A novel method for the detection of different subgroups of circulating tumor cells in patients with hepatocellular carcinoma

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Background: Using liver-derived circulating tumor cells (CTC) as “liquid biopsy” may be a promising approach for individualizing and monitoring treatment in patients with HCC. However, this is challenged by cellular heterogeneity. Therefore, we wanted to assess whether CTC subtypes are identifiable in the peripheral blood of patients with HCC and whether their distribution is associated with clinical characteristics. Methods: CTC were enriched from peripheral venous blood using density gradient centrifugation and subsequent depletion with immunomagnetic beads. CTC enriched suspensions were spun onto glass slides and stained by immunofluorescence or immunocytochemistry. Epithelial CTC were concomitantly selected for qRT-PCR based molecular analysis. Results: We were able to detect cells with epithelial, mesenchymal, stem cell-like and mixed characteristics and a remarkable variation in the size range. The distribution of these subgroups varied significantly between different patient groups and was associated with clinical outcome. Kaplan-Meier log rank test showed that a change in the ratio of epithelial to mesenchymal cells was associated with longer median time to progression (TTP; 2 months, p=0.01; [HR]=0.18). Furthermore, we were able to identify IGFBP1 as a potential molecular marker for TTP after radioembolisation (p=0.04; HR<0.1). Here, ROC analysis resulted in sensitivity and specificity of 80% (AUC=0.8; CI 0.44-0.98; p=0.03). Conclusions: Our data suggest that different CTC populations are identifiable in peripheral blood of patients with HCC and that these individual cell type profiles may have distinct clinical implications, e.g. epithelial cells as markers of aggressiveness. CTC may also be usable for identifying new molecular markers.

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... Available enrichment techniques based on various systems including the CellSearch Ò platform (Veridex, Raritan, NJ) using antibodies against EpCAM, keratin and CD45 to enumerate CTCs in breast, colon and prostate cancer as well as the filtration by size ISET (Isolation by Size of Epithelial/Throphoblastic Tumor cells) platform are described elsewhere [12][13][14][15][16][17]. However, especially the automated approaches are challenged by a lack of flexibility, needed to address the great differences in subpopulations and size that CTCs inherit before or during treatment [10,18,19]. Microscopy-based approaches allow additional morphological examinations of CTCs as well as downstream applications such as fluorescence in situ hybridization (FISH) analysis for instance employing the so-called FICTION protocol (fluorescence immunophenotyping, and interphase cytogenetics as a tool for the investigation of neoplasms) [20] or dual-colorimetric RNA-ISH assay [5] for individualized treatment decisions. ...
... In HCC, the lack of specific cell surface antigens challenges CTC detection. To date, only a few studies have been performed to identify or quantify these cells [18,[22][23][24][25][26][27][28][29][30]. Table 1 briefly summarizes the development of CTC research in the field of HCC during the last decade. ...
... Their study resulted in a detection rate of 30.5 % and a significant association of quantifiable epithelial CTCs with overall survival, BCLC staging, vascular invasion and the serum-based tumor marker AFP [30,31]. Nel et al. [18] applied a negative CTC selection technique by depleting hematopoietic cells using immunomagnetic beads directed against CD45 and subsequent multi-immunofluorescence staining to detect various CTC subpopulations in patients with HCC. The described method is based on individual CTC profiles and subsequent cell type ratios [32] analyzed the value of CTC detection in HCC and gastric cancer and pointed out limitations due to small cohorts and potential bias in the studies reported so far. ...
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Circulating tumor cells (CTC) and cancer stem cells (CSC) have been proposed as tools for detection and characterization of disease and individualization of therapy in patients with many solid tumors. Several automated and semi-automated techniques for identification and isolation of these cells from blood have been proposed and reviewed mostly focusing on their feasibility. In this mini review we summarize the recent relevant literature on this topic and discuss the clinical usability of measuring CTC and CSC in peripheral blood in patients with hepatocellular carcinoma (HCC). Besides literature, the basis for this evaluation was the authors’ experience with treating HCC and research experience on CSC and CTC. Few original reports and reviews have been published focusing on CTC and CSC in HCC. Though HCC is one of the five most common malignancies worldwide only recently these cells have come into focus for detection and characterization of this disease that is characterized by high plasticity and malignancy. A focused and prospective validation of the clinical usability of detecting these cells in HCC is still needed, but results seem promising that they may add great benefit for early detection and individualization of therapy.
... Preparation of blood samples and CTC enrichment. CTC were isolated from all patients upfront to and during treatment and examined for epithelial characteristics (Nel et al, 2012). At baseline, duplicates of 20 ml citrated peripheral venous blood were drawn (i) for ex vivo examination of Pt-(GpG) adduct persistence before systemic cisplatin-based treatment and (ii) for gene expression analysis. ...
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Background: Pt-(GpG) intrastrand crosslinks are the major DNA adducts induced by platinum-based anticancer drugs. In the cell lines and mouse models, the persistence of these lesions correlates significantly with cell damage. Here we studied Pt-(GpG) DNA adducts in circulating tumour cells (CTC) treated with cisplatin in medium upfront to systemic therapy from patients with advanced non-small-cell lung cancer (NSCLC). Methods: Blood was drawn before systemic treatment and the CD45/CD15-depleted fraction of mononuclear cells was exposed to cisplatin, verified for the presence of CTC by pan-cytokeratin (pCK) staining and immunoanalysed for the level of Pt-(GpG) in DNA. Results: Immunostaining for pCK, CD45 and subsequently for Pt-(GpG) adducts in the cisplatin-exposed cells (ex vivo) at different time points depicted distinct differences for adduct persistence in CTC between responders vs non-responders. Conclusion: Pt-(GpG) adducts can be detected in CTC from NSCLC patients and assessing their kinetics may constitute a clinically feasible biomarker for response prediction and dose individualisation of platinum-based chemotherapy. This functional pre-therapeutic test might represent a more biological approach than measuring protein factors or other molecular markers.
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