Circulating Tumor Cells In Peripheral Blood Research Articles

Comprehensive characterization of circulating tumor cells and cell-free DNA in patients with metastatic melanoma.

Advances in therapeutic approaches for melanoma urge the need for biomarkers that can identify patients at risk for recurrence and to guide treatment. The potential use of liquid biopsies in identifying biomarkers is increasingly being recognized. Here, we present a head-to-head comparison of several techniques to analyze circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in 20 patients with metastatic melanoma. In this study, we investigated whether diagnostic leukapheresis (DLA) combined with multimarker flow cytometry (FCM) increased the detection of CTCs in blood compared to the CellSearch platform. Additionally, we characterized cfDNA at the level of somatic mutations, extent of aneuploidy and genome-wide DNA methylation. Both CTCs and cfDNA measures were compared to tumor markers and extracranial tumor burden on radiological imaging. Compared to the CellSearch method applied on peripheral blood, DLA combined with FCM increased the proportion of patients with detectable CTCs from 35% to 70% (P = 0.06). However, the median percentage of cells that could be recovered by the DLA procedure was 29%. Alternatively, cfDNA mutation and methylation analysis detected tumor load in the majority of patients (90% and 93% of samples successfully analyzed, respectively). The aneuploidy score was positive in 35% of all patients. From all tumor measurements in blood, lactate dehydrogenase (LDH) levels were significantly correlated to variant allele frequency (P = 0.004). Furthermore, the presence of CTCs in DLA was associated with tumor burden (P < 0.001), whereas the presence of CTCs in peripheral blood was associated with number of lesions on radiological imaging (P < 0.001). In conclusion, DLA tended to increase the proportion of patients with detectable CTCs but was also associated with low recovery. Both cfDNA and CTCs were correlated with clinical parameters such as LDH levels and extracranial tumor burden.

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

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

Precise Capture and Dynamic Release of Circulating Liver Cancer Cells with Dual-Histidine-Based Cell Imprinted Hydrogels.

Circulating tumor cells (CTCs) detection presents significant advantages in diagnosing liver cancer due to its noninvasiveness, real-time monitoring, and dynamic tracking. However, the clinical application of CTCs-based diagnosis is largely limited by the challenges of capturing low-abundance CTCs within a complex blood environment while ensuring them alive. Here, an ultrastrong ligand, l-histidine-l-histidine (HH), specifically targeting sialylated glycans on the surface of CTCs, is designed. Furthermore, HH is integrated into a cell-imprinted polymer, constructing a hydrogel with precise CTCs imprinting, high elasticity, satisfactory blood compatibility, and robust anti-interference capacities. These features endow the hydrogel with excellent capture efficiency (>95%) for CTCs in peripheral blood, as well as the ability to release CTCs controllably and alive. Clinical tests substantiate the accurate differentiation between liver cancer, cirrhosis, and healthy groups using this method. The remarkable diagnostic accuracy (94%), lossless release of CTCs, material reversibility, and cost-effectiveness ($6.68 per sample) make the HH-based hydrogel a potentially revolutionary technology for liver cancer diagnosis and single-cell analysis.

Digital Circulating Tumor Cells Quantification.

Circulating tumor cells (CTCs) are an emerging but vital biomarker for cancer management. An efficient methodology for accurately quantifying CTCs remains challenging due to their rareness. Here, we develop a digital CTC detection strategy using partitioning instead of enrichment to quantify CTCs. By utilizing the characteristics of droplet microfluidics that can rapidly generate a large number of parallel independent reactors, combined with Poisson distribution, we realize the quantification of CTCs in the blood directly. The limit of detection of our digital CTCs quantification assay is five cells per 5 mL of whole blood. By simultaneously detecting multiple genetic mutations, our approach achieves highly sensitive and specific detection of CTCs in peripheral blood from NSCLC patients (AUC = 1). Our digital platform offers a potential approach and strategy for the quantification of CTCs, which could contribute to the advancement of cancer medical management.

The detection of circulating tumor cells indicates poor therapeutic efficacy and prognosis in patients with nonsmall cell lung cancer: A systematic review and meta-analysis.

The efficacy and prognostic value of circulating tumor cells (CTCs) in nonsmall cell lung cancer (NSCLC) are controversial based on the existing research. This systematic review and meta-analysis evaluated the significance of CTCs in NSCLC therapy monitoring and prognosis prediction, supporting their potential as clinical biomarkers. We conducted a comprehensive search of PubMed, Embase, Web of Science, The Cochrane Library, WanFang Data, CNKI, and VIP through September 20, 2023. Inclusion criteria were cohort studies involving NSCLC patients, focusing on peripheral blood CTCs, and assessing outcomes such as pre- and posttreatment CTC rates or levels, progression-free survival (PFS), and overall survival (OS). Two reviewers independently extracted the data and assessed risk of bias using the Newcastle-Ottawa Scale. We utilized Review Manager 5.4.1 for meta-analysis, calculating pooled odds ratios (ORs) for dichotomous outcomes, mean differences for continuous variables and hazard ratios (HRs) for survival data, applying fixed- or random-effects models based on heterogeneity assessed by the I2 statistic. This study was registered in PROSPERO (No. CRD42023450035). Twenty-two eligible studies with a total of 1674 NSCLC patients were included. Meta-analysis results showed that the CTCs-positive rate (OR=0.59, 95% CI 0.45 to 0.77, p=0.0001) and CTCs count (mean difference=-3.10, 95% CI -5.52 to -0.69, p=0.01) were significantly decreased after antitumor treatment. Compared with the CTCs nonreduced group, the CTC-reduced group showed better PFS (HR=1.71, 95% CI 1.35 to 2.17, p<0.00001) and OS (HR=1.50, 95% CI 1.21 to 1.86, p=0.0003) after treatment. PFS and OS in CTC-positive groups were lower than those in the CTCs-negative group pretreatment (HR=2.49, 95% CI 1.78 to 3.47, p<0.00001; HR=1.80, 95% CI 1.29 to 2.52, p=0.0006) and posttreatment (HR=3.36, 95% CI 2.12 to 5.33, p<0.00001; HR=3.31, 95% CI 1.75 to 6.27, p=0.0002). CTCs can be used as a biomarker to monitor NSCLC efficacy, predict prognosis and guide follow-up treatment.

Multi-DNA-Modified Double-Network Hydrogel with Customized Microstructure: A Novel System for Living Circulating Tumor Cells Capture and Real-Time Detection.

The precise and effective isolation of living circulating tumor cells (CTCs) from peripheral blood, followed by their real-time monitoring, is crucial for diagnosing cancer patients. In this study, a cell-imprinted double-network (DN) hydrogel modified with circular multi-DNA (CMD), coined the CMD-imprinted hydrogel with fixed cells as templates (CMD-CIDH), was developed. The hydrogel featured a customized surface for proficient capture of viable CTCs and in situ real-time fluorescent detection without subsequent release. The customized surface, constructed using polyacrylamide/chitosan DN hydrogel as the matrix on the cell template, had a dense network structure, thereby ensuring excellent stability and a low degradation rate. Optimal capture efficiencies, recorded at 93 ± 3% for MCF-7 cells and 90 ± 2% for Hela cells, were achieved by grafting the CMD and adjusting the nodule size on the customized surface. The capture efficiency remained significantly high at 67 ± 11% in simulated breast cancer patient experiments even at a minimal concentration of 5 cells mL-1. Furthermore, CMD grafted onto the surface produced a potent fluorescence signature, enabling in situ real-time fluorescent detection of the target cell's growth state even in complex environments. The customized surface is highly efficient for screening CTCs in peripheral blood and has promising potential for setting up the CTCs culture.

A novel method for detecting circulating tumor cells immunity based on micro-nano technique

This study was to develop a new method for detecting circulating tumor cells (CTCs) with high sensitivity and specificity, therefore to detect the colorectal cancer as early as possible for improving the detection rate of the disease. To this end, we prepared some micro-column structure microchips modified with graphite oxide-streptavidin (GO-SA) on the surface of microchips, further coupled with a broad-spectrum primary antibody (antibody1, Ab1), anti-epithelial cell adhesion molecule (anti-EpCAM) monoclonal antibody to capture CTCs. Besides, carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) were coupled with colorectal cancer related antibody as specific antibody 2 (Ab2) to prepare complex. The sandwich structure consisting of Ab1-CTCs-Ab2 was constructed by the microchip for capturing CTCs. And the electrochemical workstation was used to detect and verify its high sensitivity and specificity. Results showed that the combination of immunosensor and micro-nano technology has greatly improved the detection sensitivity and specificity of the immunosensor. And we also verified the feasibility of the immunosensor for clinical blood sample detection, and successfully recognitized detection and quantization of CTCs in peripheral blood of colorectal cancer patients by this immunosensor. In conclusion, the super sandwich immunosensor based on micro-nano technology provides a new way for the detection of CTCs, which has potential application value in clinical diagnosis and real-time monitoring of disease.

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

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

Analysis of Diagnostic Value of CTC and CTDNA in Early Lung Cancer.

This paper is the exploration and analysis of the diagnostic value of circulating tumor cells (CTC) and circulating tumor deoxyribonucleic acid (ctDNA) in early non-small cell lung cancer (NSCLC).A total of 125 NSCLC patients that hospitalized from March 2019 to December 2020 were selected, of which 64 subjects with stage I-II were listed as the early-stage group, and 61 patients with stage III-IV were classified as the intermediate-advanced group; In addition, 47 patients with benign pulmonary nodules hospitalized within same period were selected as a group of benign pulmonary nodules, and 50 healthy subjects were enrolled as a control group. The levels of CTC and ctDNA, serum tumor markers carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cytokeratin 21-1 (CYFRA21-1) and gastrin-releasing peptide precursor (pro-GRP) were detected. The diagnostic value of CTC and ctDNA in early NSCLC patients was analyzed by ROC curve. The area under the curve of CTC count for early-stage lung cancer was 0. 949 (P=0. 000, 95%CI: 0. 918-0. 980), and ctDNA was 0. 914 (P=0. 000, 95%CI: 0. 866~0. 963). There were significant differences in CTC count and ctDNA among subjects in each group (P<0. 05). CTC count and ctDNA of the early and intermediate-advanced group were remarkably higher than those of the control group and group of benign pulmonary nodules (P<0. 05), and CTC count and ctDNA of patients in the middle-late group were critically higher than those in the early-stage group (P<0. 05). There were significant differences in serum CEA, NSE, CYFRA21-1 and pro-GRP levels among all groups (P<0. 05). The results showed that CTC and ctDNA in NSCLC patients were significantly positively correlated with CEA, NSE, CYFRA21-1 and pro-GRP (P<0. 05). Peripheral blood CTC and ctDNA levels are significantly increased in NSCLC patients. They are positively correlated with serum tumor markers CEA, NSE, CYFRA21-1 and pro-GRP levels, and are related to tumor progression. CTC and ctDNA have high value in the clinical diagnosis of early NSCLC.

Liquid Biopsy in Hepatocellular Carcinoma: The Significance of Circulating Tumor Cells in Diagnosis, Prognosis, and Treatment Monitoring.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with poor outcomes when diagnosed at an advanced stage. Current curative treatments are most effective in early-stage HCC, highlighting the importance of early diagnosis and intervention. However, existing diagnostic methods, such as radiological imaging, alpha-fetoprotein (AFP) testing, and biopsy, have limitations that hinder early diagnosis. AFP elevation is absent in a significant portion of tumors, and imaging may have low sensitivity for smaller tumors or in the presence of cirrhosis. Additionally, as our understanding of the molecular pathogenesis of HCC grows, there is an increasing need for molecular information about the tumors. Biopsy, although informative, is invasive and may not always be feasible depending on tumor location. In this context, liquid biopsy technology has emerged as a promising approach for early diagnosis, enabling molecular characterization and genetic profiling of tumors. This technique involves analyzing circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), or tumor-derived exosomes. CTCs are cancer cells shed from the primary tumor or metastatic sites and circulate in the bloodstream. Their presence not only allows for early detection but also provides insights into tumor metastasis and recurrence. By detecting CTCs in peripheral blood, real-time tumor-related information at the DNA, RNA, and protein levels can be obtained. This article provides an overview of CTCs and explores their clinical significance for early detection, prognosis, treatment selection, and monitoring treatment response in HCC, citing relevant literature.

Exploring the Potential Driver Gene Mutations That Promote Renal Cancer Cell Metastasis and Implantation Based on Circulating Tumor Cells Culture.

Studies have shown that the circulating tumor cell (CTC) is a necessary condition for the invasion and distant metastasis of renal cell carcimona (RCC). However, few CTCs-related gene mutations have been developed which could promote the metastasis and implantation of RCC. The objective of this study is to explore the potential driver gene mutations that promote RCC metastasis and implantation based on CTCs culture. Fifteen patients with primary mRCC and three healthy subjects were included, and peripheral blood was obtained. After the preparation of synthetic biological scaffolds, peripheral blood CTCs were cultured. Successful cultured CTCs were applied to construct CTCs-derived xenograft (CDX) models, followed by DNA extraction, whole exome sequencing (WES) and bioinformatics analysis. Synthetic biological scaffolds were constructed based on previously applied techniques, and peripheral blood CTCs culture was successfully performed. We then constructed CDX models and performed WES, and explored the potential driver gene mutations that may promote RCC metastasis and implantation. Bioinformatics analysis showed that KAZN and POU6F2 may be closely related to the prognosis of RCC. We successfully performed the culture of peripheral blood CTCs and, on this basis we initially explored the potential driver mutations for the metastasis and implantation of RCC.

Analysis of different protein expression levels in peripheral blood circulating tumor cells from patients with diffuse large B-cell lymphoma and their predictive efficiency for recurrence

Objective: To analyze the expression levels of differentiation cluster 47 (CD47), signal regulatory protein α (SIRP-α), proto-oncogene (MYC) and proliferating cell associated antigen (Ki67) proteins in peripheral blood circulating tumor cells (CTC) from patients with diffuse large B-cell lymphoma and their predictive efficiency for tumor recurrence. Methods: The data of 82 patients with diffuse large B-cell lymphoma who were confirmed by histopathology and were in remission after chemotherapy in the Hematology Department of Linyi People's Hospital from January 2018 to January 2021 were retrospectively analyzed. There were 44 males and 38 females, and aged from 50 to 75 (63.8±4.6) years. The patients were divided into recurrent group (n=36) and non-recurrent group (n=46) according to their recurrence within 1 year after remission. The fasting peripheral venous blood samples (4 ml) from patients in the morning were collected, and the CTC were isolated. The expression levels of CD47, SIRP-α, MYC and Ki67 proteins in CTC were detected by Western blotting. The correlations between CD47 expression level and SIRP-α, MYC and Ki67 expression levels were analyzed by Pearson correlation analysis. The predictive efficiency of CD47, SIRP-α, MYC and Ki67 expression levels on tumor recurrence was evaluated by receiver operating characteristic (ROC) curves, and the areas under the curve (AUC) were calculated. Results: The expression levels of CD47, SIRP-α, MYC and Ki67 in recurrent group were 2.24±0.23, 1.17±0.12, 1.98±0.20 and 2.63±0.27, while those in non-recurrent group were 2.04±0.21, 1.31±0.13, 1.53±0.16 and 2.24±0.25. The expression levels of CD47, MYC and Ki67 in the recurrent group were higher than those in the non-recurrent group, while the expression levels of SIRP-α were lower than those in the non-recurrent group (all P<0.001). In 82 patients, the expression levels of CD47, SIRP-α, MYC and Ki67 were 2.13±0.22, 1.25±0.13, 1.73±0.18 and 2.41±0.26, respectively. The expression level of CD47 was negatively correlated with the expression level of SIRP-α (r=-0.308, P=0.005), but positively correlated with the expression level of MYC and Ki67 (r=0.484 and 0.332, P=0.012 and 0.003). The sensitivity of CD47, SIRP-α, MYC and Ki67 expression levels in predicting recurrence of diffuse large B-cell lymphoma was 66.7%, 72.2%, 72.2% and 66.7%, with the specificity of 67.4%, 71.7%, 67.4% and 71.7%, and AUC (95%CI) of 0.694 (0.582-0.791), 0.693 (0582-0.790), 0.714 (0.603-0.808) and 0.709 (0.598-0.804), respectively. The sensitivity of the combined detection of the above four indicators was 83.3%, with the specificity of 78.3% and the AUC (95%CI) of 0.864 (0.771-0.930), which was higher than those of the individual detection of each indicator (all P<0.05). Conclusions: The expression level of CD47 was negatively correlated with the expression level of SIRP-α, but positively correlated with the expression level of MYC and Ki67. The expression levels of CD47, SIRP-α, MYC and Ki67 have certain predictive value for tumor recurrence in patients with diffuse large B-cell lymphoma, and the predictive efficiency of combined detection is higher than single indicator detection.

Value of peripheral blood circulating tumor cell detection in the diagnosis of thoracic diseases and the prediction of severity.

Circulating tumor cell (CTC) detection, as a noninvasive liquid biopsy method, has been used in the diagnosis, prognostic indication, and monitoring of a variety of cancers. In this study, we aimed to investigate whether CTC detection could be used in the early diagnosis and prediction of severity of thoracic diseases. We enrolled 168 thoracic disease patients, all of whom underwent pathological biopsy. Carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) measurement was also performed in 146 patients. There were 131 cases of malignant thoracic diseases and 37 cases of benign lesions. We detected CTCs in a 5ml peripheral blood sample with the CTCBiopsy® system and analyzed the value of CTC count for predicting disease severity. Of 131 patients with a diagnosis of thoracic malignancy, CTCs were found in blood samples from 122 patients. However, only 2 out of 37 patients with benign thoracic disease had no detectable CTCs. There was no significant correlation between CTC count and benign and malignant lesions (P = 0.986). However, among 131 patients who had been diagnosed with malignant lesions, 33 had lymph node metastasis or distant metastasis. The presence of CTCs was significantly correlated with metastasis (P = 0.016 OR = 1.14). The area under the receiver operating characteristic (ROC) curve was 0.625 (95% confidence interval (CI), 0.519 to 0.730 P = 0.032). In addition, with stage IA1 as the cutoff, all patients were further divided into an early-stage group and a late-stage group. CTC count was significantly correlated with disease progression (P = 0.031 OR = 1.11), with an area under the curve (AUC) of 0.599 (95% CI, 0.506-0.692 P = 0.47). The sensitivity and specificity of CTC detection for the diagnosis of disease stage were 72.3% and 45.5%, respectively. In addition, the cutoff of 2.5 CTCs was the same when predicting disease metastasis and staging. Furthermore, the combination of CTC count, demographic characteristics and tumor markers had better predictive significance for disease staging. CTC count can effectively indicate the stages and metastasis of thoracic diseases, but it cannot differentiate benign and malignant diseases.

Gelatin‐Coated ZnO Array Nanoplatform for Capture, Drug Resistance Identification, and Recovery of Circulating Tumor Cells

AbstractDrug resistance causes failure of cancer treatment. Identifying drug sensitivity of tumors can guide therapeutic administration for cancer patients, however this approach lacks a facile, fast, and noninvasive evaluation platform. Herein, a gelatin‐coated ZnO array nanoplatform to isolate circulating tumor cells (CTCs) is developed, and their drug resistance for inspecting tumor sensitivity is assessed. The nanoplatform is comprised of a nanosubstrate of ZnO submicropillars (ZnO SMPs) for efficiently isolating cancer cells, and Au nanoparticles (AuNPs) based electrochemical probes for evaluating drug resistance of the captured cells. Gelatin, an extracellular matrix‐like and enzyme‐degradable material, is used to coat ZnO SMPs and AuNPs, endowing the nanoplatform with the ability to maintain cancer cell viability and realize enzyme‐responsive cell recovery for downstream bioanalysis. This nanoplatform is expected to noninvasively evaluate drug sensitivity of cancer patients through simple liquid biopsy of CTCs in peripheral blood and benefit the treatment.

Abstract P3-07-12: Allogeneic, Antigen-Presenting, GM-CSF-secreting, SV-BR-1-GM Whole Cell Therapeutic Vaccine in Advanced Metastatic Breast Cancer

Abstract Background: SV-BR-1-GM is an irradiated allogeneic cell line derived from a hormone receptor-negative (HR-) HER2-positive (HER2+) breast cancer (BC), now engineered to express HLA class I and II antigens, secrete GM-CSF and function as an antigen-presenting cell. Here we report post-hoc exploratory data for metastatic BC (MBC) patients treated with the SV-BR-1-GM regimen (SV) alone (NCT03066947) and in combination (CO) with immune checkpoint inhibitors (ICIs) (NCT03328026). Methods: SV includes cyclophosphamide 300 mg/m2 i.v. 48-72 hours prior to SV-BR-1-GM (20 × 106 cells) intradermally followed by interferon-alpha-2b at the SV-BR-1-GM inoculation sites on days 2 and 4. SV was given every 2 weeks for 3 cycles and then monthly in NCT03066947, and every 3 weeks in combination with PD-1 inhibitors in NCT03328026. Treatment was continued until disease progression or unacceptable toxicities. Results: A total of 34 refractory MBC patients (pts) were treated, including 24 with SV, and 14 with CO, including 4 who crossed over from SV to CO plus one who restarted the SV after a protocol specified administrative interruption in treatment. Median prior regimens were 5; 55% of pts had hormone receptor-positive (HR+) BC, 42% had HER2+ BC, and 16% were triple-negative BC (TNBC). Objective response rates (ORR) were 4% for monotherapy and 7% for combination. Clinical benefit rate (CBR) was 21% for monotherapy and 29% for combination. Responses and clinical benefits were seen across most BC subtypes (see table). Notably, CBR among 10 pts with HR+ BC (any HER2) who received the combination was 50% (5/10). The duration of response (DOR) among 4 patients treated with monotherapy was 49-223 days and DOR among 6 patients treated with combination was 72-292 days. Median PFS was 2.8 months for monotherapy and 4.2 months for combination. Median OS was 7.0 months for monotherapy, and 12.0 months for combination. Of 28 pts with available prior treatment data, 12 (43%) had PFS on study treatment exceeded PFS on prior treatment, of these 8/12 had &amp;gt;1 HLA match and 5/8 had ≥ 2 loci HLA-matched with SV-BR-1-GM. PFS ratio improvement was independent of the prior number of lines of therapy or BC subtypes. The SV-BR-1GM regimen was well tolerated with the most common treatment-related adverse events (AEs) being injection site reaction in 67%. There was no dose-limiting toxicity, special interest AEs such as cytokine storm, and no grade 5 observed. There were 2 non-treatment-related grade 4 AEs: worsening pleural effusion and altered mental status. Significant improvement in PFS was observed in patients with matched HLA and in association with the delayed-type hypersensitivity skin test, peripheral blood circulating tumor cells, and cancer-associated macrophages. Conclusions: SV-BR-1-GM demonstrated promising activity in patients with MBC. Treatment was well tolerated with no concerning AEs. The PFS ratio compares favorably with prior penultimate standards of care, more notably in patients with matched HLA. Clinical benefits were observed across multiple subtypes of BC, particularly in patients with HR+ disease receiving combination therapy. Phase II clinical trial is currently ongoing to evaluate the efficacy of SV-BR-1-GM in combination with ICIs. Clinical benefit and PFS on SV-BR-1-GM SV-BR-1-GM Whole Cell Therapeutic Vaccine in Heavily Pre-Treated Metastatic Breast Cancer Results Stratified by HER2 and HLA Matching Citation Format: Saranya Chumsri, William Williams, Mingjin Chang, Miguel Lopez-Lago, Charles Wiseman, Jarrod Holmes, Chaitali Nangia, Karim Mohammed, Minal Barve, Shaker Dakhil, Bonnie Guerin, Giuseppe Del Priore, Carmen Calfa. Allogeneic, Antigen-Presenting, GM-CSF-secreting, SV-BR-1-GM Whole Cell Therapeutic Vaccine in Advanced Metastatic Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-12.

Prognostic value of stem-like circulating tumor cells in patients with cancer: a systematic review and meta-analysis.

Despite increasing interest in the study of circulating tumor cells (CTC) subsets, especially epithelial-mesenchymal transition(EMT) and stem cells subsets of CTC that play a key role in tumor recurrence and metastasis, there is no evidence from meta-analyses that shows the correlation between stem-like CTCs and prognosis in cancer patients. Thus, we performed a meta-analysis to assess its prognostic value. Sixteen articles were screened by searching the PubMed, Embase, Cochrane, China National Knowledge Internet (CNKI) and Wanfang databases. The hazard ratio (HR) and 95% confidence interval (95% CI) extracted from each article were summarized. Patients with positive stem-like CTCs in peripheral blood had significantly shorter overall survival (OS, HR: 2.58, 95% CI 1.76-3.79, P < 0.00001), progression-free survival (PFS, HR: 2.21, 95% CI 1.26-3.89, P = 0.006) and disease-free survival (DFS, HR: 2.53, 95% CI: 1.12-5.70, P = 0.03). This study provides the first meta-analysis evidence for the prognostic value of stem-like CTCs, demonstrating that these cells are associated with poor prognosis in cancer patients.Systematic review registrationCRD42022322062.

Abstract PR007: Comprehensive ctDNA profiling reveals potential metastatic genomic signatures in treatment-naive early-stage breast cancer patients

Abstract Background: Genomic profiling has revolutionized precision oncology impacting the diagnosis, prognosis, and therapy decisions. Considering high spatiotemporal diversity and heterogenicity of breast tumor-cell genomes, small-gene panels often fail to capture rare but important genomic alterations. Conversely, comprehensive ctDNA sequencing approaches enable the identification of under characterized ‘long tailed driver’ genomic alterations and capture Intra and inter metastatic heterogeneity. Here, we demonstrate the clinical utility of comprehensive genome profiling with higher sensitivity to predict the possibility of metastasis in early-stage breast cancer patients. Methods: We retrospectively analyzed ctDNA and genomic DNA (gDNA) from FFPE samples as well as circulating tumor cells (CTC) in 10 treatment-naive hormone positive and HER2 negative, primary-stage breast cancer patients [GS1] using the OncoIndx comprehensive 600 gene panel. The panel captures all important cancer-relevant genomic alterations including Tumor Mutation Burden (TMB), Micro Satellite Instability (MSI), homologous recombination deficiency (HRD) prediction, and cfDNA tumor fraction (TF). CTCs were enumerated from 1.5 ml of blood using the OncoDiscover platform approved by the Drug Controller General of India having anti-EpCAM antibody-mediated immunomagnetic nanoparticles. CTCs were confirmed for cytokeratin 18+ and DAPI + markers and the absence of CD45. Results: Comprehensive genomic profile obtained from ctDNA and gDNA from FFPE of early-stage breast cancer patients predominantly exhibited the presence of alterations in PIK3CA and ESR1 signaling pathways. PIK3CA mutations were present in 77% and 44% of baseline ctDNA and gDNA samples, while ESR1 mutations were present in 44% and 22% of baseline ctDNA and gDNA, respectively. In addition, we observed about 70% additional driver mutations in ctDNA samples suggesting shedding of ctDNA together with CTC (80% positive), a likely positive biomarker of metastasis. About 50% of the patients showed higher TMB and HRD. Notably, TF representing ctDNA varied between 13% to 27% in blood samples with a corresponding ploidy range of 2.9 to 4.7. Surprisingly, ~50% of the patient population matched the mutation profile of clinically confirmed metastatic patients. All the patients harboring potential metastatic driver alterations showed the presence of CTCs in peripheral blood. Conclusions: Comprehensive ctDNA genomic profiling showed potential metastasis driving alterations suggesting the role of ctDNA-based liquid biopsy to predict metastasis in early breast cancer patients. We observed enhanced TF at the time of diagnosis, possibly due to the presence of distant metastasis, high disease burden, and aggressive tumor biology. Our results suggest that ctDNA dynamics at the time of disease presentation can predict early metastasis, and may demonstrate the divergent response of tumor heterogeneity to treatment in early-stage breast cancer. Citation Format: Gowhar Shafi, Manoj Dongare, Atul Bharde, Moubeen Fauzul, Kanchan Hariramani, Alain D’Souza, Bhagwat Jadhav, Trupti Kad, Sangeeta Prajapati, Vikas Jadhav, ManojKumar Kumaran, Sumit Haldar, Vatsal Mehra, Sujit Joshi, Gourishankar Aland, Richa Dave, Sreeja Jayant, Aravindan Vasudevan, Mohan Uttarwar, Jayant Khandare. Comprehensive ctDNA profiling reveals potential metastatic genomic signatures in treatment-naive early-stage breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR007.

Circulating tumor cells as diagnostic markers of early gastric cancer and gastric precancerous lesions.

Circulating tumor cells (CTCs) may be potential diagnostic biomarkers of various malignancies including gastric cancer. This study aimed to evaluate whether CTCs could be used to facilitate the diagnosis of early gastric cancer (EGC) or precancerous gastric lesions. The diagnostic study included consecutive patients with EGC, gastric precancerous lesions or fundic gland polyps admitted to the Gastroenterology Department, Beijing Friendship Hospital Affiliated to Capital Medical University (National Center for Digestive Diseases) between October 2016 and January 2018. A total of 92 patients were enrolled, including 57 patients with EGC, 14 patients with gastric precancerous lesions and 21 patients with fundic gland polyps (control group). CTCs were detected in 47.89% (34/71) of patients with EGC/gastric precancerous lesions and 4.76% (1/21) of patients with fundic gland polyps (P<0.001). CTC detection distinguished EGC/precancerous lesions from fundic gland polyps with an area under the ROC curve of 0.740 (95% confidence interval, 0.640-0.840; P=0.001), a sensitivity of 49.10%, a specificity of 95.00%, a positive predictive value of 97.00% and a negative predictive value of 64.90%. Peripheral blood CTCs are more common in patients with EGC or gastric precancerous lesions than in patients with fundic gland polyps. Measurement of CTCs may be a useful tool to aid the diagnosis of EGC and precancerous lesions.

Rapid metabolomic screening of cancer cells via high-throughput static droplet microfluidics

Effective isolation and in-depth analysis of Circulating Tumour Cells (CTCs) are greatly needed in diagnosis, prognosis and monitoring of the therapeutic response of cancer patients but have not been completely fulfilled by conventional approaches. The rarity of CTCs and the lack of reliable biomarkers to distinguish them from peripheral blood cells have remained outstanding challenges for their clinical implementation. Herein, we developed a high throughput Static Droplet Microfluidic (SDM) device with 38,400 chambers, capable of isolating and classifying the number of metabolically active CTCs in peripheral blood at single-cell resolution. Owing to the miniaturisation and compartmentalisation capability of our device, we first demonstrated the ability to precisely measure the lactate production of different types of cancer cells inside 125 pL droplets at single-cell resolution. Furthermore, we compared the metabolomic activity of leukocytes from healthy donors to cancer cells and showed the ability to differentiate them. To further prove the clinical relevance, we spiked cancer cell lines in human healthy blood and showed the possibility to detect the cancer cells from leukocytes. Lastly, we tested the workflow on 8 preclinical mammary mouse models including syngeneic 67NR (non-metastatic) and 4T1.2 (metastatic) models with Triple-Negative Breast Cancer (TNBC) as well as transgenic mouses (12-week-old MMTV-PyMT). The results have shown the ability to precisely distinguish metabolically active CTCs from the blood using the proposed SDM device. The workflow is simple and robust which can eliminate the need for specialised equipment and expertise required for single-cell analysis of CTCs and facilitate on-site metabolic screening of cancer cells.

RNA profiling of circulating tumor cells systemically captured from diagnostic leukapheresis products in prostate cancer patients.

The application of circulating tumor cells (CTCs) in both clinical practice and research has been continuously limited by the rare number of targets that can be found in a tube of peripheral blood. Diagnostic leukapheresis (DLA) was used to increase the sampling volume. AdnaTest was used to process the whole leukopak, and the RNAs of captured CTCs was then profiled by NanoString nCounter platform. Spike-in experiments and leukopaks from patients with metastatic prostate cancer were used to validate this new strategy. The whole leukopak was further concentrated five times to reduce the total volume from 150​mL to 30​mL, which enabled it to be processed by 3 separate AdnaTest kits. The spike-in experiment demonstrated a reliable capture when there were more than 100 cancer cells/10​mL of concentrated leukopak. In 1 out of 5 real patient samples, CTCs were only detected in the leukopak, but not in peripheral blood. The RNA profiling of DLA CTCs indicated a more aggressive phenotype of CTCs occurred when the patient was experiencing a disease relapse, even when the serum prostate specific antigen (PSA) level was still relatively low and CTCs in peripheral blood were not detectable. We established a new protocol, integrating DLA, AdnaTest and NanoString nCounter technology, to profile RNAs from CTCs captured from a large blood screening volume. The new protocol can process the whole leukopak with sensitive CTC capture. The RNA profiling of CTCs can provide valuable information for disease monitoring.