Liquid Biopsies for Neuroendocrine Tumors: Circulating Tumor Cells, DNA, and MicroRNAs

Brain tumors are the most common malignant primary intracranial tumors of the central nervous system. They are often recognized too late for successful therapy. Minimally invasive methods are needed to establish a diagnosis or monitor the response to treatment of CNS tumors. Brain tumors release molecular information into the circulation. Liquid biopsies collect and analyze tumor components in body fluids, and there is an increasing interest in the investigation of liquid biopsies as a substitute for tumor tissue. Tumor-derived biomarkers include nucleic acids, proteins, and tumor-derived extracellular vesicles that accumulate in blood or cerebrospinal fluid. In recent years, circulating tumor cells have also been identified in the blood of glioblastoma patients. In this review of the literature, the authors highlight the significance, regulation, and prevalence of molecular biomarkers such as O6-methylguanine-DNA methyltransferase, epidermal growth factor receptor, and isocitrate dehydrogenase. Herein, we critically review the available literature on plasma circulating tumor cells (CTCs), cell-free tumors (ctDNAs), circulating cell-free microRNAs (cfmiRNAs), and circulating extracellular vesicles (EVs) for the diagnosis and monitoring of brain tumor. Currently available markers have significant limitations.While much research has been conductedon these markers, there is still a significant amount that we do not yet understand, which may account for some conflicting reports in the literature.

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DIPG-60. PILOT STUDY OF CIRCULATING TUMOR CELLS IN PEDIATRIC HIGH GRADE BRAIN TUMORS

Neuro-Oncology ◽

10.1093/neuonc/noaa222.105 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii299-iii299

Author(s):

Wafik Zaky ◽

Long Dao ◽

Dristhi Ragoonanan ◽

Izhar Bath ◽

Sofia Yi ◽

...

Keyword(s):

Brain Tumor ◽

Brain Tumors ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Pediatric Brain Tumor ◽

Pediatric Brain Tumors ◽

Disease Assessment ◽

Liquid Biopsies ◽

Capture Method ◽

Pediatric Brain

Abstract BACKGROUND Despite its increasing use, circulating tumor cells (CTCs) have not been studied in pediatric brain tumors. METHODS Cell surface vimentin (CSV) is a marker for CTC detection. We developed an automated CSV-based CTC capture method for pediatric brain tumor using the Abnova Cytoquest platform. PBMCs isolated from blood samples from 52 brain tumor patients were processed to isolate CSV+ CTCs. Captured cells were then stained for CSV and CD45 and scanned to determine the number of CTCs. DIPG samples were additionally examined for H3K27M expression on CSV+ cells. Long term cancer survivors were used as a control cohort. RESULTS 86.4% of all the samples exhibited between 1–13 CSV+ CTCs, with a median of 2 CSV+ CTCs per sample. Using a value of ≥ 1 CTC as a positive result, the sensitivity and specificity of this test was 83.05% and 60.0% respectively. 19 DIPG samples were analyzed and 70% (13 samples) were positive for 1–5 CTCs. Five of these 7 positive CSV+ CTCs DIPG samples were also positive for H3K27M mutations by immunohistochemistry (71%). Mean survival in days for the CTC positive and negative DIPG samples were 114 and 211 days, respectively (p= 0.13). CONCLUSION This is the first study of CTCs in pediatric CNS tumors using an automated approach. Patients with brain tumors can exhibit CSV+ CTCs within peripheral blood. The use of specific molecular markers such as H3K27M can improve the diagnostic capability of liquid biopsies and may enable future disease assessment for personalized therapy.

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High Clinical Value of Liquid Biopsy to Detect Circulating Tumor Cells and Tumor Exosomes in Pancreatic Ductal Adenocarcinoma Patients Eligible for Up-Front Surgery

Cancers ◽

10.3390/cancers11111656 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1656 ◽

Cited By ~ 12

Author(s):

Etienne Buscail ◽

Catherine Alix-Panabières ◽

Pascaline Quincy ◽

Thomas Cauvin ◽

Alexandre Chauvet ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Liquid Biopsy ◽

Neoadjuvant Treatment ◽

Digital Pcr ◽

Portal Blood ◽

Ductal Adenocarcinoma ◽

Clinical Value ◽

Liquid Biopsies

Purpose: Expediting the diagnosis of pancreatic ductal adenocarcinoma (PDAC) would benefit care management, especially for the start of treatments requiring histological evidence. This study evaluated the combined diagnostic performance of circulating biomarkers obtained by peripheral and portal blood liquid biopsy in patients with resectable PDAC. Experimental design: Liquid biopsies were performed in a prospective translational clinical trial (PANC-CTC #NCT03032913) including 22 patients with resectable PDAC and 28 noncancer controls from February to November 2017. Circulating tumor cells (CTCs) were detected using the CellSearch® method or after RosetteSep® enrichment combined with CRISPR/Cas9-improved KRAS mutant alleles quantification by droplet digital PCR. CD63 bead-coupled Glypican-1 (GPC1)-positive exosomes were quantified by flow cytometry. Results: Liquid biopsies were positive in 7/22 (32%), 13/22 (59%), and 14/22 (64%) patients with CellSearch® or RosetteSep®-based CTC detection or GPC1-positive exosomes, respectively, in peripheral and/or portal blood. Liquid biopsy performance was improved in portal blood only with CellSearch®, reaching 45% of PDAC identification (5/11) versus 10% (2/22) in peripheral blood. Importantly, combining CTC and GPC1-positive-exosome detection displayed 100% of sensitivity and 80% of specificity, with a negative predictive value of 100%. High levels of GPC1+-exosomes and/or CTC presence were significantly correlated with progression-free survival and with overall survival when CTC clusters were found. Conclusion: This study is the first to evaluate combined CTC and exosome detection to diagnose resectable pancreatic cancers. Liquid biopsy combining several biomarkers could provide a rapid, reliable, noninvasive decision-making tool in early, potentially curable pancreatic cancer. Moreover, the prognostic value could select patients eligible for neoadjuvant treatment before surgery. This exploratory study deserves further validation.

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Prognostic Threshold for Circulating Tumor Cells in Patients With Pancreatic and Midgut Neuroendocrine Tumors

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgaa822 ◽

2020 ◽

Author(s):

Dalvinder Mandair ◽

Mohid S Khan ◽

Andre Lopes ◽

Luke Furtado O’Mahony ◽

Leah Ensell ◽

...

Keyword(s):

Regression Analysis ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Neuroendocrine Tumors ◽

Operating Characteristic ◽

Characteristic Curve ◽

Multivariate Logistic Regression Analysis ◽

Progression Free Survival ◽

Free Survival ◽

Optimum Threshold

Abstract Background Circulating tumor cells (CTCs) are detectable in patients with neuroendocrine tumors (NETs) and are accurate prognostic markers although the optimum threshold has not been defined. Objective This work aims to define optimal prognostic CTC thresholds in PanNET and midgut NETs. Patients and Methods CellSearch was used to enumerate CTCs in 199 patients with metastatic pancreatic (PanNET) (90) or midgut NETs (109). Patients were followed for progression-free survival (PFS) and overall survival (OS) for a minimum of 3 years or until death. Results The area under the receiver operating characteristic curve (AUROC) for progression at 12 months in PanNETs and midgut NETs identified the optimal CTC threshold as 1 or greater and 2 or greater, respectively. In multivariate logistic regression analysis, these thresholds were predictive for 12-month progression with an odds ratio (OR) of 6.69 (P < .01) for PanNETs and 5.88 (P < .003) for midgut NETs. The same thresholds were found to be optimal for predicting death at 36 months, with an OR of 2.87 (P < .03) and 5.09 (P < .005) for PanNETs and midgut NETs, respectively. In multivariate Cox hazard regression analysis for PFS in PanNETs, 1 or greater CTC had a hazard ratio (HR) of 2.6 (P < .01), whereas 2 or greater CTCs had an HR of 2.25 (P < .01) in midgut NETs. In multivariate analysis OS in PanNETs, 1 or greater CTCs had an HR of 3.16 (P < .01) and in midgut NETs, 2 or greater CTCs had an HR of 1.73 (P < .06). Conclusions The optimal CTC threshold to predict PFS and OS in metastatic PanNETs and midgut NETs is 1 and 2, respectively. These thresholds can be used to stratify patients in clinical practice and clinical trials.

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Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device

Nature Protocols ◽

10.1038/nprot.2017.125 ◽

2017 ◽

Vol 13 (1) ◽

pp. 34-58 ◽

Cited By ~ 49

Author(s):

Bee Luan Khoo ◽

Gianluca Grenci ◽

Ying Bena Lim ◽

Soo Chin Lee ◽

Jongyoon Han ◽

...

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Liquid Biopsies

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Functional Studies on Viable Circulating Tumor Cells

Clinical Chemistry ◽

10.1373/clinchem.2015.242537 ◽

2016 ◽

Vol 62 (2) ◽

pp. 328-334 ◽

Cited By ~ 53

Author(s):

Klaus Pantel ◽

Catherine Alix-Panabières

Keyword(s):

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Cancer Diagnostics ◽

Published Data ◽

Liquid Biopsies ◽

In Vivo Models ◽

Functional Studies

AbstractBACKGROUNDResearch on circulating tumor cells (CTCs) as new biomarkers has received great attention over the past decade. In particular, the capture and analysis of CTCs as “liquid biopsies” provides the possibility to avoid invasive tissue biopsies, with obvious implications in cancer diagnostics.CONTENTThe focus of this review is to describe and discuss how functional studies on viable CTCs can enlarge the spectrum of applications of liquid biopsies, with emphasis on breast, prostate, colon, and lung cancer as the major tumor entities in industrialized countries. The low number of CTCs in the peripheral blood of most cancer patients makes challenging the in vitro culture of CTCs. Epithelial tumor cells are difficult to culture, even when starting with millions of tumor cells. Recently, several groups have achieved important advances in the in vitro and in vivo expansion of CTCs from cancer patients at very advanced stages with higher amounts of CTCs. Here, we present current technologies to enrich and detect viable human CTCs, including positive and negative enrichment strategies that are based on antigen expression and physical properties of CTCs. We also discuss published data about functional studies on CTCs that use in vitro and in vivo models.SUMMARYFunctional analyses on CTCs offer the possibility to identify the biological properties of metastatic cells, including the identification of metastasis-initiating cells. Moreover, CTC-derived cell lines and xenografts might reveal new therapeutic targets and can be used for drug screening.

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Circulating Tumor Cells As Prognostic Markers in Neuroendocrine Tumors

Journal of Clinical Oncology ◽

10.1200/jco.2012.44.2905 ◽

2013 ◽

Vol 31 (3) ◽

pp. 365-372 ◽

Cited By ~ 92

Author(s):

Mohid S. Khan ◽

Amy Kirkwood ◽

Theodora Tsigani ◽

Jorge Garcia-Hernandez ◽

John A. Hartley ◽

...

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Neuroendocrine Tumors ◽

Prognostic Markers ◽

Prognostic Significance ◽

Elevated Serum ◽

Progression Free Survival ◽

Tumor Grade ◽

Immunomagnetic Separation ◽

Tumor Burden

Purpose To determine the prognostic significance of circulating tumor cells (CTCs) in patients with neuroendocrine cancer. Patients and Methods In this single-center prospective study, 176 patients with measurable metastatic neuroendocrine tumors (NETs) were recruited. CTCs were measured using a semiautomated technique based on immunomagnetic separation of epithelial cell adhesion molecule–expressing cells. Results Overall, 49% patients had ≥ one CTC, 42% had ≥ two CTCs, and 30% had ≥ five CTCs in 7.5 mL blood. Presence of CTCs was associated with increased burden, increased tumor grade, and elevated serum chromogranin A (CgA). Using a 90-patient training set and 85-patient validation set, we defined a cutoff of < one or ≥ one as the optimal prognostic threshold with respect to progression-free survival (PFS). Applying this threshold, the presence of ≥ one CTC was associated with worse PFS and overall survival (OS; hazard ratios [HRs], 6.6 and 8.0, respectively; both P < .001). In multivariate analysis, CTCs remained significant when other prognostic markers, grade, tumor burden, and CgA were included. Within grades, presence of CTCs was able to define a poor prognostic subgroup. For grade 1, HRs were 5.0 for PFS (P = .017) and 7.2 for OS (P = .023); for grade 2, HRs were 3.5 for PFS (P = .018) and 5.2 for OS (P = .036). Conclusion CTCs are a promising prognostic marker for patients with NETs and should be assessed in the context of clinical trials with defined tumor subtypes and therapy.

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Cell-Free DNA Variant Sequencing Using Plasma and AR-V7 Testing of Circulating Tumor Cells in Prostate Cancer Patients

Cells ◽

10.3390/cells10113223 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3223

Author(s):

Verena Lieb ◽

Amer Abdulrahman ◽

Katrin Weigelt ◽

Siegfried Hauch ◽

Michael Gombert ◽

...

Keyword(s):

Prostate Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Therapy Monitoring ◽

Brca1 Gene ◽

Sequence Variants ◽

Liquid Biopsies ◽

Treatment Change ◽

Cell Free Dna ◽

Free Dna

Prostate cancer (PCa) is the second most common malignant cancer and is a major cause of morbidity and mortality among men worldwide. There is still an urgent need for biomarkers applicable for diagnosis, prognosis, therapy prediction, or therapy monitoring in PCa. Liquid biopsies, including cell-free DNA (cfDNA) and circulating tumor cells (CTCs), are a valuable source for studying such biomarkers and are minimally invasive. In our study, we investigated the cfDNA of 34 progressive PCa patients, via targeted sequencing, for sequence variants and for the occurrence of CTCs, with a focus on androgen receptor splice variant 7 (AR-V7)-positive CTCs. The cfDNA content was associated with overall survival (OS; p = 0.014), disease-specific survival (DSS; p = 0.004), and time to treatment change (TTC; p = 0.001). Moreover, when considering all sequence variants grouped by their functional impact and allele frequency, a significant association with TTC (p = 0.017) was observed. When investigating only pathogenic or likely pathogenic gene variants, variants of the BRCA1 gene (p = 0.029) and the AR ligand-binding domain (p = 0.050) were associated with a shorter TTC. Likewise, the presence of CTCs was associated with a shorter TTC (p = 0.031). The presence of AR-V7-positive CTCs was associated with TTC (p < 0.001) in Kaplan–Meier analysis. Interestingly, all patients with AR-V7-positive CTCs also carried TP53 point mutations. Altogether, analysis of cfDNA and CTCs can provide complementary information that may support temporal and targeted treatment decisions and may elucidate the optimal choice within the variety of therapy options for advanced PCa patients.

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Detection of AR-V7 in Liquid Biopsies of Castrate Resistant Prostate Cancer Patients: A Comparison of AR-V7 Analysis in Circulating Tumor Cells, Circulating Tumor RNA and Exosomes

Cells ◽

10.3390/cells8070688 ◽

2019 ◽

Vol 8 (7) ◽

pp. 688 ◽

Cited By ~ 8

Author(s):

Mohammed Nimir ◽

Yafeng Ma ◽

Sarah A. Jeffreys ◽

Thomas Opperman ◽

Francis Young ◽

...

Keyword(s):

Prostate Cancer ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Digital Pcr ◽

Liquid Biopsies ◽

Tumor Biopsy ◽

Highly Sensitive ◽

Castrate Resistant Prostate Cancer ◽

Castrate Resistant

Detection of androgen receptor (AR) variant 7 (AR-V7) is emerging as a clinically important biomarker in castrate resistant prostate cancer (CRPC). Detection is possible from tumor tissue, which is often inaccessible in the advanced disease setting. With recent progress in detecting AR-V7 in circulating tumor cells (CTCs), circulating tumor RNA (ctRNA) and exosomes from prostate cancer patients, liquid biopsies have emerged as an alternative to tumor biopsy. Therefore, it is important to clarify whether these approaches differ in sensitivity in order to achieve the best possible biomarker characterization for the patient. In this study, blood samples from 44 prostate cancer patients were processed for CTCs and ctRNA with subsequent AR-V7 testing, while exosomal RNA was isolated from 16 samples and tested. Detection of AR and AR-V7 was performed using a highly sensitive droplet digital PCR-based assay. AR and AR-V7 RNA were detectable in CTCs, ctRNA and exosome samples. AR-V7 detection from CTCs showed higher sensitivity and has proven specificity compared to detection from ctRNA and exosomes. Considering that CTCs are almost always present in the advanced prostate cancer setting, CTC samples should be considered the liquid biopsy of choice for the detection of this clinically important biomarker.

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Circulating tumor cells as liquid biopsies of skeletal metastases

European Urology Supplements ◽

10.1016/s1569-9056(16)30433-x ◽

2016 ◽

Vol 15 (13) ◽

pp. e1637

Author(s):

A. Josefsson ◽

K. Larsson ◽

J.-E. Damber ◽

K. Welén

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Skeletal Metastases ◽

Liquid Biopsies

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