Number Of Circulating Tumor Cells Research Articles

Dually stimulative single-chain polymeric nano lock with dynamic ligands for sensitive detection of circulating tumor cells

Circulating tumor cells (CTCs) are important markers for cancer diagnosis and monitoring. However, CTCs detection remains challenging due to their scarcity, where most of the detection methods are compromised by the loss of CTCs in pre-enrichment, and by the lack of universal antibodies for capturing different kinds of cancer cells. Herein, we report a single-chain based nano lock (SCNL) polymer incorporating dually stimulative dynamic ligands that can bind with a broad spectrum of cancer cells and CTCs overexpressing sialic acid (SA) with high sensitivity and selectivity. The high sensitivity is realized by the polymeric single chain structure and the multi-valent functional moieties, which improve the accessibility and binding stability between the target cells and the SCNL. The highly selective targeting of cancer cells is achieved by the dynamic and dually stimulative nano lock structures, which can be unlocked and functionalized upon simultaneous exposure to overexpressed SA and acidic microenvironment. We applied the SCNL to detecting cancer cells and CTCs in clinical samples, where the detection threshold of SCNL reached 4 cells/mL. Besides CTCs enumeration, the SCNL approach could also be extended to metastasis assessment through monitoring the expressing level of surface SA on cancer cells.

Direct separation and enumeration of CTCs in viscous blood based on co-flow microchannel with tunable shear rate: a proof-of-principle study.

Circulating tumor cells (CTCs), which have extremely low density in whole blood, are an important indicator of primary tumor metastasis. Isolation and enumeration of these cells are critical for clinical applications. Separation of CTCs from massive blood cells without labeling and addition of synthetic polymers is challenging. Herein, a novel well-defined co-flow microfluidic device is presented and used to separate CTCs in viscous blood by applying both inertial and viscoelastic forces. Diluted blood without any synthetic polymer and buffer solution were used as viscoelastic fluid and Newtonian fluid, respectively, and they were co-flowed in the designed chip to form a sheath flow. The co-flow system provides the function of particle pre-focusing and creates a tunable shear rate region at the interface to adjust the migration of particles or cells from the sample solution to the buffer solution. Successful separation of CTCs from viscous blood was demonstrated and enumeration was also conducted by image recognition after separation. The statistical results indicated that a recovery rate of cancer cells greater than 87% was obtained using the developed method, which proved that the direct separation of CTCs from diluted blood can be achieved without the addition of any synthetic polymer to prepare viscoelastic fluid. This method holds great promise for the separation of cells in viscous biological fluid without either complicated channel structures or the addition of synthetic polymers.

Clinical applications of circulating tumor cells in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly malignant tumor and ranked as the fourth cause of cancer-related mortality. The poor clinical prognosis is due to an advanced stage and resistance to systemic treatment. There are no obvious clinical symptoms in the early stage and the early diagnosis rate remains low. Novel effective biomarkers are important for early diagnosis and tumor surveillance to improve the survival of HCC patients. Circulating tumor cells (CTCs) are cancer cells shed from primary or metastatic tumor and extravasate into the blood system. The number of CTCs is closely related to the metastasis of various solid tumors. CTCs escape from blood vessels and settle in target organs, then form micro-metastasis. Epithelial-mesenchymal transformation (EMT) plays a crucial role in distant metastasis, which confers strong invasiveness to CTCs. The fact that CTCs can provide complete cellular biological information, which allows CTCs to be one of the most promising liquid biopsy targets. Recent studies have shown that CTCs are good candidates for early diagnosis, prognosis evaluation of metastasis or recurrence, and even a potential therapeutic target in patients with HCC. It is a new indicator for clinical application in the future. In this review, we introduce the enrichment methods and mechanisms of CTCs, and focus on clinical application in patients with HCC.

Aptamer-cocktail Functionalized Nano-microfluidic Chip for Enhancing Isolation and Characterization of Circulating Cancer Cells.

Circulating tumor cells (CTCs) have been shown to have a correlation to metastasis and prognosis of patients with cancer. The enumeration and downstream gene analysis of CTCs have attracted efforts for personalized medicine. However, enumeration and phenotypic profiling in most capture devices are challenging due to the rarity and heterogeneity of CTCs. Here, we report an aptamer-cocktail strategy coupled in nano-microfluidic chip for enhancing isolation performance and characterizing the phenotypes of CTCs. Aptamer-cocktail recognizing EpCAM/Vimentin/EGFR/CD44 were bound to a nanopillar array on a nano-microfluidic chip. The recognition was validated with cancer cells by flow cytometry and the critical parameters were optimized with the nano-microfluidic chip. Finally, the system was applied to clinical samples. The proposed aptamer-cocktail showed the predominant affinity with MDA-MB-231 and SK-BR-3. When utilized to capture artificial clinical samples, it showed 71% to 83% capture efficiency. CTC detection rate was 100% in five pre-treatment and five post-treatment breast cancer patients. The enumeration data ranged from 6-33 per 2 ml. The number of CTCs in breast cancer patients before therapy was 1.27-2 times higher than that after therapy. CTCs with epithelial and mesenchymal phenotype were both detected and identified; interestingly, the mean diameter of CTCpck pos acquired in these cases was much larger than that of CTCvimentin pos Conclusion: The nano-microfluidic chip not only made it easier to phenotyping epithelial-like or mesenchymal-like CTCs, but can also be used to detect downstream genetic variation. The established platform can be applied in clinical research and facilitate auxiliary diagnosis with tumor recurrence and metastasis in advance.

Clinical relevance of the combined analysis of circulating tumor cells and anti-tumor T-cell immunity in metastatic breast cancer patients.

BackgroundMetastatic breast cancer (mBC) is a heterogeneous disease with varying responses to treatments and clinical outcomes, still requiring the identification of reliable predictive biomarkers. In this context, liquid biopsy has emerged as a powerful tool to assess in real-time the evolving landscape of cancer, which is both orchestrated by the metastatic process and immune-surveillance mechanisms. Thus, we investigated circulating tumor cells (CTCs) coupled with peripheral T-cell immunity to uncover their potential clinical relevance in mBC.MethodsA cohort of 20 mBC patients was evaluated, before and one month after starting therapy, through the following liquid biopsy approaches: CTCs enumerated by a metabolism-based assay, T-cell responses against tumor-associated antigens (TAA) characterized by interferon-γ enzyme-linked immunosorbent spot (ELISpot), and the T-cell receptor (TCR) repertoire investigated by a targeted next-generation sequencing technique. TCR repertoire features were characterized by the Morisita’s overlap and the Productive Simpson Clonality indexes, and the TCR richness. Differences between groups were calculated by Fisher’s, Mann-Whitney or Kruskal-Wallis test, as appropriate. Prognostic data analysis was estimated by Kaplan-Meier method.ResultsStratifying patients for their prognostic level of 6 CTCs before therapy, TAA specific T-cell responses were detected only in patients with a low CTC level. By analyzing the TCR repertoire, the highest TCR clonality was observed in the case of CTCs under the cut-off and a positive ELISpot response (p=0.03). Whereas, at follow-up, patients showing a good clinical response coupled with a low number of CTCs were characterized by the most elevated TCR clonality (p<0.05). The detection of CTCs≥6 in at least one time-point was associated with a lower TCR clonality (p=0.02). Intriguingly, by combining overall survival analysis with TCR repertoire, we highlighted a potential prognostic role of the TCR clonality measured at follow-up (p=0.03).ConclusionThese data, whether validated in a larger cohort of patients, suggest that the combined analysis of CTCs and circulating anti-tumor T-cell immunity could represent a valuable immune-oncological biomarker for the liquid biopsy field. The clinical application of this promising tool could improve the management of mBC patients, especially in the setting of immunotherapy, a rising approach for BC treatment requiring reliable predictive biomarkers.

Role of Circulating Tumor Cells in Determining Prognosis in Metastatic Breast Cancer.

Dinesh Chandra DovalBackground Circulating tumor cells (CTCs) in the peripheral blood may play a major role in the metastatic spread of breast cancer. This study was conducted to assess the role of CTCs to determine the prognosis in terms of survival in metastatic breast cancer patients. Methods This prospective study of 36 patients was conducted at the Hospital from April 2016 to May 2018. Details of each patient related to the demographic profile, tumor type, treatment, and follow-up information were recorded. The number of CTCs in the peripheral blood was measured by Celsee PREP 400 sample processing system and Celsee Analyzer imaging station. Results There was a positive correlation between the number of site of metastasis with number of CTCs ( p -value < 0.001). In the patients with clinical/partial response, a significant reduction in the number of CTCs after 1 month of therapy was observed ( p -value = 0.003). When the number of CTCs at baseline and 6 months were compared with the positron emission tomography response at 6 months, a statistically significant difference in CTCs in patients having partial response after 6 months was observed ( p -value = 0.001). On comparison with the responder groups, a statistically significant reduction in CTCs at baseline and 6 months was observed ( p -value = 0.001). Patients with CTCs less than 5 and more than or equal to 5 after 1 month of treatment had a mean progression-free survival of 11.1 months and 7.5 months ( p -value = 0.04) and a mean overall survival of 11.6 and 9.6 months ( p -value = 0.08), respectively. Conclusion Assessment of CTCs provides a more quantifiable response than radiographic evaluation and at a much earlier time point and is also a better predictor of survival.

Nonhematogenic circulating aneuploid cells confer inferior prognosis and therapeutic resistance in gliomas

Aneuploidy is the hallmark of malignancy. Our previous study successfully detected nonhematogenic circulating aneuploidy cells (CACs) in types of gliomas. The current prospective clinical study aims to further precisely subcategorize aneuploid CACs, including CD31− circulating tumor cells (CTCs) and CD31+ circulating tumor endothelial cells, and thoroughly investigate the clinical utilities of these different subtypes of cells. Co‐detection and analysis of CTCs and circulating tumor‐derived endothelial cells (CTECs) expressing CD133, glial fibrillary acidic protein (GFAP), or epidermal growth factor receptor variant III (EGFR vIII) were performed by integrated subtraction enrichment and immunostaining fluorescence in situ hybridization (SE‐iFISH) in 111 preoperative primary diffuse glioma patients. Aneuploid CACs could be detected in most de novo glioma patients. Among detected CACs, 45.6% were CD31−/CD45− aneuploid CTCs and the remaining 54.4% were CD31+/CD45− aneuploid CTECs. Positive detection of CTECs significantly correlated with disruption of the blood–brain barrier. The median number of large CTCs (LCTCs, >5 μm, 2) in low‐grade glioma (WHO grade 2) was less than high‐grade glioma (WHO grades 3 and 4) (3, p = 0.044), but this difference was not observed in small CTCs (SCTCs, ≤5 μm), CTECs or CACs (CTCs + CTECs). The numbers of CTCs, CTECs, or CACs in patients with contrast‐enhancing (CE) lesions considerably exceeded that of non‐CE lesions (p < 0.05). Receiver operating characteristic curves demonstrated that CD31+ CTECs, especially LCTECs, exhibited a close positive relationship with CE lesions. Survival analysis revealed that the high number of CD31− CTCs could be an adverse factor for compromised progression‐free survival and overall survival. Longitudinal surveillance of CD31− CTCs was suitable for evaluating the therapeutic response and for monitoring potential emerging treatment resistance.

Epithelial-to-mesenchymal transition of circulating tumor cells and CD133 expression on predicting prognosis of thyroid cancer patients.

The present study aimed to explore the epithelial-to-mesenchymal transition of circulating tumor cells (CTCs) and CD133 expression in determining the prognosis of patients with thyroid cancer. It enumerated different CTC subtypes and analyzed CD133 gene expression in patients with thyroid cancer to evaluate the relationship between CTC number and thyroid cancer prognosis. In total 394 patients with thyroid cancer were enrolled. Among these, 270 cases had papillary thyroid cancer (PTC), 60 had follicular thyroid cancer (FTC), 30 had medullary thyroid cancer (MTC), 15 had poorly differentiated thyroid cancer, 19 had anaplastic thyroid cancer and 10 had non-malignant thyroid nodules based on their histopathological characteristics. CTC cell counts were determined by CanPatrol CTC capture technique before treatment. The present study also performed reverse transcription-quantitative PCR for CD133 gene expression and evaluated the relationship between CD133 expression and clinical pathology. A total of 330 cases of enrolled patients were classified as differentiated thyroid cancer, which included PTC and FTC. Their prognosis was excellent. The positivity rate of CTCs at diagnosis was 95.5%. The data of the present study showed that early recurrence and metastasis rates in PTC and FTC patients with >6 CTCs and positive mesenchymal circulating tumor cells (MCTCs) were significantly higher than those in patients with <6 CTCs and MCTCs. It was also found that those patients with >6 CTCs and MCTCs had shorter overall survival. In addition, CD133 levels in patients with thyroid cancer were strongly associated with the differentiation grades of thyroid cancers. The detection of >6 CTCs and positive MCTCs in patients with differentiated thyroid cancer was an excellent biomarker for predicting the prognosis of patients. CD133 expression was also identified as a good biomarker for thyroid cancer differentiation.

Circulating tumor cell-blood cell crosstalk: Biology and clinical relevance.

Circulating tumor cells (CTCs) are the seeds of distant metastasis, and the number of CTCs detected in the blood of cancer patients is associated with a worse prognosis. CTCs face critical challenges for their survival in circulation, such as anoikis, shearing forces, and immune surveillance. Thus, understanding the mechanisms and interactions of CTCs within the blood microenvironment is crucial for better understanding of metastatic progression and the development of novel treatment strategies. CTCs interact with different hematopoietic cells, such as platelets, red blood cells, neutrophils, macrophages, natural killer (NK) cells, lymphocytes, endothelial cells, and cancer-associated fibroblasts, which can affect CTC survival in blood. This interaction may take place either via direct cell-cell contact or through secreted molecules. Here, we review interactions of CTCs with blood cells and discuss the potential clinical relevance of these interactions as biomarkers or as targets for anti-metastatic therapies.

Electrochemical impedance spectroscopy based microfluidic biosensor for the detection of circulating tumor cells

Circulating tumor cells (CTCs) can be detected using newly emerging non-invasive liquid biopsy techniques and they can be used for early diagnosis, for cancer treatment management and treatment efficiency assessment, precision medicine and to better understand the metastasis process. The number of CTCs can be as low as 1 cell/mL of blood thus there is a need for very high detection sensitivity and specificity. In this article we propose a label-free detection method based on a microfluidic device integrated with an electrochemical impedance spectroscopy biosensor capable of capturing and quantifying CTCs. We propose an interdigitated gold electrode functionalized with specific antibodies encapsulated in PDMS. The sensor was functionalized with anti-EpCAM and anti-CD36 antibodies, molecules which are expressed by CTCs and which are absent on normal blood cells. The microfluidic device was tested on MCF-7 breast cancer cells and on blood samples from canines with mammary carcinomas. The device proved to be highly sensitive to the attachment of CTCs, being able to detect the attachment of only 3 MCF-7 cells on the surface of the sensor. Thus, we report that the developed microfluidic device is a promising tool for CTCs detection, quantification and characterization.

Isolation of Circulating Tumor Cells from Seminal Fluid of Patients with Prostate Cancer Using Inertial Microfluidics.

Simple SummaryProstate cancer (PCa) is notoriously difficult to diagnose owing to the lack of reliable biomarkers and the invasiveness of obtaining a tissue biopsy from the prostate. As an alternative, we developed a liquid biopsy technique, based on isolating tumor cells from semen samples via a microfluidic device. To optimize the device, we first attempted to recover PCa cells from semen samples spiked with PCa cell lines, achieving an average efficiency of >87% cell recovery at the chosen flow rate. We then transitioned to a clinical setting using semen samples from PCa patients. The yield of isolated clinical PCa cells varied between 67 and 307 cells per mL of semen (in 15 cancer patients). These cells were stained and compared to the standard prognostic parameters such as Gleason score and PSA serum level. This study presents a potential liquid biopsy technique to augment the existing diagnosis and prognosis of PCa.Prostate cancer (PCa) diagnosis is primarily based on prostate-specific antigen (PSA) testing and prostate tissue biopsies. However, PSA testing has relatively low specificity, while tissue biopsies are highly invasive and have relatively low sensitivity at early stages of PCa. As an alternative, we developed a technique of liquid biopsy, based on isolation of circulating tumor cells (CTCs) from seminal fluid (SF). The recovery of PCa cells from SF was demonstrated using PCa cell lines, achieving an efficiency and throughput as high as 89% (±3.8%) and 1.7 mL min−1, respectively, while 99% (±0.7%) of sperm cells were disposed of. The introduced approach was further tested in a clinical setting by collecting and processing SF samples of PCa patients. The yield of isolated CTCs measured as high as 613 cells per SF sample in comparison with that of 6 cells from SF of healthy donors, holding significant promise for PCa diagnosis. The correlation analysis of the isolated CTC numbers with the standard prognostic parameters such as Gleason score and PSA serum level showed correlation coefficient values at 0.40 and 0.73, respectively. Taken together, our results show promise in the developed liquid biopsy technique to augment the existing diagnosis and prognosis of PCa.

Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

Herein we report the use of Chaperonin-Containing TCP-1 (CCT or TRiC) as a marker to detect circulating tumor cells (CTCs) that are shed from tumors during oncogenesis. Most detection methods used in liquid biopsy approaches for enumeration of CTCs from blood, employ epithelial markers like cytokeratin (CK). However, such markers provide little information on the potential of these shed tumor cells, which are normally short-lived, to seed metastatic sites. To identify a marker that could go beyond enumeration and provide actionable data on CTCs, we evaluated CCT. CCT is a protein-folding complex composed of eight subunits. Previously, we found that expression of the second subunit (CCT2 or CCTβ) inversely correlated with cancer patient survival and was essential for tumorigenesis in mice, driving tumor-promoting processes like proliferation and anchorage-independent growth. In this study, we examined CCT2 expression in cancer compared to normal tissues and found statistically significant increases in tumors. Because not all blood samples from cancer patients contain detectable CTCs, we used the approach of spiking a known number of cancer cells into blood from healthy donors to test a liquid biopsy approach using CCT2 to distinguish rare cancer cells from the large number of non-cancer cells in blood. Using a clinically validated method for capturing CTCs, we evaluated detection of intracellular CCT2 staining for visualization of breast cancer and small cell lung (SCLC) cancer cells. We demonstrated that CCT2 staining could be incorporated into a CTC capture and staining protocol, providing biologically relevant information to improve detection of cancer cells shed in blood. These results were confirmed with a pilot study of blood from SCLC patients. Our studies demonstrate that detection of CCT2 could identify rare cancer cells in blood and has application in liquid biopsy approaches to enhance the use of minimally invasive methods for cancer diagnosis.

Application of circulating tumour cells to predict response to treatment in head and neck cancer

BackgroundLocal recurrence and metastasis remain the major causes of death in head and neck cancer (HNC) patients. Circulating tumour cells (CTCs) are shed from primary and metastatic sites into the circulation system and have been reported to play critical roles in the metastasis and recurrence of HNC. Here, we explored the use of CTCs to predict the response to treatment and disease progression in HNC patients.MethodsBlood samples were collected at diagnosis from HNC patients (n = 119). CTCs were isolated using a spiral microfluidic device and were identified using immunofluorescence staining. Correlation of baseline CTC numbers to 13-week PET-CT data and multidisciplinary team consensus data were conducted.ResultsCTCs were detected in 60/119 (50.4%) of treatment naïve HNC patients at diagnosis. Baseline CTC numbers were higher in stage III vs. stage I-II p16-positive oropharyngeal cancers (OPCs) and other HNCs (p = 0.0143 and 0.032, respectively). In addition, we found that baseline CTC numbers may serve as independent predictors of treatment response, even after adjusting for other conventional prognostic factors. CTCs were detected in 10 out of 11 patients exhibiting incomplete treatment responses.ConclusionsWe found that baseline CTC numbers are correlated with treatment response in patients with HNC. The expression level of cell-surface vimentin (CSV) on CTCs was significantly higher in patients with persistent or progressive disease, thus providing additional prognostic information for stratifying the risk at diagnosis in HNC patients. The ability to detect CTCs at diagnosis allows more accurate risk stratification, which in the future may be translated into better patient selection for treatment intensification and/or de-intensification strategies.

Prognostic implication of TERT promoter mutation and circulating tumor cells in muscle-invasive bladder cancer

PurposeCurrent clinical prognostic factors are not accurate enough to identify and monitor those muscle-invasive bladder cancer (MIBC) patients at high risk of progression after radical cystectomy (RC). Here, we determined genetic alterations in the tumor and circulating tumor cell (CTC) enumeration to find biomarkers useful for the management of MIBC after RC.MethodsThirty-nine MIBC patients undergoing RC were included. Tumoral tissue DNA was analyzed by next generation sequencing. CTCs were isolated from blood collected before RC and one, four and 12 months later.ResultsSixteen (41%) patients progressed in a median time of 8.5 months and 11 (69%) of these patients harbored the TERT c.-124C > T mutation. All progressive patients harboring the TERT c.-124C > T mutation presented a significant increase in CTC number 12 months after RC compared to those without the mutation. Additionally, CTC number at 12 months was identified as an independent prognostic biomarker for tumor progression and cancer specific survival (CSS). Ten (63%) progressive patients showed an increment of CTC number with a median anticipation period of four months compared with imaging techniques.ConclusionsThe TERT c.-124C > T mutation could be considered a biomarker of aggressivity. CTC enumeration is a useful tool for identifying MIBC patients at high risk of progression and CSS after RC and for detecting tumor progression earlier than imaging techniques.

Abstract 1264: CTC-based biomarker analysis and correlation with clinical response to PSMA-TRT in mCRPC

Abstract Prostate specific membrane antigen (PSMA)-targeted radionuclide therapies (PSMA-TRTs) is one of the most promising treatments for metastatic castration-resistant prostate cancer (mCRPC) upon progression on androgen receptor signaling inhibitors (ARPi). However, recent studies show that only about half of patients respond to PSMA-TRTs. Expression of PSMA on cancer cells via PET-imaging is of some use as a predictive biomarker of response to PSMA-TRTs, but there are patients with strong PSMA imaging who do not respond and some with poor PSMA imaging that do respond. PSMA imaging does not fully capture response predictability. Currently, the lack of predictive biomarkers of response limits optimal selection of the patient population most likely to benefit from to PSMA-TRTs. PSMA-TRTs radionuclides emit α or β radiations, which induce DNA damage in targeted cells, followed by immunogenic cell death (ICD), evidenced by calreticulin (CRT) plasma membrane accumulation. To investigate the role of PSMA expression and CRT membrane localization as a biomarker of response to PSMA-TRT, we isolated circulating tumor cells (CTCs) from patients with mCRPC, enrolled in a phase I dose escalation trial of 225Ac-J591 (NCT03276572). Thirty-two (32) patients were enrolled (median age 69. 5, range 52-89; PSA 149.1, range 4.8-7168); 78% and 68% of patients received ≥ 2 prior ARPi treatments or chemotherapy, respectively. 69% and 44% of patients experienced any or &amp;gt;50% PSA decline, respectively, with a median PFS of 5.1 months and median OS of 11.1 months. CTCs were isolated before treatment (baseline) from 30/32 of enrolled patients, and 12 weeks after treatment initiation (on-treatment) from 21/32 patients. Enriched CTCs via CD45 depletion were subsequently immunostained for cytokeratin (CK, standard epithelial marker), CD45 (standard marker to identify contaminating leukocytes), PSMA, calreticulin (CRT) and DAPI. CTCs were subjected to multiplex confocal microscopy and identified as CK+/CD45-/DAPI+ cells. For each patient, at each time point, the following parameters are scored: i) total number of CTCs, ii) number of PSMA+ CTCs, iii) number of CRT+ CTCs, iv) number of CTCs with plasma membrane CRT decoration. Percent of PSMA+ CTC at baseline and changes in percentage of CRT+ CTCs and CTCs with plasma membrane CRT decoration between baseline and on treatment timepoints are correlated with clinical response and time to progression upon 225Ac-J591 treatment. Our preliminary results indicate higher expression of plasma membranous CRT in on-treatment CTCs in responders (PFS&amp;gt;12 months; n= 39 CTCs from 3 patients) than in non-responders (PFS&amp;lt;3 months; n= 20 CTCs from 2 patients). Full analysis of CRT and PSMA expression is currently being completed. Citation Format: Giuseppe Galletti, Prerna Vatsa, Joseph R. Osborne, Neil H. Bander, David M. Nanus, Scott T. Tagawa, Paraskevi Giannakakou. CTC-based biomarker analysis and correlation with clinical response to PSMA-TRT in mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1264.

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

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

Abstract LB117: Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry

Abstract Introduction: CTCs play a critical role in breast cancer (BCa) metastases. Rapid identification of live CTCs may help to predict BCa prognosis and identify which patients may derive treatment benefit, especially for those with metastatic disease. However, CTCs are present at very low concentrations in the peripheral blood, and CTC enumeration is costly and technically challenging. We previously reported that cell-size based device utilizing a microfluidic cassette could be used to isolate live blood cells (Zhang et al. 2019 AACR). Here we describe a pilot study using novel microfluidic Parsortix workflow to identify live CTCs for patients with Stage IV BCa. Methods: Four whole blood samples (7.5ml/each) were collected from each of 10 patients with stage IV BCa patients who had continued clinical progression of their disease on systemic therapy, under a Northwestern University IRB-approved study (NU20B03). All samples were stored at 15-30 °C in EDTA tubes. Live CTCs enrichment was performed using a cell size and deformability based Parsortix system (Angle) which utilizes a microfluidic based disposable cassette containing a step separation structure. The four samples from each patient were loaded onto the device and the separation procedure was started by using 4.5μm, 6.5μm 8μm and 10μm separation cassettes, respectively. After separation and cleaning, the CTCs captured by the Parsortix system were harvested by inverting the flow direction and then flushing from the cassette in 200μL PBS. The harvested CTCs were multi-stained directly by fluorescence labeled antibodies for Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes) (Menarini). Flow cytometry (FCM) was used to identify the CTCs which were classified as CK+, DAPI+ and CD45-. White blood cells from patient samples and the human breast cancer cell line MDA-MB-231 were used as negative and positive controls, respectively. Results: A total of 500 and 200 MDA-MB-231 cells were processed as controls, which were collected completely by Parsortix and identified by FCM confirming that Parsortix system could recover the CTCs effectively. CTCs were classified based on morphology and phenotype as CK+, DAPI+ and CD45-. Live CTCs (≥1) were detected in 8 out of 10 (80%), 9 out of 10 (90%), 7 out of 10 (70%) and 5 out of 10 (50%) samples by using 4.5μm (Group 1), 6.5μm (Group 2), 8μm (Group 3) and 10μm (Group 4) separation cassettes. Maximum CTCs isolated from each group were 18, 32, 12 and 8 from Group 1, 2, 3 and 4, respectively. The corresponding average CTCs isolated from each sample were 3.5 CTCs, 5.6 CTCs, 2.5 CTCs and 1.7 CTCs from Group 1, 2, 3, and 4, respectively. The 6.5μm cassettes (Group 2) using for CTCs collection had significantly higher efficacy on collection of CTCs compared to other three groups. Conclusions: In this pilot study, our findings demonstrated that the cell-sized dependent Parsortix system using multiple staining and FCM is an effective and specific approach for rapid recovery and identification of CTCs from Stage IV BCa patients. With further optimization, this strategy can help to accurately evaluate CTCs and offers a potential technique for diagnosis and treatmentmonitoring of patients with metastatic breast cancer. Citation Format: Qiang Zhang, Marwa Manai, Andrew Davis, Carolina Reduzzi, Paolo D’Amico, Saya Liz Jacob, Jianhua Jiao, Weijun Qin, Lisa Flaum, Amir Behdad, Massimo Cristofanilli, Ami Shad, Leonidas Platanias, William Gradishar. Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB117.

Abstract 5122: Identification of PD-L1 expression on circulating tumor cells as a prognostic indicator in prospective clinical trial, OMNIVORE

Abstract Despite recent therapeutic advances for patients with renal cell carcinoma (RCC) with immunotherapies, alone or in combination with VEGF tyrosine kinase inhibitors, there are no available biomarkers to predict patient benefit or evaluate mechanisms of resistance. The known heterogeneity of RCC may explain the limited utility of biomarker testing on single-site biopsies. This has led to great interest in liquid biopsy as an accessible source of tumor cells to evaluate mechanisms of response and resistance. Given the growing use of immunotherapy (IO) in patients with RCC, and the promise of their companion diagnostics in other disease settings, we evaluated the ability of liquid biopsy quantification of PD-L1 to predict patient response to IO in the OMNIVORE clinical trial, NCT03203473. Patients on trial were given nivolumab monotherapy, followed by radiographic scans at C3D1, with follow-up scans confirming CR/PR defined as “response.” Responding patients were placed on observation, while non-responders were transitioned to the dual therapy with nivolumab and ipilimumab arm. PD-L1 and HLA I expression was quantified on circulating tumor cells (CTCs) using Exclusion-Based Sample Preparation (ESP). Single-cell protein analysis of PD-L1 and HLA I on CTCs facilitated the generation of summary statistics including average expression of all CTCs, number of CTCs categorized as PD-L1 or HLA I positive, and frequency of CTCs with either positive or negative expression. ROC curves were used to compare diagnostic sensitivity and specificity of different CTC summary statistics for 30 patients with evaluable matched baseline and arm assignment data. Kaplan-Meier curve evaluation was performed on the summary statistic identified as that with the greatest clinical value. ROC curve analysis identified the metric “number of PD-L1+ CTCs” at arm assignment as having the greatest clinical value (AUC 0.8) compared to all other CTC summary statistics evaluated, with a diagnostic sensitivity/specificity of 88/60% at a cutoff of 2.5 PD-L1+ CTCs/7.5 mL. Survival analysis showed that patients with fewer PD-L1+ CTCs had a longer durable response (68 weeks vs. 16 weeks, HR 2.9) to single agent nivolumab. These results suggest that the number of PD-L1+ CTCs is a potential biomarker of treatment response that may have utility for treatment stratification and investigation of novel combination strategies for patients with poor prognostic features. This biomarker is now being tested in multiple prospective clinical trials to further study clinical utility. Citation Format: Rory M. Bade, Jennifer L. Schehr, Matthew C. Mannino, Matthew L. Bootsma, Hamid Emamekhoo, Shuang G. Zhao, Toni K. Choueiri, Sabina Signoretti, Rana R. McKay, Joshua M. Lang. Identification of PD-L1 expression on circulating tumor cells as a prognostic indicator in prospective clinical trial, OMNIVORE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5122.

Abstract 641: Single-cell RNA sequencing of rare circulating tumor cells in precursor myeloma patients reveals molecular underpinnings of tumor cell circulation

Abstract Background: Multiple Myeloma (MM) is a hematological malignancy characterized by abnormal proliferation of terminally differentiated plasma cells (PCs) in the bone marrow (BM). MM is almost always preceded by the precursor stage smoldering multiple myeloma (SMM). BM biopsies are useful to monitor disease progression, but they are invasive and not routinely collected from patients for disease monitoring during precursor stages. Profiling circulating tumor cells (CTCs) from peripheral blood (PB) could aid early detection, disease monitoring, and biomarker identification to predict patients at high risk of progression that may benefit from early therapeutic intervention. Methods: Paired PB and BM aspirates were collected from 40 SMM patients enrolled in the PCROWD study (IRB #14-174) at Dana-Farber Cancer Institute. Malignant PCs were enriched by magnetic bead-based methods and underwent 5’ single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics). Results: We analyzed 105,246 BM PCs and 33,234 PB PCs from 15 patients. To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements, assessed by concurrent scBCR-seq. A total of 86,986 BM tumor cells and 8,718 CTCs were captured. A median of 5, 26, and 47 CTCs were present per mL of blood from low, intermediate, and high-risk SMM patients as defined by the International Myeloma Working Group (IMWG) “20/2/20” criteria, suggesting sequencing-based CTC enumeration corresponds to prognosis. High levels of driver genes commonly upregulated in patients with specific translocations, including CCND1 and MAF, were detected in both BM tumor and CTC clusters in 3 patients with t(11;14) and t(14;16) confirmed by fluorescence in situ hybridization (FISH) clinical testing, and 2 additional patients with inconclusive FISH results (Wilcoxon, q &amp;lt;10-3), supporting the idea of CTC-based prognostication. Differential expression (DE) analysis revealed 8 genes that were significantly upregulated and 3 genes that were significantly downregulated in CTCs compared to BM tumor cells robustly across 15 paired samples. Gene set enrichment analysis (GSEA) revealed genes DE in CTCs are associated with TNF-α and NF-κB signaling, which are commonly induced by extrinsic factors in the bone marrow milieu, providing insight into the biology of tumor cell circulation. Conclusions: This study highlights the utility of scRNA-seq for molecular profiling of CTCs, even in asymptomatic low tumor burden disease. Additional analyses are ongoing in the expanded cohort of 40 patients with paired samples to help gain further insight into CTC heterogeneity. Overall, this study will help enable the design of new molecular liquid biopsy-based approaches to diagnosis, disease monitoring, and biological insights to improve treatment strategies for precursor myeloma patients. Citation Format: Elizabeth D. Lightbody, Danielle T. Firer, Romanos Sklavenitis-Pistofidis, Michael Agius, Ankit K. Dutta, Michelle Aranha, Jean-Baptiste Alberge, Laura Hevenor, Nang Kham Su, Cody Boehner, Erica Horowitz, Jacqueline Perry, Anna Cowan, Hadley Barr, Anna Justis, Daniel Auclair, Catherine R. Marinac, Gad Getz, Irene Ghobrial. Single-cell RNA sequencing of rare circulating tumor cells in precursor myeloma patients reveals molecular underpinnings of tumor cell circulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 641.

A Laboratory-Friendly CTC Identification: Comparable Double-Immunocytochemistry with Triple-Immunofluorescence.

Simple SummaryTumor cells that circulate in the peripheral blood of patients with solid tumors are called circulating tumor cells. Since the source of circulating tumor cells are from primary cancer sites, metastatic sites, and/or a disseminated tumor cell pool, these cells have clinical significance. The circulating tumor cells offer a rare glimpse of the evolution of the tumor and its response/resistance to treatment in a real-time non-invasive manner. Although the clinical relevance of circulating tumor cells is undeniable, the routine use of these cells remains limited due to the elusive nature of the cells, which demands highly sophisticated and costly instrumentation. We presented a specific and sensitive laboratory-friendly parallel double-detection format method for the simultaneous isolation and identification of circulating tumor cells from peripheral blood of 91 consented and enrolled patients with tumors of the lung, endometrium, ovary, esophagus, prostate, and liver. Our user-friendly cost-effective circulating tumor cells detection technique has the potency to facilitate the routine use of circulating tumor cells detection even in community-based cancer centers for prognosis, before and after surgery, which will provide a unique opportunity to move cancer diagnostics forward.The source of circulating tumor cells (CTC) in the peripheral blood of patients with solid tumors are from primary cancer, metastatic sites, and a disseminated tumor cell pool. As 90% of cancer-related deaths are caused by metastatic progression and/or resistance-associated treatment failure, the above fact justifies the undeniable predictive and prognostic value of identifying CTC in the bloodstream at stages of the disease progression and resistance to treatment. Yet enumeration of CTC remains far from a standard routine procedure either for post-surgery follow-ups or ongoing adjuvant therapy. The most compelling explanation for this paradox is the absence of a convenient, laboratory-friendly, and cost-effective method to determine CTC. We presented a specific and sensitive laboratory-friendly parallel double-detection format method for the simultaneous isolation and identification of CTC from peripheral blood of 91 consented and enrolled patients with various malignant solid tumors of the lung, endometrium, ovary, esophagus, prostate, and liver. Using a pressure-guided method, we used the size-based isolation to capture CTC on a commercially available microfilter. CTC identification was carried out by two expression marker-based independent staining methods, double-immunocytochemistry parallel to standard triple-immunofluorescence. The choice of markers included specific markers for epithelial cells, EpCAM and CK8,18,19, and exclusion markers for WBC, CD45. We tested the method’s specificity based on the validation of the staining method, which included positive and negative spiked samples, blood from the healthy age-matched donor, healthy age-matched leucopaks, and blood from metastatic patients. Our user-friendly cost-effective CTC detection technique may facilitate the regular use of CTC detection even in community-based cancer centers for prognosis, before and after surgery.