• breast cancer
• circulating tumor cells
• clinical utility
• epithelial-to-mesenchymal transition

• Liquid biopsy
• biomarkers
• circulating cell-free RNA
• circulating free or tumor DNA
• circulating tumor cells
• extracellular vesicles
• tumor-educated platelets

• Humans
• Liquid Biopsy/methods
• Neoplasms/pathology
• Neoplasms/diagnosis
• Neoplasms/metabolism
• Neoplasms/genetics
• Neoplastic Cells, Circulating/metabolism
• Neoplastic Cells, Circulating/pathology
• Biomarkers, Tumor/metabolism
• Extracellular Vesicles/metabolism
• Circulating Tumor DNA
• Cell-Free Nucleic Acids/genetics

• Breast cancer
• Circulating tumor cells
• GATA3
• Liquid biopsy

• Humans
• Female
• GATA3 Transcription Factor/analysis
• GATA3 Transcription Factor/metabolism
• Breast Neoplasms/pathology
• Breast Neoplasms/diagnosis
• Breast Neoplasms/blood
• Neoplastic Cells, Circulating/pathology
• Neoplastic Cells, Circulating/metabolism
• Aged, 80 and over
• Neoplasm Staging
• Biomarkers, Tumor/blood
• Sentinel Lymph Node Biopsy
• Biopsy, Large-Core Needle
• Mastectomy, Segmental
• Early Detection of Cancer/methods

• Circulating tumor cells isolation
• Clinical verification
• Human epidermal growth factor receptor 2
• Microfluidic device
• Prognostic

• Humans
• Neoplastic Cells, Circulating/metabolism
• Neoplastic Cells, Circulating/pathology
• Female
• Breast Neoplasms/pathology
• Breast Neoplasms/blood
• Breast Neoplasms/metabolism
• Breast Neoplasms/mortality
• Receptor, ErbB-2/metabolism
• Prognosis
• Middle Aged
• Aged
• Adult
• Biomarkers, Tumor/metabolism
• Neoplasm Metastasis
• Cell Count
• Microfluidic Analytical Techniques/instrumentation
• Microfluidic Analytical Techniques/methods
• Aged, 80 and over
• Microfluidics/methods

• Humans
• Immunoconjugates/therapeutic use
• Immunoconjugates/adverse effects
• Antigen-Antibody Complex/immunology
• Neoplasms/drug therapy
• Neoplasms/immunology
• Animals
• Receptors, IgG/metabolism
• Phagocytosis/drug effects
• Antineoplastic Agents, Immunological/adverse effects
• Antineoplastic Agents, Immunological/therapeutic use

• branch length
• membrane-receptor targeting
• nanoparticle shape
• protein corona
• single-particle tracking

• Humans
• Anisotropy
• Gold/chemistry
• Aptamers, Nucleotide/chemistry
• Aptamers, Nucleotide/metabolism
• Cell Membrane/metabolism
• Cell Membrane/chemistry
• Receptor, ErbB-2/metabolism
• Receptor, ErbB-2/chemistry
• Metal Nanoparticles/chemistry
• Cell Line, Tumor
• Ligands

• P30 CA060553 NCI NIH HHS
• P41 GM108569 NIGMS NIH HHS
• R01 GM131421 NIGMS NIH HHS
• S10 OD025194 NIH HHS

• breast cancer
• circulating tumor cells
• early detection
• prostate cancer
• tumor dormancy

• Humans
• Male
• Early Detection of Cancer
• Neoplastic Cells, Circulating
• Prostatic Neoplasms/diagnosis
• Female
• Breast Neoplasms/diagnosis

• P30 CA077598 NCI NIH HHS
• R01 CA269801 NCI NIH HHS
• R41 CA268344 NCI NIH HHS
• P30 CA077598 NCI NIH HHS
• National Cancer Institute Grant R01CA269801 NIH HHS
• National Cancer Institute
• Masonic Cancer Center, University of Minnesota
• National Institutes of Health

• FISH
• chromosomal rearrangements
• cytogenetics
• gene amplification
• polysomy

• Humans
• In Situ Hybridization, Fluorescence/methods
• Cytogenetics/methods
• Chromosome Aberrations
• Chromosome Painting
• Neoplasms/diagnosis
• Neoplasms/genetics
• Formaldehyde

• circulating tumor cell
• kidney cancer
• liquid biopsy
• microfluidic
• renal cell carcinoma

• HER2-low
• antibody–drug conjugates
• breast cancer
• human epidermal growth factor receptor 2
• sacituzumab govitecan
• sequencing
• trastuzumab deruxtecan

• Breast cancer
• Circulating tumor cell
• microRNA

Single cell technologies allow the interrogation of tumor heterogeneity, providing insights into tumor evolution and treatment resistance. To better understand whether circulating tumor cells (CTCs) could complement metastatic biopsies for tumor genomic profiling, we characterized 11 single CTCs and 10 pooled CTC samples at the mutational and copy number aberration (CNA) levels, and compared these results with matched synchronous tumor biopsies from 3 metastatic breast cancer patients with triple-negative (TNBC), HER2-positive and estrogen receptor-positive (ER+) tumors. Similar CNA profiles and the same patient-specific driver mutations were found in bulk tissue and CTCs for the HER2-positive and TNBC tumors, whereas different CNA profiles and driver mutations were identified for the ER+ tumor, which presented two distinct clones in CTCs defined by mutations in ESR1 Y537N and TP53, respectively. Furthermore, de novo mutational signatures derived from CTCs described patient-specific biological processes. These data suggest that tumor tissue and CTCs provide complementary clinically relevant information to map tumor heterogeneity and tumor evolution.

Retrieval of circulating tumor cells (CTC) has proven valuable for assessing a patient's cancer burden, evaluating response to therapy, and analyzing which drug might treat a cancer best. Although most isolation methods retrieve CTCs based on size, shape, or capture by tumor-specific antibodies, we explore here the use of small molecule tumor-specific ligands linked to magnetic beads for CTC capture. We have designed folic acid-biotin conjugates with different linkers for the capture of folate receptor (FR) + tumor cells spiked into whole blood, and application of the same technology to isolate FR + CTCs from the peripheral blood of both tumor-bearing mice and non-small cell lung patients. We demonstrate that folic acid linked via a rigid linker to a flexible PEG spacer that is in turn tethered to a magnetic bead enables optimal CTC retrieval, reaching nearly 100% capture when 100 cancer cells are spiked into 1 mL of aqueous buffer and ~ 90% capture when the same quantity of cells is diluted into whole blood. In a live animal model, the same methodology is shown to efficiently retrieve CTCs from tumor-bearing mice, yielding cancer cell counts that are proportional to total tumor burden. More importantly, the same method is shown to collect ~ 29 CTCs/8 mL peripheral blood from patients with non-small cell lung cancer. Since the ligand-presentation strategy optimized here should also prove useful in targeting other nanoparticles to other cells, the methods described below should have general applicability in the design of nanoparticles for cell-specific targeting.

• Liquid biopsy
• biomarker evaluation
• cell cultures
• circulating tumor cell cultures
• circulating tumor cells
• drug sensitivity
• liquid tumor biomarkers
• precision medicine

Circulating tumor DNA, cell-free DNA, and circulating tumor cells have been at the epitome of recent research in breast cancer. These forms of liquid biopsies have been used in monitoring disease progression, estimating the risk of relapse, and response to treatment. Much has been done in relation to serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease. Some studies have also explored their use in monitoring treatment response. As the field of liquid biopsies expands, more prospective studies are needed to tailor management in an individualistic approach. In this literature review, the authors explore the multiple uses of circulating tumor DNA and circulating tumor cells in breast cancer.

• Circulating tumor DNA
• biomarkers
• breast cancer
• cell-free DNA
• circulating tumor cells

• Accurate identification
• Cancer cell classification
• Circulating tumor cells
• Preliminary identification

• Biomarkers, Tumor/analysis
• Biosensing Techniques
• Humans
• Neoplastic Cells, Circulating/pathology

In breast cancer (BC), overexpression of HER2 on the primary tumor (PT) is determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) to stratify samples as negative, equivocal and positive to identify patients (pts) for anti-HER2 therapy. CAP/ASCO guidelines recommend FISH for analyzing HER2/neu (ERBB2) gene amplification and for resolving equivocal HER2 IHC results. However, pre-analytical and analytical aspects are often confounded by sample related limitations and tumor heterogeneity and HER2 expression may differ between the PT and circulating tumor cells (CTCs), the precursors of metastasis. We used a validation cohort of BC patients to establish a new DEPArray™-PT-HER2-FISH workflow for further application in a development cohort, characterized as PT-HER2-negative but CTC-HER2/neu-positive, to identify patients with PT-HER2 amplified cells not detected by routine pathology.

50 µm FFPE tumor curls from the validation cohort (n = 49) and the development cohort (n = 25) underwent cutting, deparaffinization and antigen retrieval followed by dissociation into a single-cell suspension. After staining for cytokeratin, vimentin, DAPI and separation via DEPArray™, single cells were processed for HER2-FISH analysis to assess the number of chromosome 17 and HER2 loci signals for comparison, either with available IHC or conventional tissue section FISH. CTC-HER2/neu status was determined using the AdnaTest BreastCancer (QIAGEN, Hilden, Germany).

Applying CAP/ASCO guidelines for HER2 evaluation of single PT cells, the comparison of routine pathology and DEPArray™-HER2-FISH analysis resulted in a concordance rate of 81.6% (40/49 pts) in the validation cohort and 84% (21/25 pts) in the development cohort, respectively. In the latter one, 4/25 patients had single HER2-positive tumor cells with 2/25 BC patients proven to be HER2-positive, despite being HER2-negative in routine pathology. The two other patients showed an equivocal HER2 status in the DEPArray™-HER2-FISH workflow but a negative result in routine pathology. Whereas all four patients with discordant HER2 results had already died, 17/21 patients with concordant HER2 results are still alive.

The DEPArray™ system allows pure tumor cell recovery for subsequent HER2/neu FISH analysis and is highly concordant with conventional pathology. For PT-HER2-negative patients, harboring HER2/neu-positive CTCs, this approach might allow caregivers to more effectively offer anti-HER2 treatment.

The online version contains supplementary material available at 10.1007/s12282-022-01330-8.

• Circulating tumor cells
• DEPArray™
• Early breast cancer
• HER2
• HER2/neu FISH
• Tumor heterogeneity

• circulating tumor cells
• companion diagnostics
• liquid biopsy
• precision medicine
• predictive biomarker

• ADN tumoral circulant
• Biopsie liquide
• Breast cancer
• Cancer du sein
• Cellule tumorale
• Circulating tumor DNA
• Circulating tumor cell
• Liquid biopsy
• circulante

• HER2 positive
• PKC/MEK/ERK
• acquired resistance
• breast cancer
• patient stratification
• phospho-profile
• signalosome

• Female
• Humans
• Breast Neoplasms/drug therapy
• Breast Neoplasms/metabolism
• MAP Kinase Signaling System
• Mitogen-Activated Protein Kinase Kinases/metabolism
• Mitogen-Activated Protein Kinase Kinases/pharmacology
• Mitogen-Activated Protein Kinase Kinases/therapeutic use
• Phosphatidylinositol 3-Kinases/metabolism
• Phosphorylation
• Trastuzumab/pharmacology
• Trastuzumab/therapeutic use
• Trastuzumab/metabolism
• Protein Kinase C/metabolism

• Research Innovation and Development Trust, University of Malta

While the concept of HER2-low expression is gaining momentum in the scientific landscape of breast cancer research, HER2-low circulating tumor cells (CTCs) also have promising relevance as biomarkers. Unveiling the biological features behind this recently highlighted evidence on CTCs might achieve both goals of speeding up the interpretation of the general understanding of HER2-low expressing breast cancer and declaring their independent biological and predictive value. If, on the one hand, studying CTCs allows to drill down more easily to a single cell resolution, on the other hand, CTC collection still remains a challenging procedure. In order to improve and standardize this process, we developed a structured pipeline for HER2-low CTC detection and collection. We defined and validated the optimal thresholds to select this specific subtype of CTCs using breast cancer cell lines of known HER2 expression. Our study represents the technical and procedural milestone that will allow a standardized collection process for future molecular investigations.

Although the detection of CTCs expressing HER2 at low intensity (HER2-low CTCs) has been shown to have a negative prognostic value in metastatic breast cancer (MBC) patients, the biological intrinsic nature of HER2-low CTCs remains unexplored. Considering the technical challenges behind the selective collection of immunophenotype-specific CTCs, we developed a pipeline to individually capture HER2-low CTCs. Four different breast cancer cell lines (MDA-MB-231, T47D, MDA-MB-453, and SKBR3), that are known to express HER2 at different immunohistochemistry levels (respectively classified as 0, 1+, 2+, and 3+), were spiked in healthy donor blood tubes (7.5 mL) and processed with the CellSearch^® (Menarini Silicon Biosystems, Bologna, Italy) for enrichment and the DEPArray NxT^™ for single cell selection. The HER2 signal-intensities of each cell line was compared using the nonparametric Mann–Whitney U test. The optimal cut-offs to distinguish HER2 1+ from 0 and 2+ cells were calculated performing the Receiver operating characteristic (ROC) curve. Median HER2 signal-intensities detected with the DEPArray NxT^™ were: 2.59 (0), 3.58 (1+), 5.23 (2+) and 38.37 (3+). DEPArray NxT efficiently differentiated each single cell line (p < 0.001). The area under the ROC curve was 0.69 and 0.70 (respectively 0 vs. 1+ and 1+ vs. 2+) and the optimal calculated cut-offs were 2.85 (lower) and 4.64 (upper). HER2-low CTCs can be detected and separately collected using predetermined intensity cut-offs. This study will allow standardized single-cell or pooled collection of HER2-low CTCs for downstream molecular analyses.

• UL1 TR001422 NCATS NIH HHS

Breast cancer is one of the common malignant tumors, and liquid biopsy has become a hot spot for clinical testing. To clarify the detection effect of liquid biopsy in breast cancer, we collected peripheral blood of HER2-positive (human epidermal growth factor receptor 2-positive) patients. Circulating tumor cells (CTCs) were isolated and analyzed. HER2 expression on CTCs was detected. The results showed that in the 198 HER2-positive samples, the CTC detection rate was 79.8% (158/198), and the mean number of CTCs was 21, ranging from 1 to 63/7.5 mL peripheral blood. Only 41.1% (65/158) of patients had histology and CTC HER2 status consistent with the remaining 58.9% (93/158) of patients, although their histological HER2 was positive, and CTC HER2 was negative. Our study confirmed the value of CTC HER2 real-time status testing in HER2-positive breast cancer patients. The inconsistency in HER2 status between CTCs and histology may be related to the time interval between CTCs and histological HER2 detection, suggesting that real-time HER2 detection is necessary for histological HER2-positive patients.

• HER2
• breast cancer
• circulating tumour cells
• liquid biopsy
• microfluidics
• overall survival

• Breast cancer
• biomarkers
• circulating tumor cells
• metastatic cascade
• prognosis

• CTC enrichment
• drug selection
• microfluidics
• molecular profiling
• precision oncology
• tumor resistance

• Delivery of Health Care
• Drug Resistance, Neoplasm/genetics
• Humans
• Neoplastic Cells, Circulating/metabolism
• Neoplastic Cells, Circulating/pathology
• Precision Medicine

• R33 CA204574 NCI NIH HHS
• FDN-148415 CIHR
• Canadian Institutes of Health Research
• Natural Sciences and Engineering Research Council of Canada

• Circulating tumor cells
• Gastric cancer
• Human epidermal growth factor receptor 2
• Trastuzumab

• Breast cancer
• CTCs
• Everolimus
• Exemestane
• mTOR inhibitor

• breast cancer
• circulating tumor DNA
• circulating tumor cells
• exosomes
• liquid biopsy
• prediction
• prognosis

• Breast Neoplasms/metabolism
• Breast Neoplasms/pathology
• Exosomes/metabolism
• Humans
• Neoplastic Cells, Circulating/metabolism
• Neoplastic Cells, Circulating/pathology
• Prognosis

The detection and analysis of circulating tumor cells (CTCs) may enable a broad range of cancer-related applications, including the identification of acquired drug resistance during treatments. However, the non-scalable fabrication, prolonged sample processing times, and the lack of automation, associated with most of the technologies developed to isolate these rare cells, have impeded their transition into the clinical practice. This work describes a novel membrane-based microfiltration device comprised of a fully automated sample processing unit and a machine-vision-enabled imaging system that allows the efficient isolation and rapid analysis of CTCs from blood. The device performance was characterized using four prostate cancer cell lines, including PC-3, VCaP, DU-145, and LNCaP, obtaining high assay reproducibility and capture efficiencies greater than 93% after processing 7.5 mL blood samples spiked with 100 cancer cells. Cancer cells remained viable after filtration due to the minimal shear stress exerted over cells during the procedure, while the identification of cancer cells by immunostaining was not affected by the number of non-specific events captured on the membrane. We were also able to identify the androgen receptor (AR) point mutation T878A from 7.5 mL blood samples spiked with 50 LNCaP cells using RT-PCR and Sanger sequencing. Finally, CTCs were detected in 8 out of 8 samples from patients diagnosed with metastatic prostate cancer (mean ± SEM = 21 ± 2.957 CTCs/mL, median = 21 CTCs/mL), demonstrating the potential clinical utility of this device.

• Circulating tumor cells (CTCs)
• Metastatic breast cancer
• Precision oncology
• Prognostic value
• Treatment monitoring

• CTC
• breast cancer metastasis
• prognostic value

• Biomarkers, Tumor/blood
• Breast Neoplasms/blood
• Breast Neoplasms/genetics
• Breast Neoplasms/pathology
• Female
• Humans
• Kaplan-Meier Estimate
• Neoplasm Metastasis
• Neoplasm Recurrence, Local/blood
• Neoplasm Recurrence, Local/genetics
• Neoplasm Recurrence, Local/pathology
• Neoplasm Staging
• Neoplastic Cells, Circulating/metabolism
• Prognosis

• 2016/21/B/NZ2/03473 Narodowe Centrum Nauki
• 2016/22/E/NZ4/00664 Narodowe Centrum Nauki

• Circulating tumor cells (CTCs)
• Clinical trials
• Early breast cancer
• Prognosis

• Biosensors
• Breast cancer cells
• Circulating tumor cells
• Electrochemical cytosensors
• Reusable cytosensors
• Tumor markers

• breast cancer
• discriminative CpG dinucleotides
• early detection
• hypermethylation
• liquid biopsy

• biomarker
• circulating tumor cells
• liquid biopsy
• overall survival
• pancreatic ductal adenocarcinoma

• Ageing
• Exposomic
• Genetics
• Inflammation
• Triple-negative breast cancer

We aimed to study the prevalence of CTCs in breast cancer (BC) patients undergoing neoadjuvant or palliative therapy with a label-free microfluidic platform (ClearCell FX), and its prognostic relevance in metastatic BC (mBC).

Peripheral blood samples were collected from 108 BC patients before starting a new line of treatment (“baseline”), majority of whom had mBC (76/108; 70.4%). CTCs were retrieved by dean flow fractionation that enriched for larger cells, and enumerated using immunofluorescence-based staining. Progression-free survival (PFS) in mBC patients was analysed using Kaplan-Meier method; cox proportional hazard models were used for univariable and multivariable analyses.

The detection rate of CTCs before starting a new line of treatment was 75.9% (n = 108; median: 8 CTCs/7.5 ml blood) at a cut off of ≥2 CTCs. PFS was inferior for mBC patients with baseline CTC count ≥5 CTCs/7.5 ml blood vs. those with < 5 CTCs/7.5 ml blood (median PFS: 4.3 vs. 7.0 months; p-value: 0.037). The prognostic relevance of CTCs was most significant in patients with HER2^- mBC (median PFS: 4.1 vs. 8.3 months; p-value: 0.032), luminal (HR+HER2-) subtype (median PFS: 4.2 vs. 8.3 months; p-value: 0.048), and patients who had one or more prior treatments (median PFS: 4.2 vs. 7.0 months; p-value: 0.02). On multivariable analysis, baseline CTC level (hazard ratio (HR): 1.84, p-value: 0.02) and pre-treatment status (HR: 1.87, p-value: 0.05) were independent predictors of PFS.

This work demonstrates the prognostic significance of CTCs in mBC detected using a label-free size-based enrichment platform.

• Adult
• Aged
• Asian People
• Breast Neoplasms/blood
• Breast Neoplasms/metabolism
• Breast Neoplasms/pathology
• Cell Count
• Female
• Humans
• Immunohistochemistry
• Kaplan-Meier Estimate
• Microfluidics
• Middle Aged
• Neoplastic Cells, Circulating/pathology
• Prognosis
• Progression-Free Survival
• Prospective Studies
• Receptor, ErbB-2/metabolism
• Singapore

Circulating tumor cells (CTCs) are important for metastatic dissemination of cancer. They can provide useful information, regarding biological features and tumor heterogeneity; however, their detection and characterization are difficult due to their limited number in the bloodstream and their mesenchymal characteristics. Therefore, new biomarkers are needed to address these questions.

Bioinformatics functional enrichment analysis revealed a subgroup of 24 genes, potentially overexpressed in CTCs. Among these genes, the chemokine receptor CXCR4 plays a central role. After prioritization according to the CXCR4 corresponding pathways, five molecules (JUNB, YWHAB, TYROBP, NFYA, and PRDX1) were selected for further analysis in biological samples. The SKBR3, MDA-MB231, and MCF7 cell lines, as well as PBMCs from normal (n = 10) blood donors, were used as controls to define the expression pattern of all the examined molecules. Consequently, 100 previously untreated metastatic breast cancer (mBC) patients (n = 100) were analyzed using the following combinations of antibodies: CK (cytokeratin)/CXCR4/JUNB, CK/NFYA/ΥWHΑΒ (14-3-3), and CK/TYROBP/PRDX1. A threshold value for every molecule was considered the mean expression in normal PBMCs.

Quantification of CXCR4 revealed overexpression of the receptor in SKBR3 and in CTCs, following the subsequent scale (SKBR3>CTCs>Hela>MCF7>MDA-MB231). JUNB was also overexpressed in CTCs (SKBR3>CTCs>MCF7>MDA-MB231>Hela). According to the defined threshold for each molecule, CXCR4-positive CTCs were identified in 90% of the patients with detectable tumor cells in their blood. In addition, 65%, 75%, 14.3%, and 12.5% of the patients harbored JUNB-, TYROBP-, NFYA-, and PRDX-positive CTCs, respectively. Conversely, none of the patients revealed YWHAB-positive CTCs. Interestingly, JUNB expression in CTCs was phenotypically and statistically enhanced compared to patients’ blood cells (p = 0.002) providing a possible new biomarker for CTCs. Furthermore, the detection of JUNB-positive CTCs in patients was associated with poorer PFS (p = 0.015) and OS (p = 0.002). Moreover, JUNB staining of 11 primary and 4 metastatic tumors from the same cohort of patients revealed a dramatic increase of JUNB expression in metastasis.

CXCR4, JUNB, and TYROBP were overexpressed in CTCs, but only the expression of JUNB was associated with poor prognosis, providing a new biomarker and a potential therapeutic target for the elimination of CTCs.

The online version of this article (10.1186/s13058-019-1166-4) contains supplementary material, which is available to authorized users.

• Bioinformatics
• Breast cancer
• CTCs
• CXCR4
• JUNB