devices in various cancers, the rarity and heterogeneity of CTCs in lung cancer make them technically challenging to isolate and analyze, hindering their clinical integration. Establishing a consensus through comparative analysis of different CTC systems is warranted. This study aimed to evaluate seven different CTC enrichment methods across five technologies using a standardized spike-in protocol: the CellMag™ (EpCAM-dependent enrichment), EasySep™ and RosetteSep™ (blood cell depletion), and the Parsortix® PR1 and the new design Parsortix® Prototype (PP) (size- and deformability-based enrichment). The Parsortix® systems were also evaluated for any differences in recovery rates between cell harvest versus in-cassette staining. Healthy donor blood (5 mL) was spiked with 100 fluorescently labeled EpCAMhigh H1975 cells, processed through each system, and the isolation efficiency was calculated. The CellMag™ had the highest recovery rate (70 ± 14%), followed by Parsortix® PR1 in-cassette staining, while the EasySep™ had the lowest recovery (18 ± 8%). Additional spike-in experiments were performed with EpCAMmoderate A549 and EpCAMlow H1299 cells using the CellMag™ and Parsortix® PR1 in-cassette staining. The recovery rate of CellMag™ significantly reduced to 35 ± 14% with A549 cells and 1 ± 1% with H1299 cells. However, the Parsortix® PR1 in-cassette staining showed cell phenotype-independent and consistent recovery rates among all lung cancer cell lines: H1975 (49 ± 2%), A549 (47 ± 10%), and H1299 (52 ± 10%). Furthermore, we demonstrated that the Parsortix® PR1 in-cassette staining method is capable of isolating heterogeneous single CTCs and cell clusters from patient samples. The Parsortix® PR1 in-cassette staining, capable of isolating different phenotypes of CTCs as either single cells or cell clusters with consistent recovery rates, is considered optimal for CTC enrichment for lung cancer, albeit needing further optimization and validation.

Combined microfluidic enrichment and staining workflow for single-cell analysis of circulating tumor cells in metastatic prostate cancer patients

Circulating tumor cells (CTCs) are precursors of cancer in the blood and provide an attractive source for dynamic monitoring of disease progression and tumor heterogeneity. However, the scarcity of CTCs in the bloodstream has limited their use in clinical practice. In this study, we present a workflow for easy detection of CTCs by cytokeratin staining using the FDA-cleared Parsortix device for size-based microfluidic enrichment. To minimize sample handling, the isolated cells are stained inside the separation cassette and harvested for subsequent single cell isolation and whole genome copy-number analysis. We validated the workflow on a panel of four prostate cancer cell lines spiked into healthy donor blood collected in CellRescue or EDTA tubes, resulting in mean recoveries of 42% (16–69%). Furthermore, we evaluated the clinical utility in a cohort of 12 metastatic prostate cancer patients and found CTCs in 67% of patients ranging from 0 to 1172 CTCs in 10 mL blood. Additionally, we isolated single patient-derived CTCs and identified genomic aberrations associated with treatment response and clinical outcome. Thus, this workflow provides a readily scalable strategy for analysis of single CTCs, applicable for use in monitoring studies to identify genomic variations important for guiding clinical therapy decision.

Mutation analysis in individual circulating tumor cells depicts intratumor heterogeneity in melanoma

Circulating tumor DNA (ctDNA) is the cornerstone of liquid biopsy diagnostics, revealing clinically relevant genomic aberrations from blood of cancer patients. Genomic analysis of single circulating tumor cells (CTCs) could provide additional insights into intra-patient heterogeneity, but it requires whole-genome amplification (WGA) of DNA, which might introduce bias. Here, we describe a novel approach based on mass spectrometry for mutation detection from individual CTCs not requiring WGA and complex bioinformatics pipelines. After establishment of our protocol on tumor cell line-derived single cells, it was validated on CTCs of 33 metastatic melanoma patients and the mutations were compared to those obtained from tumor tissue and ctDNA. Although concordance with tumor tissue was superior for ctDNA over CTC analysis, a larger number of mutations were found within CTCs compared to ctDNA (p = 0.039), including mutations in melanoma driver genes, or those associated with resistance to therapy or metastasis. Thus, our results demonstrate proof-of-principle data that CTC analysis can provide clinically relevant genomic information that is not redundant to tumor tissue or ctDNA analysis.

Longitudinal analysis of circulating tumor cell numbers improves tracking metastatic breast cancer progression

Hormone-responsive breast cancer represents the most common type and has the best prognosis, but still approximately 40% of patients with this type can develop distant metastases, dramatically worsening the patient’s survival. Monitoring metastatic breast cancer (mBC) for signs of progression is an important part of disease management. Circulating tumor cell (CTC) detection and molecular characteristics gain importance as a diagnostic tool, but do not represent a clinical standard and its value as a predictor of progression is not yet established. The main objective of this study was to estimate the prognostic value of not only the CTC numbers, but also the dynamics of the CTC numbers in the same patient during the continuous evaluation of CTCs in patients with advanced breast cancer. The other objective was to assess the molecular changes in CTCs compared to primary tumor samples by genetic analysis of the seven genes associated with estrogen signaling pathway, mutations in which are often responsible for the resistance to endocrine therapy, and subsequent progression. This approach was taken to evaluate if genetic analysis of CTCs can be used in tracking the resistance, signaling that hormonal therapy should be replaced. Consequently, this report presents the results of a longitudinal CTC study based on three subsequent blood collections from 135 patients with metastatic breast cancer, followed by molecular analysis of the isolated single CTCs. CTCs were detected and isolated using an image-based, EpCAM-independent system CytoTrack; this approach allowed evaluation of EpCAM expression in detected CTCs. Isolated CTCs were subjected to NGS analysis to assess mutational changes. The results confirm the importance of the status of the CTC for progression-free survival and overall survival and provide new data on the dynamics of the CTC during a long monitoring period and in relation to clinical progression, highlighting the advantage of constant monitoring over the single count of CTC. Furthermore, high genetic and phenotypic inter- and intrapatient heterogeneity observed in CTCs suggest that metastatic lesions are divergent. High genetic heterogeneity in the matching CTC/primary tumor samples may indicate early dissemination. The tendency towards the accumulation of activating/oncogenic mutation in CTCs, leading to anti-estrogen resistant disease, was not confirmed in this study.

Simultaneous detection of tumor-derived mutations in DNA and RNA extracted from a single circulating tumor cell based on droplet digital polymerase chain reaction.

e15053 Background: Androgen receptor splice variant 7 (AR-V7) is a constitutively activated isoform of AR and has been associated with resistance to AR-targeted therapies and development of metastatic castration-resistant prostate cancer. Confirmation of AR-V7 expression is important step in prostate cancer diagnosis and determining the appropriate drug, however, the detection of AR-V7 with tissues has limitations, such as the clinical challenge to poorly accessible tumor tissues before and after therapy. In addition, KRAS mutations serve as a prognostic biomarker for the administration of appropriate anti-cancer drugs in patients with various cancers, including non-small cell lung cancer (NSCLC). Here, we developed the process for the simultaneous detection of AR-V7 and KRAS mutation in CTCs using isolation with CytoGen’s Smart Biopsy™ System with droplet digital polymerase chain reaction (ddPCR) with high sensitivity and specificity, overcoming the limitations of tissue biopsy. Methods: ddPCR assays were performed to detect the KRAS G12C mutation using CTC gDNA and to evaluate the absolute levels of AR-V7 using CTC mRNA. Analytical validation was performed using NSCLC NCI-H358 and prostate cancer 22Rv1 cells (10 and 5 cells each), which harbor KRAS G12C and AR-V7, respectively, spiked into 5 mL of healthy blood to mimic CTC. CTCs were isolated using CytoGen's Smart Biopsy™ System. Oligo-dT magnetic beads were used to isolate mRNA from CTCs, and gDNA was isolated from the mRNA-depleted CTC lysates using the AllPrep DNA/mRNA Nano Kit. PCR and droplet reading for each assay were performed on the QX200 AutoDG Droplet Digital PCR System. Results: As a result of CTC KRAS G12C detection using the ddPCR™ KRAS G12/G13 Screening Kit, the mutated KRAS gene was successfully detected in as few as 5 NCI-H358 cells. In the results of CTC AR-V7 detection using the custom TaqMan ddPCR assay, transcripts (AR-V7 and HPRT1) were successfully detected even with only 5 22Rv1 cells. These results suggest that CytoGen's Smart Biopsy™ System could be applied to the simultaneous detection of DNA/RNA mutated genes in clinical NSCLC and prostate cancer patients with detection technology requiring high sensitivity and specificity. Conclusions: Accurately detecting multiple genetic mutations in CTCs is a challenging because CTCs are very rare in the blood. In this study, we developed the process to simultaneously extract and detect DNA mutations and RNA splicing variants in CTCs using CytoGen's Smart Biopsy™ System. This process, along with the results from our ongoing clinical trials, could provide information for the diagnosis and appropriate treatment of patients with various cancers, including NSCLC and prostate cancer.

Examination of genomic profile of true single circulating tumor cells (sCTC) from hepatocellular carcinoma and its effect on mutational features.

e15039 Background: Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer-related deaths worldwide. Despite numerous NGS studies unveiling diverse mutational patterns through tissue biopsies, the absence of tissue for biopsy and distinct biomarkers complicates both diagnosis and treatment. The utilization of sCTC genomics holds promise in furnishing clinically relevant and actionable insights to tailor personalized diagnostics and therapy for HCC. We present a comparative analysis of sCTC and paired ctDNA obtained from primary HCC patients for better Tx possibilities. Methods: Genomic profile from 24 sCTCs/CTC clusters (19 sCTCs, 79%; 5 CTC clusters, 21 %) revealed distinct mutational patterns compared to paired ctDNA. Live sCTCs/CTC clusters were isolated from peripheral blood of treatment naïve 12 Hepatobiliary Cancer with advanced and metastatic disease patients using OncoRadar technology. Whole genomes from sCTCs/CTC clusters were amplified, target enriched with hybridization capture using a comprehensive 1080 gene panel-OncoIndx to obtain sequencing libraries, and sequenced on Illumina NextSeq2000 platform in paired end mode (depth 500x). The raw sequence alignment and variant calling was performed using iCare software. Paired ctDNA samples were sequenced the same way, except at the depth of 5500x. Results: Pathogenic (P)/likely pathogenic (LP) variants were detected from all sCTCs, while ctDNA was negative for 40% samples. sCTCs showed at least twice as many P variants compared to paired ctDNA. ATM (50%), PIK3CA (42%), ARID1A /1B (42%), TP53/RB1 (34%), and IDH1 (34%) were among the most frequently mutated genes, while 42% sCTCs showed amplified CCND1/2 variants. In comparison, paired ctDNA had no distinct mutational pattern. Genomic profile of sCTC showed concordance with paired ctDNA for at least one gene mutation when compared at patient level. At pathway level, all sCTCs were mutated for at least one HRR pathway component, while 60% sCTCs had at least one mutation in receptor tyrosine kinase (RTK) pathway (FGFR1, ERBB, ALK, ROS1). sCTC population showed a strong cooccurrence of HRR mutation and epigenetic regulatory pathway mutations (OR =4, CI=0.4-60). At individual mutation level, 30 % concordance was observed in sCTC population, suggesting high mutational heterogeneity. Besides actionable targets, 50% sCTC were enriched with resistance conferring variants such as STK11, NF1, STAT5B, and RB1. Conclusions: The genomic profile of sCTCs in primary HCC pts revealed a complex mutational landscape, offering complementarity to ctDNA for clinical insights. sCTCs exhibited more actionable targets and greater heterogeneity compared to paired ctDNA, making sCTC genomics valuable for pts ineligible for tissue biopsy or lacking clinically relevant information from ctDNA.

Study of single live circulating tumor cells capture and their genomic profile and enriched mutations of PIK3CA and HRR pathway in patients with breast cancer.

e13161 Background: Capturing live single circulating tumor cells (sCTC) for intra-cellular compressive genomic profiling and concordance with ctDNA is highly challenging. Utilization of single sCTC genomics holds promise for detecting clinically relevant, actionable insights to tailor personalized therapy, especially when No Mutations Detected (NMDs) using CfDNA. Despite numerous studies providing valuable insights on mutational landscapes from breast tumors, predicting cancer progression, metastasis, and relapse continue to pose major challenges. ctDNA is suboptimal for 30-40% population with disease relapse/progression after a primary therapy. We show comprehensive genetic profile (CGP) of live sCTCs and paired ctDNA from a diverse breast cancer population. Methods: 26 live sCTCs (2 CTC clusters) were isolated from 12 patients. Live sCTCs were isolated from 12 Breast Cancer patients using OncoRadar live sCTC capture method. Whole genomes were amplified using multiple displacement amplification, target enriched with hybridization capture using OncoIndx (CGP 1080 gene panel) to obtain sequencing libraries, and sequenced on Illumina platform in paired end mode (depth 500x). The raw sequence alignment and variant calling was performed using iCare software. Paired ctDNA samples were processed the same way, except for sequencing depth (5500x). Results: Compared to paired ctNDA, genomic profiling of sCTCs revealed complex mutational patterns. Whereas sCTCs showed least two pathogenic (P) or likely pathogenic (LP) variants, paired ctDNA was negative for 25% cases (3/12). 92% sCTCs showed mutations in the PIK3CA-MTOR pathway with PIK3CA being the most prevalent (73 %). A significant association was observed between co-occurrence of HER2 amplification and PIK3CA mutations (P =0.04, OR=10). Similarly, sCTCs showed a strong correlation between HRR pathway and PIK3CA pathway mutations (r=0.76, P =0.003, OR=3.43). Genomic profile of sCTCs and paired ctDNA showed concordance with at least one gene mutation when compared at individual patient level. Additionally, at individual mutation level, PIK3CA exon 10 mutations were most frequent (61.5%) along with BRCA2 pathway variants, followed by IDH1/2(54%), ARID1A (50%), NRAS (46%), FGFR2 (42%) and TP53 (42%). Interestingly, 70% sCTCs showed the presence of therapy resistance conferring variants (NF1, ARID1A, ESR1, RB1 and PTEN), compared to only 25 % observed in paired ctDNA. Conclusions: The genomic profile of sCTCs represents true tumor heterogeneity and can be combined with ctDNA for better clinical outcome. sCTCs exhibited high mutational heterogeneity compared to paired ctDNA. Therapy resistance signatures were more prevalent in sCTCs and may offer valuable clinical insights for patients unable to provide tissue biopsy or show negative ctDNA.

Novel Isolating Approaches to Circulating Tumor Cell Enrichment Based on Microfluidics: A Review.

Circulating tumor cells (CTCs), derived from the primary tumor and carrying genetic information, contribute significantly to the process of tumor metastasis. The analysis and detection of CTCs can be used to assess the prognosis and treatment response in patients with tumors, as well as to help study the metastatic mechanisms of tumors and the development of new drugs. Since CTCs are very rare in the blood, it is a challenging problem to enrich CTCs efficiently. In this paper, we provide a comprehensive overview of microfluidics-based enrichment devices for CTCs in recent years. We explore in detail the methods of enrichment based on the physical or biological properties of CTCs; among them, physical properties cover factors such as size, density, and dielectric properties, while biological properties are mainly related to tumor-specific markers on the surface of CTCs. In addition, we provide an in-depth description of the methods for enrichment of single CTCs and illustrate the importance of single CTCs for performing tumor analyses. Future research will focus on aspects such as improving the separation efficiency, reducing costs, and increasing the detection sensitivity and accuracy.

Abstract PO5-18-04: Effect of Digoxin on clusters of circulating tumor cells in patients with metastatic breast cancer (DICCT)

Abstract Background: The presence of circulating tumor cell (CTC) clusters is associated with tumor progression and with a bad prognosis in various cancer types1,2. In breast cancer, a pre-clinical model demonstrated that Na/K-ATPase inhibitors such as digoxin could dissolve CTC clusters and reduce metastasis3. DICCT is a single-arm, therapeutic exploratory phase I study aimed to examine whether digoxin can disrupt CTC clusters in metastatic breast cancer patients (NCT03928210). Material and methods: Metastatic breast cancer patients in whom clusters of CTCs have been detected were eligible. Patients with concomitant heart disease were excluded. After progressive disease in any line of therapy, a window of opportunity before initiation of subsequent systemic therapy was used to administer digoxin treatment for seven (7) days. For CTC detection, blood samples were processed with the FDA-cleared Parsortix system (ANGLE plc, UK). Captured CTCs were stained for EPCAM, HER2, EGFR (positive CTC selection) and CD45 (exclusion marker). To gain insight into the cluster-dissolution ability of digoxin, the primary endpoint was size of CTC clusters (i.e. number of cells within each cluster) under digoxin therapy. The cumulative effect of digoxin treatment on the primary endpoint was assessed by a linear regression model. Results: Nine (9) patients with CTC clusters detected at baseline were enrolled. A target level of digoxin above 0.7 ng/ml could be confirmed at day 7 for all patients. For quantitative and qualitative analysis of CTC clusters, 5.5 to 27 ml of blood were analyzed at baseline, at day 0 pre-treatment, and post 2 hours, 3 days and 7 days of digoxin treatment. The average cluster size ranged between 2 and 11.8 cells (median 2.9) considering all samples from all patients. Digoxin treatment reduced cluster size in 8 patients (88.9%) to different extents, and the proportion of CTC cluster over single CTCs was also reduced in 6 patients (66.7%). The effect of digoxin was evident in both homotypic (cancer cells only) and heterotypic (aggregates of CTCs and white blood cells) clusters. The treatment was well tolerated, and no adverse events related to the study treatment occurred. All patients completed the treatment according to the protocol. Conclusion: DICCT provides the first-in-human proof-of-principle that digoxin induces a partial dissolution of CTC clusters in patients with metastatic breast cancer at drug levels that are safe and well tolerated. Conceptualization of follow-up trials including Na/K-ATPase inhibitors and patient outcome endpoints are underway.

Abstract SY34-01: Plasticity, dormancy, and glycosylation of circulating tumor cell clusters in response to therapy

Abstract Cellular plasticity and stemness properties enable dynamic changes of circulating tumor cells (CTCs) during cancer dissemination, such as aggregation or cohesion of single CTCs into multicellular CTC clusters with 20-100 times higher metastatic propensity than the singles. Our work demonstrated that stemness glycoproteins on breast cancer stem cells, such as CD44, CD81, and ICAM1 drive CTC aggregation in metastatic breast cancer, such as triple negative breast cancer (TNBC). To determine the glycosylation patterns in CTCs and its association with clinical outcomes, we established multiple CTC analysis approaches including CellSearch, flow cytometry, and immunohistochemistry using patient blood and tissue sections collected at the pre-treatment baseline and after-therapy timepoints. We found that chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of terminal sugar residues α2,6-sialic acids in glycoproteins. CTCs showed dynamic hypo-sialylation in the blood with loss of the sialyl-transferase ST6GAL1, promoting CTC cluster formation with cellular quiescence (proliferative dormancy) and evading chemotherapy in breast cancer. Seeded tumor cells regained ST6GAL1 to enable metastatic colonization. Many adhesion proteins and stemness drivers as glycoprotein substrates of ST6GAL1 drive CTC clustering and metastatic seeding. Neutralizing antibodies against these clustering drivers inhibit CTC cluster formation and improve therapy response, thereby blocking lung metastasis in TNBC. Citation Format: Huiping Liu. Plasticity, dormancy, and glycosylation of circulating tumor cell clusters in response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY34-01.

Abstract 7507: Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients

Abstract Background: Single circulating tumor cells (sCTC) capture without cell fixation with no blood contaminants like leukocytes are highly challenging. sCTC genome transcriptomics anticipates new avenues in new discovery, targeted therapies, and inclusion of patients in clinical trials where the tissue is inaccessible. We report, an assay to selectively capture live sCTCs at singularity with NGS-based comprehensive mutational profiling in breast cancer (BC) patients. Further, paired ctDNA outcomes using blood with mutational heterogeneity scores of sCTCs adds comprehensive genomics for establishing an accelerated treatment outcomes and resistance signatures. Methods: Retrospectively, sCTC were isolated from 70 breast cancer (BC) patients using OncoRadar platform consisting affinity-based glass beads mediated by anti EpCAM antibody. CTCs were selectively captured and released without fixing and contamination of any leukocytes. Platform possess cleavable disulfide spacer at pH 8.0, between glass beads (diam. 2 mm, area 12.57 mm2) and a ligand. Sensitivity, specificity, positive predictive value (PPV), negative prediction value (NPV), and accuracy were evaluated using MCF-7 cells and CTCs. Assay to isolate CTC has been developed in 96 well plate, for downstream NGS-based comprehensive gene panel analysis using hybridization-based target enrichment. Interestingly, no mutations detected in blood samples (~20%) for CtDNA versus CTC DNA offered non-chip implicated mutations. Results:. Analytical validation using OncoRadar showed higher cell capture efficiency (99%) with spiked MCF-7 cells/ml of blood. Sensitivity and specificity values were 97% and 98% respect, while PPV was 99% and NPV was 7.5% with the accuracy parameter of 99%. Sensitivity, specificity, PPV, and NPV parameters when assessed for CTC isolation from 70 BC patients. NGS-based mutational analysis, sCTC showed the presence of PIK3CA, ESR1, and NF1 mutations, which were absent from paired ctDNA samples. Conclusion: Detection of ctDNA obscure mutations in paired sCTC samples suggests high sensitivity of sCTC-based genomic assays compared to current standard of practice. Heterogeneity scores amongst inter and intra CTCs with MATH scores offer new dimensions in genomics. Combination of ctDNA and sCTC DNA assay offers a comprehensive realistic genomic outcome for more significant therapy decisions, especially when guideline based options may exhausts. Citation Format: Gowhar Shafi, Atul Bharde, Ganesh Khutale, Saloni Andhari, Richa Deshpande, Bhagwat Jadhav, Sangeeta Prajapati, Moubeen Fauzul, Kanchan Hariramani, Madhura Basavalingegowda, Jayant Khandare. Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer 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 7507.

Abstract 7506: Comprehensive mutational profile of true single circulating tumor cells compared with CtDNA in advanced pancreatic adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is aggressive malignancy characterized by 5-year survival rate of <10%. Comprehending PDAC's mutational landscape through NGS-based assays, acquiring representative tumor tissue biopsies remains arduous. Single circulating tumor cell (sCTC) genomics could provide clinically relevant personalized diagnostics. Here we demonstrate comparative analysis of sCTCs genomics and ctDNA outcomes in paired advanced PDAC patients. Methods: Retrospectively, 25 live sCTCs and sCTC clusters were isolated from 10 advanced PDAC patients' blood using OncoRadar platform consisting glass beads conjugated with antiEPCam antibodies, tuned for releasing CTCs, without cell fixing, an assay in 96 well plate. Whole genomes from sCTCs were amplified using random degenerate primer-based PCR. Amplified genomes and paired ctDNA were subjected to CGP using clinical assay OncoIndx® to identify patient-specific genomic alterations in sCTC and paired ctDNA samples. The raw sequence alignment and variant calling was performed using iCare software®. Results: Live 25 sCTCs and 8 CTC clusters were isolated (≥2 CTCs). Mutational profiling revealed multiple pathogenic (P) and likely pathogenic (LP) variants. sCTCs exhibited at least twice as many P variants compared to paired ctDNA samples, with CHD4, FGFR2, and HNF1α being the most common. The mutational profile of sCTCs showed concordance with at least one variant in paired ctDNA, and was enriched in dysregulated cytoskeleton and cell migration machinery. Specifically, COL7A, KIF5B, and RHOA mutations were prevalent in almost all sCTCs, with exclusive mutations (KIF5B and RHOA) in some cases. KRAS P variants were exclusive to 50% of ctDNA samples, paired sCTCs exhibited NRAS P variants. RAS isoforms were observed in 70% of sCTCs. Gene amplification occurred in CCND1/2, FGFR2, and ERBB2 in 90% of sCTCs, with CCND1 amplification more prevalent in sCTC clusters. Mutations in epigenetic repressors (HDAC1, KDM2B) were ubiquitous in sCTCs, co-occurring with DDR pathway mutations (F test, P 0.003, OR 11). Loss-of-function BRCA mutations were detected in 60% of sCTCs, and were absent in paired ctDNA. Additionally, 70% of sCTCs exhibited therapy-resistant variants (NF1, STK11, ARID1A, and PIK3CA), contrasting with a 10% frequency in paired ctDNA. Conclusions: Genomic profile of sCTCs in advanced PDAC patients revealed a complex mutational landscape, providing additional clinically relevant insights that complemented ctDNA findings. sCTCs exhibited greater tumor mutational heterogeneity compared to paired ctDNA samples, and detected additional actionable pathogenic variants. Results suggest that sCTCs may offer therapeutically relevant molecular information for advanced PDAC patients for whom tissue is unavailable and ctDNA fails to provide actionable therapy options. Citation Format: Jayant Khandare, Atul Bharde, Ganesh Khutale, Saloni Andhari, Richa Deshpande, Kanchan Hariramani, Madhura Basavalingegowda, Vikas Jadhav, Mohan Uttarwar, Gowhar Shafi. Comprehensive mutational profile of true single circulating tumor cells compared with CtDNA in advanced pancreatic adenocarcinoma [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 7506.

Abstract 3692: Genomic profiling with the novel single-cell IMPACT assay reveals evidence of genetic heterogeneity among CTCs in late-stage breast cancer patients

Abstract Introduction: Liquid biopsy enables minimally invasive molecular profiling of systemic cancer for diagnosis, therapy selection and longitudinal monitoring of minimal residual disease. Cell-based liquid biopsy methods are particularly well suited to examine the extent of genetic heterogeneity and clonal diversity among metastatic cancer cells, which are believed to be the major determinants of therapeutic failure. However, isolation and molecular profiling of circulating tumor cells (CTCs) from body fluids is challenging due to methodological constrains. Methods: To overcome these challenges, we have developed a workflow for detection, isolation, and mutational profiling of CTCs. The workflow utilizes the FDA-approved CellSearch System® and a novel method for single-cell Integrated Mutation Profiling of Actionable Cancer Targets (scIMPACT). Our workflow was optimized for reliable detection of copy number variations (CNVs) and single nucleotide variations (SNVs) in single CTCs. The method was then applied to 31 CTCs alongside with 15 matched germline samples. Results: After implementation of a customized Bayesian neural network based algorithm, accounting for biases introduced during amplification of single-cell DNA and prioritizing the detected mutations, we achieved a sensitivity of 91% and a specificity of 96%, AUC = 0.98. The CTC analysis revealed presence of CNVs affecting known oncogenes and tumor suppressor genes (including MYC, CCND1, FGF4, TP53 and RB1) as well as SNVs classified as pathogenic (e.g., ARID1A Q775*). Notably, in the examined specimens most of the chromosomal aberrations (51-82%) were clonal across all cells of a given patient. In contrast, most of the detected somatic SNVs (67-96%) were sub-clonal and thus present only in a subset of CTCs. Conclusion: In summary, the novel scIMPACT workflow enabled reliable and accurate genomic profiling of patient-derived CTCs in late-stage breast cancer patients. Our proof-of-concept study revealed evidence of genetic diversity of SNVs among the examined CTCs, which might have been acquired late in progression but relevant for therapy escape in the studied cases. Further analyses are still needed for assessing the generalizability of our finding as well as for deeper understanding of the molecular basis orchestrating the establishment and maintenance of heterogeneous genotypes of CTCs in breast cancer. Citation Format: Zbigniew T. Czyż, Clara Chaiban, Adithi Ravikumar Varadarajan, Cäcilia Köstler, Vadim Dechand, Jonas Grote, Thomas Ragg, Bernhard Polzer, Jens Warfsmann, Christoph A. Klein. Genomic profiling with the novel single-cell IMPACT assay reveals evidence of genetic heterogeneity among CTCs in late-stage breast cancer 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 3692.

Abstract 2303: Identifying and stratifying circulating tumor cell cluster subtypes in metastatic breast cancer patients and evaluating their clinical outcomes

Abstract Background: Circulating tumor cell clusters (CTCCs) are aggregated groups of tumor cells that detached from primary tumors and circulate the bloodstream. However, while Circulating Tumor Cells (CTCs) are a well studied phenomenon, CTCCs remain relatively unexplored and ill-defined, with only a few descriptive investigations evaluating their clinical utility. Adding to CTCC complexity is that various subtypes exist, including homotypic clusters consisting of a homogenous population of tumor cells and heterotypic CTCCs made of CTCs with immune/stromal white blood cells (WBCs). Furthermore, CTCs can undergo Epithelial-Mesenchymal Transition (EMT), a process characterized by loss of epithelial traits and upregulation of mesenchymal traits, and can form clustered EMTs (CEMTs). Moreover, CTCs can fuse with macrophages forming Tumor Macrophage Hybrid Cells, aka Cancer-Associated Macrophage Like cells (CAMLs) when in circulation. We enumerated all these types of cells and complex aggregates in the peripheral blood of patients with metastatic breast cancer (mBC). Methods: We enumerated the 6 populations from patient samples enrolled in IRB aproved prospective pilot studies. Whole peripheral blood (7.5mL) was filtered and stained with cytokeratin (CK) & CD45/CD14 to identify CTCs. CTCs were defined as an intact DAPI nuclei and strong filamentous CK. Homotypic CTCCs were defined as ≥2 CTCs attached together. Heterotypic CTCCs were defined as ≥1 CTC attached to ≥1 WBC. EMTs were defined as having DAPI nuclei and weak non-filamentous CK. CEMTS were defined as ≥2 EMTs. CAMLs were defined as having an enlarged polynucleated DAPI, and positive for CD45/CD14 or non-filamentous CK. Median progression free survival (mPFS) and median overall survival (mOS) over 24 months (m) were evaluated. Results: We evaluated the blood of 79 mBC patients. Single CTCs were found in 57% of patients (n=34/79), homotypic CTCCs 15% (n=12/79), heterotypic CTCCs 66% (n=27/79), EMTs 56% (n=44/79), CEMTs 23% (n=18/79) and CAMLs 97% (n=77/79). Over 24 ms, patierns with detectable heterotypic CTCCs & homotypic CTCCs had the shortest response times at mPFS=2.8 & mOS=5.8 ms, or mPFS=2.1 & mOS=4.3 ms, respectively. This was followed by CTCs mPFS=3.7 & mOS=13.7 ms, EMTs mPFS=4.3 & mOS=14.4 ms, CAMLs mPFS=4.3 & mOS=14.4 ms, and CEMT mPFS=4.7 & mOS=13.2 ms. Additionally, CTCCs were rare in HER2+ patients at 4.4% (n=1/23), but no associations to other hormone statuses were found. Conclusion: CTCCs appear to represent an array of subtypes with different biological and clinical meanings. Here we enumerated CTCC subtypes in the blood of mBC patients and stratified the various types to clinical outcomes. These data suggest CTCCs are clinically important and further understanding of their biology in the context of tumor pathogenesis is needed. Citation Format: Daniel L. Adams, Carolina Reduzzi, Aravind Aryasomayajula, R. Katherine Alpaugh, Saranya Chumsri, Cha-Mei Tang, Giuseppe Del Priore, William V. Williams, Massimo Cristofanilli. Identifying and stratifying circulating tumor cell cluster subtypes in metastatic breast cancer patients and evaluating their clinical outcomes [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 2303.