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 .