<i>Liquid biopsy</i>: from discovery to clinical implementation

Abstract

‘Liquid biopsy’ was introduced as a new diagnostic concept in 2010 [[1]] for the analysis of circulating tumor cells (CTCs) in the blood of cancer patients and has been now extended to the analysis of circulating tumor-derived factors, in particular, cell-free tumor DNA (ctDNA), as well as extracellular vesicles (EVs), cell-free microRNAs (cfmiRNAs), mRNA, long noncoding RNA, small RNA, circulating cell-free proteins, and tumor-educated platelets (TEPs). Liquid biopsy is a minimally invasive procedure usually based on sampling of blood, but also of cerebrospinal fluid, urine, sputum, ascites, and theoretically any other body fluid [[2]]. Over the past decade, various methods have been developed to detect CTCs and ctDNA in the peripheral blood of cancer patients [[3, 4]]. While reliable information can be easily obtained in patients with advanced disease, early-stage cancer patients usually present with very low concentrations of CTCs and ctDNA [[5]]. At present, most CTC assays rely on epithelial markers and the majority of CTCs detected are single isolated cells. The clinical relevance of ‘mesenchymal’ CTCs lacking any epithelial markers, as well as CTC clusters, is still under investigation. The biology of EpCAM and its role is not completely understood; however, evidence suggests that the expression of this epithelial cell-surface protein is crucial for metastasis-competent CTCs and may not be lost completely during the epithelial-to-mesenchymal transition [[6-8]]. Although most published studies have been performed on patients with carcinomas and melanomas, CTCs have been also detected in the peripheral blood of patients with primary brain tumors (glioblastomas) despite the blood–brain barrier [[9]]. Liquid biopsy assays are currently being validated for early detection of cancer, which is supposed to reduce cancer-related mortality. Despite remarkable progress, liquid biopsy-based detection of early stages of solid cancers remains a challenge. New blood-based biomarkers for early detection currently validated in clinical trials include miRNAs, exosomes, and tumor-educated platelets [[10-12]]. In patients with diagnosed cancer, CTCs and ctDNA analyses can obtain independent information on prognosis in early and advanced stages of disease. In particular, CTC counts at initial diagnosis are able to refine the current risk stratification by TNM staging in early-stage breast cancer and have been therefore included into the 2018 AJCC classification. Moreover, early detection of relapse by sequential ctDNA (or CTCs) analysis of blood samples obtained post-surgery during the follow-up is possible and may be used in future trials to stratify patients to ‘post-adjuvant’ therapies [[13]]. With the growing body of evidence concerning the prognostic value of CTCs, scientists and clinicians began to investigate the clinical utility of CTCs via interventional clinical trials. The STIC CTC METABREAST is the first study that has demonstrated the clinical utility of CTCs in assigning people with metastatic breast cancer to either chemotherapy or hormonal therapy [[14]]. In particular, patients with elevated CTC counts who were assigned to the low-risk group by the conventional physician decision profited from chemotherapy. Another key application of liquid biopsy is to identify therapeutic targets or mechanisms of resistance of metastatic cells in individual patients [[3]]. While the analysis of ctDNA focuses on mutations relevant for cancer therapy (including EGFR, KRAS, or ESR1 mutations), CTCs offer a wide spectrum of analyses at the DNA, RNA, and protein levels [[5, 15]]. Metastatic cells might have unique characteristics that can differ from the bulk of cancer cells in the primary tumor, currently used for stratification of patients to systemic therapy. Moreover, monitoring of CTCs and ctDNA before, during, and after systemic therapy (e.g., chemotherapy, hormonal therapy, antibody therapy) might provide unique information for the future clinical management of the individual cancer patient and might serve as a surrogate marker for response to therapy. In the context of recent success in antibody-mediated blockade of immune checkpoint control molecules [[16]], the expression of PD-L1 on CTCs might be of interest as potential predictive marker [[17, 18]]. Moreover, Lu et al. [[19]] reported that DNA sensing within tumor cells is essential for anti-tumor immunity triggered by DNA mismatch repair deficiency (dMMR). This recent study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy. In addition, the expression of androgen receptor variant 7 in CTCs may predict resistance to anti-androgen therapy in prostate cancer, while mutations in the estrogen receptor gene (ESR1) provide information on resistance to hormone therapy in breast cancer [[4]]. Additional therapeutic targets detected on CTCs in cancer patients include the estrogen receptor and HER-2 oncogene [[5]]. Single-cell RNAseq analysis of CTCs may provide more comprehensive information on relevant pathways [[20, 21]]. For functional analysis of CTCs, the development of in vitro and in vivo test systems has started, which might also serve as models for drug testing [[22-24]]. In particular, the development of cell lines and xenografts derived from CTCs can provide novel insights into the biology of tumor cell dissemination and may be used to discover new pathways to target specifically metastatic cells [[25, 26]]. Besides CTCs and ctDNA, the analysis of circulating microRNAs, exosomes, or tumor-educated platelets may provide complementary information as ‘liquid biopsy’. Indicatively, the integrin composition of exosomes seems to determine the organ site of metastatic niches and the RNA expression pattern of blood platelets reveals information on tumors in cancer patients [[27]]. Sensitive methods have been also developed to capture disseminated tumor cells (DTCs) in the bone marrow in cancer patients [[13]], which provide new insights into the process of ‘cancer dormancy’. The nature of dormant breast cancer cells and the mechanisms leading to their outgrowth are poorly understood. Efforts to unravel the nature of cancer dormancy have been hampered by the lack of sensitive methods to detect dormant cells in cancer patients. Very recently, Albrengues et al. [[28]] found that lung inflammation (induced by either tobacco smoke exposure or nasal instillation of lipopolysaccharide) awakened dormant cancer cells and converted them to aggressive lung metastases in the mouse model. Currently, the potential correlation between inflammation or smoking, neutrophil extracellular traps (NETs), and recurrence after dormancy in human patients needs to be tested. If such a link can be established, NETs and their downstream effectors could be targeted to reduce the risk of cancer recurrence in human patients. The development of novel therapies designed to kill dormant residual tumor cells, or maintain them in a quiescent state, represents a highly attractive approach to prevent late recurrence. Such an approach, however, would require a far more detailed understanding of tumor dormancy and recurrence than exists today, as well as biomarkers to enable monitoring of this process and predict recurrence. Analysis of DTCs leads to the discovery of new molecules relevant to the biology of metastasis such as the putative metastasis-suppressor RAI2 [[29]]. In conclusion, liquid biopsy analysis can be used to obtain new insights into metastasis biology, and as companion diagnostics to improve the stratification of therapies and to obtain insights into therapy-induced selection of cancer cells. In this context, intra-patient tumor heterogeneity may represent an important mechanism to escape the complete eradication of all tumor clones by targeted therapies [[37]]. Researchers and clinicians have known about the clinical potential of liquid biopsies for many years. To push them into widespread use, more interventional clinical trials are now needed, as well as the development of an algorithm to combine the appropriate circulating biomarkers [[38]]. The next generation of liquid biopsies is increasingly going toward the analysis of complex cancer liquid biopsy data and will require a greater role for machine learning and artificial intelligence [[39]]. Technical and clinical assay validation is very important and can be achieved in international consortia such as the European Liquid Biopsy Society (ELBS) network (www.elbs.eu). CA-P and KP received funding from the European IMI research project CANCER-ID (115749-CANCER-ID), European Union Horizon 2020 Research and Innovation program under the Marie Skłodowska-Curie grant agreement no 765492 and ERA-NET EU/TRANSCAN 2 JTC 2016 PROLIPSY. CA-P is also supported by The National Institute of Cancer (INCa, http://www.e-cancer.fr), La Fondation ARC pour la Recherche contre le Cancer, La Ligue contre le Cancer, and SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553. KP also received funding from Deutsche Krebshilfe (Nr. 70112504), Deutsche Forschungsgemeinschaft (DFG) SPP2084 µBone, and ERC Advanced Investigator Grant INJURMET (Nr. 834974). CA-P received honoraria from Menarini; KP received honoraria from Menarini, Illumina, and Agena.

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