Circulating Tumor Cells Levels Research Articles

Abstract PO1-16-02: Correlation of Circulating Tumor Cells (CTC) with Clinical Characteristics, Pathological Factors, and Treatment Response in Patients with Metastatic Breast Cancer (MBC)

Abstract Background: Circulating Tumor Cells (CTC) can be isolated in 40-80% of patients with metastatic breast cancer (MBC). High levels of CTC, defined as ≥5/7.5 ml of blood are associated with shorter progression-free survival and overall survival. CTC can be tested for clinically relevant biomarkers, such as ER, and HER2 status, and have the potential to guide therapy as well as help with disease monitoring. We investigated CTC detection rate, its correlation with clinical characteristics, pathological characteristics, and response to treatment in patients with any type of metastatic breast cancer who progressed on at least one line of therapy. Methods: Eligible patients with MBC were enrolled in the IMAGE-II study (Individualized Molecular Analyses Guide Efforts in Breast Cancer) NCT02965755, in which we obtained archival tissue as part of the standard of care, and prospectively collected serial blood samples for CTC analysis. We compared CTC levels and biomarker status with clinical factors, tissue-based pathology, and molecular biomarkers. We assessed whether changes in CTC count correlated with treatment response. Blood samples were collected at baseline (Day 1), after 1-2 weeks of therapy, after 8-12 weeks, and at subsequent restaging (every 8-12 weeks). Medical records were reviewed every 3 months for ongoing treatment response, and death. Samples were processed at the Biocept CLIA-certified Laboratory. CTC were enumerated by the presence of CD45- and DAPI+ cells. Further CTC characterization was performed by antibody staining of specific protein biomarkers (ER, HER2). Fisher’s exact test was used to evaluate the association between baseline CTC < 5 and ≥5/7.5ml with patient characteristics. McNemar’s test was used to assess discordance between tissue-based and CTC-defined markers. Results: Between 1/26/2018 and 12/31/22 baseline samples were collected from 70 women with a median age of 56 (36-82) who were enrolled at four study sites. CTC was detected in 59 (84%) of participants. Baseline CTC counts were < 5/7.5 ml in 37/59 (63%) and ≥5/7.5 ml in 22/59 (37%) of participants. Differences between breast cancer subtype and CTC-receptor status were observed (Table 1). Among the 41 patients who were ER-positive, 31 (76%) were CTC ER-negative(p< 0.001). Among 29 patients who were ER-negative, 4 (14%) were CTC ER-Positive (P< 0.001). Among the 7 patients who were HER2 positive, 2(29%) were HER2 positive on CTC. Among 63 patients who were HER2 negative, 7(11%) had HER2 expression on their CTC. The discordance in the classification of HER2 wasn't statistically significant (p=0.77). Elevated CTC at baseline was more frequently detected in younger participants (< 50 years old) (55% vs 27%; P=0.22), in Black women compared to White (60 % vs 29 %, P=0.27), and in participants with visceral vs non-visceral metastasis (52% vs 28 %, P=0.40). Patients with CTC ≥5 vs < 5/7.5mL at baseline had a shorter duration of anti-HER2-based therapy (61.5 vs 836 days, p=0.04). There were no statistically significant differences among participants who received chemotherapy agents (316.5 vs 365.5 days, P=0.88), endocrine therapy (376 vs 490 days, P=0.89), or overall therapy (272 vs 390 days, P=0.21). Conclusions: We observed significant differences in the expression of ER between tumor tissue and CTC, which can be partly due to tumor evolution over time. Additionally, participants who are young, Black, and those who have visceral metastasis may have higher CTC counts. Higher CTC counts were associated with a shorter duration of anti-HER2 therapy. Although it didn’t meet statistical significance, a similar trend was observed in patients who received chemotherapy and endocrine therapy. Table 1: MBC Tissue-Based Subtype and CTC-receptor status at baseline Citation Format: Asnakech Bayable, Tingchang Wang, Amanda Blackford, Jessica Tao, Jenna Canzoniero, Seoho Lee, Faith Too, Barbara Blouw, Mary Wilkinson, Rima Couzi, Antonio Wolff, Christie Hiton, Ben Park, Vered Stearns, Cesar Santa-Maria. Correlation of Circulating Tumor Cells (CTC) with Clinical Characteristics, Pathological Factors, and Treatment Response in Patients with Metastatic Breast Cancer (MBC) [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 PO1-16-02.

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

Abstract Background: The presence of liquid biomarkers such as circulating tumor cells (CTCs) among women undergoing neoadjuvant chemotherapy (NAC) for breast cancer may be associated with treatment response and/or an increased risk of recurrence, but limited data is available. Objectives: To detect and enumerate CTCs in blood samples from women with a new diagnosis of non-metastatic breast cancer of any subtype both i) at baseline (prior to commencing NAC), and ii) after completion of NAC and surgery using the Epic Sciences platform. Methods: Women with non-metastatic breast cancer of any subtype who have not yet commenced NAC were included, irrespective of age. Those with a prior history of another invasive cancer (apart from non-melanoma skin cancer identified 5+ years prior to enrollment) were excluded. Blood samples were obtained to measure CTCs prior to commencing NAC, and after completion of NAC and surgery (at least 4 weeks post-operatively). As previously described, CTC identification was based on immunofluorescence analysis using the Epic Sciences platform (Ueno et al 2017). Clinical/pathological data and clinical outcomes were abstracted from patients’ medical records. Associations between CTC detection and clinical/pathologic characteristics were evaluated using Fisher’s exact test for categorical variables and t-test or Wilcoxon rank sum tests for numerical variables. All analyses were performed using the R software package. Results: 50 participants who met eligibility criteria were included; a baseline blood sample was evaluable for CTC detection and enumeration for 47 patients. The median age at breast cancer diagnosis was 52 (29-75). Among them, 18 (38.3%) had HER2+ breast cancer, 17 (36.1%) had hormone receptor (HR)+/HER2- breast cancer and 12 (25.5%) had triple negative breast cancer (TNBC). The majority of patients (91%) received anthracycline and taxane-based NAC. A total of 68 samples were tested for CTC enumeration (5 mL equivalent per sample) including 47 pre-treatment and 21 post-treatment samples. CTCs were detected in 37 (79%) of patients for whom a baseline (pre-NAC) sample was available with a mean of 4.2 (SD 17.0) CTCs/mL and a range of 0 - 115.4 CTCs/mL. Detection of CTCs at baseline was highest among patients with TNBC (n=10/12, 83%), followed by those with HER2+ (n=14/18, 78%) and HR+/HER2- (n=13/17, 76%) breast cancer. CTCs were detected in 43% (n=9/21) of post-treatment samples, with a mean of 1.2 (SD 3.6) CTCs/mL and range of 0 – 16.4 CTCs/mL. Among the 20 patients for whom matched pre- and post-treatment CTC results were available, 16 (80%) had detectable CTCs pre-treatment and 8 (40%) had detectable levels post-NAC and surgery; of the 8 patients with post-treatment CTCs, 4 (50%) had HR+/HER2- breast cancer, 2 had HER2+ (25%) and 2 (25%) had TNBC. Three patients had numerically higher CTC levels after completion of NAC and surgery compared to baseline levels, 2 of whom had HR+/HER2- breast cancer and one of whom had TNBC. To-date, only 8 patients (19% of 43 who have undergone surgery) have achieved a pathological complete response (PCR) to NAC, among whom 3 had matched pre- and post-treatment CTC results available. None of these 3 patients had detectable CTCs post treatment. Conclusions: Approximately 4 in 5 women with non-metastatic breast cancer who undergo NAC have detectable CTCs at baseline (pre-treatment) using the Epic Sciences Platform. Given that CTCs remain detectable in a high proportion (40%) of patients after NAC and surgery, evaluation of CTCs as a potential measure of minimal residual disease warrants further evaluation in this patient population. Citation Format: Rania Chehade, Arushi Jain, Veronika Moravan, Giuseppe Di Caro, Megan Slade, Nadine Hartmann, Rick Wenstrup, Ana Elisa Lohmann, William Tran, Katarzyna Jerzak. A single-center prospective cohort study to evaluate circulating tumor cells as a monitoring tool in women with breast cancer treated with neoadjuvant chemotherapy: final results of baseline data [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-10.

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.

Use of circulating tumor cells and microemboli to predict diagnosis and prognosis in diffuse glioma.

Circulating tumor cell (CTC) detection is a promising noninvasive technique that can be used to diagnose cancer, monitor progression, and predict prognosis. In this study, the authors aimed to investigate the clinical utility of CTCs in the management of diffuse glioma. Sixty-three patients with newly diagnosed diffuse glioma were included in this multicenter clinical cohort. The authors used a platform based on isolation by size of epithelial tumor cells (ISET) to detect and analyze CTCs and circulating tumor microemboli (CTMs) in the peripheral blood of patients both before and after surgery. Least absolute shrinkage and selector operation (LASSO) and Cox regression analyses were used to verify whether CTCs and CTMs are independent prognostic factors for diffuse glioma. CTC levels were closely related to the degree of malignancy, WHO grade, and pathological subtypes. Receiver operating characteristic curve analysis revealed that a high CTC level was a predictor for glioblastoma. The results also showed that CTMs originate from the parental tumor rather than from the circulation and are an independent prognostic factor for diffuse glioma. The postoperative CTC level is related to the peripheral immune system and patient survival. Cox regression analysis showed that postoperative CTC levels and CTM status are independent prognostic factors for diffuse glioma, and CTC- and CTM-based survival models had high accuracy in internal validation. The authors revealed a correlation between CTCs and clinical characteristics and demonstrated that CTCs and CTMs are independent predictors for the diagnosis and prognosis of diffuse glioma. Their CTC- and CTM-based survival models can enable clinicians to evaluate patients' response to surgery as well as their outcomes.

Abstract 7500: Distribution and prognostic significance of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) in patients (pts) with lobular metastatic breast cancer (MBC)

Abstract Background: Invasive lobular carcinoma (ILC) has distinctive clinical and molecular features compared with invasive ductal carcinoma (IDC). CTCs and tdEVs are independent prognostic factors in MBC, however, liquid biopsy studies focusing on ILC are to date scarce. Higher CTC levels have been observed in ILC than IDC. One study including 28 ILC pts suggested the use of higher CTC cutoff for prognosis stratification. The clinical significance of tdEVs in ILC is unexplored. This study aimed to assess differences in the distribution and prognostic value of CTCs and tdEVs between ILC and IDC. Methods: Blood samples were collected from 304 pts with MBC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). Blood was processed with the CellSearch® system. The ACCEPT software was applied to CellSearch® images to automatically enumerate CTCs and tdEVs. Association of CTCs and tdEVs with overall survival (OS) was tested in ILC and IDC pts. A count of CTCs ≥ 5 was considered high. The cutoff levels for tdEVs were &amp;lt; 20 (low), 20-79 (intermediate), and ≥ 80 (high), as previously reported. Additional exploratory cutoffs for CTCs (≥ 20, ≥ 50, ≥ 80, and ≥ 100) and tdEVs (based on quartiles of tdEV distribution in the overall population and ILC) were also evaluated. Results: Of the 304 pts, 47 had ILC while 257 had IDC. 56% of pts received first-line therapy. Median CTC count was 8 [interquartile range (IQR) 2-35] in ILC and 1 (IQR 0-8) in IDC pts (P &amp;lt;.001). A significantly higher median tdEVs count was observed in ILC (36; IQR 18-115) than in IDC (11; IQR 2-86) pts (P =.002). High levels of CTCs (≥5) were not associated with OS in ILC (P =.88), even when higher cutoffs (≥ 20, ≥ 50, and ≥ 80) were used. Only the detection of ≥ 100 CTCs was significantly associated with OS in ILC pts (median OS 36 vs 10 months; HR 3.4; P =.028). In contrast, the prognostic effect of CTCs ≥ 5 was strong in IDC and maintained for all cutoffs. Similarly, while the prognostic value for tdEVs was confirmed for IDC using both 20-80 and exploratory cutoffs (3-105 and 18-155), no association between tdEV levels and OS was observed in ILC regardless of the cutoffs used. Conclusion: ILC is associated with higher CTCs and tdEVs compared to IDC, probably due to the impaired cell-cell adhesion that characterizes ILC. Unlike IDC, the prognostic effect for the established cutoff of ≥ 5 CTC was not observed in ILC. A higher CTC cutoff could be considered for this subtype of BC. For tdEV no association with OS was observed, regardless of the cutoff used. These findings emphasize that ILC is a distinct entity also in the liquid biopsy features. Because of the low prevalence of ILC, research efforts should be directed at combining data from ILC pts to better characterize this subtype of BC and understand the reason behind the different associations with OS as compared to IDC. Citation Format: Eleonora Nicolo, Lorenzo Gerratana, Lorenzo Foffano, Laura Munoz-Arcos, Mara S. Serafini, Maroua Manai, Letizia Pontolillo, Nadia Bayou, Elisabetta Molteni, Amanda K. Strickland, Caterina Gianni, Youbin Zhang, Paolo D'Amico, Andrew A. Davis, Jeannine Donahue, Huiping Liu, William J. Gradishar, Ami N. Shah, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Distribution and prognostic significance of circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) in patients (pts) with lobular metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7500.

Abstract 5165: Development and validation of an assay for the characterization of SSTR2 expression on CTCs from liquid biopsies

Abstract Background: Expression of Somatostatin Receptor subtype 2 (SSTR2) is observed in multiple cancers, including breast and colorectal cancer. Its role as a prognostic or predictive marker is cancer-type dependent. Recently, there has been renewed interest in assessing this biomarker since it plays a critical role as a target for radiotherapeutics (peptide receptor radionuclide therapy - PRRT) in patients with neuroendocrine tumors (NETs). Blood-based liquid biopsies are a non-invasive tool with proven effectiveness in characterizing cancer biomarker expression in patients. In this study, we describe the development and validation of a novel assay to analyze circulating tumor cells (CTCs) for expression of SSTR2 using the RareCyte platform. Experimental Procedures: Using AccuCyte®, an unbiased density-based method for collecting nucleated cells from whole blood, we transferred nucleated cells to slides and stained for SSTR2, CD45, and CK/EpCAM to identify CTCs (CK+/EpCAM+, CD45-), and evaluate SSTR2 expression. Slides were imaged with CyteFinder®, an automated multiparameter immunofluorescent (IF) microscopy system that applies machine learning algorithms for rare cell identification. Both clinical and spike-in samples were used for assay validation. Single-cell SSTR2 mean fluorescence intensities (MFI) were analyzed to determine protein expression levels. Colorectal cancer with neuroendocrine features and Merkel cell cancer (neuroendocrine tumor of the skin) patient samples were evaluated for CTC enumeration and SSTR2 expression. Results: The assay’s performance metrics for SSTR2 expression were 90+% accuracy, 85% sensitivity, 100% specificity with repeatability and intermediate precision CVs of 14.4% and 16.4%, respectively. A small cohort of colorectal cancer (1) and Merkel cell cancer (2) patient blood samples were tested using the assay. All 3 patients had high CTC levels (300+ per 7.5 mL). The percentage of SSTR2-positive CTCs ranged from 20-60%, reflective of tumor heterogeneity. Conclusions: SSTR2 is an important target now in the advent of theranostics - PRRT. Prognostic and therapeutic implications of SSTR2 expression is an area of active clinical research. SSTR2 targeted therapeutics like 177Lu-dotatate illustrate the possible utility of SSTR2 as a predictive biomarker of treatment response. To date, most of the work has been focused on assessing SSTR2 on tissue. Herein, we show how a CTC platform can be adapted to assess a clinically relevant cell surface protein biomarker. This is now included as an important correlative in a clinical trial for Merkel Cell Cancer patients called iPRRT that is actively accruing (NCT05583708). Citation Format: Erin G. Bayer, Rachel N. Ponting, Ciara M. Kelly, Areeb Lutfi, Maaz K. Afghan, Arturo B. Ramirez, Pashtoon M. Kasi. Development and validation of an assay for the characterization of SSTR2 expression on CTCs from liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5165.

Clinical value of circulating tumour cells in evaluating the efficacy of continuous hepatic arterial infusion among colorectal cancer patients

Few studies have been conducted to evaluate the efficacy of HAIC using circulating tumour cells (CTCs). In this study, a total of 100 patients who received HAIC treatment and CTC detection were selected. The results showed that after HAIC treatment, the levels of CTC, carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) decreased. Postoperative progression-free survival (PFS) rates between patients with positive and negative preoperative CTC results, and for CA19-9, CEA were significantly different. The positive rate of CTCs was 61% before chemotherapy and 23% after chemotherapy, and the correlation coefficient between the two was 0.385. Those whose CTC values increased after chemotherapy had shorter PFS rates. CTCs are an independent predictor of recurrence. Patients with CTC-positive results are more susceptible to recurrence. The CTC count in peripheral blood has a close bearing on the postoperative chemotherapy efficacy of patients with CRC and affects patients’ PFS.

Peripheral arterial rather than venous blood is a better source of circulating tumor cells in early lung cancer.

Circulating tumor cells (CTCs) play a crucial role in the early diagnosis and prognosis of lung cancer. Identification of a more suitable sample source could be a breakthrough towards enhancing CTC detectability in early-stage lung cancer. We investigated the differences in detectable CTCs between peripheral arterial and venous blood in early- and mid-stage lung cancer patients undergoing surgery and analyzed the association between clinicopathological factors and detectable CTCs in peripheral arterial and venous blood. Peripheral arterial and venous blood was collected in 5-mL samples from 56 patients with surgically resected and pathologically clear at early- or mid-stage lung cancer. Blood specimens were enriched for CTCs based on isolation by size of epithelial tumor cells. The CTCs were identified using Swiss Giemsa staining and immunohistochemistry for CD45/CD31. In stage I lung cancer, CTC-positive rate was significantly higher in peripheral arterial than in venous blood (45.45% vs. 17.39%). There was no significant difference in the number of detectable CTCs between peripheral arterial and venous blood. A low degree of differentiation was associated with a high positive rate of CTCs in peripheral venous blood. The number of circulating tumor microemboli was significantly higher in patients with tumor size >3 cm compared with ≤3 cm. CTC levels in peripheral arterial and venous blood differed little in lung cancer patients.Compared to peripheral venous blood, peripheral arterial blood had a higher CTC positivity rate in early-stage lung cancer.This study was favorable for early detection and monitoring of lung cancer.

RORα inhibits gastric cancer proliferation through attenuating G6PD and PFKFB3 induced glycolytic activity

BackgroundGlycolysis is critical for harvesting abundant energy to maintain the tumor microenvironment in malignant tumors. Retinoic acid-related orphan receptor α (RORα) has been identified as a circadian gene. However, the association of glycolysis with RORα in regulating gastric cancer (GC) proliferation remains poorly understood.MethodsBioinformatic analysis and retrospective study were utilized to explore the role of RORα in cell cycle and glycolysis in GC. The mechanisms were performed in vitro and in vivo including colony formation, Cell Counting Kit-8 (CCK-8), Epithelial- mesenchymal transition (EMT) and subcutaneous tumors of mice model assays. The key drives between RORα and glycolysis were verified through western blot and chip assays. Moreover, we constructed models of high proliferation and high glucose environments to verify a negative feedback and chemoresistance through a series of functional experiments in vitro and in vivo.ResultsRORα was found to be involved in the cell cycle and glycolysis through a gene set enrichment analysis (GSEA) algorithm. GC patients with low RORα expression were not only associated with high circulating tumor cells (CTC) and high vascular endothelial growth factor (VEGF) levels. However, it also presented a positive correlation with the standard uptake value (SUV) level. Moreover, the SUVmax levels showed a positive linear relation with CTC and VEGF levels. In addition, RORα expression levels were associated with glucose 6 phosphate dehydrogenase (G6PD) and phosphofructokinase-2/fructose-2,6-bisphosphatase (PFKFB3) expression levels, and GC patients with low RORα and high G6PD or low RORα and high PFKFB3 expression patterns had poorest disease-free survival (DFS). Functionally, RORα deletion promoted GC proliferation and drove glycolysis in vitro and in vivo. These phenomena were reversed by the RORα activator SR1078. Moreover, RORα deletion promoted GC proliferation through attenuating G6PD and PFKFB3 induced glycolytic activity in vitro and in vivo. Mechanistically, RORα was recruited to the G6PD and PFKFB3 promoters to modulate their transcription. Next, high proliferation and high glucose inhibited RORα expression, which indicated that negative feedback exists in GC. Moreover, RORα deletion improved fluorouracil chemoresistance through inhibition of glucose uptake.ConclusionRORα might be a novel biomarker and therapeutic target for GC through attenuating glycolysis.

Frequency and Prognostic Value of Circulating Tumor Cells in Cancer of Unknown Primary.

Cancer of unknown primary (CUP) is defined as a primary metastatic malignancy, in which the primary tumor remains elusive in spite of a comprehensive diagnostic workup. The frequency and prognostic value of circulating tumor cells (CTCs), which are considered to be the source of metastasis, has not yet been systematically evaluated in CUP. A total of 110 patients with a confirmed diagnosis of CUP according to the European Society for Medical Oncology (ESMO) guidelines, who presented to our clinic between July 2021 and May 2023, provided blood samples for CTC quantification using CellSearch methodology. CTC counts were correlated with demographic, clinical, and molecular data generated by comprehensive genomic profiling of tumor tissue. CTCs were detected in 26% of all patients at initial presentation to our department. The highest CTC frequency was observed among patients with unfavorable CUP (35.5%), while patients with single-site/oligometastatic CUP harbored the lowest CTC frequency (11.4%). No statistically significant association between CTC positivity and the number of affected organs (P = 0.478) or disease burden (P = 0.120) was found. High CTC levels (≥5 CTCs/7.5 mL; 12/95 analyzed patients) predicted for adverse overall survival compared to negative or low CTC counts (6-months overall survival rate 90% vs 32%, log-rank P < 0.001; HR 5.43; 95% CI 2.23-13.2). CTC dynamics were also prognostic for overall survival by landmark analysis (log-rank P < 0.001, HR 10.2, 95% CI 1.95-52.9). CTC frequency is a strong, independent predictor of survival in patients with CUP. CTC quantification provides a useful prognostic tool in the management of these patients.

Radiofrequency ablation induces tumor cell dissemination in a mouse model of hepatocellular carcinoma

BackgroundWe tested the hypothesis that radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC) promotes tumor cell release and explored a method for reducing these effects.MethodsA green fluorescent protein-transfected orthotopic HCC model was established in 99 nude mice. In vivo flow cytometry was used to monitor circulating tumor cell (CTC) dynamics. Pulmonary fluorescence imaging and pathology were performed to investigate lung metastases. First, the kinetics of CTCs during the periablation period and the survival rate of CTCs released during RFA were investigated. Next, mice were allocated to controls, sham ablation, or RFA with/without hepatic vessel blocking (ligation of the portal triads) for evaluating the postablation CTC level, lung metastases, and survival over time. Moreover, the kinetics of CTCs, lung metastases, and mice survival were evaluated for RFA with/without ethanol injection. Pathological changes in tumors and surrounding parenchyma after ethanol injection were noted. Statistical analysis included t-test, ANOVA, and Kaplan-Meier survival curves.ResultsCTC counts were 12.3-fold increased during RFA, and 73.7% of RFA-induced CTCs were viable. Pre-RFA hepatic vessel blocking prevented the increase of peripheral CTCs, reduced the number of lung metastases, and prolonged survival (all p ≤ 0.05). Similarly, pre-RFA ethanol injection remarkably decreased CTC release during RFA and further decreased lung metastases with extended survival (all p ≤ 0.05). Histopathology revealed thrombus formation in blood vessels after ethanol injection, which may clog tumor cell dissemination during RFA.ConclusionRFA induces viable tumor cell dissemination, and pre-RFA ethanol injection may provide a prophylactic strategy to reduce this underestimated effect.Relevance statementRFA for HCC promotes viable tumor cell release during ablation, while ethanol injection can prevent RFA induced tumor cell release.Key points• RFA induced the release of viable tumor cells during the ablation procedure in an animal model.• Hepatic vessel blocking can suppress tumor cells dissemination during RFA.• Ethanol injection can prevent RFA-induced tumor cell release, presumably because of the formation of thrombosis.Graphical

Biological and Clinical Significance of Undetectable Circulating Tumor Cells (CTCs) in Patients (Pts) with Multiple Myeloma (MM)

Background: There is evidence suggesting that CTCs are responsible for the spreading of clonal plasma cells (PC) inside and outside the bone marrow (BM). However, some pts unexpectedly have no CTCs at diagnosis. Here, we hypothesize that the absence of CTC defines a specific MM subtype with unique biological and clinical features. To our knowledge, this has never been investigated. Aim: Define the frequency and the characteristics of MM pts with undetectable CTCs. Methods: The presence of CTCs was assessed by NGF in 1,687 blood samples from pts with MGUS (n=467), smoldering (SMM, n=368), newly diagnosed (NDMM, n=652), and relapsed refractory MM (RRMM, n=200). All pts were enrolled in prospective PETHEMA/GEM multicenter studies. BM clonal PC from pts with (n=239) and without (n=17) detectable CTCs were isolated by FACS prior exome and RNA sequencing. Results: The frequency of pts with undetectable CTCs progressively decreased ( p &amp;lt;.001) from MGUS (54%) to SMM (21%) and NDMM (8%) and RRMM (11%). In those with detectable CTCs, the median percentage was 0.0003, 0.002, 0.01 and 0.008, respectively ( p &amp;lt;.001). The 1-log increment observed in the progression from MGUS to SMM and NDMM underpins the link between CTC levels and malignant transformation. The similarity between NDMM and RRMM is probably related with the fact that the latter were analyzed in between different lines of therapy. When compared to other SMM pts, those with undetectable CTCs were more frequently staged with low risk according to the 20/2/20 IMWG model (32% vs 49%; p &amp;lt;.001). Absence of CTCs (HR: 0.38; p =.042) and the 20/2/20 IMWG model (HR: 0.46; p =.011) had independent prognostic value in a multivariate analysis. SMM pts with undetectable vs detectable CTCs showed lower risk of transformation (progression rates at 2 years of 7% vs 24%, p =.004). When compared to other NDMM pts, those with undetectable CTCs had significantly less anemia and BM infiltration. There were no differences in the incidence of lytic lesions, renal insufficiency and hypercalcemia. Only one pt without CTCs was staged with R-ISS 3. These findings, together with a higher frequency of plasmacytomas, suggest that the MM subtype with undetectable CTCs may include pts with macrofocal disease. In transplant eligible NDMM, pts with undetectable vs detectable CTCs had PFS rates at 7 years of 90% and 44% (HR: 0.14; p &amp;lt;.001), and OS rates of 97% and 72% (HR: 0.10; p =.02). In transplant ineligible NDMM, pts with undetectable vs detectable CTCs had PFS rates at 2 years of 83% and 54% (HR: 0.31; p =.02), and OS rates of 94% and 76% (HR: 0.33; p =.13). In a multivariate analysis including the R-ISS and transplant eligibility, the absence of CTCs showed independent prognostic value for PFS (HR: 0.25; p &amp;lt;.001) and OS (HR: 0.24; p =.014). Among pts with undetectable CTCs, there were no differences in PFS and OS according to the presence of standard vs high risk cytogenetic abnormalities. In contrast with pts having detectable CTCs, the PFS and OS of those without CTCs was not significantly reduced if CR or MRD negativity were not achieved after treatment. The cytogenetic profile of BM clonal PC from pts with undetectable (n=112) vs detectable (n=846) CTCs was characterized by a lower frequency of +1q21 (16% vs 45%, p &amp;lt;.001) and del(1p32) (2% vs 8%, p =.02). Other alterations were not significant. There was no correlation between the number of mutations and the percentage of CTCs. There were 66 differentially expressed genes (DEG) between BM clonal PC from pts with detectable vs undetectable CTCs. Of note, 9 of the 66 DEG were in Chr1, with up- and downregulation of genes in 1p and 1q, respectively. There was a significant correlation between CTC levels and the expression of genes associated with MM dissemination ( FLNA, LGALS1and CXCR4). These findings, together with the modest correlation between the percentage of CTCs and BM clonal PC determined by morphology (r=.45, p &amp;lt;.001) and NGF (r=.49, p &amp;lt;.001), suggest that the selective egress of tumor cells from BM into the PB might depend on specific genetic alterations and transcriptional priming more than tumor burden. Conclusions:This study uncovered that ~8% of MM pts may progress due to mechanisms different from CTC dissemination. This subgroup showed unique features and achieved unprecedented outcomes, particularly if eligible to intensive therapy. Accordingly, our results endorse the recognition of pts with undetectable CTCs as a new biological and clinical subtype of MM.

Kinetics and Biology of Circulating Tumor Cells (CTCs) and Measurable Residual Disease (MRD): Two Dynamic High-Risk Clones in Multiple Myeloma (MM)

BACKGROUND CTCs and MRD are two small clones associated with a high risk of progression in smoldering MM (SMM) and post treatment relapse in active MM. Despite growing knowledge about their clinical significance and biological features, these questions remain unanswered: 1) What is the prognostic value of CTC and MRD kinetics? 2) Is MM dissemination and on-treatment resistance driven by specific genomic alterations? 3) Are CTCs and MRD genetically related? AIM Analyze the kinetics and molecular features of CTCs and MRD in SMM and MM patients. METHODS CTC kinetics were investigated using NGF in 164 SMM patients with ≥3 assessments collected every 6 months in the iMMunocell study. MRD kinetics were investigated using NGF in 277 MM patients with ≥4 assessments in BM aspirates after induction, transplant, consolidation, and yearly during maintenance (GEM2012MENOS65-GEM2014MAIN trials). Unsupervised clustering based on CTC and MRD kinetics was performed using CONNECTOR. Molecular features of BM tumor cells at diagnosis were compared to paired CTCs in 94 SMM and MM patients and to paired MRD in 96 MM patients. In 10 cases, genomic data from tripartite BM tumor cells, CTCs, and MRD was available. Tumor samples were isolated based on patient-specific aberrant phenotypes using FACS. Whole exome and RNA-seq were performed in 343 and 217 samples, respectively. RESULTS Based on the assessment of CTCs in 754 PB samples, SMM patients clustered into 3 groups: sustained undetectable (n = 20), stable (n = 71) and evolving (n = 73) CTC levels. With a median follow-up of 2 years, the respective 2y progression-free rates were 100%, 94% and 81% ( P =.003). Risk stratification based on CTC kinetics (C-index 0.68) outperformed a single baseline assessment (C-index 0.61) and, along with the IMWG 20/2/20 model, had independent prognostic value (HR 3.4; P = .01). Patients with high risk cytogenetics showed more frequently evolving CTC levels. Studying genetic drivers of tumor egress, we found CTCs to closely resemble BM tumor cells (88% concordance at copy number [CNA] and somatic mutational [SNV] level). No recurrent cell-specific SNV or CNA was identified. CTC's transcriptional profile revealed 268 DEGs enriched in interferon alpha, gamma response and cell-adhesion pathways. Next, the prognostic value of MRD dynamics was analyzed according to 1,759 assessments, identifying 3 patient clusters: sustained undetectable (n = 100), stable (n = 104), and evolving (n = 73) MRD levels. Respective 6-year rates of PFS after consolidation were 90%, 75% and 5% ( P &amp;lt;.001), and of OS were 94%, 95% and 66% ( P&amp;lt;.001). Risk stratification based on MRD kinetics (C-index 0.82) outperformed a single MRD assessment before maintenance (C-index 0.62), the R-ISS (C-index 0.57) and R2-ISS (C-index 0.61) at diagnosis. MRD cells showed heterogeneous genomic evolution (60% median concordance) diverging from BM tumor cells at diagnosis due to de novo CNA and SNV that emerged at a subclonal level, particularly after transplant. After induction, MM driver mutations were detected or remained clonal in 13/19 (68%) MRD, whereas after transplant, this was true for only 5/13 (38%) MRD. No recurrent cell-specific SNV or CNA was identified. The transcriptional signature of MRD was treatment dependent, with common molecular features of on-treatment resistance found in all 96 patient-paired samples analyzed after various treatment stages. The 334 DEGs were commonly enriched in KRAS signaling hallmarks. Genomic profiles of tripartite BM tumor cells, CTCs and MRD from 10 MM patients confirmed a patient-specific mutational landscape with genomic heterogeneity in MRD. For instance, one patient exhibited BRAF &amp; DIS3 mutations and del(13q) detected at baseline in BM tumor cells and CTCs, which persisted in MRD. After induction, de novo clonal SNVs, including a NEK11 mutation, emerged, followed by the appearance of 3 subclonal CNA after transplant, possibly contributing to resistance. CONCLUSIONS This is the largest dataset analyzing the kinetics and molecular features of CTCs and MRD in SMM and active MM. We showed superior prognostic value of CTC and MRD kinetics over single assessments. Tumor dissemination seems to be driven by transcriptional priming rather than acquired secondary genetic events, while on-treatment resistance is linked to genomic and transcriptional evolution, without recurrent genetic alterations and with common molecular signatures of resistance.

Single-Cell RNA Sequencing of Circulating Tumor Cells in Precursor Myeloma Patients Reveals Mechanisms of Disease Dissemination

Introduction Multiple Myeloma (MM) is preceded by the precursors Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM), where some patients with precursor disease progress to active MM faster than others. BM biopsies are useful to monitor disease progression, however, they cannot be repeated often for continuous monitoring of tumor burden especially in the precursor disease management setting. Numerous studies have identified circulating tumor cell (CTC) levels in peripheral blood (PB) are a powerful biomarker of disease aggressiveness; CTC levels have associated with disease stage, PFS and OS in various trials and even outperformed BM to assess tumor burden in the SMM setting. Genomic profiling also demonstrated CTCs harbor the same copy number abnormalities, translocations and mutations as BM tumor cells, and CTCs can be serially monitored to detect the emergence of high-risk subclones in the blood. Further investigations of the molecular profile of both normal plasma cells (NPCs) and CTCs are needed to improve our understanding of myeloma pathogenesis and mechanisms of CTC dissemination across the MM disease continuum. Methods A cohort of 309 samples including CD138-enriched paired PB and BM aspirates and matched CD138- BM aspirates were collected from 103 individuals, including patients with MGUS (n=11), SMM (n=46), NDMM (n=17) and healthy donors (n=29). Malignant PCs from BM and PB underwent 5' single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics), and CD138- BM immune cells underwent 5' scRNA-seq and single-cell T-cell receptor sequencing (scTCR-seq) to study immune alterations related the tumors' ability to circulate. To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements. Differential expression (DE) and composition analyses were conducted using Wilcoxon's rank-sum tests. Results We successfully captured and profiled 774,647 BM tumor cells and 93,878 CTCs from the PB. The percent malignant plasma cells within the PB increased with disease stage, where NDMM patients had significantly more CTCs compared to MGUS patients (p=0.017) and low-risk SMM patients (p=0.0052). A median of 13.51%, 15.53%, and 18.34% CTCs were present from low (n=20), intermediate (n=10), and high-risk (n=16) SMM patients as defined by the International Myeloma Working Group's 2/20/20 criteria, suggesting sequencing-based CTC enumeration captures prognostically relevant differences in tumor burden. High expression of driver genes upregulated in patients with translocations, including CCND1, NSD2, and MAF, were detected in both BM tumor cells and CTCs in patients with t(11;14), t(4;14), and t(14;16) as identified by fluorescence in situ hybridization (FISH). In 5 patients with normal or inconclusive FISH results, we observed high levels of CCND2 and MAF in both BM and CTCs, indicating scRNA-seq can detect missed prognostically relevant cytogenetic abnormalities. A cytogenetic classifier model was developed to determine the accuracy of translocation calling using minimal numbers of CTCs, where the classifier's performance was able to consistently identify translocations downsampled to 50 CTCs. DE analysis of CTCs vs. BM tumor cells highlighted transcriptional similarity between BM and CTCs, validating their utility as a surrogate for analyzing BM tumor cells. DE analysis also revealed 8 genes significantly upregulated and 3 genes significantly downregulated in CTCs compared to BM tumor cells, providing novel insights into genes involved in PC circulatory potential. Pathway enrichment analysis revealed genes upregulated in CTCs were associated with epithelial mesenchymal transition, interferon response, and inflammation, consistent with CTC studies on MM patients, suggesting these pathways are dysregulated earlier in the disease continuum. Conclusions In the largest scRNA-seq study on CTCs to date, we demonstrate the utility of CTC-based molecular profiling for prognostication of patients with early-stage disease and provide novel insights into PC circulatory potential. Additional analyses are ongoing to gain further insight into intra-patient CTC heterogeneity and define high-risk disease CTC signatures that emerge throughout the MM disease continuum.

EpCAM-positive circulating tumor cells and serum AFP levels predict outcome after curative resection of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has high recurrence rates exceeding 50% despite curative resection. The serum biomarker alpha-fetoprotein (AFP) is a well-known prognostic marker for HCC. EpCAM-positive circulating tumor cells (CTC) have a high predictive value for early HCC recurrence after curatively intended resection, most likely indicating micro-metastases at the time of resection. However, sensitivity remains low. The objective of this study was to evaluate a composite test comprising both CTC and AFP to identify patients at high risk for early HCC recurrence. We prospectively enrolled 58 patients undergoing curative intended resection for HCC at a tertiary referral center. Blood specimens were obtained prior to resection and analyzed for EpCAM-positive CTC and serum AFP levels. A positive result was defined as either detection of CTC or AFP levels ≥ 400 ng/ml. Eight patients tested positive for CTC, seven for AFP, and two for both markers. A positive composite test was significantly associated with shorter early recurrence-free survival (5 vs. 16 months, p = 0.005), time to recurrence (5 vs. 16 months, p = 0.011), and overall survival (37 vs. not reached, p = 0.034). Combining CTC and AFP identified patients with poor outcome after surgical resection, for whom adjuvant or neoadjuvant therapies may be particularly desirable.

Sensitivity and specificity of folate receptor α-positive circulating tumour cells in gastric cancer.

We aimed to investigate whether folate receptor α (FRα)-positive circulating tumour cells (CTCs) could be used as a noninvasive liquid biopsy approach in gastric cancer (GC). Tissue microarray and bioinformatic analyses were used to evaluate FRα expression in GC. Patients with FRα-positive CTC examinations at our institute between July 2021 and May 2022 were retrospectively evaluated. Receiver operating characteristic curves were used to evaluate the diagnostic performance of FRα-positive CTCs in GC. FRα was highly expressed and associated with poor prognosis in GC based on public database. Data for 163 patients (20 with benign disease and 143 with GC) were retrospectively collected. FRα-positive CTC levels were significantly higher in the GC group than in the benign disease group (12.15 ± 1.47 FU/3ml vs. 10.47 ± 1.63 FU/3ml, P < 0.01). FRα-positive CTC levels were also elevated in GC patients with vessel/neuron invasion or extra-nodal tumour deposits (12.31 ± 1.47 FU/3ml vs. 11.77 ± 1.38 FU/3ml, P = 0.037). Areas under the curve of FRα-positive CTC levels for GC and early GC were 0.774 (P < 0.001) and 0.736 (P = 0.005). With a cut-off value of 10.95 FU/3ml, the Youden indexes for GC and early GC were 0.502 (sensitivity = 85.2% and specificity = 65.0%) and 0.450 (sensitivity = 80.0% and specificity = 65.0%), respectively. FRα-positive CTC detection by noninvasive liquid biopsy is a useful and effective approach for screening of patients with GC.

Circulating tumor cells as a predictor and prognostic tool for metastatic clear cell renal carcinoma: An immunocytochemistry and genomic analysis

BackgroundTreatment of metastatic clear cell renal carcinoma (mccRCC) has changed dramatically over the past 20 years, without improvement in the development of biomarkers. Recently, circulating tumor cells (CTCs) have been validated as a prognostic and predictive tool for many solid tumors. ObjectiveWe evaluated CTCs in blood samples obtained from patients diagnosed with mccRCC. Comparisons of CTC counts, protein expression profiling, and DNA mutants were made in relation to overall survival and progression-free survival. MethodsCTCs were isolated from 10 mL blood samples using the ISET® system (Isolation by SizE of Tumor Cells; Rarecells, France) and counted. Protein expression was evaluated in immunocytochemistry assays. DNA mutations were identified with next generation sequencing (NGS). ResultsBlood samples (10 mL) were collected from 12 patients with mccRCC before the start of first-line systemic therapy, and again 30 and 60 days after the start of treatment. All 12 patients had CTCs detected at baseline (median, 1.5 CTCs/mL; range: 0.25–7.75). Patients with CTC counts greater than the median had two or more metastatic sites and exhibited worse progression-free survival (19.7 months) compared to those with CTC counts less than the median (31.1 months). Disease progression was observed in 7/12 patients during the study. Five of these patients had baseline CTC counts greater than the median, one had higher CTC levels at the second blood collection, and one patient had CTCs present at 1 CTC/mL which positively stained for PD-L1, N-cadherin, VEGF, and SETD2. CTC DNA from six patients with worse outcomes was subjected to NGS. However, no conclusions could be made due to the low variant allele frequencies. ConclusionDetection of CTCs in patients with mccRCC receiving first-line treatment is a feasible tool with prognostic potential since increased numbers of CTCs were found to be associated with metastasis and disease progression.

Prospective Characterization of Circulating Tumor Cells in Hormone Sensitive Oligometastatic Prostate Cancer Patients on a Metastasis-Directed Therapy Trial

Prospective data have shown that metastasis-directed therapy (MDT) can alter the natural history of oligometastatic disease. In hormone-sensitive prostate cancer (HSPC), the clinical effect of MDT has been validated by STOMP, ORIOLE and SABR-COMET phase II trials. Circulating tumor cells (CTCs) are likely the source for the formation of macroscopic metastases. CTCs may provide an approach for identifying subgroups of patients with oligometastatic HSPC (oligoHSPC) that would benefit most from MDT. Our main goal was to evaluate the feasibility of CTC detection and subtypes in oligoHSPC patients that may benefit from MDT. ORIOLE randomized men with recurrent HSPC with 1-3 metastases to observation (Obs) vs. stereotactic ablative radiotherapy (SABR) MDT. Blood samples were prospectively collected at baseline (D0) and 6-mos (D180) and shipped for analysis on Epic Sciences liquid biopsy platform (Epic Sciences, San Diego, CA). Machine learning algorithms identified CTCs and characterized androgen receptor (AR) and PSMA expression. Association with clinical factors and outcomes were examined. Biochemical failure-free survival (BFFS) event was a PSA rise of at least 2 ng/mL and 25% above nadir. Progression-free survival (PFS) was a composite endpoint including BFFS event, radiologic progression (RECIST v1.1); symptomatic progression; initiation of ADT; or death. Comparisons of patient and tumor characteristics performed by two-sample t-tests. Survival curves were generated by the Kaplan-Meier method and evaluated by the log-rank test. Effect of SABR on post-SABR on CTC levels were calculated by McNemar test. A total of 82 samples were collected in ORIOLE: 70 SABR (35 D0 and 35 D180) and 12 Obs (7 D0 and 5 D180). 30/42 men had CTCs detected on D0 (71%; AR+ = 7, PSMA+ = 13) and in 26/40 on D180 (65%; AR+ = 9, PSMA+ = 8). Median follow-up was 41.7-mos. There was no association between CTC presence or subtypes (AR+ or PSMA+) with Gleason score or PSA. PFS was significantly lower in the patients with AR+ vs. AR- CTCs on D0 in the SABR arm (p = 0.011, median PFS: AR+ = 9.3- vs. AR+ = 27.1-mos). The median BFFS trended towards a difference for AR+ = 12.9- vs. AR- = 29.2-mos (D180, p = 0.058). SABR had no effect on the presence or subtypes of CTC at D180. Baseline and dynamic CTC levels and their subtypes in oligoHSPC from the ORIOLE randomized trial of MDT was examined. AR+ CTCs at baseline and 6-mos were correlated with clinical outcomes following SABR. Longer follow-up, further analysis and a greater number of patients are needed for a more comprehensive conclusion.

Liquid biopsies in thyroid cancers: a systematic review and meta-analysis.

Thyroid cancer (TC) represents the most common endocrine malignant tumor. Liquid biopsy has been suggested as a new and accurate biomarker in cancer. This systematic review analyzes the existing literature on circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), cell-free DNA integrity index (cfDI), and their potential as biomarkers for TC, including the subtypes: differentiated (papillary and follicular), medullary, and anaplastic. A systematic search was performed in PubMed, Embase, and Cochrane databases for published articles in English between 1 January 1970 and 6 September 2022 (PROSPERO: CRD42022358592). The literature search generated a total of 635 articles. In total, 36 articles were included (patients = 2566). Four studies reported that higher levels of CTCs were associated with metastases and worse prognosis. Nineteen studies found the presence of mutated ctDNA in TC patients. The diagnostic accuracy in detecting BRAFV600E as ctDNA was determined in 11 studies regarding papillary TC. The pooled sensitivity, specificity, and diagnostic odds ratio were estimated at 56% (95% CI 36-74), 91% (95% CI 84-95) and 12 (95% CI 4.09-33.11), respectively. Four studies concluded that the cfDI was higher in patients with TC compared to benign thyroid lesions and healthy controls. The detection of CTCs, ctDNA, and cfDI may have a potential prognostic value in TC in relation to diagnosis, disease progression, and treatment efficacy. Despite the promising potential of CTCs, ctDNA, and cfDI in TC management, limitations hinder direct comparison and generalization of findings. Standardized methodologies, larger patient cohorts, and a consensus on relevant markers are needed to validate their clinical applicability and enhance TC management.

MnO2 nanosheets and gold nanoparticles supported electrochemical detection of circulating tumor cells.

The concentration of circulating tumor cells (CTCs) in peripheral blood is strongly correlated with the progress of certain metastatic cancers. In this study, we have developed a novel and facile electrochemical biosensor for the detection of CTCs based on the use of manganese dioxide nanosheets (MnO2 NSs) and gold nanoparticles (AuNPs). Aptamer sequence of target cell is modified on the surface of AuNPs for specifical recognition. With low-speed centrifugation, numerous AuNPs@DNA can be removed from the supernatant. On the other hand, MnO2 NSs are modified on the electrode surface to capture unreacted AuNPs@DNA. The declined electrochemical signal intensity can be used to reflect the level of CTCs. This biosensor achieves a wide linear range from 10 to 104 cells mL-1 and a limit of detection as low as 3 cells mL-1. Due to the specific aptamer as the recognition element, interfering cells can be successfully distinguished and this method performs satisfactorily in clinical samples. Therefore, it has great potential to be used as a powerful tool benefiting rare cells analysis and the investigation of dynamics of cellular interactions.