Circulating Tumor DNA as a Cancer Biomarker: Fact or Fiction?

The notion of utilizing cell-free DNA (cfDNA)8 in the circulation as a surrogate biomarker is not a novel concept. Mandel and Metais identified the presence of cfDNA in the blood of healthy individuals almost 60 years ago. Decades later, multiple groups were able to extend the work of Mandel and Metais into the identification of tumor-derived cfDNA—also known as circulating tumor DNA (ctDNA)—in the blood of cancer patients. These findings suggested that a “liquid biopsy” may be a feasible clinical tool because tumors seem to release fragments of DNA into the circulatory system that are both detectable and specific to the tumor. In the past decade, we have witnessed a surge in both new technologies and improvements on existing technologies for sequencing DNA that have made this once-laborious process cheaper and faster. In 2009, the cost of sequencing per genome was $100 000, whereas in 2014, this cost dropped to $5000 (taking into account labor, administration, management, utilities, reagents, and consumables). As a result, the use of ctDNA as a liquid biopsy has become ever more feasible. Clinically speaking, a ctDNA-based liquid biopsy would be the optimal mode of cancer management owing to various advantages including: (a) Retrieval of ctDNA would be minimally invasive especially compared to a tissue biopsy; (b) ctDNA could provide a full representation of the tumor (as well as any clonal metastases); and (c) ctDNA would provide a personalized snapshot of the patient's disease. Although the clinical use of ctDNA as a surrogate biomarker is still hampered by biological and technological hurdles, the implications of a liquid biopsy could be enormous as there would be numerous potential applications including (but not limited to): early detection, monitoring of minimal residual disease (MRD), assessment of treatment response, and triaging based on risk of recurrence. In this Q&A article, 4 experts offer their insight into the current state of ctDNA as a clinical tool for cancer management. Specifically, they will discuss the biological and analytical challenges this technology still faces, as well as the potential benefits of this rapidly growing area of translational research. In your opinion, what is the principal mechanism behind the release of ctDNA into the circulation? Dave S.B. Hoon: The principal mechanism can be explained by taking into consideration the multiple events associated with tumor cells. ctDNA in plasma can originate from primary, metastatic, or circulating tumor cells. It can be released from tumors by apoptosis, necrosis (programed or not), tumor cell destruction, and/or cell secretion. Likely, multiple factors affect the total ctDNA composition in the blood, which is dependent on the tumor status, burden, and histopathology. Kenneth Kinzler: One possibility is that the DNA is released by the death of circulating tumor cells. The second possibility is that the DNA is released by the death of tumor cells in the tumor bed. There are several pieces of evidence that support the latter explanation as the primary source of ctDNA. First, the majority of cases with detectable ctDNA do not have detectable circulating tumor cells. Second, in those cases where both can be detected, the level of ctDNA is 1–2 orders of magnitude greater than that present in circulating tumor cells. Third, advanced cases of cancer are often associated with increased concentrations of cfDNA from normal cells, presumably due to death and destruction of normal cells in the tumor beds. Klaus Pantel: In principle, ctDNA can be released from primary tumors, circulating tumor cells, micrometastasis, or overt metastases in cancer patients. The majority of ctDNA is most likely derived from apoptotic and necrotic tumor cells that release their fragmented DNA into the circulation. DNA is also released by nonmalignant host cells and this normal DNA dilutes the ctDNA in cancer patients, particularly in situations where tissue-damaging therapies such as chemotherapy or radiotherapy are administered. The fragment length might provide some information on the origin of the cfDNA. However, this issue is still under debate because different groups have reported different cutoff values in cancer patients. Part of these discrepancies might be due to the different technical approaches used to determine ctDNA and tumor types. Nevertheless, we still need to know more about the biology behind the release of ctDNA into the circulation. Catherine Alix-Panabières: When cancer cells die by necrosis or apoptosis, some of the released DNA ends up passively in the bloodstream, thus the release of cfDNA into the blood by dying cells is not restricted to cancer patients; in fact, cfDNA can be detected in the blood of healthy individuals, with even higher amounts in patients with benign diseases or inflammatory diseases or when subjects are aging. “Necrosis” is caused by factors external to the tumor cells or cancer tissues, which result in the unregulated digestion and release of cell components. “Apoptosis” is defined as a controlled type of cell death that can be induced by a variety of physiologic and pharmacologic agents, followed by DNA fragmentation and cell lysis. In both cases, dying tumor cells release small pieces of their fragmented DNA in the circulation. As fragmentation of ctDNA seems to be higher following apoptosis than following necrosis, it should be possible to determine how ctDNA was released in cancer patients. Specifically, ctDNA fragmented to 160–180 bp in length corresponds to nucleosome-protected DNA observed in apoptotic cells. In addition to the release of DNA by dying tumor cells, recent studies indicate that a very small amount of DNA might be released in exosomes actively by living cells but this is still controversial. How ctDNA is released into the circulation is a key question because to adopt the ctDNA as a clinical biomarker in cancer patients, it will be crucial to elucidate in the near future how the ctDNA release is related to tumor biology by understanding: (1) from which cells it is derived, (2) which specific clones contribute to total ctDNA level that reflects their clonal burden, and (3) how the ctDNA level changes over time depending on the applied cancer therapies. The source of ctDNA is of utmost importance, as it must reflect the genetic information of the clinically relevant tumor cells. As it is mainly dying tumor cells that release DNA, depending on when ctDNA is detected in blood of cancer patients, the information we can obtain from its detection may not reflect mutations of the resistant clones of tumor cells but those of sensitive subpopulations of tumor cells, mostly after the initiation of treatment—e.g., chemotherapy, targeted therapy. How is ctDNA cleared from the circulation and how does that affect its stability? Dave S.B. Hoon: Clearance of ctDNA follows similar physiological mechanisms to that of normal DNA that is released into the circulation and cleared routinely by various organs, such as tumors draining into lymph nodes, kidney, and liver. In the tumor microenvironment, lymphatic drainage is likely to clear the majority of DNA fragments that are released. The ctDNA is generally unstable except for some forms that appear to have a longer half-life for a reason that we do not fully understand. The form (i.e., exosome), size, bound molecule substance (i.e., lipid, protein), and mechanism of release collectively play a role in ctDNA clearance. Kenneth Kinzler: The rate of clearance of cfDNA from blood has been examined using exogenous DNA, fetal DNA, and tumor DNA in human and animal models. These studies all indicate that DNA is rapidly cleared from the blood. By evaluation of a single subject whose plasma was sampled at multiple times following complete tumor resection, we estimated the half-life of ctDNA after surgery to be 114 min. Dennis Lo and colleagues followed the clearance of circulating fetal DNA in 8 women after delivery and found the mean half-life for circulating fetal DNA to be 16.3 min (range 4–30 min). The mechanism of clearance for ctDNA has not been well studied but is likely to be similar to that for cfDNA from other sources and may vary with the physiological state of the patient. A combination of nuclease degradation, renal clearance, and uptake by the liver and spleen are likely to play a role. Klaus Pantel: The clearance of ctDNA is not fully understood. Previous reports indicate that ctDNA has a half-life of 16 min. However, a recent study from the same group used next generation sequencing (NGS) to study the kinetics of ctDNA, which revealed a biphasic clearance with half-lives of about 1 h for the rapid phase and 13 h for the second phase. It is assumed that the majority of ctDNA is cleared through the kidneys, which has raised recent interest to detect ctDNA in the urine, not only for cancers of the urogenital tract. It can be envisaged that the clearance rate of ctDNA is affected in patients with renal dysfunction. This aspect needs further investigation because it might be an important confounding factor modulating the amount of ctDNA in the circulation. In addition to renal clearance, DNA might be cleared by other mechanisms from the circulation. For example, DNA is sticky and may also adhere to host cells such as endothelial cells lining the blood vessels; it is not impossible that this DNA is released again into the blood. Several reports indicate that ctDNA can even be taken up by host cells and this uptake affects the biology of these cells. Thus, the mechanisms of ctDNA clearance and their effects on ctDNA stability still need further investigation. Catherine Alix-Panabières: The clearance of ctDNA, “how long it takes to be cleared and how it is done,” is currently not well understood and is still under investigation. What is known is that ctDNA has indeed a limited stability in the blood of cancer patients, as DNases also present in the blood digest it quickly. Thus, ctDNA has a short half-life of a few hours in the bloodstream, suggesting opportunities for early readouts. This genetic and fragmented material may undergo rapid changes in cancer patients and the time points of blood sampling are crucial and not easy to determine upfront when designing a clinical trial. Moreover, the release of ctDNA by dying tumor cells is most likely quite variable between cancer patients depending on the tumor type, tumor stage, response of cancer patients to therapy, tumor burden, and cell replication. ctDNA can be investigated in various regards such as point mutations, aneuploidy, rearrangements, and methylation. What are current sequencing technologies capable of investigating? Dave S.B. Hoon: There are multiple techniques available to investigate individual forms of genomic and epigenomic ctDNA aberrations. Each has its merits and some have been verified more efficiently than others in clinical trials. Most important is the specificity and sensitivity of the individual assay, which is highly related to the ctDNA clinical utility. Digital small molecule sequencing currently allows a very sensitive and specific approach for point mutation and amplification detection of ctDNA. Kenneth Kinzler: Somatic point mutations, rearrangements, aneuploidy, and methylation have all been used to identify ctDNA using NGS. These approaches for distinguishing normal from tumor DNA vary in terms of their sensitivity, specificity, and practicality when applied to ctDNA. While the specific clinical application may determine which approach is optimal, we have found that using point mutations has the best combination of attributes for most of the clinical applications that we have studied. Klaus Pantel: Highly sensitive and specific methods have been developed to detect ctDNA, such as BEAMing, Safe-SeqS, TamSeq, and digital PCR to detect single nucleotide mutations in ctDNA or whole-genome sequencing to establish copy number changes. In principle, the technologies can be divided into targeted approaches aimed to detect mutations in a set of predefined genes [e.g., KRAS proto-oncogene, GTPase (KRAS) in the context of EGFR (epidermal growth factor receptor) blockade by antibodies] or untargeted approaches (e.g., array-CGH (comparative genomic hybridization), whole-genome sequencing, or exome sequencing) aimed to screen the genome and discover new genomic aberrations, e.g., those that confer resistance to a specific targeted therapy (Murtarza et al., Nature 2013;497:108–112). The strengths and limitations of these technologies have been recently discussed in excellent reviews. In general, targeted approaches have a higher analytical sensitivity than untargeted approaches, despite strong efforts to improve detection limits. A European IMI (Innovative Medicines Initiative) consortium of more than 30 institutions from academia and industry [called CANCER-ID www.cancer-id.eu; scientific coordinator, Klaus Pantel; EFPIA (European Federation of Pharmaceutical Industries and Associations) coordinator, Thomas Schlange] is focusing now on liquid biopsies; the main goal is to validate the different cfDNA technologies side by side, first in ring experiments between different institutions and subsequently in multicenter clinical trials. Catherine Alix-Panabières: ctDNA detection among total cfDNA in plasma requires the use of molecular methods and is based on the genetic or epigenetic differences between normal and tumor-derived DNA. The targets for ctDNA detection are (1) mutations with known or predicted functional relevance (oncogene-activating mutations and tumor suppressor–inactivating mutations), (2) mutations of no or unknown functional relevance (somatic chromosomal rearrangements, noncoding DNA mutations and DNA variants), and (3) epigenetic modifications (methylation status of key sequences and histone modifications). Some of the current technologies capable of investigating ctDNA include: (i) technology combining pyrophosphorolysis-activated polymerization and allele-specific amplification during PCR; (ii) a set of digital genomic methods that improve identification of genetic alterations in ctDNA; (iii) a PCR-based method that allows single-molecule PCR reactions to be performed on magnetic beads in water-in-oil emulsions, the BEAMing approach; (iv) Other digital PCR technologies that include droplet digital PCR and PCR amplification followed by NGS. cfDNA by can determine (a) the presence of a mutation and its a and (b) the whole-genome of the of mutations in a to an about the sensitivity of technologies for ctDNA detection, it has been reported that digital PCR and BEAMing can detect point mutations with from to In addition to investigating point mutations and copy number the evaluation of the methylation status of tumor and genes in ctDNA can be by PCR these technologies have been reported with various in using cancer cell clinical and their evaluation are still in in clinical trials. we have an of new sequencing technologies such as sequencing and by What are the advantages of these technologies compared to the Dave S.B. Hoon: The techniques offer more but are not than approaches of NGS. in digital small molecule is a more highly sensitive and specific approach than NGS. is and of detection are rapidly and approaches can only be when clinical is Kenneth Kinzler: methods can detect ctDNA in advanced cases where the of ctDNA can more than of the total DNA. However, the of cases with detectable ctDNA, especially in the of early requires methods that can detect a few when they of the total DNA. digital approaches (e.g., digital BEAMing, and detection and even at these Klaus Pantel: The main of these technologies is their higher technologies have been used in studies the blood of patients with advanced cancer where the ctDNA amount is very and can of the total amount of cfDNA. However, these technologies are not able to detect amounts of ctDNA in the of normal circulating which is a clear in we extend to patients with or early detection of For these an number of more sensitive technologies have been developed that can detect ctDNA at concentrations such as for the However, now the total amount of cfDNA and the available of genome might become an important Thus, to the of these new the of the plasma needs to be increased to obtain a amount of ctDNA for In this ctDNA in early cancer patients the same as circulating tumor cell Thus, the that ctDNA will detect cancer from a of blood is Catherine Alix-Panabières: have a limited sensitivity and detect mutations that to of the total of they particularly the ctDNA amounts are whereas new technologies are able to detect very amounts of ctDNA. of genetic and epigenetic now has been by the of highly specific and sensitive techniques (e.g., from to for digital PCR and This different clinical with more sensitive we can the in the of the detection of and the detection of whereas with only advanced and diseases could be detected and What is the technical that we are still with regards to Dave S.B. Hoon: The technical or is the of ctDNA from small amounts of This of will we all the sensitivity a The most important technical is to identify what level of ctDNA is of clinical utility. This will likely vary cancer depending on the clinical status of the and what question is by the specific biomarker ctDNA. Kenneth Kinzler: technology has made detection of ctDNA feasible and future are likely to such even more and However, these current are likely to be limited by and biological than technical the side, we are limited by the number of cell present in a plasma For example, 1 of plasma about of any to detect ctDNA to 1 in a plasma the biological side, only a small of benign tumors and some tumor (e.g., release detectable concentrations of ctDNA, even when with very sensitive under near optimal Some of the limitations of using ctDNA as a biomarker may be by and or for blood tumor DNA has been to be a cancer biomarker in other available clinical such as urine, and Klaus Pantel: The technical is the identification of very amounts of ctDNA in blood variable amounts of cfDNA and the of the of genomic aberrations. the genomic are defined by the of available targeted therapy, this seems to be with the existing However, ctDNA is used for of in cancer patients or even for early detection of primary disease in the genomic in an individual are In patients with the primary tumor at a might be available for Nevertheless, the of these is highly For early detection, there is no tumor material available and the of possible genomic is for most Moreover, it is well known that mutations with even in individuals that cancer during their For example, a recent study has that up to of healthy individuals over the of years mutations but was only in a of these patients. Thus, the detection of mutations on cfDNA might not indicate that the individual has cancer or will cancer in but it might such as and to for a unknown tumor Catherine Alix-Panabières: First, need to be ctDNA in an e.g., blood plasma or blood specific to of DNA, DNA through and to the most and a complete evaluation of the analytical (e.g., sensitivity, specificity, and external as well as the time from the blood to the result to the cancer is these technical points need to be more we need to the clinical relevance of ctDNA at different time points to different key applications such as of patients for therapy, evaluation of treatment early of or treatment Moreover, for early of we have to in that (1) specific mutations must be known to detect ctDNA [e.g., KRAS or proto-oncogene, mutation in and (2) have benign tumors (e.g., which mutations that can with mutations and may findings ctDNA is used for cancer How long do it will be we the of ctDNA into clinical Dave S.B. Hoon: are currently a time ctDNA are in The ctDNA are by and also by individual patients. in a few years we may and of ctDNA for specific Kenneth Kinzler: While it is to when any new technology will be into clinical there is a growing number of studies that indicate that ctDNA can currently used clinical cancer in specific clinical would and that ctDNA will be used to clinical in an of situations over the next Klaus Pantel: The of targeted therapies is the for the of ctDNA into clinical the first ctDNA for growth factor mutations in cell cancer has been by the for the when no tumor tissue can be for mutations in genes targets and/or the resistance genes will in the near is an tumor for this application because various mutations specific targeted therapies in small of patients have been recently identified and are not in a number of patients. However, it needs to be that DNA of ctDNA tumor tissue will that the will have a and response to the The clinical of ctDNA any other needs to be in clinical studies in which therapy are based on ctDNA can the ctDNA the treatment in an individual cancer and does this to an It may years these studies are and by the The recent of the controlled phase were the use of targeted their not improve compared with treatment at in patients with ctDNA for mutations resistance to targeted therapies might be also hampered by the that we still know about the biology of ctDNA ctDNA mainly the genome of dying tumor cells but tumor cells cancer and therapy Thus, the of the time points for ctDNA will be crucial to detect those ctDNA that are derived from the resistant tumor cell In this the of circulating tumor cells as early of to therapy to tumor used in the might be Catherine Alix-Panabières: The of molecular for treatment with technological has become these years and we still that liquid biopsy will to and sensitive of and monitoring cancer in patients. However, the of ctDNA into clinical may several years as we need to what will be the best technology for ctDNA and detection, what will be the and ctDNA will be more and of current such as tissue As we need to the clinical relevance of ctDNA its clinical the key question for personalized In for primary very of cancer patients and individuals need to be including patients with benign diseases as a other approaches [e.g., for cancer or for this requires of patients and and of years or by this Q&A was the sequencing the of a new with a goal to blood that would cost than and have the to detect of cancer at The will be based on “liquid and sequencing of ctDNA. The has raised $100 and it such as and that these will by and they will be available in cell-free DNA circulating tumor DNA minimal residual disease next generation sequencing sequencing by and cell KRAS proto-oncogene, GTPase proto-oncogene, growth factor

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