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Liquid Biopsies, What We Do Not Know (Yet)
Cancer Cell
Perspective Liquid Biopsies, What We Do Not Know (Yet) Alberto Bardelli1,2,* and Klaus Pantel3,* 1University
of Torino, Department of Oncology, SP 142, Km 3.95, 10060 Candiolo, Torino, Italy Cancer Institute – FPO, IRCCS, Candiolo, Torino, Italy 3Department of Tumor Biology, Center of Experimental Medicine, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Martinistrabe 52, 20246 Hamburg, Germany *Correspondence: [email protected] (A.B.), [email protected] (K.P.) http://dx.doi.org/10.1016/j.ccell.2017.01.002 2Candiolo
The inherent molecular heterogeneity of metastatic tumors and the ability of cancer genomes to dynamically evolve are not properly captured by tissue specimens. Analysis of cell-free DNA and circulating tumor cells has the potential to change clinical practice by exploiting blood rather than tissue as a source of information. Liquid biopsies are already used to monitor disease response and track the emergence of drug resistance. The suitability of blood-based molecular profiles for early detection and monitoring minimal residual disease is being evaluated. In this review, we address open questions in this fast-evolving field of research. Introduction: The Biological Basis of Liquid Biopsies A new diagnostic concept referred to as ‘‘liquid biopsy’’ has received considerable attention over the past years (Alix-Panabie`res and Pantel, 2016; Diaz and Bardelli, 2014). Circulating tumor DNA (ctDNA) fragments are released by tumor cells into the bloodstream and in principle contain genetic defects identical to the tumor cells they originate from. Accordingly, molecular alterations, which can be detected in cell-free DNA (cfDNA) of plasma, span the types of genomic alterations identified in tumors and include point mutations, rearrangements, amplifications, and even gene copy variations (Figure 1). Although fragmented, free DNA is stable in the circulation. On the contrary, free RNA molecules do not generally survive in the bloodstream. The exceptions are cell-free microRNAs that can be detected in blood plasma or serum of cancer patients (Schwarzenbach et al., 2014). Relevant molecular information may also be obtained by analyzing RNA molecules present in extracellular vesicles such as exosomes (both coding and non-coding) or in platelets (Best et al., 2015; Joosse and Pantel, 2015). Exosomes, first isolated 30 years ago, are nanoscale vesicles shed by most types of cells. The nucleic-acid- and protein-rich content of these nanoparticles, floating in virtually all bodily fluids, has potential for non-invasive molecular diagnostics and may represent a novel therapeutic delivery system (Melo et al., 2015; Skog et al., 2008). Moreover, exosomes can also affect tumor biology, and their integrin composition might promote metastasis by determining organ-specific metastatic niches (Hoshino et al., 2015). However, current isolation techniques, such as ultracentrifugation, are not convenient and do not result in high-purity isolation. This represents an interesting challenge that can be addressed with microfluidic technologies (Liga et al., 2015), such as nano-plasmonic exosome technology, in which sensing is based on surface plasmon resonance to achieve label-free exosome detection (Im et al., 2014). In particular, the enrichment and analysis of proteins, RNA, and DNA from tumor-derived exosomes might provide unique information on viable tumor cells. Encouraging proof-of-principle studies demonstrating clinical relevance have been performed in patients with solid tumors such as pancreatic or ovarian cancer (Melo et al., 2015). 172 Cancer Cell 31, February 13, 2017 ª 2017 Elsevier Inc.
Recently, tumor-educated blood platelets (TEPs) were proposed as an alternative source of tumor-related biological information (Best et al., 2015). mRNA sequencing of TEPs from patients with different types of localized and metastasized tumors and healthy individuals detected tumors with 96% accuracy. The biology behind the potential diagnostic role of TEPs is related to the interaction between blood platelets and tumor cells that alters the RNA profile of platelets and affects tumor growth and dissemination (Kuznetsov et al., 2012). The initial studies on TEPs should now be validated in larger cohorts of individuals. In particular, confounding factors affecting the mRNA content in cancer patients undergoing treatment and suffering from comorbidities need to be evaluated. Over the past 10 years, large-scale clinical studies have focused on the use of circulating tumor cell (CTC) counts as predictors of prognosis and response to therapy, particularly in breast and prostate cancer (Alix-Panabie`res and Pantel, 2016; Krebs et al., 2014). Although the capture of CTCs from whole blood is more cumbersome than the isolation of cfDNA in plasma samples, CTCs offer the opportunity to obtain information at the DNA, RNA, and protein level. Moreover, proof-of-principle studies have shown that functional in vitro and in vivo analyses are possible (Baccelli et al., 2013; Cayrefourcq et al., 2015; Hodgkinson et al., 2014; Yu et al., 2013) in a subset of patients. In the present review, we focus on the clinical applications of ctDNA and CTCs as key components of liquid biopsies and address open questions in this fast-evolving field of research. Capturing Molecular Heterogeneity and Cancer Evolution in the ctDNA Molecular and cellular heterogeneity are hallmarks of cancer and one of the greatest challenges of tumor diagnostics and therapy (Wang et al., 2014). The genomic instability and phenotypic plasticity of cancer cells are responsible for lesion-specific genomic landscapes (de Bruin et al., 2014; Gerlinger et al., 2012; Russo et al., 2016; Siravegna et al., 2015). In contrast to single tissue biopsies, blood carries DNA derived from cancer cells located at distinct metastatic sites. (Bettegowda et al., 2014; Murtaza et al., 2015). While current evidence suggests that ctDNA and CTCs likely represent a molecular proxy of the overall disease, it
Cancer Cell
Perspective Figure 1. Liquid Biopsies for Solid Tumors The figure highlights the biological basis of liquid biopsies based on circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs). Key applications of liquid biopsies in the clinical setting are also indicated, these include tumor genotyping, assessing drug response, tracking minimal residual disease, and monitoring clonal evolution.
remains to be formally proven that multiple metastatic lesions located in different organs shed ctDNA and CTCs homogeneously. Analyses of tissue and blood from autopsies could further address how comprehensively ctDNA interrogates the heterogeneity of metastatic cancers (Alsop et al., 2016; Gerlinger et al., 2012). What are the Mechanisms through which DNA Is Released by Normal and Tumor Cells? Cancer patients have much higher levels of normal circulating cfDNA than healthy individuals. When tumors increase in volume, so too does the cellular turnover and hence the number of apoptotic and necrotic cells. Most cfDNA fragments measure between 180 and 200 bp, suggesting that apoptosis likely produces the majority of cfDNA in the circulation. Interestingly, shorter ctDNA fragments have been reported in at least some tumor types (e.g., hepatocellular carcinomas) as well as large cfDNA fragments of thousands of base pairs, which are probably the result of necrosis (Jiang and Lo, 2016). Liquid biopsies are rapidly being integrated into the clinical management of cancer patients, yet the exact nature and origin of cfDNA remains to be clarified (Thakur et al., 2014). Further progress in this area can be derived from animal models and from coupling ctDNA analyses with autopsy studies. Which Body Fluids Contain Circulating Tumor DNA? Most studies focus on cfDNA that is released in the blood (plasma or serum) of cancer patients. Notably, the categories of body fluids that can be profiled have recently expanded well beyond blood and now include urine, cerebrospinal fluid (CSF), and saliva (De Mattos-Arruda et al., 2015; Melkonyan et al., 2008; Wang et al., 2015a, 2015b). For example, recent studies have shown that trans-renal DNA can be used to detect point mutations associated with drug resistance, and accurate molecular profiles of brain tumors can be successfully obtained from CSF (De Mattos-Arruda et al., 2015; Wang et al., 2015b). The molecular landscape of head and neck cancers can be derived from saliva (Wang et al., 2015a). Urine and saliva
can be obtained daily thus offering the possibility to monitor the profile of cancer patients longitudinally. In some instances, the biology of the tumor and its anatomical location dictates which fluid is most suited for liquid biopsies. For example, while ctDNA can be found in the blood of the majority of metastatic cancers patients, the amount of cfDNA in the circulation of patients with brain tumors, such as gliomas and medulloblastomas, is often limited (Bettegowda et al., 2014). On the contrary, CSF (which is secreted by the choroid plexuses) can be successfully used to profile ctDNA in patients with tumor masses localized in the brain (De Mattos-Arruda et al., 2015; Wang et al., 2015b). Clinical Applications of Liquid Biopsy Early Detection and Genotyping of Solid Tumors: in Blood Veritas? There is an urgent need for reliable indicators of early cancer, particularly in tumor types that are usually discovered at later stages (e.g., lung and pancreatic cancer). Both CTCs and ctDNA are released by early tumor lesions and, therefore, have generated expectations by research groups and companies to develop a blood-based cancer test. Despite recent development of very sensitive technologies, developing a reliable test for early cancer detection remains challenging. In particular, applying a blood test for widespread population screening requires an exquisite specificity. Cancer-associated mutations occur with increasing age even in individuals that never develop cancer during their lifetime (Genovese et al., 2014), which can cause false-positive findings. Studies on cancer screening start usually with comparisons of cancer patients with controls (healthy individuals or patients with benign diseases). Focusing on patients with a high risk of developing cancer (e.g., chronic obstructive pulmonary disease [COPD] patients) is a valuable strategy to speed up this validation process. Ilie et al. (2014) reported that CTCs were detected in COPD patients without clinically detectable lung cancer. The study included 168 COPD patients and 77 subjects without COPD matched for smoking habits. CTCs were detected in 5 of 168 patients, while no CTCs were detected in the control group. The annual surveillance of the CTC-positive COPD patients by computed tomography (CT) scan screening detected lung nodules 1–4 years after CTC detection, leading to prompt surgical resection and histopathological diagnosis of earlystage lung cancer. These preliminary findings need to be validated in larger cohorts, and events that may lead to unspecific findings in non-cancer patients, such as the release of Cancer Cell 31, February 13, 2017 173
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Perspective non-malignant epithelial cells into the blood of patients, need to be excluded. Detection of cancer by monitoring ctDNA is also an area of active investigation (Oncology, 2016). The identification of very low amounts of ctDNA in blood samples with variable amounts of cfDNA remains, however, challenging. cfDNA is composed primarily of DNA that originates from normal cells and by a relatively small fraction derived from tumor cells. The sensitivity of standard DNA analysis approaches, such as Sanger sequencing, does not allow the detection of ctDNA (Leung et al., 2016; Siravegna and Bardelli, 2016). Besides sensitivity, the specificity of ctDNA-based tests is also challenging. Cancer-associated driver mutations occur with increasing age even in individuals who never develop cancer during their lifetime. For example, mutations in the TP53 gene have been detected in more than 10% of non-cancer controls (Fernandez-Cuesta et al., 2016; Pantel, 2016), and clonal hematopoiesis with somatic mutations was observed in 10% of individuals older than 65 years of age and was a strong risk factor for subsequent hematologic cancer. However, the absolute risk of conversion from clonal hematopoiesis to hematologic cancer was modest (1.0% per year) (Genovese et al., 2014). Recently, Kato et al. (2016) outlined the conundrum of genetic ‘‘drivers’’ in benign conditions such as BRAF mutations in nevi or FGFR3 mutations in seborrheic keratosis. Thus, the detection of cancer-associated mutations on cfDNA might not indicate that the individual tested has cancer already or will develop cancer in her/his lifetime. The interest in the liquid biopsy approach has surged remarkably and a plethora of assays are currently available. The European consortium CANCER-ID (www. cancer-id.eu) encompasses 37 institutions and provides a new platform for international assay validation. Can Liquid Biopsy Detect and Monitor Minimal Residual Disease in Early-Stage Patients? Reliable detection and quantification of minimal residual disease (MRD) is effectively employed in the management of patients with hematological malignancies but not in patients with solid tumors. Early detection of small micrometastatic lesions that are currently undetectable by clinical imaging procedures (CT or MRI scans) would largely increase the chances to prevent full-blown, incurable metastatic disease. Although the clinical relevance of ctDNA and CTCs for disease monitoring in metastatic patients is well established, the role of these biomarkers in early-stage patients remains to be established. MRD may be one key area of application for liquid biopsies (Beaver et al., 2014; Diehl et al., 2008; Tie et al., 2016). In a large study on 1,493 non-metastatic breast cancer patients, Rack et al. (2014) demonstrated that the presence of persisting CTCs after chemotherapy showed a negative influence on progression-free and overall survival. In cancer types for which adjuvant therapy has marginal benefit in a pathologically staged population or in lower-risk populations where adjuvant therapy is not currently offered, patients with detectable ctDNA or CTCs might be recruited into clinical trials to assess the benefit of intervening with adjuvant therapy. This might extend the benefit of adjuvant therapy as opposed to only monitoring its utility in populations who are already going to receive adjuvant therapy as a current standard. Randomized trials are needed to demonstrate clinical utility of this approach. 174 Cancer Cell 31, February 13, 2017
In patients with non-metastatic cancers, ctDNA-based liquid biopsies could be optimized to capture and monitor genomic markers of MRD following curative resection, possibly preceding the development of clinical or radiologic recurrence. Analogously, ctDNA analyses could be used to stratify patients who are at high risk for recurrence and spare low-risk patients from the toxicities of unnecessary systemic therapies. For example, recent studies have shown that the identification of tumor genomic alterations in the plasma of non-metastatic breast and colorectal cancer patients anticipates the diagnosis of clinical metastatic relapses (Garcia-Murillas et al., 2015; Olsson et al., 2015; Reinert et al., 2016). It was also found that post-surgery increases in ctDNA were correlated with the development of metastases at distant sites except in the brain (Bettegowda et al., 2014). In conclusion, CTC- and ctDNA-based screening of patients with higher risk of relapse may create opportunities for therapeutic interventions before the development of clinical metastasis. Monitoring stratification of high-risk patients with MRD to more intense therapy is another important application. For example, patients with Dukes B colon cancer do not receive adjuvant chemotherapy after surgical resection of the primary tumor. However, a significant portion of these patients (10%–20%) will relapse within 5 years post-surgery, and CTC detection by RT-PCR analysis of plastin-3 RNA in the peripheral blood helps to identify these high-risk patients upfront (Yokobori et al., 2013). Can We Use Liquid Biopsy to Guide Therapy? Current decision making for targeted therapy in patients with metastatic disease is based on the analysis of the primary tumor. Furthermore pharmacological therapy (chemo or targeted) is known to affect the molecular landscape of cancers. Importantly, metastatic relapse can occur many years after primary tumor resection, and the information obtained from the resected primary tumor might be outdated. Although re-staging of metastatic lesions has started to become more acceptable, biopsies of metastatic lesions are invasive procedures. On the contrary, liquid biopsy allows repeated analyses over the course of treatment, which can inform on treatment responses or the emergence of resistance in real time. In this section, we focus on the question of how molecular analyses of ctDNA and CTCs can contribute to better understand therapy resistance and improve personalized anti-cancer therapy. Breast Cancer. Patients with estrogen receptor (ER)-positive breast tumors receive endocrine therapy, but they can harbor ER-negative CTCs (Paoletti et al., 2015). This might be a potential mechanism of resistance and is currently being evaluated in clinical trials. Mutations of the ER gene are found in approximately 20% of biopsies from metastatic breast cancer patients (Toy et al., 2013). Somatic mutations in the ER gene can be readily identified in ctDNA (Chu et al., 2016). ER mutations can be detected in ctDNA of breast cancer patients in association with resistance to endocrine therapy (Chu et al., 2016; Guttery et al., 2015; Schiavon et al., 2015). These data further suggest that blood can be used to identify additional ER mutations not found by sequencing of a single metastatic lesion (Chu et al., 2016). The gene encoding the p110a catalytic domain of phosphatidylinositol 3-kinase (PI3K), PIK3CA, is the most commonly mutated oncogene in breast cancer, and more than 80% of somatic PIK3CA mutations occur in one of three recurrent hotspot
Cancer Cell
Perspective locations. Several agents targeting PIK3CA are in clinical use. Analysis of plasma-derived ctDNA for the detection of PIK3CA mutations in patients with metastatic breast cancer is feasible (Higgins et al., 2012). Notably, PIK3CA mutational status can change upon disease recurrence. PIK3CA mutations can also be detected in enriched CTC pools of metastatic breast cancer patients (Schneck et al., 2013). Analysis of isolated single CTCs resulted in higher detection rates of PIK3CA mutations and showed strong intra-patient heterogeneity in the PIK3CA mutational status (Neves et al., 2014; Pestrin et al., 2015; Polzer et al., 2014). The HER2 oncogene is amplified and overexpressed in approximately 20% of primary breast carcinomas and can be effectively blockaded pharmacologically. Although primary tumors are currently used to stratify patients to HER2-directed therapy, overt distant metastases and CTCs have discrepant HER2 statuses compared with the primary tumor in up to 30% of cases (Fehm et al., 2010). CTCs in women with advanced ER-positive/HER2-negative breast cancer can acquire a HER2positive subpopulation after multiple courses of therapy (Arteaga and Engelman, 2014). Notably, HER2+ and HER2 CTCs can interconvert spontaneously, with cells of one phenotype producing daughters of the opposite (Jordan et al., 2016), suggesting dynamic functional states within CTCs that may contribute to escape from HER2-targeted therapy and require novel therapeutic strategies. Prostate Cancer. Prostate-specific antigen (PSA) and prostatespecific membrane antigen (PSMA) are upregulated following androgen receptor (AR) activation and AR suppression, respectively. Interestingly, PSA/PSMA-based measurements on CTCs appear to be surrogates for AR signaling in CTCs, and this information might help to predict the outcome of AR-based therapy (Gorges et al., 2016b; Miyamoto et al., 2012). Recent studies focusing on prostate cancer also demonstrated that mRNA analysis of CTCs could reveal substantial information on drug sensitivity and resistance. For example, mRNA expression of AR-V7, a truncated form of AR that lacks the ligand-binding domain but remains constitutively active, in CTCs could predict failure of anti-androgen therapy with enzalutamide and abiraterone (Antonarakis et al., 2014; Steinestel et al., 2015). Mutations in the AR gene can be effectively identified in cfDNA of prostate cancer patients (Azad et al., 2015; Wyatt et al., 2016). Genomic profiling of the AR in cfDNA in metastatic castrationresistant prostate cancer (mCRPC) can provide insights into enzalutamide response and resistance (Wyatt et al., 2016). Mutations in the AR gene can also be identified in CTC-enriched peripheral blood samples from patients with castration-resistant prostate cancer (Jiang et al., 2010). AR mutations were detected in 20 of 35 CRPC patients: 19 missense mutations, 2 silent mutations, 5 deletions, and 1 insertion were observed. Recently, whole-exome sequencing of CTCs from prostate cancer patients has provided a window into tumor progression (Lohr et al., 2014). Lung Cancer. Several studies have compared the epidermal growth factor receptor (EGFR) mutational status in ctDNA and matched tissue samples in biopsies of lung cancer patients (Kuang et al., 2009; Nakamura et al., 2012; Taniguchi et al., 2011). The concordance between EGFR status in tumor and plasma/serum samples was high in the initial trials, and the US
Food and Drug Administration and the European Medicines Agency approved tests to detect EGFR mutations in this clinical setting directly from plasma if no biopsy material is available (www.accessdata.fda.gov/cdrh_docs/pdf12/P120022c.pdf; www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ ucm504488.htm;www.accessdata.fda.gov/cdrh_docs/pdf12/ P120019S007c.pdf). Analysis of CTCs from patients with metastatic non-smallcell lung cancer identified the expected EGFR-activating mutation in CTCs from 11 of 12 patients (92%) and in matched free plasma DNA from 4 of 12 patients (33%) (p = 0.009) (Maheswaran et al., 2008). The T790M mutation, which confers drug resistance, was revealed in CTCs from patients who had received tyrosine kinase inhibitors. Serial increases in CTC counts was associated with tumor progression, with the emergence of additional EGFR mutations in some cases. Recently, mutations in the KRAS and EGFR genes relevant for treatment decisions could be detected in CTCs captured by in vivo isolation and were confirmed in the primary tumors of the same patients (Gorges et al., 2016a). Colorectal Cancer. ‘‘RAS’’ wild-type colorectal cancers often respond to EGFR blockade based on the antibodies cetuximab and panitumumab (Karapetis et al., 2008; Van Cutsem et al., 2011). Several groups have reported high concordance between the mutational status of KRAS in the tissue and ctDNA of colorectal cancer patients (Bettegowda et al., 2014; Mouliere et al., 2013; Siravegna et al., 2015). The same is true for NRAS and BRAF mutations; of note, in several instances blood-based analyses detected KRAS mutations that were not detected in the surgical specimen (Siravegna et al., 2015). This likely reflects tumor heterogeneity that was missed in the tissue biopsy but captured in ctDNA and illustrates the problem of using tissue as the gold standard. ctDNA analyses were central in showing that KRAS and NRAS mutations rapidly emerge during EGFR blockade in vitro and in vivo and can often be detected before relapse is detected radiologically (Figure 2). Longitudinal ctDNA profiles of patients treated with cetuximab and panitumumab revealed that KRAS clones, which emerge during EGFR blockade, decline upon withdrawal of EGFR-specific antibodies, indicating that clonal evolution continues beyond clinical progression (Siravegna et al., 2015). In the majority of cases, the initial steps in the development of CRC involves acquisition of a mutation in adenomatous polyposis coli (APC). Mutations in APC can therefore be considered truncal (stem) events that are present in all the cells in the primary tumor and in the metastases. Detection of APC mutations in the blood of CRC patients can be used to track response to therapy and to estimate the levels of molecular heterogeneity that occur when resistance to targeted drugs emerges in distinct clones (Russo et al., 2016; Tie et al., 2015; Morelli et al., 2015). Conclusions: What Is Required to Bring Liquid Biopsy into Routine Clinical Diagnostics? The first two companion diagnostic tests for the determination of EGFR mutations in ctDNA have been approved by the regulatory agencies in Europe and in the USA. These interventional tests can now be used to guide anti-EGFR treatment in EGFRmutated non-small-cell lung cancer patients using blood when Cancer Cell 31, February 13, 2017 175
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Perspective
Figure 2. Liquid Biopsies to Monitor Cancer Evolution during Targeted Therapy Due to the intrinsic heterogeneity of human tumors, distinct drug resistance mechanisms can evolve within a single patient and even within individual metastatic lesions. Tissue biopsies are inherently unable to capture this heterogeneity while liquid biopsies can profile more comprehensively clonal evolution in space and time. As an example, we describe a patient with metastatic colorectal cancer: upon treatment with anti-EGFR antibodies, resistant clones progressively emerge carrying resistance mutations. Circulating free DNA allows identification, tracking, and quantification of clones bearing distinct alleles. Monitoring of a stem (truncal) mutation in the TP53 gene tracks tumor burden, while lesion-specific mutations (KRAS, MAPK) provide a measure of clonal evolution during therapy.
access to tissue is impaired. While these examples demonstrate the clinical applicability of ctDNA, additional interventional studies are required to demonstrate the clinical utility of a liquid biopsy, i.e., its capacity inform a decision to adopt or to reject a therapeutic action. Tissue biopsies presently remain the gold standard or reference for liquid biopsy analyses, and this concept is even adopted by regulatory agencies to approve tests. However, the use of tissue biopsies as a reference standard is questionable. Besides the intra-tumor heterogeneity of the lesion biopsied and used as a reference, ctDNA or CTCs can be derived from lesions that were not biopsied and may contain a divergent genomic composition. Even in patients with no metastatic lesions detected by current imaging modalities (stage M0), occult micrometastases may contribute to the pool of ctDNA or CTCs and, therefore, lead to different genomic landscapes than the primary tumor used as a reference. Until now, most, if not all, ctDNA-based investigations have been in essence proof-of-concept reports. Altogether these represent the foundation of future studies that will aim at using liquid biopsies to change clinical practice. Among these, the first 176 Cancer Cell 31, February 13, 2017
will likely be diagnostic assays that will exploit blood rather than tissue as a primary source of information for optimizing personalized therapy. These will be paralleled by analyses aimed at using liquid biopsies to monitor MRD in patients with solid cancers. The possibility of exploiting liquid biopsies for screening purposes (non-invasive early detection) is also fascinating and likely to attract prominent interest in the future. ACKNOWLEDGMENTS This study was supported by the European Community’s Program under grant agreement no. 602901 MErCuRIC (A.B.); the European Community‘s Horizon 2020 Program under grant agreement no. 635342-2 MoTriColor (A.B.); IMI contract no. 115749 CANCER-ID (A.B. and K.P.); the Fondazione Piemontese per la Ricerca sul Cancro-ONLUS 5 per mille 2011 Ministero della Salute (A.B.); AIRC 2010 Special Program Molecular Clinical Oncology 5 per mille, project no. 9970 Extension program (A.B.) and AIRC IG no. 16788 (A.B.); GOI-Merck’s Grant for Oncology Innovation Research Project (A.B.); the European Research Council Advanced Investigator grant 269081 DISSECT (K.P.); the European Research Council Proof-of-Concept Grant Capture-CTC (K.P.). We thank Giulia Siravegna for helpful suggestions and critical reading of the manuscript, Elizabeth Cook for drafting the figures. We apologize for all of the excellent reports that we could not cite because of space restrictions.
Cancer Cell
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Perspective Liquid Biopsies, What We Do Not Know (Yet) Alberto Bardelli1,2,* and Klaus Pantel3,* 1University
of Torino, Department of Oncology, SP 142, Km 3.95, 10060 Candiolo, Torino, Italy Cancer Institute – FPO, IRCCS, Candiolo, Torino, Italy 3Department of Tumor Biology, Center of Experimental Medicine, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Martinistrabe 52, 20246 Hamburg, Germany *Correspondence: [email protected] (A.B.), [email protected] (K.P.) http://dx.doi.org/10.1016/j.ccell.2017.01.002 2Candiolo
The inherent molecular heterogeneity of metastatic tumors and the ability of cancer genomes to dynamically evolve are not properly captured by tissue specimens. Analysis of cell-free DNA and circulating tumor cells has the potential to change clinical practice by exploiting blood rather than tissue as a source of information. Liquid biopsies are already used to monitor disease response and track the emergence of drug resistance. The suitability of blood-based molecular profiles for early detection and monitoring minimal residual disease is being evaluated. In this review, we address open questions in this fast-evolving field of research. Introduction: The Biological Basis of Liquid Biopsies A new diagnostic concept referred to as ‘‘liquid biopsy’’ has received considerable attention over the past years (Alix-Panabie`res and Pantel, 2016; Diaz and Bardelli, 2014). Circulating tumor DNA (ctDNA) fragments are released by tumor cells into the bloodstream and in principle contain genetic defects identical to the tumor cells they originate from. Accordingly, molecular alterations, which can be detected in cell-free DNA (cfDNA) of plasma, span the types of genomic alterations identified in tumors and include point mutations, rearrangements, amplifications, and even gene copy variations (Figure 1). Although fragmented, free DNA is stable in the circulation. On the contrary, free RNA molecules do not generally survive in the bloodstream. The exceptions are cell-free microRNAs that can be detected in blood plasma or serum of cancer patients (Schwarzenbach et al., 2014). Relevant molecular information may also be obtained by analyzing RNA molecules present in extracellular vesicles such as exosomes (both coding and non-coding) or in platelets (Best et al., 2015; Joosse and Pantel, 2015). Exosomes, first isolated 30 years ago, are nanoscale vesicles shed by most types of cells. The nucleic-acid- and protein-rich content of these nanoparticles, floating in virtually all bodily fluids, has potential for non-invasive molecular diagnostics and may represent a novel therapeutic delivery system (Melo et al., 2015; Skog et al., 2008). Moreover, exosomes can also affect tumor biology, and their integrin composition might promote metastasis by determining organ-specific metastatic niches (Hoshino et al., 2015). However, current isolation techniques, such as ultracentrifugation, are not convenient and do not result in high-purity isolation. This represents an interesting challenge that can be addressed with microfluidic technologies (Liga et al., 2015), such as nano-plasmonic exosome technology, in which sensing is based on surface plasmon resonance to achieve label-free exosome detection (Im et al., 2014). In particular, the enrichment and analysis of proteins, RNA, and DNA from tumor-derived exosomes might provide unique information on viable tumor cells. Encouraging proof-of-principle studies demonstrating clinical relevance have been performed in patients with solid tumors such as pancreatic or ovarian cancer (Melo et al., 2015). 172 Cancer Cell 31, February 13, 2017 ª 2017 Elsevier Inc.
Recently, tumor-educated blood platelets (TEPs) were proposed as an alternative source of tumor-related biological information (Best et al., 2015). mRNA sequencing of TEPs from patients with different types of localized and metastasized tumors and healthy individuals detected tumors with 96% accuracy. The biology behind the potential diagnostic role of TEPs is related to the interaction between blood platelets and tumor cells that alters the RNA profile of platelets and affects tumor growth and dissemination (Kuznetsov et al., 2012). The initial studies on TEPs should now be validated in larger cohorts of individuals. In particular, confounding factors affecting the mRNA content in cancer patients undergoing treatment and suffering from comorbidities need to be evaluated. Over the past 10 years, large-scale clinical studies have focused on the use of circulating tumor cell (CTC) counts as predictors of prognosis and response to therapy, particularly in breast and prostate cancer (Alix-Panabie`res and Pantel, 2016; Krebs et al., 2014). Although the capture of CTCs from whole blood is more cumbersome than the isolation of cfDNA in plasma samples, CTCs offer the opportunity to obtain information at the DNA, RNA, and protein level. Moreover, proof-of-principle studies have shown that functional in vitro and in vivo analyses are possible (Baccelli et al., 2013; Cayrefourcq et al., 2015; Hodgkinson et al., 2014; Yu et al., 2013) in a subset of patients. In the present review, we focus on the clinical applications of ctDNA and CTCs as key components of liquid biopsies and address open questions in this fast-evolving field of research. Capturing Molecular Heterogeneity and Cancer Evolution in the ctDNA Molecular and cellular heterogeneity are hallmarks of cancer and one of the greatest challenges of tumor diagnostics and therapy (Wang et al., 2014). The genomic instability and phenotypic plasticity of cancer cells are responsible for lesion-specific genomic landscapes (de Bruin et al., 2014; Gerlinger et al., 2012; Russo et al., 2016; Siravegna et al., 2015). In contrast to single tissue biopsies, blood carries DNA derived from cancer cells located at distinct metastatic sites. (Bettegowda et al., 2014; Murtaza et al., 2015). While current evidence suggests that ctDNA and CTCs likely represent a molecular proxy of the overall disease, it
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Perspective Figure 1. Liquid Biopsies for Solid Tumors The figure highlights the biological basis of liquid biopsies based on circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs). Key applications of liquid biopsies in the clinical setting are also indicated, these include tumor genotyping, assessing drug response, tracking minimal residual disease, and monitoring clonal evolution.
remains to be formally proven that multiple metastatic lesions located in different organs shed ctDNA and CTCs homogeneously. Analyses of tissue and blood from autopsies could further address how comprehensively ctDNA interrogates the heterogeneity of metastatic cancers (Alsop et al., 2016; Gerlinger et al., 2012). What are the Mechanisms through which DNA Is Released by Normal and Tumor Cells? Cancer patients have much higher levels of normal circulating cfDNA than healthy individuals. When tumors increase in volume, so too does the cellular turnover and hence the number of apoptotic and necrotic cells. Most cfDNA fragments measure between 180 and 200 bp, suggesting that apoptosis likely produces the majority of cfDNA in the circulation. Interestingly, shorter ctDNA fragments have been reported in at least some tumor types (e.g., hepatocellular carcinomas) as well as large cfDNA fragments of thousands of base pairs, which are probably the result of necrosis (Jiang and Lo, 2016). Liquid biopsies are rapidly being integrated into the clinical management of cancer patients, yet the exact nature and origin of cfDNA remains to be clarified (Thakur et al., 2014). Further progress in this area can be derived from animal models and from coupling ctDNA analyses with autopsy studies. Which Body Fluids Contain Circulating Tumor DNA? Most studies focus on cfDNA that is released in the blood (plasma or serum) of cancer patients. Notably, the categories of body fluids that can be profiled have recently expanded well beyond blood and now include urine, cerebrospinal fluid (CSF), and saliva (De Mattos-Arruda et al., 2015; Melkonyan et al., 2008; Wang et al., 2015a, 2015b). For example, recent studies have shown that trans-renal DNA can be used to detect point mutations associated with drug resistance, and accurate molecular profiles of brain tumors can be successfully obtained from CSF (De Mattos-Arruda et al., 2015; Wang et al., 2015b). The molecular landscape of head and neck cancers can be derived from saliva (Wang et al., 2015a). Urine and saliva
can be obtained daily thus offering the possibility to monitor the profile of cancer patients longitudinally. In some instances, the biology of the tumor and its anatomical location dictates which fluid is most suited for liquid biopsies. For example, while ctDNA can be found in the blood of the majority of metastatic cancers patients, the amount of cfDNA in the circulation of patients with brain tumors, such as gliomas and medulloblastomas, is often limited (Bettegowda et al., 2014). On the contrary, CSF (which is secreted by the choroid plexuses) can be successfully used to profile ctDNA in patients with tumor masses localized in the brain (De Mattos-Arruda et al., 2015; Wang et al., 2015b). Clinical Applications of Liquid Biopsy Early Detection and Genotyping of Solid Tumors: in Blood Veritas? There is an urgent need for reliable indicators of early cancer, particularly in tumor types that are usually discovered at later stages (e.g., lung and pancreatic cancer). Both CTCs and ctDNA are released by early tumor lesions and, therefore, have generated expectations by research groups and companies to develop a blood-based cancer test. Despite recent development of very sensitive technologies, developing a reliable test for early cancer detection remains challenging. In particular, applying a blood test for widespread population screening requires an exquisite specificity. Cancer-associated mutations occur with increasing age even in individuals that never develop cancer during their lifetime (Genovese et al., 2014), which can cause false-positive findings. Studies on cancer screening start usually with comparisons of cancer patients with controls (healthy individuals or patients with benign diseases). Focusing on patients with a high risk of developing cancer (e.g., chronic obstructive pulmonary disease [COPD] patients) is a valuable strategy to speed up this validation process. Ilie et al. (2014) reported that CTCs were detected in COPD patients without clinically detectable lung cancer. The study included 168 COPD patients and 77 subjects without COPD matched for smoking habits. CTCs were detected in 5 of 168 patients, while no CTCs were detected in the control group. The annual surveillance of the CTC-positive COPD patients by computed tomography (CT) scan screening detected lung nodules 1–4 years after CTC detection, leading to prompt surgical resection and histopathological diagnosis of earlystage lung cancer. These preliminary findings need to be validated in larger cohorts, and events that may lead to unspecific findings in non-cancer patients, such as the release of Cancer Cell 31, February 13, 2017 173
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Perspective non-malignant epithelial cells into the blood of patients, need to be excluded. Detection of cancer by monitoring ctDNA is also an area of active investigation (Oncology, 2016). The identification of very low amounts of ctDNA in blood samples with variable amounts of cfDNA remains, however, challenging. cfDNA is composed primarily of DNA that originates from normal cells and by a relatively small fraction derived from tumor cells. The sensitivity of standard DNA analysis approaches, such as Sanger sequencing, does not allow the detection of ctDNA (Leung et al., 2016; Siravegna and Bardelli, 2016). Besides sensitivity, the specificity of ctDNA-based tests is also challenging. Cancer-associated driver mutations occur with increasing age even in individuals who never develop cancer during their lifetime. For example, mutations in the TP53 gene have been detected in more than 10% of non-cancer controls (Fernandez-Cuesta et al., 2016; Pantel, 2016), and clonal hematopoiesis with somatic mutations was observed in 10% of individuals older than 65 years of age and was a strong risk factor for subsequent hematologic cancer. However, the absolute risk of conversion from clonal hematopoiesis to hematologic cancer was modest (1.0% per year) (Genovese et al., 2014). Recently, Kato et al. (2016) outlined the conundrum of genetic ‘‘drivers’’ in benign conditions such as BRAF mutations in nevi or FGFR3 mutations in seborrheic keratosis. Thus, the detection of cancer-associated mutations on cfDNA might not indicate that the individual tested has cancer already or will develop cancer in her/his lifetime. The interest in the liquid biopsy approach has surged remarkably and a plethora of assays are currently available. The European consortium CANCER-ID (www. cancer-id.eu) encompasses 37 institutions and provides a new platform for international assay validation. Can Liquid Biopsy Detect and Monitor Minimal Residual Disease in Early-Stage Patients? Reliable detection and quantification of minimal residual disease (MRD) is effectively employed in the management of patients with hematological malignancies but not in patients with solid tumors. Early detection of small micrometastatic lesions that are currently undetectable by clinical imaging procedures (CT or MRI scans) would largely increase the chances to prevent full-blown, incurable metastatic disease. Although the clinical relevance of ctDNA and CTCs for disease monitoring in metastatic patients is well established, the role of these biomarkers in early-stage patients remains to be established. MRD may be one key area of application for liquid biopsies (Beaver et al., 2014; Diehl et al., 2008; Tie et al., 2016). In a large study on 1,493 non-metastatic breast cancer patients, Rack et al. (2014) demonstrated that the presence of persisting CTCs after chemotherapy showed a negative influence on progression-free and overall survival. In cancer types for which adjuvant therapy has marginal benefit in a pathologically staged population or in lower-risk populations where adjuvant therapy is not currently offered, patients with detectable ctDNA or CTCs might be recruited into clinical trials to assess the benefit of intervening with adjuvant therapy. This might extend the benefit of adjuvant therapy as opposed to only monitoring its utility in populations who are already going to receive adjuvant therapy as a current standard. Randomized trials are needed to demonstrate clinical utility of this approach. 174 Cancer Cell 31, February 13, 2017
In patients with non-metastatic cancers, ctDNA-based liquid biopsies could be optimized to capture and monitor genomic markers of MRD following curative resection, possibly preceding the development of clinical or radiologic recurrence. Analogously, ctDNA analyses could be used to stratify patients who are at high risk for recurrence and spare low-risk patients from the toxicities of unnecessary systemic therapies. For example, recent studies have shown that the identification of tumor genomic alterations in the plasma of non-metastatic breast and colorectal cancer patients anticipates the diagnosis of clinical metastatic relapses (Garcia-Murillas et al., 2015; Olsson et al., 2015; Reinert et al., 2016). It was also found that post-surgery increases in ctDNA were correlated with the development of metastases at distant sites except in the brain (Bettegowda et al., 2014). In conclusion, CTC- and ctDNA-based screening of patients with higher risk of relapse may create opportunities for therapeutic interventions before the development of clinical metastasis. Monitoring stratification of high-risk patients with MRD to more intense therapy is another important application. For example, patients with Dukes B colon cancer do not receive adjuvant chemotherapy after surgical resection of the primary tumor. However, a significant portion of these patients (10%–20%) will relapse within 5 years post-surgery, and CTC detection by RT-PCR analysis of plastin-3 RNA in the peripheral blood helps to identify these high-risk patients upfront (Yokobori et al., 2013). Can We Use Liquid Biopsy to Guide Therapy? Current decision making for targeted therapy in patients with metastatic disease is based on the analysis of the primary tumor. Furthermore pharmacological therapy (chemo or targeted) is known to affect the molecular landscape of cancers. Importantly, metastatic relapse can occur many years after primary tumor resection, and the information obtained from the resected primary tumor might be outdated. Although re-staging of metastatic lesions has started to become more acceptable, biopsies of metastatic lesions are invasive procedures. On the contrary, liquid biopsy allows repeated analyses over the course of treatment, which can inform on treatment responses or the emergence of resistance in real time. In this section, we focus on the question of how molecular analyses of ctDNA and CTCs can contribute to better understand therapy resistance and improve personalized anti-cancer therapy. Breast Cancer. Patients with estrogen receptor (ER)-positive breast tumors receive endocrine therapy, but they can harbor ER-negative CTCs (Paoletti et al., 2015). This might be a potential mechanism of resistance and is currently being evaluated in clinical trials. Mutations of the ER gene are found in approximately 20% of biopsies from metastatic breast cancer patients (Toy et al., 2013). Somatic mutations in the ER gene can be readily identified in ctDNA (Chu et al., 2016). ER mutations can be detected in ctDNA of breast cancer patients in association with resistance to endocrine therapy (Chu et al., 2016; Guttery et al., 2015; Schiavon et al., 2015). These data further suggest that blood can be used to identify additional ER mutations not found by sequencing of a single metastatic lesion (Chu et al., 2016). The gene encoding the p110a catalytic domain of phosphatidylinositol 3-kinase (PI3K), PIK3CA, is the most commonly mutated oncogene in breast cancer, and more than 80% of somatic PIK3CA mutations occur in one of three recurrent hotspot
Cancer Cell
Perspective locations. Several agents targeting PIK3CA are in clinical use. Analysis of plasma-derived ctDNA for the detection of PIK3CA mutations in patients with metastatic breast cancer is feasible (Higgins et al., 2012). Notably, PIK3CA mutational status can change upon disease recurrence. PIK3CA mutations can also be detected in enriched CTC pools of metastatic breast cancer patients (Schneck et al., 2013). Analysis of isolated single CTCs resulted in higher detection rates of PIK3CA mutations and showed strong intra-patient heterogeneity in the PIK3CA mutational status (Neves et al., 2014; Pestrin et al., 2015; Polzer et al., 2014). The HER2 oncogene is amplified and overexpressed in approximately 20% of primary breast carcinomas and can be effectively blockaded pharmacologically. Although primary tumors are currently used to stratify patients to HER2-directed therapy, overt distant metastases and CTCs have discrepant HER2 statuses compared with the primary tumor in up to 30% of cases (Fehm et al., 2010). CTCs in women with advanced ER-positive/HER2-negative breast cancer can acquire a HER2positive subpopulation after multiple courses of therapy (Arteaga and Engelman, 2014). Notably, HER2+ and HER2 CTCs can interconvert spontaneously, with cells of one phenotype producing daughters of the opposite (Jordan et al., 2016), suggesting dynamic functional states within CTCs that may contribute to escape from HER2-targeted therapy and require novel therapeutic strategies. Prostate Cancer. Prostate-specific antigen (PSA) and prostatespecific membrane antigen (PSMA) are upregulated following androgen receptor (AR) activation and AR suppression, respectively. Interestingly, PSA/PSMA-based measurements on CTCs appear to be surrogates for AR signaling in CTCs, and this information might help to predict the outcome of AR-based therapy (Gorges et al., 2016b; Miyamoto et al., 2012). Recent studies focusing on prostate cancer also demonstrated that mRNA analysis of CTCs could reveal substantial information on drug sensitivity and resistance. For example, mRNA expression of AR-V7, a truncated form of AR that lacks the ligand-binding domain but remains constitutively active, in CTCs could predict failure of anti-androgen therapy with enzalutamide and abiraterone (Antonarakis et al., 2014; Steinestel et al., 2015). Mutations in the AR gene can be effectively identified in cfDNA of prostate cancer patients (Azad et al., 2015; Wyatt et al., 2016). Genomic profiling of the AR in cfDNA in metastatic castrationresistant prostate cancer (mCRPC) can provide insights into enzalutamide response and resistance (Wyatt et al., 2016). Mutations in the AR gene can also be identified in CTC-enriched peripheral blood samples from patients with castration-resistant prostate cancer (Jiang et al., 2010). AR mutations were detected in 20 of 35 CRPC patients: 19 missense mutations, 2 silent mutations, 5 deletions, and 1 insertion were observed. Recently, whole-exome sequencing of CTCs from prostate cancer patients has provided a window into tumor progression (Lohr et al., 2014). Lung Cancer. Several studies have compared the epidermal growth factor receptor (EGFR) mutational status in ctDNA and matched tissue samples in biopsies of lung cancer patients (Kuang et al., 2009; Nakamura et al., 2012; Taniguchi et al., 2011). The concordance between EGFR status in tumor and plasma/serum samples was high in the initial trials, and the US
Food and Drug Administration and the European Medicines Agency approved tests to detect EGFR mutations in this clinical setting directly from plasma if no biopsy material is available (www.accessdata.fda.gov/cdrh_docs/pdf12/P120022c.pdf; www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ ucm504488.htm;www.accessdata.fda.gov/cdrh_docs/pdf12/ P120019S007c.pdf). Analysis of CTCs from patients with metastatic non-smallcell lung cancer identified the expected EGFR-activating mutation in CTCs from 11 of 12 patients (92%) and in matched free plasma DNA from 4 of 12 patients (33%) (p = 0.009) (Maheswaran et al., 2008). The T790M mutation, which confers drug resistance, was revealed in CTCs from patients who had received tyrosine kinase inhibitors. Serial increases in CTC counts was associated with tumor progression, with the emergence of additional EGFR mutations in some cases. Recently, mutations in the KRAS and EGFR genes relevant for treatment decisions could be detected in CTCs captured by in vivo isolation and were confirmed in the primary tumors of the same patients (Gorges et al., 2016a). Colorectal Cancer. ‘‘RAS’’ wild-type colorectal cancers often respond to EGFR blockade based on the antibodies cetuximab and panitumumab (Karapetis et al., 2008; Van Cutsem et al., 2011). Several groups have reported high concordance between the mutational status of KRAS in the tissue and ctDNA of colorectal cancer patients (Bettegowda et al., 2014; Mouliere et al., 2013; Siravegna et al., 2015). The same is true for NRAS and BRAF mutations; of note, in several instances blood-based analyses detected KRAS mutations that were not detected in the surgical specimen (Siravegna et al., 2015). This likely reflects tumor heterogeneity that was missed in the tissue biopsy but captured in ctDNA and illustrates the problem of using tissue as the gold standard. ctDNA analyses were central in showing that KRAS and NRAS mutations rapidly emerge during EGFR blockade in vitro and in vivo and can often be detected before relapse is detected radiologically (Figure 2). Longitudinal ctDNA profiles of patients treated with cetuximab and panitumumab revealed that KRAS clones, which emerge during EGFR blockade, decline upon withdrawal of EGFR-specific antibodies, indicating that clonal evolution continues beyond clinical progression (Siravegna et al., 2015). In the majority of cases, the initial steps in the development of CRC involves acquisition of a mutation in adenomatous polyposis coli (APC). Mutations in APC can therefore be considered truncal (stem) events that are present in all the cells in the primary tumor and in the metastases. Detection of APC mutations in the blood of CRC patients can be used to track response to therapy and to estimate the levels of molecular heterogeneity that occur when resistance to targeted drugs emerges in distinct clones (Russo et al., 2016; Tie et al., 2015; Morelli et al., 2015). Conclusions: What Is Required to Bring Liquid Biopsy into Routine Clinical Diagnostics? The first two companion diagnostic tests for the determination of EGFR mutations in ctDNA have been approved by the regulatory agencies in Europe and in the USA. These interventional tests can now be used to guide anti-EGFR treatment in EGFRmutated non-small-cell lung cancer patients using blood when Cancer Cell 31, February 13, 2017 175
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Perspective
Figure 2. Liquid Biopsies to Monitor Cancer Evolution during Targeted Therapy Due to the intrinsic heterogeneity of human tumors, distinct drug resistance mechanisms can evolve within a single patient and even within individual metastatic lesions. Tissue biopsies are inherently unable to capture this heterogeneity while liquid biopsies can profile more comprehensively clonal evolution in space and time. As an example, we describe a patient with metastatic colorectal cancer: upon treatment with anti-EGFR antibodies, resistant clones progressively emerge carrying resistance mutations. Circulating free DNA allows identification, tracking, and quantification of clones bearing distinct alleles. Monitoring of a stem (truncal) mutation in the TP53 gene tracks tumor burden, while lesion-specific mutations (KRAS, MAPK) provide a measure of clonal evolution during therapy.
access to tissue is impaired. While these examples demonstrate the clinical applicability of ctDNA, additional interventional studies are required to demonstrate the clinical utility of a liquid biopsy, i.e., its capacity inform a decision to adopt or to reject a therapeutic action. Tissue biopsies presently remain the gold standard or reference for liquid biopsy analyses, and this concept is even adopted by regulatory agencies to approve tests. However, the use of tissue biopsies as a reference standard is questionable. Besides the intra-tumor heterogeneity of the lesion biopsied and used as a reference, ctDNA or CTCs can be derived from lesions that were not biopsied and may contain a divergent genomic composition. Even in patients with no metastatic lesions detected by current imaging modalities (stage M0), occult micrometastases may contribute to the pool of ctDNA or CTCs and, therefore, lead to different genomic landscapes than the primary tumor used as a reference. Until now, most, if not all, ctDNA-based investigations have been in essence proof-of-concept reports. Altogether these represent the foundation of future studies that will aim at using liquid biopsies to change clinical practice. Among these, the first 176 Cancer Cell 31, February 13, 2017
will likely be diagnostic assays that will exploit blood rather than tissue as a primary source of information for optimizing personalized therapy. These will be paralleled by analyses aimed at using liquid biopsies to monitor MRD in patients with solid cancers. The possibility of exploiting liquid biopsies for screening purposes (non-invasive early detection) is also fascinating and likely to attract prominent interest in the future. ACKNOWLEDGMENTS This study was supported by the European Community’s Program under grant agreement no. 602901 MErCuRIC (A.B.); the European Community‘s Horizon 2020 Program under grant agreement no. 635342-2 MoTriColor (A.B.); IMI contract no. 115749 CANCER-ID (A.B. and K.P.); the Fondazione Piemontese per la Ricerca sul Cancro-ONLUS 5 per mille 2011 Ministero della Salute (A.B.); AIRC 2010 Special Program Molecular Clinical Oncology 5 per mille, project no. 9970 Extension program (A.B.) and AIRC IG no. 16788 (A.B.); GOI-Merck’s Grant for Oncology Innovation Research Project (A.B.); the European Research Council Advanced Investigator grant 269081 DISSECT (K.P.); the European Research Council Proof-of-Concept Grant Capture-CTC (K.P.). We thank Giulia Siravegna for helpful suggestions and critical reading of the manuscript, Elizabeth Cook for drafting the figures. We apologize for all of the excellent reports that we could not cite because of space restrictions.
Cancer Cell
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