Epithelial Circulating Tumor Cells Research Articles

Abstract A046: Development of a scoring system to classify HER2 protein expression in circulating tumor cells through immunofluorescence following isolation using Parsortix® instruments

Abstract Background. HER2 testing through immunohistochemistry (IHC) is routinely performed on tissue biopsy samples from metastatic breast cancer (MBC) patients, resulting in a HER2 score that is used in patient stratification and treatment decisions. However, tissue biopsy is invasive, with little opportunity for re-examination while liquid biopsy is minimally invasive and allows monitoring of disease progression through recurrent sample collection. This study aimed to establish an assay (Research Use Only) allowing scoring of HER2 protein expression in circulating tumor cells (CTCs), by immunofluorescence (IF) staining, using cancer cell lines of known HER2 expression. Method. Cultured cancer cell lines with HER2 expression comparable to tissue samples (i.e. HER2 0, 1+, 2+ and 3+ by IHC score) were used in this study (SKBR3 [3+], HCC1954 [3+], SKOV-3 [2+/3+], ZR-75-1 [2+], MCF-7 [0 – 1+] and Hs 578T [0]). Cultured cancer cells were spun onto CellKeep™ slides and stained with ANGLE’s Portrait HER2 IF panel, comprising a nuclear dye, an anti-HER2 antibody, epithelial, mesenchymal and leukocyte markers. Slides were imaged on a BioView Allegro Plus, a platform equipped with artificial intelligence for automated imaging, CTC candidate identification and reporting. The workflow was implemented using samples from 5 MBC patients with known HER2 IHC scores, whose blood was drawn into Streck Cell-Free DNA tubes and processed on ANGLE's Parsortix® instrument, a marker-independent microfluidic device which isolates CTCs based on size and deformability, before being stained with the Portrait HER2 assay. The cohort is being expanded and currently includes three HER2 0 and two HER2 3+ patients as diagnosed by tissue IHC. Results. By observing mean fluorescence intensity (MFI) of each cancer cell line reported to be reflective of each IHC score, thresholds were set to establish a protein scoring system. 95%, 95% and 91% of SKBR3, HCC1954 and SKOV-3, respectively, fell in the 3+ class, 83% of ZR-75-1 fell in the 2+ class, 69% of MCF-7 fell in the 1+ class and 25% and 89% of MCF-7 and Hs 578T, respectively, fell in the 0 class. ≥1 CTC were found in all 5 subjects (mean=405, median=146), with 60% donors presenting mesenchymal CTCs, 20% epithelial CTCs and 20% epithelial and mesenchymal CTCs. HER2+ CTCs were found in both HER2 3+ donors, but not in the HER2 0 donors. HER2+ CTCs from both donors fell in the 3+ class based on MFI, despite one donor receiving Herceptin treatment (1 CTC) and the other being treatment naïve (435 CTCs). Conclusion. This study showed it is possible to establish a HER2 protein scoring system using cultured cancer cell lines stained with the Portrait HER2 assay and demonstrates the potential utility of liquid biopsy in tracking HER2 status. This scoring system will be applied to CTCs isolated from an extended cohort of MBC patients with known tissue biopsy HER2 IHC results to test the robustness of the system and assess concordance of CTC and tissue biopsy scores. FISH will be performed on the samples from MBC patients to confirm results. Citation Format: Alex Young, Laila Vlietinck, Bethan Edwards, Liam Cook, Chassidy Johnson, Mariacristina Ciccioli, Anne-Sophie Pailhes-Jimenez. Development of a scoring system to classify HER2 protein expression in circulating tumor cells through immunofluorescence following isolation using Parsortix® instruments [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 A046.

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.

Combination of circulating tumor cells and 18F-FDG PET/CT for precision diagnosis in patients with non-small cell lung cancer.

To investigate the value of 2-deoxy-18f-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) and circulating tumor cells (CTCs) for the differential diagnosis of patients with benign lung diseases and those with NSCLC. To explore the phenotypic heterogeneity of CTCs and their correlation with FDG uptake in patients with Stage I-IV NSCLC. Blood specimens from patients with benign lung diseases and patients with primary NSCLC were collected for the detection of CTCs and their subtypes (epithelial, mixed, and mesenchymal) and analyzed for 18F-FDG PET/CT tumor metabolic parameters, including the maximum standardized uptake value (SUVmax), standard uptake value (SUL), metabolic tumor volume of primary lesion (MTV), total lesion glycolysis of primary lesion (TLG). Clinical data including age, gender, smoking history, tumor size, TNM stage and pathology type were also collected. The value of the two method alone and in combination for the differential diagnosis of benign and malignant was comparatively analyzed. Finally, the differences in CTC and its subtypes in different stages of NSCLC were compared, and FDG metabolic parameters were correlated with CTC subtypes. There were a total of 65 patients with pulmonary diseases, including 12 patients with benign pulmonary diseases and 53 patients with NSCLC. The mean age was 67 ± 10 (38-89 years), 27 were females and 38 were males. 31 (22 males and 9 females) had a long history of smoking. The mean size of the largest diameter of all single lesions was 36 ± 22 mm with a range of 10-108 mm. Seven out of 12 benign diseases were inflammatory granulomatous lesions and 5 were inflammatory pseudotumours. Twenty-four out of 53 NSCLC were adenocarcinomas and 29 were squamous carcinomas. Twelve out of 53 patients with NSCLC were in Stage I, 10 were in Stage II, 17 were in Stage III and 14 were in Stage IV. SUVmax, SUL, MTV, TLG, total CTCs, epithelial CTCs, and mixed CTCs were all valuable in the differential diagnosis of benign and malignant. TLG combined with mixed CTCs was statistically different from all other diagnostic methods (p < 0.05) and higher than any other diagnostic criteria. In the differential diagnosis of benign and Stage I NSCLC, only total CTC (Z = -2.188 p = 0.039) and mixed CTCs (Z = -3.020 p = 0.014) had certain diagnostic efficacy, and there was no statistical difference between them (p = 0.480). Only mesenchymal CTCs differed in Stage I-IV NSCLC, with a higher number of those who developed distant metastases than those who had non-distant metastases. Epithelial CTCs correlated with SUVmax (r = 0.333, p = 0.015) and SUL (r = 0.374, p = 0.006). Mmesenchymal CTCs correlated with MTV (r = 0.342, p = 0.018) and TLG (r = 0.319, p = 0.02). Further subgroup analyses revealed epithelial CTCs were correlated with SUVmax (r = 0.543, p = 0.009) and SUL (r = 0.552, p = 0.008), and the total CTCs was correlated with SUVmax (r = 0.622, p = 0.003), SUL (r = 0.652, p = 0.003), MTV (r = 0.460, p = 0.031), and TLG (r = 0.472, p = 0.027) in the early group (Stage I-II). Only mesenchymal CTCs was associated with MTV (r = 0.369, p = 0.041), and TLG (r = 0.415, p = 0.02) in the intermediate-late group (Stage III-IV). Both FDG PET metabolic parameters and CTCs demonstrated diagnostic value for NSCLC, and combining TLG with mixed CTCs could enhance their diagnostic efficacy. The total CTCs and mixed CTCs showed greater diagnostic value than FDG PET in distinguishing benign lesions from Stage I NSCLC. In NSCLC patients, the epithelial CTCs exhibited a positive correlation with SUVmax and SUL, while mesenchymal CTCs correlated with MTV, and TLG. Besides, epithelial CTCs showed stronger correlations with SUVmax and SUL, and total CTCs showed stronger correlations with SUVmax, SUL, MTV, and TLG in Stage I-II NSCLC. Only mesenchymal CTCs in Stage III-IV NSCLC showed correlations with MTV and TLG. Stage IV NSCLC cases displayed a higher number of mesenchymal CTCs.

Fluorescent identification of immunomagnetically captured CTCs using triplex-aptamer-targeted dendritic SiO2@Fe3O4 nanocomposite.

The enumeration of circulating tumor cells (CTCs) in peripheral blood plays a crucial role in the early diagnosis, recurrence monitoring, and prognosis assessment of cancer patients. There is a compelling need to develop an efficient technique for the capture and identification of these rare CTCs. However, the exclusive reliance on a single criterion, such as the epithelial cell adhesion molecule (EpCAM) antibody or aptamer, for the specific recognition of epithelial CTCs is not universally suitable for clinical applications, as it usually falls short in identifying EpCAM-negative CTCs. To address this limitation, we propose a straightforward and cost-effective method involving triplex fluorescently labelled aptamers (FAM-EpCAM, Cy5-PTK7, and Texas Red-CSV) to modify Fe3O4-loaded dendritic SiO2 nanocomposite (dmSiO2@Fe3O4/Apt). This multi-recognition-based strategy not only enhanced the efficiency in capturing heterogeneous CTCs, but also facilitated the rapid and accurate identification of CTCs. The capture efficiency of heterogenous CTCs reached up to 93.33%, with a detection limit as low as 5 cells/mL. Notably, the developed dmSiO2@Fe3O4/Apt nanoprobe enabled the swift identification of captured cells in just 30min, relying solely on the fluorescently modified aptamers, which reduced the identification time by approximately 90% compared withthe conventional immunocytochemistry (ICC) technique. Finally, these nanoprobe characteristics were validated using blood samples from patients with various types of cancers.

Abstract PO2-24-02: Molecular characterization of primary tumors associated with various CTCs’ subpopulation

Abstract Background: Circulating tumor cells (CTCs) are prognostic in primary breast cancer (BC) patients and represent a heterogeneous population of cells with different phenotypes and biological value. However, molecular characterization of primary tumors associated with various CTCs’ subpopulation is lacking. This study aimed to identify expression of genes associated with the presence of different CTCs subpopulations in primary BC patients using a comprehensive genomics approach. Methods: This prospective ongoing study included 312 patients (pts) with non-metastatic BC enrolled from March 2012 to February 2015. CTCs were detected on day -1 to 0 before surgery. Isolated peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSepTM kit negative selection with anti-CD45 antibody. RNA extracted from CD45-depleted (CD45-) PBMC was interrogated for expression of EMT-inducing transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC of patients were compared to those of CD45- PBMC of 60 healthy donors. Formalin-fixed paraffin-embedded tumor tissue was subject for gene expression analysis performed by HTG EdgeSeq Oncology Biomarker panel (HTG Molecular Diagnostics, Inc., Tucson, USA). Results: CTCs with epithelial phenotype (CTC_EP) were detected in 30 (9.6%) pts, CTC with EMT (epithelial-to-mesenchymal transition) were detected in 54 (17.3%), while blood of 79 (25.3%) pts exhibit at least one type of CTC. Patients with detectable CTC in peripheral blood had inferior disease-free survival compared to patients without detectable CTC (HR = 0.62, 95% CI 0.37-1.06, P=0.048). There was no association between epithelial CTCs and analysed patients/tumour variables. Genes expression analysis identified 7 differentially expressed genes in patients with the CTC_EP phenotype (CHGA, MESP1, COL2A1, MT2A, COMP, MMP7 and ORM2), 200 genes associated with CTC_EMT phenotype (top 10 genes included CHGA, CECR6, SMAD2, HUS1, FGF10, HNF1A, SEC61G, CDK4, CD44 and F3). Analysis identified that low expression of CHGA and MMP7 was consistently associated with any CTC subpopulations. Conclusions: In this study for the first-time revealed gene expressions associated with various CTC subpopulations. We suppose that these genes could represent potential therapeutic target aimed to inhibit metastatic cascade in breast cancer. Citation Format: Michal Mego, Gabriel Minarik, Milan Hucko, Katarina Kalavska, Marian Karaba, Juraj Benca, Tatiana Sedlackova, Lucia Kucerova, Jozef Mardiak, Lubos Klucar, Zuzana Cierna. Molecular characterization of primary tumors associated with various CTCs’ subpopulation [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-02.

The correlation study between TOP2A gene expression in circulating tumor cells and chemotherapeutic drug resistance of patients with breast cancer

BackgroundPatients with breast cancer (BC) at advanced stages have poor outcomes because of high rate of recurrence and metastasis. Biomarkers for predicting prognosis remain to be explored. This study aimed to evaluate the relationships between circulating tumor cells (CTCs) and outcomes of BC patients.Patients and methodsA total of 50 female were enrolled in this study. Their diagnoses were determined by clinical characteristics, image data, and clinical pathology. CTC subtypes and TOP2A gene expression on CTCs were detected by CanPatrol™ technology and triple color in situ RNA hybridization (RNA-ISH), which divided into epithelial CTCs (eCTCs), mesenchymal CTCs (MCTCs), and hybrid CTCs (HCTCs) based on their surface markers. Hormone receptor, including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) expression, was measured by immunohistochemistry (IHC) method before treatment. The risk factors for predicting recurrence and metastasis were calculated by COX risk regression model. The progression-free survival (PFS) of patients was determined using Kaplan–Meier survival curve.ResultsThe patients with a large tumor size (≥ 3 cm) and advanced tumor node metastasis (TNM) stages had high total CTCs (TCTCs) (P < 0.05). These patients also had high TOP2A expression level. COX risk regression analysis indicated that TOP2A expression levels in TCTCs, ER + , HER-2 + , and TNM stages were critical risk factors for recurrence and metastasis of patients (P < 0.05). The PFS of patients with ≥ 5 TCTCs, ≥ 3 HCTCs, and positive TOP2A expression in ≥ 3 TCTCs was significantly longer than that in patient with < 5 TCTCs, < 3 HCTCs, and TOP2A expression in < 3 TCTCs (P < 0.05). In contrast, the PFS of patients with positive hormone receptors (ER + , PR + , HER-2 +) also was dramatically lived longer than that in patients with negative hormone receptor expression.ConclusionsHigh TCTC, HCTCs, and positive TOP2A gene expression on CTCs were critical biomarkers for predicting outcomes of BC patients. Positive hormone receptor expression in BC patients has significant favor PFS.

Isolation of Viable Epithelial and Mesenchymal Circulating Tumor Cells from Breast Cancer Patients.

Circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT) may exhibit more aggressive features than epithelial CTCs and are more frequently observed during disease progression. Therefore, detection and characterization of both epithelial and mesenchymal CTCs in cancer patients are urgently needed to allow for a better understanding of the metastatic process and more effective treatment. Here we describe a method for detection and isolation of viable epithelial and mesenchymal CTCs from peripheral blood of breast cancer patients. The method is based on density gradient centrifugation, multiplex immunofluorescent staining, and negative anti-CD45 selection. Cells obtained after the procedure are suitable for genomic or transcriptomic profiling, and they can also be isolated by micromanipulation for single-cell analysis.

Significance of circulating tumor cells detection in tumor diagnosis and monitoring

To detect circulating tumor cells (CTCs) in the peripheral blood of patients with tumor, and to analyze the significance of CTC detection in tumor diagnosis and monitoring. In the present study, peripheral blood was collected from 125 patients with tumor, and CTCs were isolated and identified. Differences in CTC number and subtype detection were analyzed for different tumor diseases and stages. CTCs were detected in 122 of the 125 patients with tumor, with a positive rate of 97.6%. The number of CTCs increases in patients with vascular metastasis. The number of mesenchymal CTCs increases in patients with lymph node or vascular metastasis. The average ratio of epithelial CTCs in each positive sample decreases in the later stages of cancer compared with the earlier stages, while the average ratio of mesenchymal CTCs increases in the later stages of cancer compared with the earlier stages. The results showed that CTCs with mesenchymal phenotypes are closely related to lymph node or vascular metastasis. CTC detection can help with early diagnosis of tumor diseases. Continuous monitoring of changes in CTCs number and subtypes can assist clinical judgment of tumor disease development status and prognosis.

Effect of various hepatectomy procedures on circulating tumor cells in postoperative patients: a case-matched comparative study.

The objective of this study is to elucidate the prevalence of systemic circulating tumor cells (CTCs) prior to and following resection of hepatocellular carcinoma (HCC), and to compare the disparities in postoperative CTCs in terms of quantity and classifications between the open liver resection (OPEN) and laparoscopic liver resection (LAP) cohorts. From September 2015 to May 2022, 32 consecutive HCC patients who underwent laparoscopic liver resection at Southwest Hospital were retrospectively enrolled in this study. The clinicopathological data were retrieved from a prospectively collected computer database. Patients in the OPEN group matched at a 1:1 ratio with patients who underwent open liver resection during the study period on age, gender, tumor size, number of tumors, tumor location, hepatitis B surface antigen (HBsAg) positivity, alpha-fetoprotein (AFP) level, TNM and Child-Pugh staging from the database of patients to form the control group. The Can-Patrol CTC enrichment technique was used to enrich and classify CTCS based on epithelial-mesenchymal transformation phenotypes. The endpoint was disease-free survival (DFS), and the Kaplan-Meier method and multiple Cox proportional risk model were used to analyze the influence of clinicopathological factors such as total CTCs and CTC phenotype on prognosis. The mean age of the 64 patients with primary liver cancer was 52.92 years (23-71), and 89.1% were male. The postoperative CTC clearance rate was more significant in the OPEN group. The total residual CTC and phenotypic CTC of the LAP group were significantly higher than those of the OPEN group (p = 0.017, 0.012, 0.049, and 0.030, respectively), which may increase the possibility of metastasis (p = 0.042). In Kaplan-Meier analysis, DFS was associated with several clinicopathological risk factors, including Barcelona Clinical Liver Cancer (BCLC) stage, tumor size, and vascular invasion. Of these analyses, BCLC Stage [p = 0.043, HR (95% CI) =2.03(1.022-4.034)], AFP [p = 0.007, HR (95% CI) =1.947 (1.238-3.062)], the number of positive CTCs [p = 0.004, HR (95% CI) =9.607 (2.085-44.269)] and vascular invasion [p = 0.046, HR (95% CI) =0.475 (0.22-1.023)] were significantly associated with DFS. In comparison to conventional OPEN technology, LAP technology has the capacity to augment the quantity of epithelial, mixed, and mesenchymal circulating tumor cells (CTCs). Following the surgical procedure, there was a notable increase in the total CTCs, epithelial CTCs, and mixed CTCs within the LAP group, indicating a potential drawback of LAP in facilitating the release of CTCs.

Circulating Tumor Cell Subpopulations Predict Treatment Outcome in Pancreatic Ductal Adenocarcinoma (PDAC) Patients.

There is a high clinical unmet need to improve outcomes for pancreatic ductal adenocarcinoma (PDAC) patients, either with the discovery of new therapies or biomarkers that can track response to treatment more efficiently than imaging. We report an innovative approach that will generate renewed interest in using circulating tumor cells (CTCs) to monitor treatment efficacy, which, in this case, used PDAC patients receiving an exploratory new therapy, poly ADP-ribose polymerase inhibitor (PARPi)-niraparib-as a case study. CTCs were enumerated from whole blood using a microfluidic approach that affinity captures epithelial and mesenchymal CTCs using anti-EpCAM and anti-FAPα monoclonal antibodies, respectively. These antibodies were poised on the surface of two separate microfluidic devices to discretely capture each subpopulation for interrogation. The isolated CTCs were enumerated using immunophenotyping to produce a numerical ratio consisting of the number of mesenchymal to epithelial CTCs (denoted "Φ"), which was used as an indicator of response to therapy, as determined using computed tomography (CT). A decreasing value of Φ during treatment was indicative of tumor response to the PARPi and was observed in 88% of the enrolled patients (n = 31). Changes in Φ during longitudinal testing were a better predictor of treatment response than the current standard CA19-9. We were able to differentiate between responders and non-responders using ΔΦ (p = 0.0093) with higher confidence than CA19-9 (p = 0.033). For CA19-9 non-producers, ΔΦ correctly predicted the outcome in 72% of the PDAC patients. Sequencing of the gDNA extracted from affinity-selected CTC subpopulations provided information that could be used for patient enrollment into the clinical trial based on their tumor mutational status in DNA repair genes.

Cell State and Cell Type: Deconvoluting Circulating Tumor Cell Populations in Liquid Biopsies by Multi-Omics.

Bi-directional crosstalk between the tumor and the tumor microenvironment (TME) has been shown to increase the rate of tumor evolution and to play a key role in neoplastic progression, therapeutic resistance, and a patient's overall survival. Here, we set out to use a comprehensive liquid-biopsy analysis to study cancer and specific TME cells in circulation and their association with disease status. Cytokeratin+, CD45- circulating rare cells (CRCs) from nine breast and four prostate cancer patients were characterized through morphometrics, single-cell copy number analysis, and targeted multiplexed proteomics to delineate cancer cell lineage from other rare cells originating in the TME. We show that we can detect epithelial circulating tumor cells (EPI.CTC), CTCs undergoing epithelial-to-mesenchymal transition (EMT.CTC) and circulating endothelial cells (CECs) using a universal rare event detection platform (HDSCA). Longitudinal analysis of an index patient finds that CTCs are present at the time of disease progression, while CECs are predominately present at the time of stable disease. In a small cohort of prostate and breast cancer patients, we find high inter-patient and temporal intra-patient variability in the expression of tissue specific markers such as ER, HER2, AR, PSA and PSMA and EpCAM. Our study stresses the importance of the multi-omic characterization of circulating rare cells in patients with breast and prostate carcinomas, specifically highlighting overlapping and cell type defining proteo-genomic characteristics of CTCs and CECs.

Correlation analysis of circulating tumor cells and Claudin-4 in breast cancer.

Objective: We aimed to explore the relationship between peripheral blood circulating tumor cells (CTCs) and the expression of Claudin-4 in patients with breast cancer, and further explore the potential impact on clinical prognosis and risk assessment. Methods: We classified and enumerated circulating tumor cells in the blood of breast cancer patients by CTC-enriched in situ hybridization and the detection of Claudin-4 expression by immunohistochemistry. We carried out an analysis of the correlation between the two and the comparison of their impact on clinical parameters and prognosis. Results: There were 38 patients with a low expression of Claudin-4 and 27 patients with a high expression of Claudin-4. Compared with Claudin-4 low-expression patients, the number of CTCs was higher in patients with high Claudin-4 expression (11.7 vs. 7.4, p < 0.001). High Claudin-4 expression was associated with a lower count of epithelial CTCs (E-CTCs) (3.4 vs. 5.0, p = 0.033), higher counts of mesenchymal CTCs (M-CTC) (4.4 vs. 1.1, p < 0.001), and epithelial/mesenchymal CTCs (E/M-CTCs) (4.0 vs. 3.5, p = 0.021). The intensity of Claudin-4 was positively correlated with CTC (rs = 0.43, p = 0.001). Multivariate COX regression analysis showed that CTC counts (HR = 1.3, p < 0.001), Claudin-4 (HR = 4.6, p = 0.008), and Lymphatic metastasis (HR = 12.9, p = 0.001) were independent factors for poor prognosis. COX regression of CTC classification showed that epithelial/mesenchymal CTCs (E/M-CTC) (HR = 1.9, p = 0.001) and mesenchymal CTCs (M-CTC) (HR = 1.5, p = 0.001) were independent influencing factors of adverse reactions in breast cancer patients. Conclusion: The number of CTC in breast cancer is positively correlated with the expression of Claudin-4. High CTC counts and a high proportion of M-CTCs correlated with Claudin-4 expression. CTC counts and Claudin-4 expression were independent predictors of poor prognosis in breast cancer patients.

Abstract 3364: Combined enrichment of plasma EpCAM-positive extracellular vesicles (EVs) and circulating tumor cells (CTCs) from a single tube of cancer patient blood samples, for subsequent molecular analysis

Abstract Background: EVs are secreted by cells in physiological and pathological conditions and carry selected molecules, such as proteins, DNA and RNA; specific EVs released by neoplastic cells are defined as “tumor-derived extracellular vesicles (tdEVs)”. tdEVs have been demonstrated to be involved in promoting cell growth and survival, shaping the tumor microenvironment and increasing invasiveness and metastatic activity. Thanks to their systemic availability in body fluids and their role in tumor pathogenesis, tdEVs have gained attention in the liquid biopsy area, as a possible biomarker for tumor diagnosis, prognosis and monitoring and also as possible therapeutic targets. Beyond being a target for the capture of CTCs, the Epithelial Cell Adhesion Molecule (EpCAM) was also recently employed for the detection of EVs released by tumors. Here we present a method (for research use only) based on the CellMag™ technology (Menarini Silicon Biosystems, MSB) to carry out parallel enrichment of EVs and CTCs from whole blood through an EpCAM-based magnetic enrichment. Method: Whole blood samples from stage III/IV prostate (n=5) or breast cancer patients (n=6) and healthy donors (n=4) were collected in CellRescue™ tubes (MSB). After centrifugation, EpCAM+ EVs and CTCs were enriched from the plasma and cell fraction respectively, using the CellMag™ Epithelial CTC kit (MSB); CTCs were counted at the CellTracks Analyzer II (MSB). EVs were enriched for qualitative evaluation by Transmission Electron Microscopy (TEM) and for miRNA analysis by Real-time PCR, using the TaqMan™ Advanced miRNA Human Serum/Plasma panel. Results: CTCs counts ranged from 0 to 68; TEM analysis showed a higher density of EpCAM+ extracellular microvesicles (0,1-1µm) in breast cancer patients’ samples (n=2) compared to controls (n=2). Endogenous hsa-miR-16-5p control was detected in all analyzed samples (n=9 patients’ samples, n=2 controls). One control and two patients’ samples were tested for a panel of 188 serum/plasma miRNAs. In both the breast and the prostate cancer patients a higher percentage of miRNAs was found with respect to healthy control (31%, 32% vs. 17%). Among these, several oncogenic miRNAs with diagnostic and prognostic value (such as hsa-miR-125b-5p or hsa-miR-26a-5p) were exclusively found in patients’ samples, and further confirmed to be related to ErbB2 pathway in breast cancer using the miRPathDB 2.0 database, despite the CTCs count was 0 for both these patients’ samples. Conclusions: The described multi-target liquid biopsy approach enabled the combined enrichment of EpCAM+ CTCs and plasma EpCAM+ EVs from a single blood sample, along with their molecular analysis. Citation Format: Giulia Bregola, Martina Masotti, Valentina Mattei, Martina Moras, Claudio Ratti, Francesco Picardo, Cecilia Simonelli, Nicolò Manaresi, Francesca Fontana. Combined enrichment of plasma EpCAM-positive extracellular vesicles (EVs) and circulating tumor cells (CTCs) from a single tube of cancer patient blood samples, for subsequent molecular analysis [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 3364.

Nano-magnetic aptamer sensor incorporating AND logic recognition-launched hybridization chain reaction for organ origin identification of circulating tumor cells

Circulating tumor cells (CTCs) are effective bio-markers for early diagnosis and disease monitoring of cancer, but there is lack of simple and effective strategy to achieve the detection of multiple markers on CTCs while enriching, especially the identification of organ origin, limits their broad clinical applications. Here we present a nano-magnetic aptamer sensor platform to capture broad spectrum of epithelial CTCs and simultaneously identify their multiple organ origins by AND logic recognition-launched hybridization chain reaction. The platform exhibited excellent capture and enrichment capabilities for epithelial CTCs, and showed the ability to precisely identify their organ origin markers (GPC3 for liver and TTF1 for lung), even able to trace metastatic cancer to related organs. The developed nano-magnetic aptamer sensor platform provides a feasible strategy for multi-marker accurate detection of CTCs and a promising auxiliary tool for the precise treatment of cancer.

Abstract P1-05-35: Identification of Circulating Tumor Cells captured by the FDA Cleared Parsortix® PC1 System from the Peripheral Blood of Metastatic Breast Cancer Patients using Immunofluorescence and Cytopathological Evaluations

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. The Parsortix® PC1 System, the first FDA-cleared medical device for the capture and harvest of CTCs from peripheral blood of metastatic breast cancer (MBC) patients for use in subsequent user-validated downstream analyses, enables the epitope independent capture of CTCs with diverse phenotypes based on cell size and deformability. In this study, CTCs isolated from the blood of MBC patients by the Parsortix® PC1 System were identified using an immunofluorescence (IF) based assay for detection of epithelial CTCs followed by Wright-Giemsa staining and cytomorphological review. The aim of this study was to determine the proportion of MBC patients and self-declared female healthy volunteers (HVs) that had one or more CTCs identified in the population of cells harvested from their peripheral blood by the Parsortix® PC1 System. Methods: Peripheral blood from 75 HVs and 77 patients with MBC was prospectively collected into K2EDTA tubes at the University of Rochester Medical Center. The blood collected from each subject (8.6±1.2mL) was processed on a Parsortix® PC1 System within 8 hours of collection. The cells harvested by the system were cytospun onto a charged slide and IF stained using an optimised antibody panel. The IF panel consisted of a nuclear dye (DAPI), positive selection markers targeting epithelial CTCs (Cytokeratins (CK) and EpCAM), and negative selection markers targeting white blood cells, such as lymphocytes, macrophages, granulocytes, monocytes, fibroblasts, and cells of megakaryoblastic potential. The stained slides were imaged using fluorescence microscopy and CTCs were defined as nucleated cells (DAPI+) that were positive for CK and/or EpCAM and negative for the blood lineage markers. The IF slides were subsequently stained with Wright-Giemsa and analysed by a qualified pathologist using light microscopy. Morphological features of malignant cells were used to define and identify CTCs. All laboratory testing and analysis was performed by operators blinded to the clinical status of each subject. Results: On the evaluable IF-stained slides, cells classified as CTCs based on their IF staining pattern were identified in 45.3% (34/75) of the MBC patients (range = 0 – 125 CTCs, mean = 7) and in 5.6% (4/71) of the HVs (range = 0 – 8 CTCs, mean = 0). No EpCAM+, CK- CTCs were identified in either MBC patients or HVs. In the 34 MBC patients with one or more CTCs observed, 70.6% had only CK+, EpCAM- cells, while the remaining 29.4% had at least one CK+, EpCAM+ cell. In the HVs, one out of the four CTC-positive donors had only CK+, EpCAM+ cells while the other three had only CK+, EPCAM- cells. On the evaluable Wright-Giemsa stained slides, cells classified as CTCs by the pathologist were identified in 57.1% (40/70) of the MBC patients (range = 0 – 41 CTCs, mean = 4) and in 4.4% (3/68) of the HVs (range = 0 – 14, mean = 0). Conclusions: This study demonstrated the ability of ANGLE’s Parsortix® PC1 System to capture and harvest CTCs from a significantly larger proportion of MBC patients compared to HV subjects. The presence of epithelial cells in subjects without diagnosed disease has been previously described in the literature, with their significance being unclear. This study also demonstrated that the cells harvested by the Parsortix® PC1 System can be successfully evaluated using both IF staining and Wright-Giemsa cytomorphological assessment for the identification of CTCs. Interestingly, a high proportion of the identified CTCs did not express EpCAM, further highlighting the limitations of using EpCAM-based approaches to capture CTCs. Citation Format: Mariacristina Ciccioli, Richard Moore, Kyu Kwang Kim, Negar Khazan, Michael C. Miller, Anne-Sophie Pailhes-Jimenez. Identification of Circulating Tumor Cells captured by the FDA Cleared Parsortix® PC1 System from the Peripheral Blood of Metastatic Breast Cancer Patients using Immunofluorescence and Cytopathological Evaluations [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 P1-05-35.

μ-opioid receptor agonist facilitates circulating tumor cell formation in bladder cancer via the MOR/AKT/Slug pathway: a comprehensive study including randomized controlled trial.

μ-opioid receptor agonists (MORAs) are indispensable for analgesia in bladder cancer (BC) patients, both during surgery and for chronic pain treatment. Whether MORAs affect BC progression and metastasis remains largely unknown. This study focused on the effects of MORAs on the formation of circulating tumor cells (CTCs) in BC and aimed to provide potential therapeutic targets, which would retain the pain-relieving effects of MORAs in BC patients without sacrificing their long-term prognosis. Different preclinical models were used to identify the effects of MORAs on the progression of BC. A novel immunocapture microfluidic chip was utilized to analyze whether MORAs affected the number of CTCs in mouse models and clinical BC patients. Bioinformatic analyses, total transcriptome sequencing, and molecular biology methods were then used to investigate the underlying mechanisms in these models and in BC cell lines. Mouse models of hematogenous metastasis and in situ BC demonstrated that tumor metastasis was significantly increased after MORA treatment. A significant increase in the number of mesenchymal and/or epithelial CTCs was detected after MORA treatment in both the mouse models and clinical trial patients. Mechanistically, MORAs facilitated the formation of CTCs by activating the MOR/PI3K/AKT/Slug signaling pathway, hereby promoting the epithelial-mesenchymal transition (EMT) of BC cells, as knockdown of MOR, Slug or blockade of PI3K inhibited the EMT process and CTC formation. MORAs promoted BC metastasis by facilitating CTC formation. The EMT-CTC axis could be targeted for preventive measures during MORA treatment to inhibit the associated tumor metastasis or recurrence in BC patients.

The mesenchymal circulating tumor cells as biomarker for prognosis prediction and supervision in hepatocellular carcinoma

PurposeHepatocellular carcinoma (HCC) is one of the most common cancers and a leading cause of death worldwide. Accurate prognosis prediction tools are urgently needed. While the use of circulating tumor cells (CTCs) as prognostic prediction tool has a clear potential.MethodsWe established a comprehensive, negative enrichment-based strategy for CTCs analysis in patients with HCC, involving identification of epithelial CTCs (E-CTCs) and mesenchymal CTCs (M-CTCs) through specific biomarker. This strategy was performed in 127 HCC cases, 21 nonmalignant liver disease (NMLD) patients and 42 health control to analyze the relevance between CTCs and tumor recurrence.ResultsThe total CTC number and M-CTC percent were positively correlated with tumor malignancy and high recurrence risk. Individually, preoperative total CTC number and M-CTC percent could robustly distinguish relapse cases from those with no relapse, with sensitivity of 80.95% and 90.48%, specificity of 74.12% and 84.71%, respectively. Levels of preoperative total CTC number and M-CTC percent can both be regarded as independent risk factors for HCC with early recurrence (P = 0.0053, P < 0.0001), and are both significantly correlated with worse recurrence-free survival (RFS) (log rank P < 0.0001; HR 7.78, 95% CI = 3.59–16.87; log rank P < 0.0001; HR 24.4, 95% CI = 8.67–68.77). The levels of total CTC number and M-CTC number had higher effectiveness than alpha fetal protein (AFP) in HCC longitudinal supervision (77.78% vs 88.89% vs 22.22%).ConclusionPreoperative and postoperative CTCs with higher effectiveness than AFP in prognosis prediction and recurrence supervision, indicating that CTCs could work as the biomarker for HCC clinical management.

Liquid Biopsy Detecting Circulating Tumor Cells in Patients with Non-Small Cell Lung Cancer: Preliminary Results of a Pilot Study.

Lung cancer is still the leading cause of cancer-related death worldwide. Interest is growing towards early detection and advances in liquid biopsy to isolate circulating tumor cells (CTCs). This pilot study aimed to detect epithelial CTCs in the peripheral blood of early-stage non-small cell lung cancer (NSCLC) patients. We used Smart BioSurface® (SBS) slide, a nanoparticle-coated slide able to immobilize viable nucleated cellular fraction without pre-selection and preserve cell integrity. Forty patients undergoing lung resection for NSCLC were included; they were divided into two groups according to CTC value, with a cut-off of three CTCs/mL. All patients were positive for CTCs. The mean CTC value was 4.7(± 5.8 S.D.) per ml/blood. In one patient, next generation sequencing (NGS) analysis of CTCs revealed v-raf murine sarcoma viral oncogene homolog B(BRAF) V600E mutation, which has also been identified in tissue biopsy. CTCs count affected neither overall survival (OS, p = 0.74) nor progression-free survival (p = 0.829). Multivariable analysis confirmed age (p = 0.020) and pNodal-stage (p = 0.028) as negative predictors of OS. Preliminary results of this pilot study suggest the capability of this method in detecting CTCs in all early-stage NSCLC patients. NGS on single cell, identified as CTC by immunofluorescence staining, is a powerful tool for investigating the molecular landscape of cancer, with the aim of personalized therapies.

Gene expression profiling of circulating tumor cells captured by MicroCavity Array is superior to enumeration in demonstrating therapy response in patients with newly diagnosed advanced and locally advanced non-small cell lung cancer.

Circulating tumor cells (CTCs) are a promising non-invasive tool for monitoring therapy response. The only Food and Drug Administration (FDA)-approved test is limited to enumeration of epithelial CTC without further characterization and is not approved for the management of non-small cell lung cancer (NSCLC). Here we use a MicroCavity Array (MCA) system to capture CTC agnostic of epithelial markers for further molecular testing in NSCLC. CTCs were enumerated by fluorescent microscopy as longitudinal sampling throughout disease management from 213 NSCLC patients. CTC-enriched samples from a subset of 127 patients were interrogated for gene expression by reverse transcription polymerase chain reaction (RT-PCR) using a customized pre-selected panel of 20 genes. At least 1 CTC was detected by enumeration in 53.8% of samples. Most patients had fewer than 5 CTCs (91%) and the highest observed count was 35 CTCs. Enumeration of single CTCs was not prognostic, although detection of CTC clusters at any time point was associated with increased risk of progression [hazard ratio (HR) 3.00, 95% confidence interval (CI): 1.1-8.2, P=0.0318]. In contrast, 124 (97.6%) patients with samples interrogated for gene expression had at least 1 gene detectable in at least 1 sample, and 101 (79.5%) had at least one elevated epithelial gene in at least one timepoint. High expression of BCL2, CD274 [programmed death-ligand 1 (PD-L1)], CDH1, EPCAM, FGFR1, FN1, KRT18, MET and MUC1 were associated with poor prognosis. Patients with CTCs positive for at least 3 epithelial genes at baseline all progressed within 10 months (HR 8.2, P<0.001, 95% CI: 3.2-21.1). BCL2, CD274 (PD-L1), EPCAM and MUC1 remained significant independent prognostic factors in multivariate, time-dependent analyses of progression and death. The selective profile of CTC genes and identification of CTC clusters better correlated with prognosis than enumeration of enriched CTC in NSCLC patients in this study.

Capture of Heterogeneous Circulating Tumor Cells in Colorectal Cancer Patients on an Immunomagnetic and Anti-Nonspecific Adsorption Platform.

Circulating tumor cells (CTC) have been represented by different phenotypes due to the epithelial-mesenchymal transition (EMT), which are epithelial CTC (E-CTC), mesenchymal CTC (M-CTC), and mixed (epithelial, mesenchymal) CTC (EM-CTC). Limited work has systematically discussed the associations of CTC number, especially proportions of E-CTC, M-CTC, and EM-CTC in total CTC, with colorectal cancer (CRC) progressions via a simple method with high performances. To achieve this goal, this paper presents the fabrication of a novel anti-nonspecific adsorption immunomagnetic platform called Fe3O4@SiO2@PTMAO@Aptamer, which was obtained by modifying polymeric trimethylamine N-oxide (PTMAO) on magnetic Fe3O4@SiO2, which was then linked with dual aptamers of an epithelial cellular adhesion molecule (EpCAM) and cell surface vimentin (CSV), targeting different CTC phenotypes. Results demonstrated that the abundant coating of PTMAO on Fe3O4@SiO2 improved the anti-nonspecific adsorption and noncell adhesion abilities of the immunomagnetic particles and could capture heterogeneous CTC with higher efficiency within 10 min. These excellent performances of Fe3O4@SiO2@PTMAO@Aptamer allowed us to inspect the correlations of numbers of E-CTC, M-CTC, EM-CTC, or proportions of them in total CTC with clinical information on CRC patients in detail. Our data innovatively and clearly revealed that the captured CTC, especially M-CTC proportion, displayed more close associations with progression, diagnosis, surgery, and chemotherapeutic effects for CRC patients. Overall, we believe that our approach will bring a new understanding of CTC-based liquid biopsy for cancer diagnosis and treatment.