21 marzo 2018

Diverse subtypes of CTCs with cytogenetic abnormalities in hepatocellular carcinoma patients determine cancer relapse

Detection of hepatocellular carcinoma (HCC) circulating tumor cells (CTCs) performed with conventional strategies, is significantly limited due to inherently heterogeneous and dynamic expression of EpCAM, as well as degradation of cytokeratins during epithelial-to-mesenchymal transition, which inevitably lead to non-negligible false negative detection of such “uncapturable and invisible” CTCs. A novel SE-iFISH (SE, subtraction enrichment; iFISH, immunostaining-fluorescence in situ hybridization) strategy was applied by L.Wang and colleagues to comprehensively detect, in situ phenotypically and karyotypically characterize hepatocellular and cholangiocarcinoma CTCs in HCC patients subjected to surgical resection. Clinical significance of diverse subtypes of CTC was systematically investigated.


Global morbidity of hepatobiliary malignancies, mainly including hepatocellular carcinoma (HCC), and cholangiocarcinoma (CC) has recently increased (McGlynn KA & London WT, 2011; Mittal S & El-Serag HB, 2013; Razumilava N & Gores GJ, 2014) and conventional clinical approaches, such as imaging, pathology and serum tumor marker detection, show very limited prognostic efficacy. Circulating tumor cells (CTCs) play a fundamental role in tumor metastasis (Aceto N et al., 2015), thus they are worldwide considered as ‘liquid biopsy’ due to their tremendous prognostic and diagnostic significance in cancer. It has been recently reported that considerable amount of CTCs remains in circulation following resection of primary tumor in cancer patients (Bayarri-Lara C et al., 2016; Sandri MT et al., 2010). Surgical resection may not be effective to significantly reduce quantity of CTCs in all the cases, suggesting some of those CTCs might not be derived from the resected primary malignant tumors, instead, they may pre-exist in circulation even at very early stage of carcinoma formation and contribute to postsurgical cancer metastasis and relapse (Wang L et al., 2018). In addition, circulating tumor microemboli (CTM, a cluster of ≥2 CTCs), are derived from oligoclonal clumps of primary malignant carcinoma cells and constitute a very highly metastasis-competent subset of CTCs due to 50 times greater metastasis potential than that of single CTC (Aceto N et al., 2014; Cheung et al.,2016). Conventional CTC detection strategies are restricted and biased to only both cytokeratin (CK) and EpCAM double positive CTCs, showing overall positive detection of 1030% for CTCs shed from limited types of solid tumor (Pantel, 2016). In view of dynamic (Gires O & Stoecklein NH, 2014) or absent (Went PT et al., 2004) expression of EpCAM, as well as degradation of both CK and EpCAM in CTCs during epithelial-mesenchymal transition (EMT) (Willipinski-Stapelfeldt B et al., 2015; Yu M et al., 2013), conventional CTC detection methods are not only unable to effectively detect multi-functional mesenchymal CTCs (Tsai JH & Yang J, 2013), which are usually deficient in epithelial marker expression as well as in small cell size (≤ 5 mm of WBCs) (Ito H et al., 2014), but also lead to a significant false negative detection of CTCs, particularly with HCC CTCs expressing limited amount of stemness marker EpCAM (Sun YF et al., 2013; Terris B et al., 2010; Wu LJ et a., 2012). Based on the well accepted concept that aneuploidy of chromosome, leading to genomic instability (Passerini V et al., 2016), is the most common characteristic of malignant cells (Gordon DJ et al., 2012; Gops GJ et al., 2005), a novel CTC detection strategy, integrating subtraction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) has been recently established (Li YL et al., 2014; Lin PP et al., 2017). This EpCAM-independent technology greatly increases CTC detection efficiency in terms of both sensitivity and specificity (Lin PP, 2015) and allows for classifying CTCs into diverse subtypes that are relevant to predict patient prognosis and neoplasm recurrence (Wang L et a., 2018).

In the current study, Wang and colleagues from Tsinghua University (Beijing, China) investigated clinical significance of the post-operative EpCAM+ circulating tumor stem cells (CTSCs) and EpCAM CTC subtypes in cancer metastasis and recurrence in patients with hepatobiliary malignancies. Obtained results indicate that postsurgical quantity of CTM (cut-off: ≥ 1 in 6 ml blood), EpCAM+ CTSCs with pentasomy of chromosome 8 (≥ 1 cell in 6 ml of blood), and EpCAM small CTCs with trisomy 8 (≥ 5 cells), as well as increasing of small triploid CTCs (cut-off ≥ 5 CTCs / 6 ml of blood) compared to those quantified in pre-operative patients, significantly correlate to post-operative HCC patients’ poor prognosis and cancer relapse.

In this clinical scenario, CellDynamics has developed a disruptive technology, CellViewer, that allows microfluidics-based isolation of rare cells in suspension with intact genomic heritage thus ensuring cell viability and integrity for downstream molecular analysis. In addition, CellViewer  technology reproduces an in vivo-like environment for CTC culture, real-time immunofluorescence staining and drug administration in order to get detailed information on dynamic biological processes at the cellular and multicellular level.



Fig. 1. Detection and in situ phenotypic and karyotypic characterization of hepatocellular carcinoma and cholangiocarcinoma cells, CTCs and CTMs by SE-iFISH (from L. Wang et al. 412 (2018) 99-107). (A) Immunofluorescence staining of tumor biomarkers on cancer cells. Cancer cell line cells HepG2 and SW480 on coverslips were immunostained with the indicated monoclonal antibodies. CK18 and vimentin show intracellular staining in HepG2 cells, whereas PD-L1 (HepG2) and EpCAM (SW480) have a positive plasma membrane staining. (B) Tumor biomarkers-iFISH. Both protein expression of the indicated tumor biomarkers and aneuploid chromosome 8 in tumor cells enriched from blood are revealed. WBC (CD45+, red arrow) does not show positive staining of each tumor biomarker. (C) A non-hematopoietic multiploid EpCAM+ CTSC (DAPI+, CD45-, EpCAM+, CEP8+) enriched from a HCC patient blood was detected by EpCAM-iFISH. WBC (CD45+, red arrow) is negative for EpCAM staining. (D) An EpCAM+ triploid CTC from a cholangiocarcinoma patient. (E) An EpCAM+ CTM from a HCC patient. (F) An EpCAMmultiploid CTC from a HCC patient. (G) An EpCAM- triploid CTC from a HCC patient. (H) An EpCAM- CTM from a cholangiocarcinoma patient. WBCs (CD45+/EpCAM-) in (HeG) are indicated by red arrows. Bars, 5 mm.




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