Circulating Tumor Cells Assay Research Articles

Acoustic Enrichment of Heterogeneous Circulating Tumor Cells and Clusters from Metastatic Prostate Cancer Patients.

There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on the microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility), resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogeneous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding a higher number of CTCs using acoustophoresis. Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Liquid Biopsy for Detection of Pancreaticobiliary Cancers by Functional Enrichment and Immunofluorescent Profiling of Circulating Tumor Cells and Their Clusters.

Circulating tumor cells (CTCs) have historically been used for prognostication in oncology. We evaluate the performance of liquid biopsy CTC assay as a diagnostic tool in suspected pancreaticobiliary cancers (PBC). The assay utilizes functional enrichment of CTCs followed by immunofluorescent profiling of organ-specific markers. The performance of the assay was first evaluated in a multicentric case-control study of blood samples from 360 participants, including 188 PBC cases (pre-biopsy samples) and 172 healthy individuals. A subsequent prospective observational study included pre-biopsy blood samples from 88 individuals with suspicion of PBC and no prior diagnosis of cancer. CTCs were harvested using a unique functional enrichment method and used for immunofluorescent profiling for CA19.9, Maspin, EpCAM, CK, and CD45, blinded to the tissue histopathological diagnosis. TruBlood® malignant or non-malignant predictions were compared with tissue diagnoses to establish sensitivity and specificity. The test had 95.9% overall sensitivity (95% CI: 86.0-99.5%) and 92.3% specificity (95% CI: 79.13% to 98.38%) to differentiate PBC (n = 49) from benign conditions (n = 39). The high accuracy of the CTC-based TruBlood test demonstrates its potential clinical application as a diagnostic tool to assist the effective detection of PBC when tissue sampling is unviable or inconclusive.

Abstract 7497: Advancing multi-biomarker CTC assay and CDx development through the automated GenoCTC system

Abstract Introduction: The successful outcomes observed in clinical trials emphasize the effectiveness of therapeutic approaches relying on biomarker tests, particularly in the development of cancer drugs. The parallel development of a drug and its associated predictive biomarker is considered an ideal strategy for achieving rapid and successful drug development in precision medicine. In this context, we employ an automated circulating tumor cell (CTC) isolation platform, known as GenoCTC, to facilitate the development of biomarker-driven CTC assays, with the ultimate goal of developing companion diagnostic (CDx) tests. Method: This study employed the 5th generation GenoCTC device, a fully automated system designed for marker-based CTC enrichment using immune magnetophoresis. We confirmed the isolation of CTCs through spiking tests, utilizing cancer biomarkers such as cMET, Mesothelin (MSLN), and Claudin 3 (CLDN3). cMET is a biomarker extensively studied in various cancers, particularly non-small cell lung cancer. We previously showed the prognostic significance of cMET expressing CTCs in HR+ and HER2- metastatic breast cancer patients. MSLN and CLDN3 are tumor-associated proteins showing overexpression across diverse cancer types, with a potential for the development of antibody-based cytotoxic drugs. The GenoCTC device allows efficient separation of CTCs expressing these biomarkers, ensuring high accuracy, precision, specificity, and sensitivity. Simultaneously, therapeutic drugs directed at these markers are progressing through phase trials or preclinical studies. Conclusion: Our results show that cMET, MSLN, and CLDN3 are potential markers for the development of biomarker tests, that can improve the patient selection process for personalized therapy targeting these biomarkers. Citation Format: Chaithanya Chelakkot, Jieun Park, Chae Rin Kim, Yeonu Lee, Yu Rim Lee, Saehyung Lee, Jun Young Choi, Young Ho Moon, Yoon-La Choi, Young Kee Shin, Hun Seok Lee. Advancing multi-biomarker CTC assay and CDx development through the automated GenoCTC system [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 7497.

Abstract 7486: Development and analytic validation of an ARv7/PSMA dual biomarker circulating tumor cell assay

Abstract Background Prostate cancer drug targets and biomarkers may be investigated non-invasively on circulating tumor cells (CTCs) obtained via liquid biopsies. PSMA is an important drug target in prostate cancer, and ARv7 is a splice variant that confers resistance to second-generation androgen receptor inhibitors. In this study, we describe the development and analytic validation of a novel assay to quantify ARv7 and PSMA protein expression on CTCs. The assay was validated with blood samples from patients with Stage IV prostate cancer to demonstrate clinical feasibility. To our knowledge, a PSMA/ARv7 dual protein assay for CTCs has not previously been reported. Methods LNCaP (PSMA positive), PC3 (PSMA negative), 22Rv1 (ARv7 positive), and BT474 (ARv7 negative) cells were spiked into healthy donor blood to generate model CTCs. Nucleated cells were collected from these spike-in samples using the AccuCyte® sample preparation kit and spread onto microscope slides. Slides were fixed and stained with antibodies to cytokeratin, EpCAM, and CD45 to detect CTCs and exclude white blood cells (WBCs), and with PSMA and ARv7 to evaluate protein expression on identified CTCs. The slides were imaged with the CyteFinder® automated immunofluorescence scanning microscope that identifies CTCs for confirmation by trained reviewers based on machine learning algorithms and quantitatively measures biomarker signal mean fluorescence intensity (MFI). Results For ARv7/PSMA expression classification of the model CTCs, the dual biomarker assay showed analytic sensitivity of 83%/97% and specificity 98%/99%, yielding accuracy of 90.2%/97.9%, respectively. The dynamic range of MFI for both biomarkers was approximately three orders of magnitude. Inter-run reproducibility CV was 13.6% (ARv7) and 6.9% (PSMA). Slide repeatability mean CV was 11.1% (ARv7) and 12.0% (PSMA). Clinical feasibility of the assay was investigated in 9 progressing Stage IV prostate cancer patient blood samples. CTCs were identified in all clinical samples (median 4, range 1 - 384). Thresholds for biomarker positivity were established based on MFI measurements of positive and negative control cell lines. ARv7 positivity in clinical samples ranged from 33% to 100%, and PSMA positivity ranged from 0% to 75%. Conclusions We have developed a dual ARv7/PSMA assay that simultaneously measures protein expression of both biomarkers on individual CTCs. The assay is accurate and reproducible for the assessment of ARv7 and PSMA expression by liquid biopsy in clinical studies. Citation Format: Jon Ladd, Erin Bayer, Brock Bartels, Eric Kaldjian, Arturo Ramirez. Development and analytic validation of an ARv7/PSMA dual biomarker circulating tumor cell assay [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 7486.

Abstract 6562: Comprehensive liquid biopsy: Multi-analyte evaluation of cellular and plasma components of blood in prostate cancer from a single blood collection tube

Abstract Background Liquid biopsy samples offer a non-invasive method for investigating drug targets and biomarkers in solid tumors. In this study, we describe a comprehensive liquid biopsy analysis of whole blood to identify circulating tumor cells (CTCs), large extracellular vesicles and CTC fragments, and CTC-white blood cell hybrids; quantify protein biomarker expression on CTCs; perform targeted mutation profiling on CTCs and cell-free DNA (cfDNA) from plasma; and analyze protein expression on white blood cells (WBCs) from a single 7.5 mL blood sample. This array of tests was applied to blood samples from patients with Stage IV prostate cancer to demonstrate clinical feasibility. Methods A novel dual biomarker CTC assay was developed to measure protein expression of PSMA and ARv7 on CTCs from stage IV prostate cancer patients. Nucleated cells from clinical samples were processed to microscope slides using AccuCyte®, an unbiased, density-based separation method that also allows for plasma collection for downstream analysis. Slides were fixed and stained with antibodies to cytokeratin, EpCAM, and CD45 to detect CTCs and exclude WBCs, and with PSMA and ARv7 to evaluate protein biomarker expression on identified CTCs. The slides were imaged with the CyteFinder® automated immunofluorescence scanning microscope that identifies CTCs for confirmation by trained reviewers and quantitatively measures protein mean fluorescence intensity (MFI) on any cells detected on the slides (CTCs, CTC-WBC hybrids, large extracellular vesicles and CTC fragments and WBCs). Individual CTCs and control pools of WBCs from clinical samples with at least 4 CTCs were picked using CytePicker®, a needle-based system used to isolate single cells. cfDNA was isolated from plasma removed during the AccuCyte® process using a QIAamp MinElute ccfDNA kit. Picked CTCs, WBC pools, and isolated ctDNA were sequenced using the OncoZoom Cancer HotSpot panel from Paragon Genomics. Results Nine progressing Stage IV prostate cancer patient blood samples were stained with a novel PSMA/ARv7 dual biomarker assay. CTCs were identified in all clinical samples (median 4, range 1 - 384). PSMA positivity in clinical samples ranged from 0% to 75%, and ARv7 positivity ranged from 33% to 100%, and. Additional analysis using QuPath was performed to determine PSMA/ARv7 protein expression on WBCs and CTC fragments. ctDNA analysis from 5 individual samples was compared to sequencing results from individual CTCs from the same samples. Conclusions Using a single 7.5 mL blood liquid biopsy, we can measure protein expression on enumerated CTCs and quantify these same protein biomarkers on WBCs and CTC fragments/oncosomes, determine the genetic makeup of the tumor through the sequencing of cfDNA, and monitor genetic mutations in intact CTCs in circulation. Citation Format: Jon Ladd, Erin Bayer, Brock Bartels, Arista Tischner, Rachel Ponting, Eric Kaldjian, Arturo Ramirez. Comprehensive liquid biopsy: Multi-analyte evaluation of cellular and plasma components of blood in prostate cancer from a single blood collection tube [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 6562.

Validation of a single-cell sequencing assay for the detection of circulating tumor cells in metastatic breast cancer.

1053 Background: Blood Biopsy and the evaluation of Circulating Tumor Cells (CTC) has provided valuable clinical information for patients with metastatic breast cancer, specifically where a tissue biopsy is not feasible. A critical problem in the field is that most assays cannot distinguish true CTCs from other types of rare cells. By combining established protein biomarkers associated with circulating epithelial cells with single-cell whole-genome sequencing to assess copy number variations (CNVs, a signature for genomic instability - GI), we differentiate rare cells from true CTCs. Additionally, we can determine the presence or absence of regions that contain gene amplifications (e.g., ERBB2/HER2). Here we report the validation of a clinical blood biopsy CTC assay that can enumerate CTCs, determine if those CTCs have GI and if selected CTCs are expressing HER2 or ER antigens. Methods: Relevant cell line controls of epithelial origin and known expression levels of ER or HER2 were spiked into healthy donor whole blood at varying concentrations. The nucleated cells were deposited on microscope glass slides at high density. Immunofluorescence staining of slides was performed to identify the presence and localization of nuclei (via DAPI), pan-CK, HER2, ER, CD45 and CD31. Nucleated cells were classified as CTC candidates if they were pan-cytokeratin(+) and CD45/CD31(-), and were further characterized for expression of ER and HER2 protein. Defined CTC candidates were imaged and located by specific coordinates allowing single-CTC isolation for whole genome amplification. Single-cell sequencing data was then analyzed with the Epic CTC DNA copy number analysis pipeline to detect GI and amplification at the ERBB2 locus. Results: The limit of detection for CTCs was determined to be 1 CTC in 6 million white blood cells with a linear range of 1- 300 CTC per slide. The sensitivity and specificity for HER2 detection was determined to be 94% and 97%, respectively. ER assay sensitivity and specificity were 91% and 100%, respectively. Overall accuracy was 100% for HER2 and 94% for ER detection. Overall precision across all relevant variables produced %CVs <10% for HER2 and ER. A genome possessed GI if more than 12 large-scale transitions (LSTs) were detected. For detection of 2-fold amplification at the ERBB2 locus, gene amplification was detected with 85% sensitivity and 94% specificity. Tested healthy donor blood samples were absent of detected CTCs and lacked LST/GI or ERBB2 amplification in all (46) isolated WBCs. Conclusions: These results reflect the validation of a novel clinical assay combining CTC detection with single-cell GI and amplification of the ERBB2 locus. This assay better discriminates CTC detection with the addition of an assessment of GI. Additionally, along with protein expression measures of ER and HER2, it provides the ability to assess if HER2 expression is driven by ERBB2 amplification.

A combination of circulating tumor cell (CTC) and cancer stem cell (CSC) markers to predict the prognosis of breast cancer.

e13007 Background: CTCs are tumor cells that shed from either primary tumor or metastatic foci and can provide more information about the tumor's biological entity than circulating tumor DNA (ctDNA), or exosomes do. The current FDA-approved CTC assay misses the CSC phenotypes, which are pivotal in driving tumorigenesis, metastasis, resistance to treatment, and cancer recurrence. Therefore, we combine CTC and CSC markers to determine the prognosis. We hypothesized that the CSC markers on CTC correlate with poorer outcomes. Methods: Peripheral blood samples of qualified breast cancer patients were collected at diagnosis and every 6 months during follow-up. EpCAM, MUC-1, and HER2 are used as CTC markers, while TWIST, AKT, PI3K, and ALDH-1 are used to detect CSC. According to the expression level of the above markers, patients were categorized into four groups: NN negative with both CTC and CSC markers, NP expressing CSC markers, PN expressing CTC markers, and PP positive with both CTC and CSC markers. The progression-free survival (PFS) and overall survival (OS) of all four groups were calculated by Kaplan-Meier model from the date of surgery until the disease progression and death/the last follow-up of the patients, respectively. Results: We screened 100 patients aged 18 years or older with confirmed stage II to stage IV breast cancer and selected 81 patients for the study. Fifteen patients dropped out of the study due to loss of follow-up or incomplete clinical information leaving 66 evaluable patients. There are 55 Caucasians (83%), 5 African Americans (7.5%), 5 Hispanics (7.5%), and 1 Asian (1.5%). The median age of the patients is 52.5 years old (29-81), and the median follow-up is 22.6 months (2.5-65). Most patients have stage III disease (60 and 91%) followed by 4 stage IV (6%) and 2 Stage II (3%). The median time between the first sample date and surgery is 172 days (-148 to 270). The NN group has the most patients (41 and 62%) compared to NP group (16 and 24%) and PN group (6 and 9%). The PP group has the least number of patients (2 and 3%) and is not observed in patients with positive hormonal receptors (HR). The PN group has a higher ratio of HR-HER2+ patients than NP group does. The HR and HER2 statuses were checked in the four groups. Interestingly, the NN and NP groups are mostly HR+HER2- patients, while the PN group is mainly HR- patients regardless of the HER2 status. The PP group only has HR- patients. The PFS curves start to flatten at 12 months and separate among the four groups, with the highest PFS percentage in the NP (81.2%) and NN (77.8%) followed by the PN (62.5%) and PP (50%) group. The OS percentage is 70% in NN, 66% in PN, 60% in NP, and 50% in PP, similar to the PFS pattern. Conclusions: Checking both CTC and CSC markers help triage the prognosis of breast cancer patients. CTC and CSC markers observed in breast cancer patients with different HR or HER2 status can be further investigated for potential correlations.

High-performance assay of HepG2 cells through inertial separation in a microfluidic chip coupled with fluorescence and photoelectrochemical detection

As a non-invasive, highly specific and sensitive assay strategy, liquid biopsy plays an important role in the diagnosis, the prognosis and the adjuvant therapy of cancer. Circulating tumor cell (CTC) have been considered as a crucial biomarker for liquid biopsy. However, CTCs are difficult to be detected accurately due to their very low content in blood. Herein, HepG2 cells stained with Hcy-716 fluorescence probe were separated and enriched from blood through inertial separation in a microfluidic chip, and then were detected by fluorescent and photoelectrochemical strategies simultaneously. Sulfhydryl functionalized TLS11a aptamers were fixed on a gold nanorods modified screen printing electrode surface to supply a photoelectrochemical cytosensing platform for HepG2 cells. Under optimized conditions, the separation efficiency and the purity of HepG2 cells can reach 82.6% and 73.4%, respectively. A linear response was obtained in the range of 1 × 102 ∼ 5 × 105 cells mL−1, and the detection limit was calculated to be 15 cells mL−1. The separation with microfluid chip and detection with fluorescent imaging and photoelectrochemical aptasensing dual-model strategy provide a practical tool for CTCs assay, thus benefiting for liquid biopsy in cancer diagnosis.

Utilization of Circulating Tumor Cells in the Management of Solid Tumors.

Circulating tumor cells (CTCs) are tumor cells shed from the primary tumor into circulation, with clusters of CTCs responsible for cancer metastases. CTC detection and isolation from the bloodstream are based on properties distinguishing CTCs from normal blood cells. Current CTC detection techniques can be divided into two main categories: label dependent, which depends upon antibodies that selectively bind cell surface antigens present on CTCs, or label-independent detection, which is detection based on the size, deformability, and biophysical properties of CTCs. CTCs may play significant roles in cancer screening, diagnosis, treatment navigation, including prognostication and precision medicine, and surveillance. In cancer screening, capturing and evaluating CTCs from peripheral blood could be a strategy to detect cancer at its earliest stage. Cancer diagnosis using liquid biopsy could also have tremendous benefits. Full utilization of CTCs in the clinical management of malignancies may be feasible in the near future; however, several challenges still exist. CTC assays currently lack adequate sensitivity, especially in early-stage solid malignancies, due to low numbers of detectable CTCs. As assays improve and more trials evaluate the clinical utility of CTC detection in guiding therapies, we anticipate increased use in cancer management.

Abstract 2296: Engineering and evaluation of a novel circulating tumor cell capture assay for urothelial carcinoma

Abstract Background: Circulating tumor cells (CTCs) are promising real-time, noninvasive biomarkers to assess therapy response and to monitor disease. However clinical applications of CTCs in urothelial carcinomas (UC) remain limited. There is a need for sensitive and disease-specific CTC assay for urothelial cancer. As CTCs are very rare in blood compared to blood cells and may vary due to tumor heterogeneity, detection and isolation of them solely based on one biomarker (e.g., EpCAM) would be insufficient. To achieve better results and increase clinical relevance, we designed a CTC capture device that was functionalized with multiple antibodies specific to UC. Methods: We engineered a novel UC-CTC capture device using nanotechnology and biomimetic cell rolling. To capture UC cells, we examined 4 biomarkers: EpCAM, Nectin-4, Trop-2, and FGFR3, that are highly expressed in UC. We engineered CTC assays using single and multi-biomarker capture surfaces. The capture sensitivity and selectivity of the device were investigated using four bladder cancer cell lines as well as a leukocyte model, HL-60, as negative control. CTCs are defined as CK+/DAPI+/CD45- cells. The capture sensitivity was compared between the different surfaces with each of the different antibodies and the final UC-CTC chip with optimized antibody mix was chosen to detect CTCs from UC patients. Results: We examined the CTC capture using 4 UC cell lines: UM-UC-1, T-24, HT1376, RT4. The devices that utilized single antibodies were much less sensitive (capture efficiencies less than 82%). In comparison, combination antibody capture improved sensitivity to >90%. Negative control using the HL-60 cells showed minimal non-specific binding, <0.3%. We were also able to validate the CTC assay using patient samples and demonstrated high sensitivity. For example, we captured ~302 CTCs/mL in a patient who had early-stage T2 muscle invasive UC. Conclusion: We developed a novel CTC capture device using four UC-specific capture antibodies for the detection and isolation of CTCs in UC. Compared to the configuration of EpCAM alone, the mixture of multiple antibodies improved the capture efficiency up to 1.4-fold for in vitro bladder cancer cell spiking assays. High specificity was also achieved, with low levels of non-specific leukocyte detection. High sensitivity and specificity suggest that this platform has potential impact to detect CTCs from patients with UC. Citation Format: Qinhan Zhou, Michael Poellmann, Nikhila Reddy Sultanpuram, Damla Gunenc, Krista English, Luke Chen, Runze Zhao, Seungpyo Hong, Andrew Wang, Tian Zhang. Engineering and evaluation of a novel circulating tumor cell capture assay for urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2296.

Abstract 2300: Non-invasive liquid biopsy assay for the detection of ASCL1 expression in CTCs of patients with lung cancer

Abstract Background: Lung cancer (LC) is the deadliest cancer worldwide, with small cell LC (SCLC) and neuroendocrine (NE) LC being the most aggressive and lethal forms. The transcription factor, achaete-scute homolog 1 (ASCL1), regulates neuronal and NE cell development, and is a hallmark of the ASCL1(+) SCLC subtype. Further, investigating subtype has become increasingly important as new drug candidates emerge for SCLC that may be efficacious in only some patients. ASCL1 by IHC tissue biopsy is highly specific, detecting patients who have transformed from NSCLC to SCLC and have acquired resistance to EGFR tyrosine kinase inhibitor therapy in NSCLC. Quantification of ASCL1 on circulating tumor cells (CTCs) is a blood-based, non-invasive alternative to tissue biopsy that provides functional protein-level expression data to monitor lung cancer patients across the disease continuum, informing patient treatment. Methods: A 4-color immunofluorescence (IF) assay was developed to detect ASCL1 expression in CTCs from routine blood samples, comprising detection of nucleated cells (DAPI), cytokeratin positive CTCs, CD45(+) white blood cells (WBC), and ASCL1(+) cells. During validation, cancer cell lines (SHP-77 [ASCL(+)] and U2-OS [ASCL1(-)]) were spiked into normal blood at a ratio of 1:10,000 (CLC/WBC) and plated onto coated glass slides. Slides were stained, imaged, and CTC candidates were identified with a proprietary imaging algorithm. The threshold of ASCL1 positivity (316 MFI) included 99% of all ASCL1(-), U2-OS cells. Assay specificity, sensitivity, overall accuracy, and subcellular localization of biomarker expression were assessed. The ability of the assay to quantify ASCL1 expression in CTCs was confirmed using 31 samples from 11 Extensive Stage SCLC patients. Results: The ASCL1 CTC assay achieved an overall accuracy of 0.992 (95% CI 0.081, 0.993), and an analytical specificity and sensitivity of 0.990 and 0.995, respectively. The assay displayed a positive predictive value and a negative predictive value of 0.979 and 0.998, respectively, determined by evaluating ASCL1 expression in SHP-77 (N=4062) and U2-OS (N=8645). ASCL1 expression was detected in the nucleus of all SHP-77 and majority of SCLC patient CTCs. Of the 11 Extensive Stage SCLC patients tested, 9 (82%) showed at least one conventional CTC (0.02 - 9.63 CTCs/mL), and all 9 (100%) had at least 1 ASCL1(+) CTC (0.06 - 5.5 CTCs/mL). Conclusion: This blood based ASCL1 assay is a highly specific and sensitive approach to monitor ASCL1 expression on CTCs isolated from the blood of lung cancer patients. Citation Format: Luisa Fernandez, Andrew Kunihiro, Mark Frilles, Alisa Tubbs, Jason Christiansen, David Bourdon. Non-invasive liquid biopsy assay for the detection of ASCL1 expression in CTCs of patients with lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2300.

Abstract P5-06-01: A Multi-center Clinical Study to Harvest and Characterize Circulating Tumor Cells from Patients with Metastatic Breast Cancer Using the Parsortix® PC1 System in support of FDA clearance

Abstract Background: Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a non-invasive surrogate source of tumor material to investigate tumor characteristics in real-time. However, the only FDA-cleared CTC assay is limited to the enumeration of surface marker-defined epithelial cells and not designed for further characterization of the CTCs identified. The Parsortix® PC1 system is a semi-automated microfluidic device capable of capturing and harvesting CTCs from peripheral blood based on cell size and deformability, making it cell-surface marker agnostic. Here, we demonstrate that the Parsortix® PC1 system enables the enrichment and capture of CTCs from the blood of patients with metastatic breast cancer (MBC) and their interrogation using evaluation techniques commonly available in clinical laboratories. Methods: As part of a multicenter clinical trial (NCT03427450), peripheral blood samples from 216 patients with MBC and 205 healthy volunteers (HVs) were prospectively collected at four different clinical sites located throughout the United States. Each subject provided two separate blood samples collected into K2EDTA Vacutainer® tubes to be processed using the Parsortix® PC1 system on the same day. The cells harvested from one of the blood samples collected from each subject by the Parsortix® PC1 system were deposited onto cytology slides using a cytocentrifugation method and stained with Wright-Giemsa reagents using an automated stainer. The stained slides were subjected to cytopathological evaluation by a board-certified pathologist to enumerate CTCs. As proof of principle, cells harvested from the second blood sample were evaluated using one of three additional techniques: molecular profiling by qRT-PCR, RNA sequencing, or cytogenetic analysis of HER2 amplification by FISH. Results: Cytologic examination identified one or more cells as a CTC in 48.5% (95% CI of 41.5 – 55.4%) of the 194 patients with MBC and 9.9% (95% CI of 6.4 – 14.9%) of the 192 HVs. The results from the qRT-PCR evaluation (102 HVs and 74 MBC patients) showed differential expression of cancer-related genes (KRT19, EPCAM, and TWIST1) in the patients with MBC compared to the HVs. Results from the RNA sequencing (53 HVs and 16 MBC patients) showed differential expression of several genes involved in the Kegg Cancer Pathway in the patients with MBC compared to the HVs. The results from the HER2 FISH evaluation (38 HVs and 101 MBC patients) showed that while the majority of the CTC identified had normal HER2/CEP17 ratios, detection of HER2 amplification was possible. Conclusions: The Parsortix PC1 system is capable of capturing and harvesting CTCs from the peripheral blood of patients with MBC. Harvested cells can be evaluated using standard orthogonal methodologies such as gene expression and FISH to identify and characterize CTCs. Based in part on the above results, the FDA granted a De Novo classification request (DEN200062) for the Parsortix PC1 device in May of 2022. Citation Format: Evan Cohen, Gitanjali Jayachandran, Richard Moore, Massimo Cristofanilli, Julie E. Lang, Joseph Khoury, Michael F. Press, Heather McBride, Kyu Kwang Kim, Negar Khazan, Qiang Zhang, Youbin Zhang, Roberta Guzman, Michael C. Miller, James Reuben, Naoto T. Ueno. A Multi-center Clinical Study to Harvest and Characterize Circulating Tumor Cells from Patients with Metastatic Breast Cancer Using the Parsortix® PC1 System in support of FDA clearance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-01.

Performance of a blood-based liquid biopsy test to detect PSMA expression on circulating tumor cells in men with metastatic prostate cancer.

256 Background: PSMA-targeted radioligand therapy has dramatically improved clinical outcomes in men with mCRPC. A blood-based liquid biopsy assay to detect PSMA expression on circulating tumor cells (CTCs) could be helpful to optimize therapeutic decisions and identify patients most likely to benefit from PSMA-targeted therapies. Here we describe the development of this PSMA blood-based assay and the application of the single cell sequencing (SCS) pipeline to identify copy number alterations and further characterize heterogeneity in late-stage disease. Methods: A 4-color immunofluorescence (IF) assay was developed to detect PSMA expression on CTCs on the Epic Sciences liquid biopsy platform. CTCs were identified as DAPI+, cytokeratin (CK)+, and CD45- with appropriate cell morphology. Cells were plated onto glass slides and subjected to IF staining followed by CTC identification using a high throughput scanning platform with digital pathology machine learning algorithms. Cell line cells (LNCaP, PC3s, 22Rv1) of varying PSMA expression levels were spiked into normal blood (CLC/WBC; 1:10000) to create laboratory derived slides. Assay specificity, sensitivity, limit of detection (LOD), precision, and overall accuracy, as well as subcellular localization of biomarker expression were reported. Clinical validation was performed in a cohort of high-risk mCRPC patients (N=24) with previously demonstrated PSMA expression by tissue biopsy, utilizing 3ml of blood. Additionally, a subset of CTCs (36 cells from 11 patients) underwent single-cell low-pass whole genome sequencing to identify copy number alterations. Results: The PSMA CTC assay (Epic Sciences) achieved analytical specificity, sensitivity, and overall accuracy of over 99%. Precision was evaluated using intra-class correlation (ICC) (0.98). The assay showed an LOD of 1 CLC/technical replicate. PSMA expression was specific to staining outside the nucleus and distinguished from CK cytoplasmic staining. Analysis of 24 mCRPC samples revealed that 21/24 (88%) had at least one conventional CTC/mL (range: 0-30 CTCs/mL), of which 16/21 (76%) were PSMA positive (range: 0-27 CTCs/mL) with considerable tumor heterogeneity. 36 sequenced CTCs (N=11 pts) exhibited genomic heterogeneity and frequent AR (3/11), MYC (7/11), and MYCN (5/11) gains or losses of PTEN (4/11), TP53 (7/11), BRCA2 (5/11), CHD1 (6/11), and/or RB1(5/11 pts). Conclusions: The PSMA CTC assay analytical results firmly support its feasibility to detect PSMA expression in CTCs from mCRPC patient samples. The assay demonstrates high sensitivity, specificity, and overall accuracy. Many ongoing trials are designed to target PSMA-positive tumors, positioning this assay as a robust research tool to identify and longitudinally monitor PSMA expression and explore heterogeneity, response, and resistance in these patient populations.

A Multi-Center Clinical Study to Harvest and Characterize Circulating Tumor Cells from Patients with Metastatic Breast Cancer Using the Parsortix® PC1 System.

Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a surrogate source of tumor material that can be obtained via a venipuncture (also known as a liquid biopsy) and used to better understand tumor characteristics. However, the only FDA-cleared CTC assay has been limited to the enumeration of surface marker-defined cells and not further characterization of the CTCs. In this study, we tested the ability of a semi-automated device capable of capturing and harvesting CTCs from peripheral blood based on cell size and deformability, agnostic of cell-surface markers (the Parsortix® PC1 System), to yield CTCs for evaluation by downstream techniques commonly available in clinical laboratories. The data generated from this study were used to support a De Novo request (DEN200062) for the classification of this device, which the FDA recently granted. As part of a multicenter clinical trial, peripheral blood samples from 216 patients with metastatic breast cancer (MBC) and 205 healthy volunteers were subjected to CTC enrichment. A board-certified pathologist enumerated the CTCs from each participant by cytologic evaluation of Wright-Giemsa-stained slides. As proof of principle, cells harvested from a concurrent parallel sample provided by each participant were evaluated using one of three additional evaluation techniques: molecular profiling by qRT-PCR, RNA sequencing, or cytogenetic analysis of HER2 amplification by FISH. The study demonstrated that the Parsortix® PC1 System can effectively capture and harvest CTCs from the peripheral blood of MBC patients and that the harvested cells can be evaluated using orthogonal methodologies such as gene expression and/or Fluorescence In Situ Hybridization (FISH).

1475P Association of circulating tumor cells (CTCs) with immune checkpoint inhibitor (ICI) responses in metastatic renal cell carcinoma (mRCC)

mRCC treatment has expanded to include several ICIs and combinations with targeted kinase inhibitors. However, only clinical criteria has been validated to predict for ICI treatment responses. We previously described a highly sensitive CTC assay with multivalent binding, e-selectin induced cell rolling, and multiple antibody ligands. We prospectively evaluated whether CTC change would predict for ICI response in mRCC.

Circulating tumor cell assay to non-invasively evaluate PD-L1 and other therapeutic targets in multiple cancers

Biomarker directed selection of targeted anti-neoplastic agents such as immune checkpoint inhibitors, small molecule inhibitors and monoclonal antibodies form an important aspect of cancer treatment. Immunohistochemistry (IHC) analysis of the tumor tissue is the method of choice to evaluate the presence of these biomarkers. However, a significant barrier to biomarker testing on tissue is the availability of an adequate amount of tissue and need for repetitive sampling due to tumor evolution. Also, tumor tissue testing is not immune to inter- and intra-tumor heterogeneity. We describe the analytical and clinical validation of a Circulating Tumor Cell (CTC) assay to accurately assess the presence of PD-L1 22C3 and PD-L1 28.8, ER, PR and HER2, from patients with solid tumors to guide the choice of suitable targeted therapies. Analytically, the test has high sensitivity, specificity, linearity and precision. Based on a blinded case control study, the clinical sensitivity and specificity for PD-L1 (22C3 and 28.8) was determined to be 90% and 100% respectively. The clinical sensitivity and specificity was 83% and 89% for ER; 80% and 94% for PR; 63% and 89% for HER2 (by ICC); and 100% and 92% for HER2 (by FISH), respectively. The performance characteristics of the test support its suitability and adaptability for routine clinical use.

Abstract 1950: A novel ESR1 mutation assay for single circulating tumor cell by application of DEPArray࣪ System and Digital Droplet PCR

Abstract Introduction: Although circulating tumor cells (CTC) display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. ESR1 mutations are a major mechanism of acquired endocrine resistance in metastatic breast cancer (MBC). We previously reported that ESR1 mutations in circulating tumor DNA (ctDNA) was associated with worse prognosis in MBC (2020 ASCO). Herein, we report a new ultra-high sensitivity approach of ESR1 mutations assay for single CTC by using DEPArray system and Droplet Digital PCR (ddPCR). Methods: Whole blood sample (7.5ml/each) was collected from stage IV MBC patients and then CTC enumeration was performed in CellSearch™ System by targeting the EpCAM antigen (2020 AACR #3120). The single CTC and single white blood cell (WBC) were then isolated from CellSearch cartridges by using DEPArray࣪ System (Menarini). The single CTC DNA was isolated by QIAamp DNA Micro Kit (Qiagen). ESR1 mutations hotspots including p.E380Q, p.Y537S and p.D538G were analyzed by using QX200™ ddPCR System (Annealing/Extension=58 °C, 40 cycles) and the corresponding probes (Mutation-FAM and Wild type-HEX mixed) for p.E380Q-dHsaMDS500014536, p.Y537-dHsaMDS975379796, p.D538G- dHsaMDS460485301 respectively (Bio-Rad). Meanwhile, corresponding plasma ctDNA was analyzed by Guardant 360 Health NGS-based assay for a 73 genes panel for single nucleotide variants. Results: Positive controls (using 0.00000325ng pure positive DNA) and negative control (using water) were performed in each ddPCR assay. In cohort 1: total of 8 samples including 4 single CTC samples and 4 single WBC samples were collected using the DEPArray system. Wild type ESR1 (p.E380Q, p.Y537S and p.D538G) was detected in all 4 single CTC samples and 4 single WBC samples. There were 3 copies, 3.4 copies, 1.6 copies and 1.8 copies of p.E380Q mutation droplets found in 4 single CTC samples when there were no p.Y537S or p.D538G mutations found in these samples. There was no ESR1 mutation found in the corresponding ctDNA. In cohort 2: Two samples including 4 CTCs and 100 CTCs were collected from CellSearch cartridges. There were 0.09 vs 1 copies (8.25%) and 0.1 vs 0.31 copies (24.39%) of p.E380Q mutation droplets found in these two samples respectively, compared to ctDNA results of 1.80% and 0 respectively. Furthermore, there was 0.08 vs 0.67 copies (10.66%) of p.D538G mutation droplets were found in the sample including 100 CTCs compared to 12.70% mutations detected in the corresponding ctDNA. Conclusion: Our new approach verified the feasibility of ESR1 mutations assay in very low amount DNA from single CTCs with high sensitivity even when ESR1 mutation could be found in CTCs but not ctDNA. This approach may offer a strong evidence for early disease metastasis, efficacy of individualized treatment monitoring and personalizing cancer therapy for precision medicine. Citation Format: Qiang Zhang, Paolo D'Amico, Marwa Manai, Carolina Reduzzi, Andrew A. Davis, Lorenzo Gerratana, Weijun Qin, Jianhua Jiao, Saya L. Jacob, Jeannine Donahue, Youbin Zhang, Lisa Flaum, Massimo Cristofanilli, Leonidas C. Platanias, Ami N. Shah, William Gradishar. A novel ESR1 mutation assay for single circulating tumor cell by application of DEPArray࣪ System and Digital Droplet PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1950.

Abstract 5994: Separating cancer cells from neutrophils by gradient acoustic focusing

Abstract Introduction: A subset of aggressive prostate cancers (PCa) progress to invade surrounding tissue to form distant metastases and shed of circulating tumor cells (CTCs). However, due to a scarcity of CTC among the millions of nucleated blood cells, there is an unmet need to improve CTC assays. Acoustophoresis uses standing ultrasound waves in a flow-through microfluidic chamber to discriminate cells based on the size, density, and compressibility. The technique is label-free as opposed to assays that rely on the cell surface marker EpCAM to detect CTCs. We will use label-free two-step acoustophoresis method as the initial separation step followed by a density medium purging step, to obtain efficient recovery of CTC of very high purity. Experimental procedure: Acoustic cell separation of CTCs from blood is challenging in reference to overlap of acoustic properties between small-sized CTCs and densely granular WBC. To obtain high recovery of CTC, we must apply a sufficiently large acoustic focusing amplitude which leads to a contaminating cell population comprising 2% white blood cells, (WBCs) that mainly contain eosinophil cells. To obtain high purity cancer cell fractions there is a critical need for an additional cleaning step. Therefore, we used the density medium iodixanol in a purging step where the optimal iodixanol medium concentration is intended to provide one cell type with positive, and the other with negative acoustic contrast. Preliminary results: show that the granulocytes can be transferred by the ultrasound field into the high-density medium (iodixanol) and can thereby be removed through a first outlet. The cancer cells do not transfer into the iodixanol medium and will be collected in a pure PCa cell fraction in the low-density medium through a second outlet. The samples were processed at a flow rate of 75 μl/min. The limiting factor regarding flow rate when purging the sample from granulocytes is overheating caused by the ultrasound transducer. The separation efficiency for cancer cells was 99.5% with 2.2% contaminating granulocytes. Theoretically, by combining the two separation steps only 0,044% of the WBC would remain in the cancer cell fraction. Conclusion: It is unclear which are the functional properties that enable a primary tumor to shed CTCs, and comprehensive characterization is urgently needed to identify key features of these processes. CTC fractions of high purity will enable single cell analysis with validated Fluidigm-based RT-PCR panels, mass spectrometry, and RNA-sequencing. We propose to use a two-step acoustic separation method, with a primary acoustic separation step and a second high-density medium purging step. Citation Format: Cecilia Magnusson, Mahdi Rezayati Charan, Hans Lilja, Per Augustsson. Separating cancer cells from neutrophils by gradient acoustic focusing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5994.

Circulating tumor cells: A step toward precision medicine in hepatocellular carcinoma.

Serum alpha-fetoprotein and radiologic imaging are the most commonly used tests for early diagnosis and dynamic monitoring of treatment response in hepatocellular carcinoma (HCC). However, the accuracy of these tests is limited, and they may not reflect the underlying biology of the tumor. Thus, developing highly accurate novel HCC biomarkers reflecting tumor biology is a clinically unmet need. Circulating tumor cells (CTCs) have long been proposed as a noninvasive biomarker in clinical oncology. Most CTC assays utilize immunoaffinity-based, size-based, and/or enrichment-free mechanisms followed by immunocytochemical staining to characterize CTCs. The prognostic value of HCC CTC enumeration has been extensively validated. Subsets of CTCs expressing mesenchymal markers are also reported to have clinical significance. In addition, researchers have been devoting their efforts to molecular characterizations of CTCs (e.g. genetics and transcriptomics) as molecular profiling can offer a more accurate readout and provide biological insights. As new molecular profiling techniques, such as digital polymerase chain reaction, are developed to detect minimal amounts of DNA/RNA, several research groups have established HCC CTC digital scoring systems to quantify clinically relevant gene panels. Given the versatility of CTCs to provide intact molecular and functional data that reflects the underlying tumor, CTCs have great potential as a noninvasive biomarker in HCC. Large-scale, prospective studies for HCC CTCs with a standardized protocol are necessary for successful clinical translation.

Liquid Biopsy Assessment of Circulating Tumor Cell PD-L1 and IRF-1 Expression in Patients with Advanced Solid Tumors Receiving Immune Checkpoint Inhibitor.

Reliable biomarkers that can be serially monitored to predict treatment response to immune checkpoint inhibitors (ICIs) are still an unmet need. Here, we present a multiplex immunofluorescence (IF) assay that simultaneously detects circulating tumor cells (CTCs) and assesses CTC expression of programmed death ligand-1 (PD-L1) and interferon regulatory factor 1 (IRF-1) as a candidate biomarker related to ICI use. To assess the potential of CTC PD-L1 and IRF-1 expression as candidate biomarkers for patients with advanced epithelial solid tumors receiving ICIs. We tested the IF CTC assay in a pilot study of 28 patients with advanced solid tumors who were starting ICI. Blood for CTC evaluation was obtained prior to starting ICI, after a single cycle of therapy, and at the time of radiographic assessment or treatment discontinuation. At baseline, patients with 0-1 CTCs had longer progression-free survival (PFS) compared to patients with ≥ 2 CTCs (4.3 vs 1.3 months, p = 0.01). The presence of any PD-L1+ CTCs after a single dose of ICI portended shorter PFS compared to patients with no CTCs or PD-L1- CTCs (1.2 vs 4.2 months, p = 0.02); the presence of any PD-L1+ or IRF-1+ CTCs at time of imaging assessment or treatment discontinuation also was associated with shorter PFS (1.9 vs 5.5 months, p < 0.01; 1.6 vs 4.7 months, p = 0.05). CTC PD-L1 and IRF-1 expression did not correlate with tumor tissue PD-L1 or IRF-1 expression. Strong IRF-1 expression in tumor tissue was associated with durable (≥1 year) radiographic response (p = 0.02). Based on these results, CTC PD-L1 and IRF-1 expression is of interest in identifying ICI resistance and warrants further study.