Number Of Circulating Tumor Cells Research Articles

Electrochemical cytosensor utilizing tetrahedral DNA/bimetallic AuPd holothurian-shaped nanoparticles for ultrasensitive non-destructive detection of circulating tumor cells.

As a real-time fluid biopsy method, the detection of circulating tumor cells (CTCs) provides important information for the early diagnosis, precise treatment, and prognosis of cancer. However, the low density of CTCs in the peripheral blood hampers their capture and detection with high sensitivity and selectivity using currently available methods. Hence, we designed a sandwich-type electrochemical aptasensor that utilizes holothurian-shaped AuPd nanoparticles (AuPd HSs), tetrahedral DNA nanostructures (TDNs), and CuPdPt nanowire networks (NWs) interwoven with a graphdiyne (GDY) sheet for ultrasensitive non-destructive detection of MCF-7 breast cancer cells. CuPdPt NW-GDY effectively enhanced the electron transfer rate and coupled with the loaded TDNs. The TDNs could capture MCF-7 cells with precision and firmness, and the resulting composite complex was combined with AuPd HSs to form a sandwich-type structure. This novel aptasensor showed a linear range between 10 and 106 cells mL-1 and an ultralow detection limit of 7 cells mL-1. The specificity, stability, and repeatability of the measurements were successfully verified. Moreover, we used benzonase nuclease to achieve non-destructive recovery of cells for further clinical studies. According to the results, our aptasensor was more sensitive measuring the number of CTCs than other approaches because of the employment of TDNs, CuPdPt NW-GDY, and AuPd HSs. We designed a reliable sensor system for the detection of CTCs in the peripheral blood, which could serve as a new approach for cancer diagnosis at an early stage.

Abstract PO3-14-10: A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy: final results of baseline data

Abstract Background: The presence of liquid biomarkers such as circulating tumor cells (CTCs) among women undergoing neoadjuvant chemotherapy (NAC) for breast cancer may be associated with treatment response and/or an increased risk of recurrence, but limited data is available. Objectives: To detect and enumerate CTCs in blood samples from women with a new diagnosis of non-metastatic breast cancer of any subtype both i) at baseline (prior to commencing NAC), and ii) after completion of NAC and surgery using the Epic Sciences platform. Methods: Women with non-metastatic breast cancer of any subtype who have not yet commenced NAC were included, irrespective of age. Those with a prior history of another invasive cancer (apart from non-melanoma skin cancer identified 5+ years prior to enrollment) were excluded. Blood samples were obtained to measure CTCs prior to commencing NAC, and after completion of NAC and surgery (at least 4 weeks post-operatively). As previously described, CTC identification was based on immunofluorescence analysis using the Epic Sciences platform (Ueno et al 2017). Clinical/pathological data and clinical outcomes were abstracted from patients’ medical records. Associations between CTC detection and clinical/pathologic characteristics were evaluated using Fisher’s exact test for categorical variables and t-test or Wilcoxon rank sum tests for numerical variables. All analyses were performed using the R software package. Results: 50 participants who met eligibility criteria were included; a baseline blood sample was evaluable for CTC detection and enumeration for 47 patients. The median age at breast cancer diagnosis was 52 (29-75). Among them, 18 (38.3%) had HER2+ breast cancer, 17 (36.1%) had hormone receptor (HR)+/HER2- breast cancer and 12 (25.5%) had triple negative breast cancer (TNBC). The majority of patients (91%) received anthracycline and taxane-based NAC. A total of 68 samples were tested for CTC enumeration (5 mL equivalent per sample) including 47 pre-treatment and 21 post-treatment samples. CTCs were detected in 37 (79%) of patients for whom a baseline (pre-NAC) sample was available with a mean of 4.2 (SD 17.0) CTCs/mL and a range of 0 - 115.4 CTCs/mL. Detection of CTCs at baseline was highest among patients with TNBC (n=10/12, 83%), followed by those with HER2+ (n=14/18, 78%) and HR+/HER2- (n=13/17, 76%) breast cancer. CTCs were detected in 43% (n=9/21) of post-treatment samples, with a mean of 1.2 (SD 3.6) CTCs/mL and range of 0 – 16.4 CTCs/mL. Among the 20 patients for whom matched pre- and post-treatment CTC results were available, 16 (80%) had detectable CTCs pre-treatment and 8 (40%) had detectable levels post-NAC and surgery; of the 8 patients with post-treatment CTCs, 4 (50%) had HR+/HER2- breast cancer, 2 had HER2+ (25%) and 2 (25%) had TNBC. Three patients had numerically higher CTC levels after completion of NAC and surgery compared to baseline levels, 2 of whom had HR+/HER2- breast cancer and one of whom had TNBC. To-date, only 8 patients (19% of 43 who have undergone surgery) have achieved a pathological complete response (PCR) to NAC, among whom 3 had matched pre- and post-treatment CTC results available. None of these 3 patients had detectable CTCs post treatment. Conclusions: Approximately 4 in 5 women with non-metastatic breast cancer who undergo NAC have detectable CTCs at baseline (pre-treatment) using the Epic Sciences Platform. Given that CTCs remain detectable in a high proportion (40%) of patients after NAC and surgery, evaluation of CTCs as a potential measure of minimal residual disease warrants further evaluation in this patient population. Citation Format: Rania Chehade, Arushi Jain, Veronika Moravan, Giuseppe Di Caro, Megan Slade, Nadine Hartmann, Rick Wenstrup, Ana Elisa Lohmann, William Tran, Katarzyna Jerzak. A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy: final results of baseline data [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 PO3-14-10.

Abstract PO3-16-02: Trastuzumab and taxane chemotherapy in the first-line in MBC patients with a HER2-negative primary tumor and HER2-positive circulating tumor cells: a phase II trial

Abstract Purpose: HER2 overexpressing circulating tumor cells (CTCs) are observed in up to 20% of HER2-negative metastatic breast cancer patients. Since targeted anti-HER2 therapy has drastically increased the outcome of patients with HER2 positive breast cancer, we hypothesized that patients with HER2 overexpressing CTCs might benefit from the addition of trastuzumab to chemotherapy. Methods: In this single-arm, phase II trial, HER2 negative patients with HER2-positive CTCs received trastuzumab as addition to first-line treatment with taxane chemotherapy (trastuzumab group). Patients with detectable CTCs but without HER2 overexpression and that received taxane chemotherapy only, were used as internal control group (taxane monotherapy group). The primary outcome measure was progression-free rate at 6 months (PFR6). The study was powered to reach a PFR6 of 80%; in this case this strategy was considered worth further investigation. In November 2022, the study was terminated early due to slow patient accrual. Results: 63 HER2 negative patients with metastatic disease were screened, of which 8 patients had HER2-positive CTCs and were treated in the first line with trastuzumab in addition a taxane. Patients receiving taxane monotherapy were used as a control group (n = 27). The median number of CTCs was 15 (range 1 – 131) in patients with HER2-positive CTCs, compared to median 5 (range 1-1047) in the control group. PFR6 was 50% in the trastuzumab group and 64% in the taxane monotherapy group, with no significant difference in median PFS (8 versus 9 months, p=0.51). Excluding the patients that had no measurable CTCs, there was a borderline significant difference between median CTC count in the patients that did and that did not have HER2 positive CTCs (p=0.05). We confirmed a significant higher CTC-count in samples that had at least one HER2-postive CTC in an independent patient set (n=233, p< 0.001). Conclusions: Although this study was performed in a limited number of patients, no clinical benefit of trastuzumab was observed. Firstly, we conclude that our strategy was not feasible for clinical implementation due to high numbers needed to screen. And, second, due to the strong correlation between CTC numbers and detected CTC HER2 positivity, the prognostic value of the CTC numbers precludes judgement of any predictive value of the HER2 status of CTCs. Citation Format: Noortje Verschoor, Manouk Bos, Inge de Kruijff, Ngoc M Van, Jaco Kraan, Jan Drooger, Johanna Zuetenhorst, Saskia M Wilting, Stefan Sleijfer, Agnes Jager, John WM Martens. Trastuzumab and taxane chemotherapy in the first-line in MBC patients with a HER2-negative primary tumor and HER2-positive circulating tumor cells: a phase II trial [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 PO3-16-02.

Circulating Tumor Cell Phenotype Detection and Epithelial-Mesenchymal Transition Tracking Based on Dual Biomarker Co-Recognition in an Integrated PDMS Chip.

Circulating tumor cells (CTCs) are widely considered as a reliable and promising class of markers in the field of liquid biopsy. As CTCs undergo epithelial-mesenchymal transition (EMT), phenotype detection of heterogeneous CTCs based on EMT markers is of great significance. In this report, an integrated analytical strategy that can simultaneously capture and differentially detect epithelial- and mesenchymal-expressed CTCs in bloods of non-small cell lung cancer (NSCLS) patients is proposed. First, a commercial biomimetic polycarbonate (PCTE) microfiltration membrane is employed as the capture interface for heterogenous CTCs. Meanwhile, differential detection of the captured CTCs is realized by preparing two distinct CdTe quantum dots (QDs) with red and green emissions, attached with EpCAM and Vimentin aptamers, respectively. For combined analysis, a polydimethylsiloxane (PDMS) chip with simple structure is designed, which integrates the membrane capture and QDs-based phenotype detection of CTCs. This chip not only implements the analysis of the number of CTCs down to 2 cellsmL-1, but enables EMT process tracking according to the specific signals of the two QDs. Finally, this method is successfully applied to inspect the correlations of numbers or proportions of heterogenous CTCs in 94 NSCLS patients with disease stage and whether there is distant metastasis.

Abstract PO5-14-02: Diagnostic leukapheresis to increase CTC detection rate in breast cancer patients after 5 years of adjuvant endocrine treatment

Abstract Purpose: Patients with early hormone receptor positive, HER2 negative breast cancer have a risk of recurrence for up to 20 years after the initial diagnosis and is therefore treated with adjuvant endocrine treatment (ET) for 5 to 10 years. As a result there is a large proportion of patients that overtreated, hence, an urgent need for better biomarkers to guide treatment decisions. In previous research, it was shown that circulating tumor cells (CTCs) are detected in 5% of patient after 5 years of adjuvant treatment and that this indicated a 13-fold higher risk of recurrence, with a median lead time of 2.8 years. However, the sensitivity and specificity of the test were moderate. This could be caused by stochastic variation, given the low numbers of CTCs detected in 7.5 ml peripheral blood. We hypothesized that increasing the screening volume, increases the test statistics. Methods: In this study, we investigated whether increasing the screening volume by diagnostic leukapheresis (DLA) can increase the detection rate of CTCs (CellSearch platform). With DLA, 5 liters of peripheral blood was screened, which in previous studies had shown to increase the CTC-yield per ml by 25-fold. To this end, we included early hormone receptor positive, HER2 negative breast cancer patients that were treated with ET for 4.5-5.5 years with no signs of disease recurrence. We compared the number of detected CTCs in the standard fraction of 7,5 mL peripheral blood (PB), to buffy coat (30 ml), to DLA sample (200 × 109 white blood cells (WBC), equal to 25-50 mL PB) and to DLA after WBC depletion. Results: Thirty two hormone receptor positive/HER2 negative breast cancer patients were included in this study after a median duration of ET of 60 months, of which 3 (9%) patients completed ET according to clinical guidelines and the remaining patients were still on treatment. Twenty eight patients (88%) had 1-3 positive lymph nodes at diagnosis, and 3 patients had 4-9 positive lymph nodes. The Clinical Treatment Score at 5 years (CTS5 score) was intermediate or high in 75%, and unknown in 19%, indicating a recurrence risk of >5% at years 5-10 after diagnosis. In none of the included patients, CTCs were detected in 7,5 ml PB, nor in any of the other tested blood volumes. To date, 2 patients have been diagnosed with overt metastatic disease during follow-up. DLA was well tolerated and did not cause adverse events. Conclusions: We conclude that DLA was unable to increase the detection rate of CTCs in hormone receptor positive, HER2 negative patients with early breast cancer being on treatment for a median duration of 5 years. Additional circulating tumor DNA analyses are planned. Citation Format: Noortje Verschoor, Jaco Kraan, Johanna Zuetenhorst, Mijntje Vastbinder, Jan Drooger, Stefan Sleijfer, Saskia M Wilting, John WM Martens, Agnes Jager. Diagnostic leukapheresis to increase CTC detection rate in breast cancer patients after 5 years of adjuvant endocrine treatment [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 PO5-14-02.

Abstract PO4-13-08: Mechanical conditioning (MeCo) score is higher in circulating tumor cells (CTCs) compared to primary tumors in early-stage breast cancer and even higher in metastases compared to CTCs in metastatic breast cancer

Abstract Background: The mechanical conditioning (MeCo) score is a gene expression signature that is acquired early by cancer cells in the primary breast tumor and is reflective of their responsiveness to matrix stiffness (fibrotic-like matrix rigidity). Further, chromatin remodeling in response to stiffness allows cancer cells to retain their aggressive features even in the absence of mechanical stimulation by the primary tumor microenvironment, for instance, during their dissemination through the circulation in metastasis. Importantly, patients who have high MeCo score tumors are at higher risk of developing metastatic breast cancer, compared to low MeCo scores (p< 0.0001; HR=2.2, 95%CI 1.7-2.7; Watson et al 2021, PMID: 34192535). Moreover, circulating tumor cells (CTCs) are associated with higher rate of metastatic dissemination, making CTC detection in the circulation of breast cancer patients a significant prognostic biomarker for breast cancer metastasis. Beyond their enumeration per blood volume units, specific prognostic features of CTCs are not fully explored. We sought to determine whether, compared to primary tumors, CTCs retain high MeCo scores and whether these scores are maintained during late-stage breast cancer in the metastatic sites. Methods: CTCs were isolated from peripheral blood of two patient cohorts: stage II-III breast cancer patients using immunomagnetic enrichment/FACS methodology (Lang et al 2018, PMID: 29868978) and of metastatic breast cancer patients using ANGLE Parsortix microfluidics system (Ring et al 2022, PMID: 35000083). Gene expression profiling using RNA-seq was performed in CTCs and in matching primary tumors (PT) and metastases (MET) for the early-stage and late-stage cohorts, respectively. A quantile normalization approach was used to allow comparison across cohorts. MeCo scores were computed for all samples as per Watson et al 2021. Wilcoxon matched-pairs signed rank test was performed for comparison of MeCo scores from matching samples within each cohort; Mann-Whitney unpaired test was used to compared MeCo scores of CTCs across cohorts. Results: In 12 pairs of early-stage breast cancer patient CTCs and PT, MeCo scores of CTCs were significantly higher than their matched PTs (p=0.026). Furthermore, in 26 pairs of metastatic patient’s CTCs and MET, MeCo scores of METs were significantly higher than matching CTCs (p=0.0004). MeCo scores of CTCs were similar between early- and late-stage breast cancer despite differing CTC isolation strategies (epitope-dependent and microfluidics size gradient). 98% of genes in the MeCo score were present in the evaluable CTC, MET and PT samples. Conclusions: Our results show that the MeCo score is higher in CTCs compared to PTs and in METs compared to CTCs in early- and late-stage breast cancer, respectively. Therefore, the MeCo score is progressively higher throughout the metastatic cascade in breast cancer. These findings demonstrate that mechanical conditioning is retained during metastatic progression, even as cancer cells transition through the circulation where the initial induction by matrix stiffness is lost. Further, these findings support that cancer cells with higher MeCo scores are more competent with–and potentially selected for by–metastatic progression. Importantly, these findings provide a novel feature of CTCs, mechanical conditioning (MeCo), which is associated with higher capacity for metastasis. Further, since the CTC MeCo score is elevated even in early-stage breast cancer, it could provide, in addition to CTC enumeration, a potential prognostic tool to predict metastatic risk in breast cancer patients. Citation Format: Gus Mouneimne, Adam Watson, Adam Grant, Daniel Campo, Alexander Ring, Pushpinder Bains, Julie Lang. Mechanical conditioning (MeCo) score is higher in circulating tumor cells (CTCs) compared to primary tumors in early-stage breast cancer and even higher in metastases compared to CTCs in metastatic breast cancer [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 PO4-13-08.

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.

Microfluidic-SERS Technologies for CTC: A Perspective on Clinical Translation.

Enumeration and phenotypic profiling of circulating tumor cells (CTCs) provide critical information for clinical diagnosis and treatment monitoring in cancer. To achieve this goal, an integrated system is needed to efficiently isolate CTCs from patient samples and sensitively evaluate their phenotypes. Such integration would comprise a high-throughput single-cell processing unit for the isolation and manipulation of CTCs and a sensitive and multiplexed quantitation unit to detect clinically relevant signals from these cells. Surface-enhanced Raman scattering (SERS) has been used as an analytical method for molecular profiling and in vitro cancer diagnosis. More recently, its multiplexing capability and power to create distinct molecular signatures against their targets have garnered attention. Here, we share our insights into the combined power of microfluidics and SERS in realizing CTC isolation, enumeration, and detection from a clinical translation perspective. We highlight the key operational factors in CTC microfluidic processing and SERS detection from patient samples. We further discuss microfluidic-SERS integration and its clinical utility as a paradigm shift in clinical CTC-based cancer diagnosis and prognostication. Finally, we summarize the challenges and attempt to look forward to what lies ahead of us in potentially translating the technique into real clinical applications.

Trajectory analysis of circulating tumor cells through contorted deterministic lateral displacement array for unruptured trapping: a simulation study

During the multistage process of cancer spread, fragments of cells branch out from the primitive tumor tissue and scatter throughout the body by blood flow stream or lymph, which are termed as circulating tumor cells (CTCs), regarded as an important biomarker for early cancer diagnosis. Deterministic lateral displacement (DLD) is one of the most efficient passive type particle isolation techniques which functions on asymmetrical laminar flow diversion around the placed microposts forming an array, based on the critical dimeter of the device. In this simulation study, complete trajectory analysis of CTCs and WBCs has been performed using COMSOL Multiphysics software with respect to the angular orientation of sample inlet channel. A simulated model of an asymmetric DLD array-based microfluidic device with latest empirical expression for critical diameter has been presented with optimized sample inlet orientation so as to maintain perfect separation efficiency of CTCs along with arrangement for unruptured trapping of CTCs and WBCs. The approximate CTC isolation efficiency was obtained above 95% for sample inlet inclinations up to 0.5°, and further tilt showed reduction in efficiency. Migration angle of CTC trajectory for the modeled device was obtained around 15° for both the sample inlet orientations leading to least intermingling of CTCs and WBCs. A smaller number of CTC–WBC interaction points was observed for clockwise sample inlet tilt compared to the anticlockwise displacement reducing possibilities of cell rupture. The shear rate around central microposts was found to be higher as well in clockwise sample inlet orientation. This simulation study establishes that sample inlet clockwise tilting of maximum around 0.5°–0.7° leads to better CTC isolation and provides better chances of unruptured trapping of cells at the designated outlets. The results of this study provide an approach toward further optimization of DLD devices' functioning and, thus, could help fabricate better DLD-based microfluidic devices for efficient trapping of CTCs.

Orthotopic model for the analysis of melanoma circulating tumor cells

Metastatic melanoma, a highly lethal form of skin cancer, presents significant clinical challenges due to limited therapeutic options and high metastatic capacity. Recent studies have demonstrated that cancer dissemination can occur earlier, before the diagnosis of the primary tumor. The progress in understanding the kinetics of cancer dissemination is limited by the lack of animal models that accurately mimic disease progression. We have established a xenograft model of human melanoma that spontaneously metastasizes to lymph nodes and lungs. This model allows precise monitoring of melanoma progression and is suitable for the quantitative and qualitative analysis of circulating tumor cells (CTCs). We have validated a flow cytometry-based protocol for CTCs enumeration and isolation. We could demonstrate that (i) CTCs were detectable in the bloodstream from the fourth week after tumor initiation, coinciding with the lymph node metastases appearance, (ii) excision of the primary tumor accelerated the formation of metastases in lymph nodes and lungs as early as one-week post-surgery, accompanied by the increased numbers of CTCs, and (iii) CTCs change their surface protein signature. In summary, we present a model of human melanoma that can be effectively utilized for future drug efficacy studies.

Detection of γ-OHPdG in circulating tumor cells of patients with hepatocellular carcinoma as a potential prognostic biomarker of recurrence

Background and AimsBlood-based biomarkers for hepatocellular carcinoma (HCC) and its recurrence is lacking. We previously showed that hepatic γ-hydroxy-1,N2-propano-2′-deoxyguanosine (γ-OHPdG), an endogenous DNA adduct derived from acrolein by lipid peroxidation, increased during hepatocarcinogenesis. Additionally, higher hepatic γ-OHPdG from HCC patients after surgery were strongly associated with poor survival (P < .0001) and recurrence-free survival (P = .007) (Fu Y et al., Hepatology, 2018). These findings suggest that γ-OHPdG is a potential prognostic biomarker for HCC and its recurrence. To attain the goal of using γ-OHPdG as a biomarker in future preventive and therapeutic trials, we developed a blood-based method to detect γ-OHPdG in circulating liver tumor cells from HCC patient blood. MethodsWe first established the specificity of anti-γ-OHPdG antibody by determining its dose-response in HepG2 cells treated with acrolein. Then, HepG2 cells in spiked blood of healthy volunteers (HV) and circulating tumor cells (CTCs) from 32 HCC patients were isolated using a RosetteSep™ CD45 Depletion Cocktail and Ficoll Paque. The HCC CTCs identified with anti-asialoglycoprotein receptor 1, a surface protein expressed solely in hepatocytes, were stained with an anti-γ-OHPdG antibody. The number of total HCC CTCs and γ-OHPdG-positive CTCs, as well as the staining intensity, were quantified using a MetaMorph software. As an initial effort toward its clinical application, we also evaluated γ-OHPdG in CTCs from these patients along with certain clinical features. ResultsThe γ-OHPdG antibody specificity was demonstrated by an acrolein concentration-dependent increase of γ-OHPdG-positive HepG2 cells and the intensity of γ-OHPdG staining. The recovery of HepG2 cells from spiked blood was ∼50-60%, and the positivity rate of CTCs in blood from 32 patients with advanced HCC was 97%. The MetaMorph analysis showed a wide variation among patients in total number of CTCs, γ-OHPdG positivity, and staining intensity. Statistical analysis revealed γ-OHPdG in CTCs of these patients appear to be associated with multifocality and poor differentiation. ConclusionsA blood-based method was developed and applied to HCC patients to evaluate the potential of γ-OHPdG in CTCs as a prognostic biomarker.

Prognostic Value of Liquid-Biopsy-Based Biomarkers in Upper Tract Urothelial Carcinoma.

Currently, there are no reliable prognostic factors to determine which upper tract urothelial carcinoma (UTUC) patients will progress after radical nephroureterectomy (RNU). We aim to evaluate whether liquid-biopsy-based biomarkers (circulating tumor cells (CTCs), cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA)) were able to predict clinical outcomes in localized UTUC patients undergoing RNU. Twenty patients were prospectively enrolled between 2021 and 2023. Two blood samples were collected before RNU and three months later. CTCs and cfDNA were isolated and evaluated using the IsoFlux system and Quant-iT PicoGreen dsDNA kit, respectively. Droplet digital PCR was performed to determine ctDNA status. Cox regression analysis was performed on CTCs, cfDNA, and ctDNA at two different follow-up time points to examine their influence on tumor progression and cancer-specific survival (CSS). During a median follow-up of 18 months, seven (35%) patients progressed and three (15%) died. Multivariate analysis demonstrated that cfDNA levels three months after RNU are a significant predictor of tumor progression (HR = 1.085; p = 0.006) and CSS (HR = 1.168; p = 0.029). No associations were found between CTC enumeration and ctDNA status with any of the clinical outcomes evaluated. The evaluation of cfDNA levels in clinical practice could improve the disease management of UTUC patients.

Circulating tumor cells help differentiate benign ovarian lesions from cancer before surgery: A literature review and proof of concept study using flow cytometry with fluorescence imaging.

Current tools are insufficient for distinguishing patients with ovarian cancer from those with benign ovarian lesions before extensive surgery. The present study utilized a readily accessible platform employing a negative selection strategy, followed by flow cytometry, to enumerate circulating tumor cells (CTCs) in patients with ovarian cancer. These counts were compared with those from patients with benign ovarian lesions. CTC counts at baseline, before and after anticancer therapy, and across various clinical scenarios involving ovarian lesions were assessed. A negative-selection protocol we proposed was applied to patients with suspected ovarian cancer and prospectively utilized in those subsequently confirmed to have malignancy. The protocol was implemented before anticancer therapy and at months 3, 6, 9 and 12 post-treatment. A cut-off value for CTC number at 4.75 cells/ml was established to distinguish ovarian malignancy from benign lesions, with an area under the curve of 0.900 (P<0.001). In patients with ovarian cancer, multivariate Cox regression analysis revealed that baseline CTC counts and the decline in CTCs within the first three months post-therapy were significant predictors of prolonged progression-free survival. Additionally, baseline CTC counts independently prognosticated overall survival. CTC counts obtained with the proposed platform, used in the present study, suggest that pre-operative CTC testing may be able to differentiate between malignant and benign tumors. Moreover, CTC counts may indicate oncologic outcomes in patients with ovarian cancer who have undergone cancer therapies.

Abstract 7499: Circulating tumor cell enumeration and protein expression by CapioCyte and Allegro Plus imaging

Abstract Radiation or chemoradiation followed by surgical resection is the current standard of therapy for many diseases. These treatments are increasingly supplemented with monoclonal antibodies, such as epidermal growth factor receptor (EGFR)-targeting and programmed death ligand 1 (PD-L1) checkpoint-blocking antibodies. The use of these therapies is typically dependent on solid tissue biopsy to confirm elevated expression of these markers in the solid tumor, which carries complications and may only be sampled at diagnosis. In contrast, a liquid biopsy may detect these markers with minimal invasiveness and at multiple points throughout treatment and recovery. Here, we describe the development of an automated and integrated workflow for circulating tumor cell (CTC) analysis. We previously demonstrated that the CapioCyte method is a highly sensitive approach for enrichment of CTCs using a combination of biomimetic rolling on recombinant E-selectin and antibody-mediated capture on a nanostructured polymer surface within a flow chamber for capture. Here we report a fully automated CapioCyte instrument for CTC purification and immunofluorescence staining using epithelial, white blood cell and nuclear makers to identify and distinguish CTCs from WBCs while also quantifying EGFR or PD-L1 presence on respective CTCs. The immunostaining conditions were developed and validated for high analytical sensitivity and specificity using cell lines and health donor white blood cells with known biomarker status. The final assay conditions were transferred to the CapioCyte for automated immunostaining. CapioCyte enriched and stained CTC samples are paired with the BioView Allegro Plus platform, an imaging and analysis solution using machine learning and customized algorithms for automated imaging and CTC candidate identification based on high-resolution Z-stack images. The reported comprehensive and automated workflow was used to enumerate CTCs in patients with non-small cell lung carcinoma (NSCLC) in a feasibility cohort. Median CTC counts from NSCLC patients was 7.5 CTC/ml (n=26, mean 51.7, range 0.3 - 406.2). Studies are on-going to expand the cohort including testing blood from head and neck cancer patients and respective EGFR and PD-L1 status. The combined and automated CapioCyte-Allegro Plus workflow shows great potential for routine quantification of CTCs and EGFR and PD-L1 status. Citation Format: Ju-heon Lim, Chassidy Johnson, Tal Kaplan, Michael J. Poellmann, Yuval Harari, Elad Kfir, Andrew Z. Wang, Seungpyo Hong. Circulating tumor cell enumeration and protein expression by CapioCyte and Allegro Plus imaging [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 7499.

Abstract 7503: Non-clinical study of circulating tumor cells in humanized mice: Post-xenograft effects in immunodeficient mice

Abstract Circulating tumor cells (CTCs) possess the potential to serve as innovative markers in clinical trials, contributing to the understanding of cancer metastasis mechanisms, diagnosis, prognosis, the development of personalized treatment strategies, and enabling the monitoring of treatment efficacy. NSG mice, which are a strain of immunodeficient mice frequently utilized as a non-clinical model for cancer research, exhibit diminished levels of natural killer (NK) cells and abnormal monocytes in their bloodstream. In a previous study, a positive correlation was observed between the duration of injection and the levels of white blood cells (WBC) in mice that were injected with various cancer cell lines derived from humans. To evaluate the correlation among the primary tumor and circulating tumor cells (CTCs), as well as the impact on immune cells, we assessed changes in immune cell composition, primary tumor size, and enumeration of CTCs in xenografted mice over a period of time. This investigation was carried out using CytoGen's Smart Biopsy™ systems. The results, which were obtained using equipment including CytoGen's Smart Biopsy™ Systems, showed that the distribution of white blood cell (WBC) components exhibited variations based on age, primary tumor burden, and circulating tumor cell (CTC) count following injection. A notable association was observed between the augmentation of immune cells and the quantity of circulating tumor cells (CTCs) present in the bloodstream. Therefore, alterations in immune cells may potentially impact the potential to affect the ability to influence the efficacy of cancer immunotherapy, metabolic therapy, and anti-metastatic therapy. When conducting non-clinical investigations with the objective of developing cancer treatment protocols, it is crucial to meticulously select humanized or immunodeficient mice that are appropriate for the study. Therefore, it is advisable to perform regular monitoring of murine blood samples in order to observe any variations in the counts of circulating tumor cells (CTCs), while considering a standardized blood volume per individual. In the immunodeficient mouse model, the existence of circulating tumor cells (CTCs) results in an increase in the quantity of cells present in the bloodstream. The observed rise in circulating tumor cells (CTCs) is found to be concurrent with the progression and development of the primary tumor. The consideration of this aspect is of utmost importance in the formulation of non-clinical studies aimed at the development of cancer treatment protocols. Citation Format: Sehyung Pak, Hajin Pak, Hee Jin Yoo, Sra Min, Hui Ji Kim, JaeYoon Lee, Min-Ji Song, Jaesung Choi, Aejin Jeong, Hanbyeol Kim, Jung Won Kim, Hai-Chon Lee. Non-clinical study of circulating tumor cells in humanized mice: Post-xenograft effects in immunodeficient mice [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 7503.

Abstract 3017: Lack of MCPIP1 enzymatic activity leads to neoplastic transformation of normal kidney cells

Abstract Introduction: The MCPIP1 protein (Monocyte Chemotactic Protein-1 Induced Protein) is involved in the negative regulation of inflammation due to its RNase activity. A growing number of publications suggest that the MCPIP1 protein may influence the development of cancer by regulation of factors involved in angiogenesis, proliferation and cell death. We believe, that MCPIP1 may be a potential tumor suppressor. Our recent results show that lack of enzymatic activity of MCPIP1 leads to increased tumor growth, angiogenesis and metastasis. The main aim of our research is to study the importance of MCPIP1 for neoplastic transformation of normal, epithelial kidney cells, as well as acquirement of stemness features. Material and Methods: To examine the effect of MCPIP1 in normal epithelial cell lines TCMK-1, RPTEC-Tert1 and HK-2 were transduced to overexpress mutated, inactive form of MCPIP1 (pLIX D141N) with proper control (pLIX PURO). We analyzed cells clonogenicity, as well as the levels of CSCs markers by western blot, NGS and qPCR. Next, cells were injected subcutaneously into NOD-SCID mice to check if mutation of MCPIP1 will induce tumor growth in vivo. Mice blood was analyzed for the presence of circulating tumor cells (CTCs), and secretion of cytokines by Proteome Profiler assay. Tumors were evaluated by multiple IHC stainings, as well as western blot and qPCR. Results and Discussion: We have shown that the mutation of MCPIP1 increases the clonogenicity of kidney cells. Moreover, pLIX D141N cells were characterized by higher levels of the c-Met receptor, vimentin, Twist, CD44 and c-Myc, which are the markers of epithelial to mesenchymal transition and cancer stemness. The obtained results suggest that the loss of the MCPIP1 protein causes the acquisition of the characteristics of neoplastic cells. Next, we checked whether MCPIP1 mutation in normal cells predisposes them to proliferate when administered to mice. We found that, D141N mutation caused the growth of large tumors with higher expression of Cd44, Klf4 and Myc and number of CTCs, while the control cells injected into mice developed only local fibrosis with high expression of aSMA. pLIX D141N tumors secreted CXCL16 into mice plasma, and were characterized by increased phosphorylation of c-Met, Src kinase and c-Myc. Conclusions: We believe that the MCPIP1 protein may be a marker of tumor initiation and play a key role in neoplastic transformation by regulating the changes in cell phenotype and levels of CSCs markers. This study was supported by National Science Center grants no. 2022/47/B/NZ5/02724, 2022/45/B/NZ5/01973 and MNS 15/2021. Citation Format: Paulina Marona, Iga Piasecka, Judyta Gorka, Oliwia Kwapisz, Rafal Myrczek, Ewelina Pospiech, Jolanta Jura, Katarzyna Miekus. Lack of MCPIP1 enzymatic activity leads to neoplastic transformation of normal kidney cells [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 3017.

Abstract 7493: Real-time monitoring of CTC number, biomarker expression, and kinetics as prognostic and predictive markers in patients with GI malignancies

Abstract Background: Most of the utility in the clinic regarding liquid biopsies has been around circulating tumor DNA (ctDNA). ctDNA analysis provides valuable insight into mutational burden and therapeutic targets, but the speed and cost of analysis are prohibitive especially when it comes to serial testing. Using a cohort of patients with advanced or metastatic gastrointestinal (GI) cancers, we herein show the feasibility of using circulating tumor cell (CTC) enumeration, biomarker analysis, and kinetics as complementary to or as an alternative to ctDNA testing. Methods: Using the RareCyte platform, nucleated cells from blood were collected, spread on slides, stained, and imaged with CyteFinder® for CTC identification (CK/EpCAM+, CD45-) and quantification of biomarkers (PD-L1/HER2 or Ki67/EGFR). CTC enumeration (CLIA-accredited CTC test) and biomarker status for each patient were tracked over time and compared with ctDNA mutational analysis and tumor burden. Results: CTCs were detected in 60/70 samples (86%) and in 36/38 unique patients (95%). On average, more CTCs were detected in baseline samples from colorectal cancer patients compared to other subtypes (Table 1). Moreover, multiple patients showed pronounced changes in CTC number that correlated with treatment response, as well as strong correlation between CTC and tissue biomarker expression e.g. HER2. Rapid decline and/or CTC clearance was seen as early as a few days into starting effective systemic therapy. The average turnaround time for CTC testing was 5-7 days. Table 1. CTC values in initial baseline blood draw sample Gastrointestinal cancer subtype # of patients Average # of CTCs per 7.5mL blood Median # of CTCs per 7.5mL blood Minimum # of CTCs per 7.5mL blood Maximum # of CTCs per 7.5mL blood Colorectal carcinoma 27 84 3 0 1378 Cholangiocarcinoma 3 8 3 1 23 Pancreatic cancer 3 2 2 0 3 Hepatocellular carcinoma 2 4 5 0 10 Gallbladder cancer 1 6 6 3 8 Appendix cancer 1 2 2 1 3 Anal cancer 1 0 0 0 0 Conclusions: Our study emphasizes that real-time monitoring of CTCs is complementary to traditional tumor burden monitoring via imaging and ctDNA analysis. CTCs offer multiple unique advantages vs ctDNA including early response assessment within days of initiating therapy, a substantial decrease in cost of serial testing, and the ability to assess clinically relevant cell surface protein biomarkers e.g. HER2 and PD-L1. Citation Format: Erin G. Bayer, Brock M. Bartels, Rachel N. Ponting, Areeb Lutfi, Maaz K. Afghan, Arturo B. Ramirez, Pashtoon M. Kasi. Real-time monitoring of CTC number, biomarker expression, and kinetics as prognostic and predictive markers in patients with GI malignancies [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 7493.

Abstract 3750: Dual isolation and profiling of circulating tumor cells and circulating tumor DNA from a single liquid sample using the Genesis Cell Isolation System and Droplet Digital PCR

Abstract Introduction: Recent advances in technology support the use of liquid biopsy as a noninvasive method for cancer monitoring and diagnosis. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are both regarded as key liquid biopsy biomarkers, offering significant disease-related molecular insights. Despite their potential, dual isolation and profiling have been challenged in a clinical setting by the lack of established platforms and optimized protocols that support this approach. The Genesis System with Celselect SlidesTM captures CTCs based on size and deformability, then the enriched cells can be recovered for subsequent downstream analysis. Concurrently, Droplet Digital PCR (ddPCRTM) can be utilized to interrogate cell-free DNA (cfDNA) to determine the presence of a range of DNA aberrations present in ctDNA. This study, using a model lung CTC-ctDNA sample, illustrates the feasibility of this dual-biomarker approach to detect cancer-associated nucleic acid mutations simultaneously with CTC enumeration, using commercially available kits and instruments. Methods: To model lung cancer samples, blood samples were prepared by spiking in two model circulating markers, cultured cancer cells (model CTCs, human ㅣlung cancer cell line A549) and fragmented genomic DNA containing EGFR mutations (model ctDNA, multiplexed 23 ctDNA [~145 bp band &amp;gt; 95%]), into whole blood from a healthy donor prior to enrichment/extraction. Plasma isolation from the whole blood model samples was performed using either two-step centrifugation or Ficoll-Paque PLUS solution for plasma/buffy coat separation. The extracted plasma layer was used for ctDNA separation, while the remaining cell layer was used for CTC isolation on the Genesis System with a modified protocol. Results: The Genesis System isolated 78-79% of spiked cancer cells in whole blood at a concentration of 40 cells/ml. Despite additional plasma separation steps and liquid handling, the system isolated CTCs spiked in with minimal cell loss (&amp;lt;6%). With an optimized ctDNA extraction, more than 70% of spiked ctDNA in blood samples in the size range of 120-220 bp was extracted. Using the Bio-Rad QX200 ddPCR System and ddPCR EGFR T790M Mutation Detection Assay, blood samples with the model ctDNA spiked in showed positive droplets (17 copies) for EGFR T790M while control blood samples without model ctDNA spiked in showed no positive droplets. Discussion and Conclusions: This work illustrates an optimized protocol for the dual isolation and profiling of CTCs and ctDNA from a single blood sample by using an automated CTC isolation platform and high-sensitivity ddPCR based ctDNA mutation detection. This protocol enables comprehensive liquid biopsy studies using a single blood sample from cancer patients with readily available instruments and kits, which can be applied to many other cancers. Citation Format: Yoon-Tae Kang, Abiodun Bodunrin, Errile Pusod, Kris Simonyi, Adam Corner, David Coe, Michelle Racey, Elizabeth J. Dreskin. Dual isolation and profiling of circulating tumor cells and circulating tumor DNA from a single liquid sample using the Genesis Cell Isolation System and Droplet Digital PCR [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 3750.

Abstract 3696: Liquid biopsy biomarker analysis during treatment with 177Lu-PSMA-617 in castrate resistant prostate cancer

Abstract Background: Prostate-Specific Membrane Antigen (PSMA) is a highly expressed prostate cancer (PCa) cell surface protein regulated by androgen receptor signaling and the target of the recently approved radioligand therapy 177Lu-PSMA-617. While some patients have dramatic responses to this therapy, rates of upfront or acquired resistance are high even in patients with PSMA-PET-avid disease, and better biomarkers of 177Lu PSMA-617 response and resistance are needed to select patients more effectively for this therapy. To address this need, we evaluated the relationship between longitudinal circulating tumor cell (CTC) androgen signaling and PSMA expression to 177Lu-PSMA-617 response or resistance in patients with metastatic prostate cancer. We leveraged a CTC androgen signaling signature that we have previously shown is prognostic for patients with metastatic prostate cancer treated with AR signaling inhibitors. Methods: Longitudinal blood samples were collected from 20 patients for circulating tumor cell (CTC) analysis at C1D1, C2D1, C4D1, and C6D1. CTCs were isolated with antibodies to EpCAM followed by parallel enumeration, analysis of cell surface expression of PSMA and RNA extraction for AR or neuroendocrine marker transcriptional profiling. Results: CTC samples were assessed for PSMA cell surface protein expression prior to treatment and on treatment. FOLH1 (PSMA) gene expression was detected from CTCs at baseline that persisted through treatment. Gene expression representative of AR pathway activation was present in 70% of the cohort at baseline and 50% of patients at C2D1. Concordant with these observations, no significant decrease in CTC number or PSMA protein expression was seen from baseline to C2D1. However, increased CTC AR pathway activation at C2D1 was associated with increased mean serum PSA (155 vs 21.93 ng/ml) and was associated with decreased duration of response. Conclusions: Improved tools to predict who will respond to 177Lu-PSMA-617 and detect early treatment resistance and disease progression are needed. Here, we show that persistent CTC burden, CTC AR signaling, and CTC PSMA expression are common early in treatment with 177Lu-PSMA-617 and may be associated with lower likelihood of clinical response. Further ongoing studies are needed to elucidate the relationship between these liquid biopsy results and clinical outcomes. Citation Format: Jamie M. Sperger, Marina N. Sharifi, Luke Nunamaker, Viridiana Carreno, Alex H. Chang, Shannon R. Reese, Charlotte Linebarger, Amy K. Taylor, Grace C. Bliitzer, John Floberg, Shuang G. Zhao, Joshua M. Lang. Liquid biopsy biomarker analysis during treatment with 177Lu-PSMA-617 in castrate resistant prostate cancer [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 3696.

Abstract 533: Attenuating EZH2-mediated metastasis and tumor growth by pharmacological-based approaches using three-dimensional in vitro culture models

Abstract Triple Negative breast cancer (TNBC) accounts for 10-15% of all breast cancers where patients encounter poor clinical outcomes. Treatment modalities for TNBC are conventional cytotoxic chemotherapy and radiation leads to 35%-40% relapse within 5 years of diagnosis. There is a growing need for more effective targeted therapeutics to inhibit TNBC growth. The role of EZH2 in maintaining tumor growth and metastasis in TNBC is well documented. EZH2 promotes breast tumor-initiating cell expansion (BITC) and cancer progression by impairing the DNA damage repair process favoring activation of oncogenes. Despite the advancement in the discovery of inhibitors for EZH2 that attenuate its catalytic activity, resistance to these small molecules limits their use in solid tumors. The neurotransmitter dopamine via its D1 receptor activation in TNBC cell lines induces apoptosis and autophagy, as well as inhibits the invasion and regress in mammary tumors in vivo. Here we hypothesize that the presence of dopamine D1 receptor agonist (A77636) enhances the efficacy of EZH2 inhibitors (GSK126) to inhibit in vitro TNBC tumor growth and metastasis. To test the efficacy of the combination we employed a 3D culture system of MDA-MB-231 cells encapsulated in calcium-alginate microgels seeded from a microfluidic droplet generator. We also employed a 3D organ-on-chip-based microphysiological (MPS) platform (SynTumor) to study the effect of the drug combination on metastasis in vitro. The SynTumor MPS devices replicate the pathophysiological architecture of native vascularized breast tumors. The combination treatment enhanced a significant reduction of tumor spheroid diameter compared to the vehicle and indicated spheroid regression over treatment. Furthermore, the combination therapy induced necrosis and caused complete inhibition of EZH2 expression. The knockdown of EZH2 inhibited tumor spheroid formation and displayed cytoskeleton de-arrangement. In the microfluidic SynTumor model, circulating tumor cell numbers were reduced by half at 96 hrs after EZH2 combination treatment. Our data indicate that the combinatorial effect of DRD1 agonist and EZH2 inhibitor efficiently attenuates the EZH2-mediated in vitro tumor growth and metastasis (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: xilal Rima, Gautam Sarathy, Chunyu Hu, Divya S. Patel, Deborah Ramsey, Lara Rizotto, Dario Palmeri, Gwen Fewell, Bhuvaneswari Ramaswamy, Eduardo Reátegu, Eswar Shankar. Attenuating EZH2-mediated metastasis and tumor growth by pharmacological-based approaches using three-dimensional in vitro culture models [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 533.