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HER-2 status of circulating tumor cells in a metastatic breast cancer cohort: A comparative study on characterization techniques.

ABSTRACT: BACKGROUND:Personalized targeted treatment in metastatic breast cancer relies on accurate assessment of molecular aberrations, e.g. overexpression of Human Epidermal growth factor Receptor 2 (HER-2). Molecular interrogation of circulating tumor cells (CTCs) can provide an attractive alternative for real-time biomarker assessment. However, implementation of CellSearch-based HER-2 analysis has been limited. Immunofluorescent (IF) image interpretation is crucial, as different HER-2 categories have been described. Major questions in CTC research are how these IF categories reflect gene expression and amplification, and if we should consider 'medium' HER-2 expressing CTCs for patient selection. METHODS:Tumor cells from spiked cell lines (n = 8) and CTCs (n = 116 samples) of 85 metastatic breast cancer patients were enriched using CellSearch. Comparative analysis of HER-2 expression by IF imaging (ACCEPT, DEPArray, and visual scoring) with qRT-PCR and HER-2/neu FISH was performed. RESULTS:Automated IF HER-2-profiling by DEPArray and ACCEPT delivered comparable results. There was a 98% agreement between 17 trained observers (visual scoring) and ACCEPT considering HER-2neg and HER-2high expressing CTCs. However, 89% of HER-2med expressing CTCs by ACCEPT were scored negative by observers. HER-2high expressing tumor cells demonstrated HER-2/neu gene amplification, whereas HER-2neg and HER-2med expressing tumor cells and CTCs by ACCEPT were copy-number neutral. All patients with HER-2-positive archival tumors had ?1 HER-2high expressing CTCs, while 80% of HER-2-negative patients did not. High relative gene expression of HER-2 measured on enriched CTC lysates correlated with having ?1 HER-2high expressing CTCs. CONCLUSION:Automated images analysis has enormous potential for clinical implementation. HER-2 characterization and clinical trial design should be focused on HER-2high expressing CTCs.

SUBMITTER: Brouwer A

PROVIDER: S-EPMC6726188 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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HER-2 status of circulating tumor cells in a metastatic breast cancer cohort: A comparative study on characterization techniques.

Brouwer Anja A De Laere Bram B van Dam Pieter-Jan PJ Peeters Dieter D Van Haver Jasper J Sluydts Ellen E El Moussaoui Ali A Mendelaar Pauline P Kraan Jaco J Peeters Marc M Van Laere Steven S Dirix Luc L

PloS one 20190904 9

<h4>Background</h4>Personalized targeted treatment in metastatic breast cancer relies on accurate assessment of molecular aberrations, e.g. overexpression of Human Epidermal growth factor Receptor 2 (HER-2). Molecular interrogation of circulating tumor cells (CTCs) can provide an attractive alternative for real-time biomarker assessment. However, implementation of CellSearch-based HER-2 analysis has been limited. Immunofluorescent (IF) image interpretation is crucial, as different HER-2 categori ...[more]

PMID: 31483799

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Project description:PurposeAndrogen receptor (AR) is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+) breast cancer. However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood. Circulating tumor cells (CTCs) can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer.MethodsBlood samples from patients with metastatic breast cancer were deposited on glass slides, subjected to nuclear staining with DAPI, and reacted with fluorescent-labeled antibodies to detect CD45, cytokeratin (CK), and biomarkers of interest (AR, estrogen receptor [ER], and HER2) on all nucleated cells. The stained slides were scanned and enumerated by non-enrichment-based non-biased approach independent of cell surface epithelial cell adhesion molecule (EpCAM) using the Epic Sciences CTC platform. Data were analyzed using established digital pathology algorithms.ResultsOf 68 patients, 51 (75%) had at least 1 CTC, and 49 of these 51 (96%) had hormone-receptor-positive (HR+)/HER2-negative primary tumors. AR was expressed in CK+ CTCs in 10 patients. Of these 10 patients, 3 also had ER expression in CK+ CTCs. Single cell genomic analysis of 78 CTCs from 1 of these 3 patients identified three distinct copy number patterns. AR+ cells had a lower frequency of chromosomal changes than ER+ and HER2+ cells.ConclusionsCTC enumeration and analysis using no enrichment or selection provides a non-biased approach to detect AR expression and chromosomal aberrations in CTCs in patients with metastatic breast cancer. The heterogeneity of intrapatient AR expression in CTCs leads to the new hypothesis that patients with AR+ CTCs have heterogeneous disease with multiple drivers. Further studies are warranted to investigate the clinical applicability of AR+ CTCs and their heterogeneity.

Project description:Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.

Project description:Circulating Tumor Cells (CTCs) represent a "liquid biopsy of the tumor" which might allow real-time monitoring of cancer biology and therapies in individual patients. CTCs are extremely rare in the blood stream and their analysis is technically challenging. The CellSearch(®) system provides the enumeration of CTCs with prognostic significance in patients with metastatic breast cancer (mBC), but it does not allow their molecular characterization, which might be useful to identify therapeutically relevant targets for individualized treatment. Combining the CellSearch(®) and DEPArray™ technologies allows the recovery of single CTCs as a pure sample for molecular analysis. The purpose of the study was to investigate the heterogeneity of PIK3CA mutational status within single CTCs isolated from individual mBC patients. CTCs were enriched and enumerated by CellSearch(®) in blood samples collected from 39 mBC patients. In 20 out of 39 patients enriched samples with ≥5 CTCs were sorted using DEParray™ to isolate single CTCs or pools of CTCs to be submitted to Whole Genome Amplification (WGA) before sequencing analysis. In 18 out of 20 patients, it was possible to perform PIK3CA sequencing on exons 9 and 20. Twelve subjects were wild type (wt) for the PIK3CA gene. PIK3CA status could also be assessed in pools of CTCs in seven of these patients, with consistent wt status found. Six patients (33%) had a PIK3CA mutation identified. In 2 of the six patients, molecular heterogeneity was detected when mutational analysis was performed on more than one single CTC, including one patient with loss of heterozygosity on both single and pooled CTCs, and one patient with three different PIK3CA variants on single CTCs but PIK3CA wt status on pooled CTC samples. In six out of the 18 cases PIK3CA status was also evaluable on a primary tumor sample. In one of the six cases a discordance in PIK3CA status between the primary (wild-type) and the matched CTC (exon 20 mutation) was observed. This study demonstrates the feasibility of a non-invasive approach based on the liquid biopsy in mBC patients. Moreover, our data suggest the importance of characterizing CTCs at the single cell level in order to investigate the molecular heterogeneity within cells from the same patient.

Dataset Information

HER-2 status of circulating tumor cells in a metastatic breast cancer cohort: A comparative study on characterization techniques.

Publications

HER-2 status of circulating tumor cells in a metastatic breast cancer cohort: A comparative study on characterization techniques.

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