generate false-positive signals, thus decreasing the overall accuracy. According to previous work, not all the detection targets are tumour specific, because the same targets can be found in blood cells. 45, 46 Pantel et al have reported that CK-19, a major marker for CTC detection, is also present in immune cells. 47 Multiplex PCR, such as the AdnaTest kit( AdnaGen AG), could overcome this limitation. 45, 48 Moreover, considering the genetic heterogeneity of CTCs, a multiplex PCR assay might provide improved sensitivity and specificity rates.
Functional assays Functional assays exploit aspects of live cellular activity for CTC detection. Interestingly, these assays have the particularity to focus on the discovery of‘ metastasis competent cells’. In order to detect only viable CTCs, the functional Epithelial ImmunoSPOT( EPISPOT) assay, was introduced for in vitro CTC detection. 49 This technology assesses the presence of CTCs based on secretion, shed or release of specific proteins during 24 – 48 hours of short-term culture. 50 EPISPOT has been applied to blood and bone marrow samples, and validated in several different cancers, for example, breast and colon with the CK19 EPISPOT assay, or prostate with the PSA-EPISPOT assay. 51 – 54 This test is currently being further developed into a liquid format with micro-droplets( called EPIDROP) that allows capture of single viable CTCs and subsequent molecular characterisation.
Another in vitro functional assay, Vita-Assay™( Vitatex), exploits the preferential adhesion of invasive rare blood cells to a specialised matrix to enrich viable CTCs from blood up to one millionfold. 55 This method has also been tested in metastatic prostate 56 and breast cancer. 57
In vivo, important information can be obtained by transplantation of patient-derived CTCs into immunodeficient mice: metastases that were grown after xenotransplantation of enriched CTCs have the most characteristics of metastasis-initiator cells. 8 A report on patients with small-cell lung cancer showed that CTCs from patients with either chemosensitive or chemorefractory tumours are tumourigenic in immunocompromised mice, and the resultant CTC-derived explants mirrored the response of the donor patient to platinum and etoposide chemotherapy. 58 However, at present, these in vivo assays require very high CTC yields in the transplanted blood sample, which have so far only been achieved in a few patients.
Strategies for CTC characterisation CTCs hold the key to understand the biology of metastasis and provide a biomarker to non-invasively measure the evolution of tumour subclone during treatment and disease progression. Improvements in technologies to yield purer CTC populations might now enable better cellular and molecular investigations. Characterisation of CTCs allows better insights into tumour heterogeneity, within most assays, including immunofluorescence, array CGH, next generation sequencing of both DNA and RNA, and fluorescence in situ hybridisation( FISH).
Protein analyses on single CTCs are currently performed by immunostaining with antibodies
CTCs, as liquid biopsy, hold the key to understanding the biology of metastasis and provide a biomarker for personalised treatment in cancer patients
144 HHE 2018 | hospitalhealthcare. com directed against protein of interest. Multiple labelling is possible but usually restricted to a few proteins of interest for tumour cell biology and cancer therapy. This may help to identify signaling pathways relevant to metastasis development and treatment responses. In breast cancer patients, the HER2 status of CTCs could be assessed and show discrepancies with primary tumour status. 59, 60 More recently, immune checkpoint regulators such as programmed death-ligand 1 have become exciting new therapeutic targets and could be used for liquid biopsy in future clinical trials on patients
61, 62
undergoing immune checkpoint blockage. For single-cell sequencing to identify genomic and transcriptomic characteristics of CTCs, most studies have focused on genomic analyses and carried out whole genome amplifications( WGAs) to increase the amount of DNA, which is subsequently subjected to the analyses of specific mutations and copy number variations using conventional and next-generation sequencing technologies. 37, 63, 64 As an example, CTCs with mutated KRAS genes will escape anti-EGFR therapy and their early detection might help to guide therapy in individual patients, although it noteworthy that WGA has the inherent risk of inducing a bias and the results need to be therefore carefully validated. Besides isolation of single CTCs, enrichment by 3 – 4 log units might be sufficient to obtain a concentration of one CTC in 1000 blood cells, which is in the range that is suitable for highly sensitive mutation analyses technologies such as digital PCR. 65
Another approach is FISH analysis of single
66, 67
CTCs identified by immunocytochemistry. Such an immuno-FISH approach can be combined with automated detection of CTCs and might be easier to implement in future clinical diagnostics.
Conclusions The recent explosion in the field of CTC biology is reflected in the myriad of CTC technologies developed within the last decades. New technologies have arisen to address new challenges as our understanding of CTC biology evolves permanently. Recent focuses on the epithelial-to-mesenchymal transition and stem cell markers in CTCs illuminated the potential values of new biomarkers on CTCs and they may provide information of clinical interest. While clinical studies using CellSearch™ and other CTC technologies have affirmed that CTC enumeration provides relevant prognostic information and clinical validity, the potential for liquid biopsy to address clinical utility is still under investigation. In conclusion, liquid biopsy diagnostics might help to focus the current cancer screening modalities, which could potentially reduce health care costs.
Acknowledgments The authors received support from DGOS, the National Institute of Cancer( INCA), ARC Foundation and CANCER-ID, an Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115749, based on financial contributions from the European Union’ s Seventh Framework Program( FP7 / 2007-2013), and EFPIA companies’ in-kind contribution.