Detection Of Circulating Tumor Cells Research Articles

Clinical Validation of a Size-Based Microfluidic Device for Circulating Tumor Cell Isolation and Analysis in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) presents as metastatic disease in one third of cases. Research on circulating tumor cells (CTCs) and liquid biopsies is improving the understanding of RCC biology and metastases formation. However, a standardized, sensitive, specific, and cost-effective CTC detection technique is lacking. The use of platforms solely relying on epithelial markers is inappropriate in RCC due to the frequent epithelial-mesenchymal transition that CTCs undergo. This study aimed to test and clinically validate RUBYchip™, a microfluidic label-free CTC detection platform, in RCC patients. The average CTC capture efficiency of the device was 74.9% in spiking experiments using three different RCC cell lines. Clinical validation was performed in a cohort of 18 patients, eight non-metastatic (M0), five metastatic treatment-naïve (M1TN), and five metastatic progressing-under-treatment (M1TP). An average CTC detection rate of 77.8% was found and the average (range) total CTC count was 6.4 (0-27), 101.8 (0-255), and 3.2 (0-10), and the average mesenchymal CTC count (both single and clustered cells) was zero, 97.6 (0-255), and 0.2 (0-1) for M0, M1TN, and M1TP, respectively. CTC clusters were detected in 25% and 60% of M0 and M1TN patients, respectively. These results show that RUBYchip™ is an effective CTC detection platform in RCC.

Aneuploid subtypes of circulating tumor cells and circulating tumor-derived endothelial cells predict the overall survival of advanced lung cancer.

This study aimed to detect circulating tumor cells (CTCs) and circulating tumor-derived endothelial cells (CTECs) in patients with advanced lung cancer, for describing the distribution characteristics of CTC and CTEC subtypes, exploring the correlation between CTC/CTEC subtypes and novel prognostic biomarkers. A total of 52 patients with advanced lung cancer were enrolled in this study. Using the subtraction enrichment-immunofluorescence in situ hybridization (SE-iFISH) system, CTCs and CTECs derived from these patients were identified. Based on cell size, there were 49.3% small and 50.7% large CTCs, and 23.0% small and 77.0% large CTECs. Triploidy, tetraploidy, and multiploidy varied in the small and large CTCs/CTECs. Besides these three aneuploid subtypes, monoploidy was found in the small and large CTECs. Triploid and multiploid small CTCs and tetraploid large CTCs were associated with shorter overall survival (OS) in patients with advanced lung cancer. However, none of the CTECs subtypes showed a significant correlation with patient prognosis. In addition, we found strong positive correlations (P<0.0001) in the four groups including triploid small cell size CTCs and multiploid small cell size CTECs, and multiploid small cell size CTCs and monoploid small cell size CTECs. Furthermore, combined detection of the specific subtypes, including triploid small CTC and monoploid small CTEC, triploid small CTC and triploid small CTEC, and multiploid small CTC and monoploid small CTEC, were associated with poor prognosis in advanced lung cancer. Aneuploid small CTCs are associated with the outcome of patients with advanced lung cancer. In particular, the combined detection of triploid small CTCs and monoploid small CTECs, triploid small CTCs and triploid small CTECs, and multiploid small CTCs and monoploid small CTECs has clinical significance for predicting prognosis in patients with advanced lung cancer.

Mg-based micromotors for efficient electrochemical detection of circulating tumor cells

The efficient capture and detection of circulating tumor cells (CTCs) in blood is very important for the early detection of cancer. In this work, efficient detection of CTCs through electrochemical means utilizing Mg-Based micromotors was realized, the prepared Fe3O4/P/anti-E nanoparticle was subjected to modification using an aldehyde-amine condensation reaction to attach it to the Mg ball. Utilizing the asymmetrical structure of micromotor, we find that the movement range of the magnesium microsphere has obviously improved. On this basis, efficient capture of CTCs can be realized upon the modification of Fe3O4/P/anti-E with capturing cyclability ability. Such a behavior can be attributed to the anti-E modified on the micromotor can target the epithelial cell adhesion factors on CTCs. Furthermore, A funnel-shaped electrochemical detection device was developed to realize the electrochemical detection of captured CTCs based on the insularity of CTCs. The strategy can potentially provide new visions for the early detection and timely treatment of cancer.

Highly sensitive SERS cytosensor based on catalytic hairpin assembly-mediated zipper-like structures

Circulating tumor cells (CTCs) are cancer biomarkers for the diagnosis at early stage. However, it’s still a great challenge to develop the cytosensor for CTCs detection due to its ultralow counts in human peripheral blood. Herein, a highly sensitive cytosensor based on surface-enhanced Raman scattering (SERS) was developed. The tumor cells were first identified by a strand replacement reaction (SDR) that released the initiator of catalytic hairpin assembly (CHA) reaction. Subsequently, the initiator opened the residue hairpin DNA outside the silver coated gold nanoparticles (Au@Ag NPs). The double strand DNA (dsDNA) CHA products drove the Au@Ag NPs towards the same side of dsDNA to form a zipper-like structure. This structure brought about hotspots area for SERS quantitative analysis of A549 tumor cells. The designed cytosensor exhibited good linearity ranging from 10 to 500 cells/mL with a calculated limit of detection (LOD) of 8 cells/mL. A SERS cytosensor with high sensitivity and good reproducibility was obtained in this work. In addition, this research presents a new method for rapid tumor cell detection in biological samples for cancer diagnosis.

Combined measurement of circulating tumor cells and magnetic nanoparticles-assisted multiple blood markers improves the diagnostic efficiency of early-stage lung adenocarcinoma

The study aims to evaluate the efficiency and clinical value of circulating tumor cells (CTCs), blood tumor markers, and serum microRNAs for diagnosing lung adenocarcinoma in its early stages. The study included 40 patients with early-stage lung adenocarcinoma, 40 individuals with benign pulmonary nodules, and 40 healthy volunteers in groups 1, 2, and 3, respectively. The negative enrichment–fluorescence in situ hybridization was used to classify and quantify the aneuploidy and its number distribution in CTCs in groups 1–3. Magnetic nanoparticles were used to extract DNA. The levels of miR-223-3p and miR-486-5p in the serum of groups 1–3 were then determined using reverse transcription quantitative real-time polymerase chain reaction. CTC aneuploidy and its number distribution was detected and the detection of various blood markers was performed. The diagnostic accuracy was determined through receiver operating characteristic curve (ROC) drawn among the groups. The total number of CTCs and aneuploidies in patients with early-stage lung adenocarcinoma was higher than in patients with benign pulmonary nodules and healthy people. Patients with early-stage lung adenocarcinoma had significantly higher levels of miR-223-3p and miR-486-5p than patients with benign pulmonary nodules and healthy people. ROC analysis found that the sensitivity and area under the ROC curve of CTCs combined with multiple blood markers for distinguishing patients with early-stage lung adenocarcinoma from those without lung nodules were 80.12% and 0.945%, respectively. The sensitivity and area under the ROC curve of the patients were 85.38% and 0.948%, respectively. The detection of CTCs, carcinoembryonic antigen, cytokeratin 19 fragment, miR-223-3p, and miR-486-5p was effective for early-stage lung adenocarcinoma and benign lung nodules. The diagnosis of miR-223-3p is clinically significant, as the accurate diagnostic index of miR-223-3p is better than that of CTCs and other blood indicators. However, CTCs combined with blood markers have the highest efficiency in diagnosing early-stage lung adenocarcinoma.

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

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

Cytomorphologic visualization of circulating tumor cells in urinary bladder cancer patients using ScreenCell™ technology: Potential as a simple cytology test.

Circulating tumor cells (CTC) are a recent technique which is a potentially important prognostic factor in many solid tumors. There are many techniques of detecting CTCs, but they usually implement costly techniques like EpCAM targeted detection, fluorescence-based diagnosis, or magnetic bead based positive or negative selection. The diagnostic utility of simple cytomorphological diagnosis after routine staining of CTCs have been rarely studied. We aimed to detect CTCs in 24 patients clinically suspected to have Urinary Bladder Cancer using a simple but efficient patented filtration technology (ScreenCell™), followed by optical microscopic visualization after routine May-Grunwald-Giemsa (MGG) staining. The detected CTCs were then tested for association with the histologic type, lamina propria invasion, deep muscle invasion and the T-stage. Out of the 24 patients tested, one was found to have papilloma, nine had low grade urothelial carcinoma, 13 had high grade urothelial carcinoma and one had poorly differentiated adenocarcinoma. Of these, two LGUC, eight HGUC and one adenocarcinoma had detectable CTC. Presence of CTCs had a statistically significant association with Lamina propria invasion (P = .006) and T-stage (P = .02), and a trend toward significance for differentiating LGUC from HGUC (P = .10). These results suggest that cytomorphological detection of CTC is likely to be clinically useful in diagnosis and prognostication of urinary blader cancers. These findings need to be confirmed on studies with larger sample sizes.

Clinical implications and perspectives of portal venous circulating tumor cells in pancreatic cancer

Despite recent improvements in the diagnosis and treatment of pancreatic cancer (PC), clinical outcomes remain dismal. Moreover, there are no effective prognostic or predictive biomarkers or options beyond carbohydrate antigen 19-9 for personalized and precise treatment. Circulating tumor cells (CTCs), as a member of the liquid biopsy family, could be a promising biomarker; however, the rarity of CTCs in peripheral venous blood limits their clinical use. Because the first venous drainage of PC is portal circulation, the portal vein can be a more suitable location for the detection of CTCs. Endoscopic ultrasound-guided portal venous sampling of CTCs is both feasible and safe. Several studies have suggested that the detection rate and number of CTCs may be higher in the portal blood than in the peripheral blood. CTC counts in the portal blood are highly associated with hepatic metastasis, recurrence after surgery, and survival. The phenotypic and genotypic properties measured in the captured portal CTCs can help us to understand tumor heterogeneity and predict the prognosis of PC. Small sample sizes and heterogeneous CTC detection methods limit the studies to date. Therefore, a large number of prospective studies are needed to corroborate portal CTCs as a valid biomarker in PC.

A Multiple Targeting Nanoprobe for Identifying Cancer Metastatic Sites Based on Detection of Various mRNAs in Circulating Tumor Cells.

Identification of cancer metastatic sites is of importance for adjusting therapeutic interventions and treatment choice. However, identifying the location of metastatic lesions with easy accessibility and high safety is challenging. Here we demonstrate that cancer metastatic sites can be accurately detected by a triple targeting nanoprobe. Through coencapsulating molecular beacons probing a cancer biomarker (CXCR4 mRNA), a lung metastatic biomarker (CTSC mRNA), and a bone metastatic biomarker (JAG1 mRNA), the nanoprobe decorated by SYL3C conjugated hyaluronic acid and ICAM-1 specific aptamer conjugated hyaluronic acid can target diverse phenotyped circulating tumor cells (CTCs) during epithelial-mesenchymal and mesenchymal-epithelial transitions in whole blood for sensitive probing. The detection of CTCs from cancer patients shows that the nanoprobe can provide accurate information to distinguish different cancer metastasis statuses including nonmetastasis, lung metastasis, and bone metastasis. This study proposes an efficient screening tool for identifying the location of distant metastatic lesions via facile blood biopsy.

Abstract 1026: A novel detection system of circulating tumor cells based on size-dependent enrichment with immunofluorescent staining of CK8/18 in advanced non-small cell lung cancer

Abstract Background: Detection of circulating tumor cells (CTCs) in the blood is a potentially noninvasive tool for cancer diagnosis, monitoring and prognosis evaluation. However, the CTC detection is difficult because of the rarity of CTCs, especially in non-small cell lung cancer (NSCLC), and the detection sensitivity of CTCs was reported to be less than 50% in NSCLC patients. Herein, we developed a novel CTC detection system and evaluated its performance in advanced NSCLC patients. Methods: Whole blood samples were collected from stage IV or recurrence NSCLC patients. CTC detection was performed with a novel system (Sysmex Corporation, Hyogo, Japan) by using size-dependent enrichment for capturing CTCs and immunofluorescent staining reagents including anti-cytokeratin (CK, AE1/AE3 or CK8/18) (specific for epithelial cells), anti-CD45 (specific for leukocyte) and Hoechst (for nucleus). Fluorescence intensity and cell images of each marker were obtained by Molecular Imaging Flow Cytometry (MI-FCM). Circulating cells that were positive for Hoechst and anti-CK but negative for anti-CD45 were defined as CTCs. To confirm whether CTCs detected were derived from lung cancer origin, CTCs obtained from patients with EGFR mutations were subjected to single cell sorting, whole genome amplification and PCR analysis for the mutation detection. Results: A total of 20 NSCLC patients (10 EGFR mutant, 10 EGFR wild-type) were enrolled in this study. Of these, CTCs were detected in 16 (80%) patients (number of CTCs, 1 to 66 in 4.5ml whole blood) when CK8/18 was used as epithelial cell staining, but in only 11 (55%) patients (1 to 6 CTCs) when using AE1/AE3. The detection sensitivity of CTCs based on CK8/18 was significantly higher compared to that based on AE1/AE3 in our novel system (80% vs. 50%, p = 0.03). The number of CTCs was significantly lower in EGFR mutation-positive patients than in EGFR wild-type patients (median number of CTCs, 3 vs. 22, p = 0.01). Furthermore, among 10 EGFR mutation-positive patients, EGFR mutations were detected in CTCs derived from 6 (60%) patients. All the mutation types detected in CTCs were matched with those identified in the corresponding primary tumors. Conclusion: Our novel CTC detection system demonstrated that the detection rate of CTCs (80%) was higher than those in previous studies. Further studies are needed for the clinical application of this system. Citation Format: Tetsuya Sakai, Yoshitaka Zenke, Mami Onishi, Eri Morita, Masatoshi Yanagida, Yuji Shibata, Hiroki Izumi, Kaname Nosaki, Hibiki Udagawa, Shingo Matsumoto, Kiyotaka Yoh, Koichi Goto. A novel detection system of circulating tumor cells based on size-dependent enrichment with immunofluorescent staining of CK8/18 in advanced non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1026.

Abstract 3382: Detection of CTCs using a micro-channel combined fluid dynamics and electrophoresis in colorectal cancer

Abstract Background: Circulating tumor cells (CTCs) are tumor cells that can be isolated from peripheral blood. Investigation of CTCs might allow comprehensive studies at the DNA, RNA, and protein. However, fewer CTCs generally can be collected from patients with colorectal cancer, limiting the possibility of clinical applications of CTC analysis despite the development of many different methods. We have developed a micro-channel device that integrates hydrodynamic filtration (HDF) capable of size separation and dielectrophoresis (DEP) capable of electrical property separation, using peripheral blood from colorectal cancer patients. Method: A separation test was performed using a whole peripheral blood sample obtained from colorectal cancer patients with KRASmutation in the tumor tissue. The sample blood was diluted 2-fold with PBS, and red blood cells and plasma were removed using HDF. After that, Leukocytes and CTCs were separated in the same channel by DEP. DNA was extracted from CTCs (port 1) and leucocyte fraction (port 2), and KRAS mutations were detected using droplet digital PCR. In order to evaluate the separated fractions, microwell slide was used for evaluation by nuclear staining, CD45 and EpCAM antibody staining. Results: 52 colorectal cancer patients were participated. The median age of patients was 71 years, 38 (73%) were male. Twenty-eight (53.9%) had colon cancers. Four stage III and 48 stage IV patients were included. The quality of DNA extracted from all patients were suitable for molecular genetic analysis. The DNA concentration of port 1 was significantly lower than that of port 2 (371 ng/mL vs 36667 ng/mL, P&amp;lt;0.001). Using droplet digital PCR, KRAS mutations were detected in the port 1 of 8 (15.4%) patients and in port 2 of 15 (28.8%) patients. In 35.5% of patients, KRAS mutations were detected in either port 1 or port 2. Conclusion: This device does not require pretreatment and is easy to extract CTCs. Since CTCs are contaminated with the leukocyte fraction, it is necessary to improve the accuracy of CTCs collection by adjusting the conditions of DEP. Citation Format: Ryo Ohta, Takeshi Yamada, Nobuhiko Taniai, Hiroshi Yoshida. Detection of CTCs using a micro-channel combined fluid dynamics and electrophoresis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3382.

Metastasis risk stratification and response prediction through dynamic viable circulating tumor cell counts for rectal cancer in a neoadjuvant setting.

Distant metastasis (DM) and neoadjuvant treatment response prediction remain critical challenges in the management of locally advanced rectal cancer (LARC). The aim of this study was to investigate the clinical relevance of viable circulating tumor cells (CTCs) for DM or response in patients with LARC in a neoadjuvant setting. The detection of viable CTCs at different stages of treatment was planned for consecutive patients from a prospective trial. The Kaplan-Meier method, Cox proportional hazards model, and logistic regression model were utilized to analyze factors associated with DM or pathological complete response (pCR) and clinical complete response (cCR). Between December 2016 and July 2018, peripheral blood samples from 83 patients were collected before any treatment (median follow-up time, 49.3 months). CTCs were present in 76 of 83 patients (91.6%) at baseline, and more than three CTCs detected in the blood sample was considered high risk. Only the CTC risk group was significantly associated with 3-year metastasis-free survival (MFS) (high risk vs. low risk, 57.1% (95% CI, 41.6-72.6) vs. 78.3% (95% CI, 65.8-90.8), p = 0.018, log-rank test). When all the important variables were entered into the Cox model, the CTC risk group remained the only significant independent factor for DM (hazard ratio (HR), 2.74; 95% CI, 1.17-6.45, p = 0.021). The pCR and continuous cCR rates were higher in patients with a decreased number of CTCs of more than one after radiotherapy (HR, 4.00; 95% CI, 1.09-14.71, P = 0.037). The dynamic detection of viable CTCs may strengthen pretreatment risk assessment and postradiotherapy decision making for LARC. This observation requires further validation in a prospective study.

Abstract 1033: Evaluation of HER2 status in circulating tumor cells isolated using the Parsortix® system

Abstract Background: Evaluation of HER2 status in tissue from breast cancer patients is a standard test for clinical diagnosis and treatment. However, tissue biopsy is invasive and repeated testing is generally not possible. Liquid biopsy allows minimally invasive, repeat sample collection for regular monitoring. This study combined ANGLE’s Parsortix® PR1 Research System*, an epitope-independent microfluidic device that isolates and harvests rare cells from blood based on size and deformability, with two downstream assays: an Immunofluorescence (IF) assay for HER2 protein detection and an IF assay combined with FISH targeting HER2 gene amplification, to determine HER2 status on circulating tumor cells (CTCs). Methods: Blood was collected from healthy volunteers into Streck Cell-Free DNA tubes. Two aliquots of blood from each donor were spiked with cultured SKBR3 (HER2+) and Hs 578T (HER2-) breast cancer cells and then processed using Parsortix® PR1 instruments. Captured cells were harvested and spun onto positively charged slides. One slide per donor was stained using ANGLE’s IF panel for CTC detection, consisting of a nuclear dye, antibodies targeting epithelial and mesenchymal markers, and antibodies targeting blood cell markers. After imaging, slides were de-stained and processed using a commercially available HER2 FISH kit. Cancer cells identified by the IF panel were re-imaged to examine FISH signal. Cells were considered HER2+ if the ratio of HER2 foci to CEP17 foci was ≥2. The second slide was stained using the same IF CTC panel in combination with an antibody targeting HER2 protein. HER2 expression was analyzed based on intensity of fluorescence signal. Same workflow was implemented in a small cohort of breast cancer patients with blood processed 72-144 hours post-draw using Parsortix® PC1 Clinical System. Results: The numbers of epithelial and mesenchymal CTCs detected on each slide were comparable in both assays. In HER2 FISH-stained samples, 97.3% of the SKBR3 cells and 13.3% of the Hs 578T cells harvested showed HER2 amplification. This data was consistent with the literature (~20% of Hs 578T cells tend to form isochromosomes, altering the HER2/CEP17 ratio). In IF-only stained samples, 96.8% of the SKBR3 cells and 0% of the Hs578T cells harvested overexpressed HER2 protein. In patient samples, CTCs were identified in 80% of the donors (mean: 21; median: 6; range: 0-200), with 17% of the CTC-positive donors having ≥1 CTC overexpressing HER2 protein. Conclusions: This study demonstrates the feasibility of HER2 evaluation using IF for protein expression and HER2 FISH for gene amplification on CTCs enriched from blood using the Parsortix® systems. It also demonstrates that blood collection in Streck tubes is suitable for both assays, with the advantage of allowing longer blood sample storage and facilitating batch shipping and processing of clinical samples. *Research Use Only. Not for use in diagnostic procedures. Citation Format: Nerea Borreguero-Munoz, Alex Young, Jessica Kimber, Mariacristina Ciccioli, Anne-Sophie Pailhes-Jimenez. Evaluation of HER2 status in circulating tumor cells isolated using the Parsortix® system [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 1033.

Abstract 6610: Increasing blood volumes to detect minimal residual disease in neoadjuvant-treated early breast cancer patients

Abstract Breast cancer (BC) is the most frequent neoplasia affecting women worldwide normally detected at early stages. In this regard, early diagnosis drastically decreases mortality, however, around 20% of these patients will later relapse. This is mainly caused by undetectable molecular residual disease (MRD) not eliminated by standard primary treatments. Therefore, it is crucial to detect the after-treatment MRD to stratify the patients by their risk of relapse. Liquid biopsies have emerged as non-invasive method to obtain information about tumors and improve clinical cancer management. Regarding this, much has been hypothesized about utilizing high blood volumes to overcome the necessity of complex and resource-intensive next generation sequencing (NGS) methodologies to detect highly diluted blood tumor components in localized cancers. Herein, we employed a combined analysis of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) together with high blood volumes and single-assay droplet digital PCR (ddPCR) to detect MRD with ultra-high sensitivity. We prospectively assayed 124 samples extracted at baseline, post-neoadjuvant therapy (NAT), post-surgery and a follow-up on a six-monthly basis. A median of 76.40 mL of blood to detect CTCs and 40 mL of plasma to detect ctDNA per patient from 19 BC women were used in this study. ddPCR assays were performed with a median of 14 partitions per determination to detect ctDNA and 12 partitions for CTCs. Overall, ctDNA, CTCs and ctDNA and/or CTCs were detected in 84.21%, 66.66% and 89.47% respectively in the pre-treatment blood samples. MRD (ctDNA and/or CTCs) was detected in 73.68% of the after NAT blood samples. On the other hand, it was detected in 46.66% and 70.00% of the post-surgery and follow-up samples respectively. Post-NAT MRD was detected in 57.14% (4/7) and 83.33% (10/12) of patients with and without pathological complete response pCR respectively. To note, the discordant patients achieving pCR in tissue with detectable MRD in blood were high-risk BC. Importantly, in one of the two patients without pCR and no MRD detected, not enough sample were available to complete the analysis. The other discordant patient presented a localized disease with residual cancer burden value of 1 and no lymph nodes affected. In 1 out of 19 (5.26%) patient clinically relapsed with a positive MRD detection 6 months earlier. Applying this methodology, we observed a sensitivity of 0.004% in ctDNA detection and 0.224 CTCs per mL of blood. Overall, this novel methodology greatly improves sensitivity for ctDNA and CTCs detection in treatment-naïve early BC. In addition, MRD was successfully detected in post-treatment samples antedating clinical relapse by 6 months in one patient. This prospective study is potentially demonstrating that using high blood volumes and a single-assay ddPCR is a cost-effective strategy to monitor localized BC and predict relapses. Citation Format: Alfonso Alba-Bernal, Ana Godoy-Ortiz, María Emilia Domínguez-Recio, Begoña Jimenez-Rodriguez, María Elena Quirós-Ortega, Esperanza López-López, Guillermo Carbajosa-Antona, Jesús Peralta-Linero, Luis Vicioso, Estefanía Bellagarza-García, Guadalupe Dolores Garrido-Ruiz, Cynthia Robles-Podadera, Alicia Garrido-Aranda, María Dunia Roldán-Díaz, Jesús Velasco-Suelto, Rocío Lavado-Valenzuela, Martina Álvarez, Nuria Ribelles, Javier Pascual, Emilio Alba, Iñaki Comino-Méndez. Increasing blood volumes to detect minimal residual disease in neoadjuvant-treated early breast cancer patients. [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 6610.

Abstract 2315: Temporal changes in circulating tumor cells and circulating tumor DNA in patients with resectable pancreatic ductal adenocarcinoma

Abstract Background: Circulating tumor cells (CTCs), circulating cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA) are minimally invasive biomarkers that carry vital tumor onset, progression, and metastasis information, which is crucial for cancer diagnosis, prognosis, and management. We report here our longitudinal study of these circulating biomarkers in resectable pancreatic ductal adenocarcinoma (PDAC) patients. Although the overall 5-year survival rate of PDAC is low (~6%), truly resectable patients are expected to have better outcomes than non-resectable PDAC patients, especially if liquid biopsy can help guide anticancer treatment selection. Method: We evaluated temporal changes in CTCs, cfDNA, and ctDNA in peripheral blood samples in a cohort of patients with resectable PDAC treated with pre-operative chemotherapy and surgical excision. Five PDAC survivors in complete remission for three years without evidence of disease served as negative controls. Samples of peripheral blood were collected prior to the start of treatment and every two weeks while patients underwent neoadjuvant chemotherapy, followed by surgical excision of the pancreatic tumor. CTCs were enumerated using a previously reported microfluidic device [1] that integrates immunoaffinity-based isolation with size-based filtration, while cfDNA and ctDNA were measured using quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR). Results: Among the initial seven patients who have completed the treatment sequences [2], CTCs were detectable in all patients (100%) at some points during treatment, but only 50% of patients had detectable CTCs before the start of chemotherapy. Median cfDNA concentrations were comparable to those negative controls throughout treatment. Using ddPCR to analyze peripheral blood samples, KRAS mutations were detected in in 71% of patients during anticancer therapy, but only in 29% patients prior to treatment. Overall, the majority of circulating biomarkers (81% for CTCs and 91% for ctDNA) were detected in blood samples collected after the start of neoadjuvant therapy and before surgery. Conclusion: This study suggests that a longitudinal study of circulating biomarkers provides additional data compared to single-point liquid biopsy, and that biologic “release” of biomarkers due to therapy can increase detectability. -Further study is needed to determine the clinical utility of liquid biopsy for resectable PDAC patients. [1] Chen, et al., Angew. Chem. Int. Ed., 58, 2019, 7606-7610. [2] Dopico, et al., Biosensors, 12, 2022, 206. Citation Format: Yang Zhang, Pablo J. Dopico, Minh-Chau N. Le, Zhijie Yang, Youxiang Wang, Sherise C. Rogers, Thomas J. George, Hugh Z. Fan. Temporal changes in circulating tumor cells and circulating tumor DNA in patients with resectable pancreatic ductal adenocarcinoma [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 2315.

Abstract 3354: Isolation and characterization of circulating tumor cells from ductal carcinoma in situ patients using label free technology

Abstract Ductal carcinoma in situ (DCIS) is an early form of breast cancer that has traditionally been characterized by neoplastic cells that are still confined to the mammary ducts. The 20-year breast cancer mortality rate following a DCIS diagnosis, with or without treatment, remains at 3.3%. Despite the overall success in DCIS treatments preventing local invasive breast cancer recurrence, there are still a substantial number of women who, after a DCIS diagnosis, die from metastatic breast cancer. We currently lack predictive tools to identify the high-risk patients who would benefit most from treatment. Emerging clinical and experimental evidence suggest that breast cancer cells may disseminate throughout the body very early in carcinogenesis. These circulating tumor cells (CTCs), rare cancer cells that are shed from the primary tumor into blood, may offer insights into early cancer progression and help identify high-risk patients. Because CTCs can extravasate from blood into tissue and form secondary tumor sites or dormant niches, they are potential biomarkers of metastasis and recurrence. The labyrinth, a high-throughput inertial microfluidic device, has previously demonstrated success in isolating CTCs through inertial separation due to their larger size when compared to white blood cells. Compared to antigen-based isolation technology, inertial microfluidic devices can isolate heterogeneous CTCs with a variety of surface proteins. Using this technology, we isolated and identified CTCs (DAPI+/Pan cytokeratin+/CD45- cells) in 63.2% (12/19) of DCIS patients with an average of 1.27 CTCs per five mls of blood compared to 0.259 CTCs per five mls of blood found in healthy controls (p =0.0078, unpaired t-test). The majority of healthy controls (15/19) had no detectable CTCs. Additionally, immunofluorescence staining showed that 10.5% of CTCs were EpCAM (Epithelial cell adhesion molecule) positive and 26.3% were positive for vimentin, a mesenchymal marker. Single-cell qPCR revealed potential CTCs with no gene expression of traditional white blood cell markers such as CD45, CD20, CD11B, and CD3D. The majority of these cells expressed proliferation (PKM2, TIMP1) and mesenchymal (SPARC, TGFβ1) markers, which can contribute to the cells’ ability to migrate and metastasize. Preliminary targeted DNA sequencing of eight CTC-enriched blood samples identified copy number variations previously associated with higher grade and poorly differentiated DCIS, and single nucleotide variations previously found in DCIS samples (NOTCH1 c.7541_7542del). Ultimately, this study suggests that DCIS generates CTCs prior to microinvasion. CTC isolation and detection may serve as a biomarker for high-risk DCIS and may also be used to explore mechanisms of early cancer dissemination. Citation Format: Brittany Rupp, Brooke Thanasiu, Neha Nagpal, Yan Hong, Dean E. Brenner, Kirsten Tuck, Kirk Herman, Fariba Behbod, Justin Colacino, Max Wicha, Sunitha Nagrath. Isolation and characterization of circulating tumor cells from ductal carcinoma in situ patients using label free technology [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 3354.

Abstract 2292: Novel method for the detection and evaluation of disease biomarkers on white blood cells from liquid biopsies

Abstract Purpose: Blood based liquid biopsies of circulating tumor cells (CTCs) are a non-invasive tool with proven effectiveness in the prognosis, treatment selection, and stratification of cancer patients. In this study we demonstrate a novel application of the RareCyte platform, which was developed to find and analyze rare CTCs from blood samples, in evaluating white blood cell (WBC) sub-populations for disease biomarkers. Experimental Procedures: Using AccuCyte®, an unbiased density-based method for collecting nucleated cells from whole blood, we transferred the nucleated cells to slides and stained for CD14, CD66b, and CK/EpCAM to identify monocytes (CD14+/CD66b-), granulocytes (CD14-/CD66b+), and CTCs (CK/EpCAM+), as well as a drug-target biomarker to evaluate its expression across all cells. These slides were then imaged with CyteFinder®, an automated multiparameter immunofluorescent (IF) microscopy system that applies machine learning algorithms for cell identification. Each slide contains millions of cells, so we developed novel scanning and analysis algorithms to quantify WBC sub-populations in addition to rare CTCs. Results: In addition to detection and enumeration of CTCs, the RareCyte system was able to detect individual white blood cells and quantify monocyte and granulocyte subpopulations. Our results correlated very well with standard CBC (Complete Blood Count) readings from multiple donors. Additionally, the monocyte and granulocyte populations were evaluated for expression of a drug-target biomarker. While expression was observed on both granulocytes and monocytes, monocytes had a significantly higher expression of the drug-target biomarker, a critical finding for future implementation of this drug. Conclusions: Our results show that through modifications to the RareCyte platform we can identify rare and non-rare cell populations simultaneously and evaluate expression of biomarkers and drug-targets across multiple cells of interest. This new application combines the benefits of high throughput data analysis of flow cytometry with the specificity and sensitivity of rare cell detection. Additionally, high-resolution multiplexed immunofluorescent imaging provides cell morphology and biomarker localization information. Our ability to add multiple biomarkers in conjunction with CTC and other rare cell detection provides multiple avenues for future research. One possible application is characterizing white blood cells that cluster with CTCs, which are known to portend a worse prognosis for patients. Our modified workflow could be implemented to precisely characterize these WBCs that contribute to the metastatic process. Utilization of the RareCyte platform for both rare and non-rare cell analysis presents an exciting opportunity for a deeper evaluation of patients and their prognosis through liquid biopsies. Citation Format: Erin Bayer, Jon Ladd, Joshua Nordberg, Arturo B. Ramirez, Jessica A. Baker, Thomas Daly. Novel method for the detection and evaluation of disease biomarkers on white blood cells from liquid biopsies [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 2292.

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

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

Abstract 3373: Deep learning for circulating tumor cell (CTC) identification with the CELLSEARCH system: achieving top human-level performance

Abstract Background: Enumeration of CTCs in blood samples with the CELLSEARCH® system is a prognostic biomarker in metastatic breast, prostate and colorectal cancer (full intended use documents.cellsearchctc.com). Currently, the final CTC identification is performed by human reviewers through visual assessment, which is a time-consuming procedure that could be affected by subjective interpretations. Modern Artificial Intelligence (AI) methods can provide a solution by automating the process of CTC identification, thus eliminating subjectivity and producing faster and more reproducible results. We have recently presented an automated algorithm (for research use only) based on deep learning models for cell segmentation and classification which allows the automated identification of CTCs in CELLSEARCH® fluorescent images (Ansaloni et al., EACR 2022). The purpose of this study was to assess inter-rater variability among human reviewers and to compare the AI performance against them. Methods: Blood samples derived from metastatic cancer patients were processed with CELLTRACKS® AUTOPREP®. The AI algorithm was trained on fluorescent images of 7255 CTCs and 32876 non-CTC events (training set) acquired by CELLTRACKS ANALYZER II® (CTAII) from 90 breast, 122 prostate and 54 colorectal cancer samples. 10 human reviewers, qualified for CTC image analysis, performed blind labeling of a separate dataset independently (test set: 55 samples taken from 27 breast and 28 prostate cancer). We studied inter-rater agreement and variability using Fleiss Kappa and Coefficient of Variation (CV). The labels provided by 5 experienced reviewers (average experience ≅ 15 years) were used to generate the ground truth (GT) by majority voting. The CTC detection performance of the AI was ranked against the other 5 reviewers (average experience ≅ 5 years). Results: In the test dataset, 2990 out of 9554 events were identified as CTCs by the GT review on CTAII images. In the task of classifying each event as CTC or non-CTC, the inter-rater agreement among the 10 human reviewers measured by Fleiss Kappa was 0.85. These classification results provided the number of CTCs per patient counted by each reviewer. The inter-rater variability on the CTC enumeration among the 10 reviewers was evaluated for each sample (median CV = 17.9% and interquartile range = 17.6% , excluding 27 samples with mode = 0 CTC). Measured against the GT classification, the AI performance (accuracy = 94.7%; F1 = 91.6%) was at the top of the human reviewers’ ranking (accuracy = 92.4 - 93.6 - 93.8 - 94.0 - 95.8%; F1 = 86.8 - 89.2 - 89.2 - 89.5 - 93.1%). Conclusion: The AI algorithm reached top-ranking performance in CTC identification, surpassing 4 out of 5 expert reviewers. These results show the applicability of AI to cell classification to remove human subjectivity from the review process and to maximize standardization among different research centers. Citation Format: Luca Biasiolli, Pietro Ansaloni, Nicolò Gentili, Ramona Miserendino, Chiara Rossi, Giulio Signorini, Gianni Medoro. Deep learning for circulating tumor cell (CTC) identification with the CELLSEARCH system: achieving top human-level performance [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 3373.

Abstract 5602: Markers of hypoxic cells: Testing a pre-clinical detection assay

Abstract The proposed project aims to test the applicability of a novel set of patented organic compounds as markers of hypoxic circulating tumor cells (H-CTC) with potential pre-clinical relevance. Circulating tumor cells (CTC) are individual cells or clusters that can move from tumors to circulation invading other tissues. Often cells in the tumor need to adapt to low oxygen levels, generating a population of cells known as hypoxic cells that can survive low oxygen levels. These hypoxic cells can produce more cell junction proteins, augmenting connections between cells causing them to break away in clusters rather than individually. Clustered CTCs are much more efficient at metastasis formation. Release of CTC can also be stimulated by cancer treatments such as radiation and chemotherapy. To date, our research has generated in-vitro studies with a set of 3-nitrobenzazolo[3,2- a]quinolinium chloride salts (NBQS) demonstrating its capacity as markers of hypoxic human cancer cells in vitro. Comparison with the control Pimonidazole demonstrated the advantages of novel NBQ-TOM compounds as hypoxic marker. We here, aim to test the applicability of NBQS to identify hypoxic cells among circulating tumor cells (H-CTC). To date limited commercially CTC detection methods are available. Our application can be described as pre-clinical detection technology of hypoxic cancer cells serving also in cancer management. To determine the capacity of novel compound, NBQ-345 TOM as hypoxic fluorescent marker, colon tumor cells (COLO 205) are culture under hypoxic and aerobic environment and treated with NBQ-345TOM (25uM) for 24 hours. After treatment fluorescence is measured with an OPTIMA BMG Fluorimeter. Fluorescence emission results demonstrated significant formation of the NBQ-234 TOM fluorescent metabolite on hypoxic tumor cells in contrast to cells treated under aerobic environment. Also, fluorescence microscopy analysis of colon cancer cells treated for 24 hours with NBQ-345 TOM at hypoxic and aerobic conditions confirmed the stronger fluorescence generation at hypoxic conditions in contrast to cells at aerobic conditions. Citation Format: Beatriz Zayas, Karoline Rios, Luis Ortiz, Osvaldo Cox. Markers of hypoxic cells: Testing a pre-clinical detection assay. [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 5602.