Liquid biopsy: Potential and challenges (2016)

Current molecular diagnostics in cancer patients is based on needle biopsies, which, however, face serious limitations which can lead to false decisions. Individual tumors consist of diverse subpopulations, and the small amount of tissue obtained by needle biopsies may not represent the most aggressive subclones. Moreover, some tumor entities such as lung cancer are located at remote sites and a needle biopsy can be very difficult and at high risk. Finally, the mere analysis of the resected primary tumor alone (current standard practise in oncology) may provide misleading information with regard to the characteristics of metastases, the key target for systemic anticancer therapy. Distant metastases can harbor unique genomic characteristics not detectable in the corresponding primary tumor of the same patient and metastases located at different sites show a considerable intra-patient heterogeneity (Gerlinger et al., 2013). Thus, even if re-evaluation of tumor characteristics based on the biopsy of the most accessible metastasis is possible, it may not reveal sufficient information for treatment decisions. This dilemma has led to a new diagnostic concept: the Liquid Biopsy, i.e., analysis of circulating tumor cells (CTCs) and cell-free circulating nucleic acids (in particular circulating tumor DNA -ctDNA) released into the peripheral blood from the primary tumor and/or metastatic deposits. In this special issue, we discuss the current challenges and future perspectives of CTCs and ctDNA as biomarkers in clinical oncology. Both CTCs and ctDNA are interesting complementary biomarkers that can be used in future trials assessing new drugs or drug combinations. Sensitive methods have been developed to capture CTCs in the peripheral blood and disseminated tumor cells (DTCs) in the bone marrow at the single cell level in cancer patients (Alix-Panabières and Pantel, 2014). The analysis of CTCs may provide clinically relevant information as “liquid biopsy” (Heitzer et al., 2013; Pantel and Alix-Panabières, 2013; Pantel et al., 2013; Schwarzenbach et al., 2014). Moreover, analysis of DTCs provide new insights into the process of “cancer dormancy” (LeBleu et al., 2014; Lu et al., 2011; Uhr and Pantel, 2011) and also lead to the discovery of new molecules relevant to the biology of metastasis such as the putative metastasis-suppressor RAI2 (Werner et al., 2015). For clinical use, blood analyses are much more acceptable than invasive bone marrow aspirations. At present, most CTC assays rely on epithelial markers and miss CTCs undergoing an epithelial–mesenchymal transition (EMT). New markers such as the actin bundling protein plastin-3 (Yokobori et al., 2013) are not downregulated during EMT and might therefore overcome this important limitation. Although the majority of CTC studies have been performed on patients with carcinomas, CTCs have been also recently detected in the peripheral blood of patients with primary brain tumors (Muller et al., 2014). CTC enumeration and characterization with certified systems provides reliable information on prognosis and may serve as liquid biopsy to identify therapeutic targets or mechanisms of resistance on metastatic cells such as mutations in KRAS or expression of the androgen receptor variant 7 (Alix-Panabières and Pantel, 2014). 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 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 surrogate marker for response to therapy (Bidard et al., 2014; Scher et al., 2015). In the context of recent success in antibody-mediated blockade of immune checkpoint control molecules, expression of the PD-L1 on CTCs (Mazel et al., 2015) might be of interest as potential predictive marker. Functional characterization using specialized in vitro and in vivo test systems has started (Baccelli et al., 2013; Cayrefourcq et al., 2015; Hodgkinson et al., 2014), which might provide novel insights into the biology of CTCs and serve as models for drug testing. Elevated concentrations of cell-free ctDNA fragments have been found in blood plasma and serum of cancer patients with various tumor types and associated with unfavorable outcome (Schwarzenbach and Pantel, 2015). ctDNA fragments mainly originate from apoptotic or necrotic tumor cells which discharge their DNA into the blood circulation (Schwarzenbach et al., 2011). With the development of next generation sequencing technologies, the field of ctDNA analysis – which originally started more than 20 years ago– has revived and focused on genomic aberrations relevant to therapy resistance in patients with metastatic cancer (e.g., k-ras mutations for EGFR inhibition in colorectal cancer) (Diaz et al., 2012). Besides the development of ultrasensitive technologies targeting specific gene mutations on DNA fragments present at very low concentrations (Newman et al., 2014), recent studies have used sequential ctDNA assessments to unravel the emergence of resistant tumor cell clones in cancer patients receiving systemic therapy (Dawson et al., 2013; Siravegna et al., 2015). A better understanding of resistance to therapy will eventually lead to personalized therapeutic strategies adapted to the individual needs of cancer patients. More recently, circulating microRNAs (miRNA) have extended the spectrum of liquid biopsy (Schwarzenbach et al., 2014) and even tumor-derived blood platelets appear to be promising blood-based biomarkers (Best et al., 2015; Joosse and Pantel, 2015). Microvesicles such as exosomes contain miRNAs, proteins and probably even DNA and have become interesting diagnostic tools of liquid biopsy (Melo et al., 2015) but, moreover, also affect cancer biology, e.g., the integrin composition of exosomes can govern site-directed cancer metastasis (Hoshino et al., 2015). Thus, liquid biopsy analyses will contribute to a better understanding of cancer biology and improved clinical management of cancer patients. In conclusion, the molecular and functional analysis of CTCs and circulating nucleic acids can be used as companion diagnostics to improve the stratification of therapies and to obtain insights into therapy-induced selection of cancer cells. The current issue summarizes the state-of-the-art in the rapidly emerging field of liquid biopsy focusing in particular on CTCs and ctDNA.