CHAPTER THREE
`
`Advances in Circulating Tumor
`DNA Analysis
`Samantha Perakis*, Martina Auer*, Jelena Belic*, Ellen Heitzer*,1
`*Institute of Human Genetics, Medical University of Graz, Graz, Austria
`1Corresponding author: e-mail address: ellen.heitzer@medunigraz.at
`
`Contents
`
`1.
`Introduction
`2. History and Landmark Developments
`2.1
`ctDNA as a Potential Cancer Biomarker
`2.2
`Technological Improvements
`3. The Biology of Circulating Tumor DNA
`3.1
`The Nature of Circulating Tumor DNA
`3.2
`Release and Clearance
`3.3 Origin of cfDNA
`3.4 Nucleosome Occupancy
`4. Preanalytical and Analytical Considerations
`4.1
`Blood Sampling and Storage
`4.2
`Serum vs Plasma
`4.3
`Extraction and Quantification of cfDNA
`5. Methods
`5.1 High Resolution Methods
`5.2
`Error Suppression in NGS Data
`5.3
`Panel Sequencing for the Detection of Point Mutations
`5.4
`Exome Sequencing
`5.5
`Comprehensive Genome-Wide Approaches
`6. Clinical Use of ctDNA
`7. ctDNA in Breast Cancer
`7.1
`Early Detection of Breast Cancer
`7.2
`Assessment of ctDNA at Baseline as a Prognostic Marker in Breast Cancer
`7.3
`Longitudinal Monitoring of Therapy Response
`7.4
`Early Detection of Recurrence
`7.5 Detection of Subclonal Mutations in Plasma DNA
`8. ctDNA in Ovarian Cancer
`9. ctDNA in CRC
`9.1
`ctDNA as a Prognostic Maker
`9.2 Monitoring Treatment Response
`9.3
`Concordance Between Mutations Tumor and Plasma DNA
`
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`Advances in Clinical Chemistry, Volume 80
`ISSN 0065-2423
`http://dx.doi.org/10.1016/bs.acc.2016.11.005
`
`# 2017 Elsevier Inc.
`All rights reserved.
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`10. Prostate Cancer
`10.1 Application of ctDNA in PCa
`10.2 Monitoring Antiandrogen Treatments
`11. Lung Cancer
`11.1 Mutation Detection in ctDNA of Lung Cancer Patients
`11.2 Clinical Utility of ctDNA for the Detection of Resistance in NSCLC
`12. Melanoma
`12.1 Detection of BRAF Mutations in ctDNA
`13. Conclusion and Outlook
`References
`
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`Abstract
`
`The analysis of cell-free circulating tumor DNA (ctDNA) is a very promising tool and
`might revolutionize cancer care with respect to early detection, identification of mini-
`mal residual disease, assessment of treatment response, and monitoring tumor evolu-
`tion. ctDNA analysis, often referred to as “liquid biopsy” offers what tissue biopsies
`cannot—a continuous monitoring of tumor-specific changes during the entire course
`of the disease. Owing to technological improvements, efforts for the establishment of
`preanalytical and analytical benchmark, and the inclusion of ctDNA analyses in clinical
`trial, an actual clinical implementation has come within easy reach. In this chapter,
`recent advances of the analysis of ctDNA are summarized starting from the discovery
`of cell-free DNA, to methodological approaches and the clinical applicability.
`
`ABBREVIATIONS
`ARMS amplification refractory mutation system
`BEAMing beads, emulsion, amplification, and magnetics
`CAPP-Seq cancer personalized profiling by deep sequencing
`castPCR competitive allele-specific TaqMan PCR
`cfDNA cell-free DNA
`cffDNA cell-free fetal DNA
`ctDNA cell-free circulating tumor DNA
`CTCs circulating tumor cells
`COLD-PCR coamplification at lower denaturation temperature-PCR
`CRC colorectal cancer
`DISSECT differential strand separation at critical temperature
`dPCR digital PCR
`ddPCR digital droplet PCR
`EQA external quality assessment
`gDNA genomic DNA
`GE genome equivalent
`iDES integrated digital error suppression
`LNA locked nucleic acid
`LOD limit of detection
`LOH loss of heterozygosity
`mCRC metastatic CRC
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`MRD minimal residual disease
`MSI microsatellite instability
`NGS next-generation sequencing
`NSCLC nonsmall cell lung cancer
`OS overall survival
`PCR polymerase chain reaction
`PFS progression-free survival
`PNA peptide nucleic acids
`QPCR quantitative polymerase chain reaction
`SCNA somatic copy number alteration
`SOP standard operating procedure
`UV ultraviolet
`RCA rolling circle amplification
`TAm-Seq tagged-amplicon deep sequencing
`TKIs tyrosine kinase inhibitors
`UID unique identifier
`
`1. INTRODUCTION
`
`“Written in blood—DNA circulating in the bloodstream could guide cancer
`treatment—if researchers can work out how best to use it”: this is what Nature fea-
`tured in July 2014 [1]. In this chapter, the potential of cell-free circulating
`tumor DNA (ctDNA) was discussed with experts in the field. All experts
`agreed that the analysis of ctDNA is a very promising tool and might
`revolutionize cancer care with respect to early detection, identification of
`minimal residual disease (MRD), assessment of treatment response, and
`monitoring tumor evolution. Since then, many hurdles have been over-
`come while other issues have remained. On the one hand, technological
`improvements now allow the analysis of extremely rare alleles and a number
`of clinical trials have implemented ctDNA in their designs. Likewise, efforts
`are being made in order to establish benchmarks for the analysis of ctDNA,
`which is a crucial point for implementation in clinical routine. On the other
`hand, we lack a consensus on how to best use the available methods and the
`actual benefit for patients in terms of survival has yet to be proven. Finally,
`we still have much to learn about the biology and dynamics of ctDNA.
`What exactly is the actual benefit of the so-called “liquid biopsy,”
`i.e., the analysis of ctDNA from blood? Targeted therapies have dramatically
`improved response rates, survival and time to therapy failure in the last years,
`yet cancer is one of the most common causes of death worldwide [2].
`Molecular profiling of tumors is a central element in the management of
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`many patients with cancer and is used to determine molecular targets at a
`single time point before treatment commences. Obtaining tumor material
`requires an invasive intervention, which not only carries some risks for
`patients, but also is a costly and time-consuming procedure. Moreover, a
`tissue biopsy only provides a snapshot of the molecular aberration in the
`tumor and may not be a true representation of the molecular profile. In addi-
`tion to tumor heterogeneity at the time of diagnosis/biopsy, tumors are con-
`stantly evolving during the course of disease or under the selective pressure
`of a specific treatment. A liquid biopsy offers what tissue biopsies cannot—a
`continuous monitoring of tumor-specific changes during the entire course
`of the disease. In the last few years, it has been shown that ctDNA reflects the
`molecular landscape of a tumor and its metastases and therefore can reflect
`the efficiency and relevance of the chosen treatment specific for a molecular
`target or indicate the emergence of any resistance-conferring mechanisms.
`Furthermore, progression or recurrence can be predicted before it is clini-
`cally or radiologically obvious.
`The potential and benefit of ctDNA analyses are discussed extensively
`elsewhere [3–10]. The purpose of this chapter is to summarize recent advances
`in the analysis of ctDNA. First, we give a brief overview about the discovery
`of ctDNA and associated landmark developments. Second, we present the
`current knowledge of the biology of DNA. Third, we summarize pre-
`analytical and analytical considerations and highlight some of the new meth-
`odological developments. Finally, we present recent data on the clinical utility
`of ctDNA analysis for the most common tumor entities. Owing to the vast
`amount of published data, we were not able to include all available studies.
`Moreover, although there are also many studies on the analysis of epigenetic
`changes in ctDNA, we mainly focused on genetic changes.
`
`2. HISTORY AND LANDMARK DEVELOPMENTS
`
`Although the structure of DNA was only first described by Watson
`and Crick [11], the presence of DNA in human plasma of healthy and sick
`individuals was already described by Mandel and Metais [12]. However, this
`finding did not gain too much attention and it took almost 30 years until the
`discovery was revived. After a long period of silence, Tan et al. reported
`about high levels of circulating DNA in patients with systemic lupus
`erythematosus
`(SLE) using both the diphenylamine reaction and
`gel-diffusion studies [13]. This observation finally drew attention to the fact
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`that cell-free DNA (cfDNA) circulates in plasma/serum. Moreover, in a
`pioneering work, Stroun and Anker demonstrated the spontaneous release
`of nucleic acids by living frog auricles, which helped promote further inter-
`est in this field [14]. Shortly after, Koffler et al. demonstrated increased levels
`of cfDNA in the serum of patients suffering from SLE or rheumatoid arthri-
`tis [15]. Steinman initially challenged these findings since he was unable to
`identify any DNA in plasma of healthy individuals [16]. He assumed that the
`appearance of cfDNA in the circulation is truly pathological; therefore, he
`suggested that only plasma should be measured [16]. Similar results were
`reported by Davis and Davis, who stated that serum is not suitable for the
`analysis of circulating DNA since genomic DNA (gDNA) is sporadically
`released into serum during the clotting process [17]. In 1977, Leon et al. first
`demonstrated a prognostic value of the cfDNA in rheumatoid arthritis
`patients [18]. High levels of cfDNA were commonly found in patients with
`more severe symptoms, who had active rheumatoid arthritis for less than
`10 years, whereas patients with longer duration of disease showed lower
`levels of DNA [19]. In contrast, using a highly sensitive and specific radio-
`assay, Cox and Gokcen found DNA as a normal constituent of both serum
`and plasma [20]. In the same year, Anker et al. postulated that human blood
`leucocytes spontaneously release DNA when incubated in vitro [21]. In the
`early 1980s, the value of circulating DNA concentrations in the diagnosis of
`pulmonary embolism (PE) or acute myocardial infarction was investigated
`[22–24], but these studies revealed discrepant results. Some of these land-
`mark developments are displayed in Fig. 1.
`
`2.1 ctDNA as a Potential Cancer Biomarker
`The use of circulating DNA as a potential cancer biomarker was discovered
`in 1977 when Leon et al. reported elevated levels of cfDNA in the circu-
`lation of cancer patients (Fig. 1). In some patients, even a decrease of
`cfDNA after successful anticancer therapy was observed [19]. Using a radio-
`immunoassay for quantification, the absolute amounts of cfDNA were deter-
`mined in the serum of 173 patients with various types of cancer and in
`55 healthy individuals. The authors found significantly higher DNA levels
`in the serum of patients with metastatic disease, although no correlation
`between DNA levels and the size or location of the primary tumor could
`be seen. Those patients with decreasing cfDNA levels under therapy, how-
`ever, showed shrinkage of tumor size and a reduction of pain. On the con-
`trary, when cfDNA levels either increased or remained unchanged, a lack
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`Fig. 1 Timeline of selected landmark developments and recent advances in ctDNA
`analysis.
`
`of response to the treatment was noted [19]. A few years later, Stroun et al.
`provided the first evidence that DNA found in the circulation of
`cancer patients was derived from cancer cells using a method based on the
`decreased strand stability of cancer cell DNA (Fig. 1) [25]. Consistent with
`the study from Leon et al., the authors reported elevated cfDNA levels in can-
`cer patients compared to healthy individuals; however, it turned out that the
`presence of increased amounts of cfDNA alone is not a sufficiently strong
`biomarker for the diagnosis of cancer [25]. Soon it became clear that the
`detection of tumor-specific changes could be a potentially successful appli-
`cation of cfDNA as a clinical biomarker. The first direct detection of
`tumor-derived DNA was demonstrated in a study by Sorensen et al.
`(Fig. 1) [26]. Using allele-specific amplification and direct sequencing of
`amplified polymerase chain reaction (PCR) products, the authors demon-
`strated the occurrence of mutant KRAS fragments in the plasma of patients
`with pancreatic adenocarcinoma. Shortly after, it was recognized that plasma
`DNA is likely to be associated with apoptosis and might be used as a cell death
`marker since it circulates mainly as mononucleosomes (Fig. 1) [27].
`All these studies formed the foundation for the clinical use of ctDNA for
`detection of malignant tumors, evaluating the efficacy of treatment, and for
`detection of tumor recurrences. Nevertheless, the major breakthrough of
`circulating tumor DNA analyses was delayed in comparison to the
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`applications based on cell-free fetal DNA (cffDNA). Although several
`groups were able to detect a variety of tumor-specific alterations such as
`microsatellite instability (MSI), loss of heterozygosity (LOH), point muta-
`tions, and aberrant methylation in plasma, and it was suggested that the level
`of tumor-specific DNA reflects the tumor burden [28–35], the first evidence
`for the presence of fetal DNA in the circulation of pregnant women attracted
`much more attention (Fig. 1) [36]. This discovery followed a series of further
`studies and developments of tests for fetal sex [36], Rh factor [37], genetic
`disorders like ß-thalassemia [38], and trisomy [39]. This pioneering work
`mainly from Dennis Lo’s group has revolutionized prenatal genetic testing
`by providing a noninvasive source of fetal genetic material and today a vari-
`ety of commercially available noninvasive prenatal tests are on the market
`and several million pregnant women have already made use of these tests.
`
`2.2 Technological Improvements
`Progress in the analysis of cfDNA in cancer patients has been made at the
`turn of the millennium on the one hand due to technical improvements,
`and on the other hand due to the recognition of the clinical utility of ctDNA
`analyses with respect to predictive, preventive, and personalized medicine.
`Previously, targeted approaches, i.e., the analysis of single or a few targets
`that are commonly mutated, have mostly been used. Several groups were
`able to detect tumor-specific mutation in known driver genes such as
`APC, KRAS, and TP53 mutations in the plasma of cancer patients with
`different tumor entities [40–45]. In some studies, it turned out that, despite
`the assumption that ctDNA levels correlate with tumor burden, occasion-
`ally, patients with large tumors had no detectable amounts of ctDNA in
`their circulation. Although the reasons for these variable results remained
`unknown, the need for the development of more sensitive assays arose.
`Owing to technological advances, the analytical sensitivity of ctDNA
`detection improved dramatically in recent years and today new techno-
`logies including ARMS (amplification refractory mutation system) [46],
`BEAMing (beads, emulsion, amplification, and magnetics) [47], digital
`PCR (dPCR) [48,49], or molecular barcoding and error suppression strat-
`egies [50,51] allow for the identification of mutant alleles frequencies far
`below 1%. For example, using BEAMing, Diehl et al. were able to detect
`mutant allele frequencies down to 0.01% (Fig. 1) [47]. In a follow-up study,
`the authors suggested ctDNA measurements could be used to reliably mon-
`itor tumor dynamics [52]. In breast cancer patients, Dawson et al. were able
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`to demonstrate that ctDNA levels showed a that greater dynamic range and
`greater correlation with changes in tumor burden than the conventional
`tumor marker CA15.3 or circulating tumor cells (CTCs) (Fig. 1) [53]. In
`a follow-up study performed by same group, the first exome-wide sequenc-
`ing analysis of tumor cfDNA was performed and the authors were able to
`track acquired resistance to cancer therapy in almost all patients (Fig. 1).
`In 2012, the first study showing that molecular alterations of KRAS are
`causally associated with the onset of acquired resistance to anti-EGFR treat-
`ment in colorectal cancers (CRCs) and that these alterations can be detected
`noninvasively months before radiographic progression was published [54].
`The first whole-genome analyses of cfDNA were conducted by Leary
`et al., who demonstrated the feasibility of directly detecting chromosomal
`alterations in the plasma of cancer patients (Fig. 1) [55]. The reconstruction
`of genome-wide somatic copy number alterations (SCNAs) was shortly after
`demonstrated by two other groups [56–58], whereas the Dennis Lo group
`further developed the low-coverage WGS approach by a combined assess-
`ment of hypomethylation and cancer-associated copy number aberrations
`(Fig. 1) [59]. These developments enabled, in addition to the analysis of
`point mutations, the detection of resistance mechanisms based on SCNAs
`such as acquired gains of KRAS, MET, or ERBB2 that occurred either as
`novel focal amplifications or as high-level polysomy of chromosomes [60].
`The relevance of noninvasive analyses of acquired resistance to cancer
`therapy by sequencing of plasma DNA has ever since increased in the past
`few years and some noninvasive tests, e.g., detection of resistance conferring
`the T790M mutation in EGFR, are already in clinical use.
`In 2003, the pioneers of the cfDNA field Anker and Stroun suggested
`that it would be worthwhile to initiate large-scale clinical trials in order
`to prove the clinical benefit of cfDNA analyses [61]. However, the concept
`of ctDNA as a “liquid biopsy” has only recently been continuously imple-
`mented in clinical trials in order to evaluate the actual clinical utility of liquid
`tumor profiling [62–68]. Several prospective, randomized trials are currently
`ongoing in order to truly demonstrate the long-term clinical benefit of
`ctDNA analyses for cancer patients (Table 1). To this end, large patient
`populations under homogeneous types of therapy have to be analyzed pro-
`spectively with objectifiable, favorable, and nonfavorable outcomes. Only
`then will we know with certainty whether the analysis of ctDNA can main-
`tain its simple and reasonable promises.
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`Table 1 Selected Randomized Trials Which Include ctDNA Analysis
`
`Trial (Official Title)
`
`Description
`
`Condition
`
`Study Design
`
`ClinicalTrials.
`gov Identifier
`
`Status
`
`References
`
`A Phase III Randomized,
`Double-Blind Placebo-Controlled
`Study of BKM120 With
`Fulvestrant, in Postmenopausal
`Women With Hormone
`Receptor-positive HER2-negative
`Locally Advanced or Metastatic
`Breast Cancer Which Progressed on
`or After Aromatase Inhibitor
`Treatment
`
`Phase II Study of Pemetrexed and
`Gemcitabine for Treatment
`Resistant Patients With Metastatic
`Colorectal Cancer and KRAS
`Mutations
`
`PIK3CA status in ctDNA for
`prediction of buparlisib plus
`fulvestrant efficacy in
`postmenopausal women with
`
`endocrine-resistant HR +/HER2
`
`advanced breast cancer
`
`Use of quantitative levels of cfDNA
`for prognosis and evaluation of
`safety and efficacy of pemetrexed
`with gemcitabine in heavily
`pretreated, chemotherapy
`refractory, KRAS mutated
`colorectal cancer
`
`Assessment of the Minimal Residual
`Disease in Diffuse Large B-Cell
`Lymphomas (DLBCL) From
`Cell-free Circulating DNA by
`Next-Generation Sequencing
`(NGS)
`
`Assessment of clonal evolution of
`cfDNA mutation pattern in patient
`cohort with Diffuse Large B-Cell
`Non-Hodgkin Lymphomas
`(DLBCL) before, during, and after
`standard treatment
`
`Detection of KRAS, NRAS et
`BRAF Mutations in Plasma
`Circulating DNA From Patients
`With Metastatic Colorectal Cancer
`(ColoBEAM)
`
`Comparison of RAS and BRAF
`genotyping results from cfDNA
`using OncoBEAM™ technique
`with results of standard genotyping
`techniques and FFPE samples
`
`Breast cancer
`
`Randomized
`
`NCT01610284 Ongoing
`
`Masking: Double Blind
`(Subject, Caregiver,
`Investigator, Outcomes
`Assessor)
`
`Primary Purpose:
`Treatment
`
`Ciruelos et al. [361] and
`Yamamoto-Ibusuki et al.
`[362]
`
`Metastatic colorectal cancer Masking: Open Label
`
`NCT01109615 Terminated Spindler et al. [69]
`
`Primary Purpose:
`Treatment
`
`Diffuse large B-cell
`non-Hodgkin lymphomas
`(DLBCL)
`
`Masking: Open Label
`
`NCT02339805 Ongoing
`
`Bohers et al. [363]
`
`Metastatic colorectal cancer Masking: Open Label
`
`Primary Purpose:
`Diagnostic
`
`NCT02751177 Recruiting
`patients
`
`Continued
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`Table 1 Selected Randomized Trials Which Include ctDNA Analysis—cont’d
`
`Trial (Official Title)
`
`Description
`
`Condition
`
`Study Design
`
`ClinicalTrials.
`gov Identifier
`
`Status
`
`References
`
`A Randomized, Double-Blind
`Placebo-Controlled Phase II Study
`of the MEK Inhibitor GSK1120212
`Plus Gemcitabine vs Placebo Plus
`Gemcitabine in Subjects With
`Metastatic Pancreatic Cancer
`
`Evaluation of OS, PFS, ORR, and
`DOR in patients with pancreatic
`cancer treated with trametinib and
`gemcitabine and assessment of
`outcome based on KRAS mutations
`determined by cfDNA
`
`Metastatic pancreatic
`cancer
`
`Randomized
`
`NCT01231581 Completed Infante et al. [364]
`
`Masking: Double Blind
`(Subject, Outcomes
`Assessor)
`
`Primary Purpose:
`Treatment
`
`Assessment of genomic variation in
`metastases and primary tumors and
`confirmation of these aberrations
`CTCs and cfDNA
`
`Metastatic breast cancer
`
`Observational Model:
`Case-Only
`
`NCT02626039 Recruiting
`patients
`
`Detection of biomarkers for
`prediction of PFS in
`postmenopausal patients with
`advanced metastatic breast cancer
`who have progressed on anastrozole
`or letrozole
`
`Hormone receptor positive
`malignant neoplasm of
`breast, metastatic breast
`cancer
`
`Intervention Model:
`Single Group Assignment
`
`NCT02109913 Recruiting
`patients
`
`Masking: Open Label
`
`Characterization and Comparison
`of Drugable Mutations in Primary
`Tumors, Metastatic Tissue,
`Circulating Tumor Cells and
`Cell-Free Circulating DNA in
`Metastatic Breast Cancer Patients
`
`Phosphatidylinositol 3-kinase
`(PI3K) Pathway Analysis in Tumor
`Tissue and Circulating DNA to
`Obtain Further Insight in the
`Efficacy of Everolimus When
`Combined With Exemestane:
`A Side-study Protocol Attached to
`Standard Treatment With
`Everolimus and Exemestane for
`Postmenopausal Patients With
`Hormone Receptor-positive
`Advanced Metastatic Breast Cancer,
`Who Have Progressed on
`Anastrozole or Letrozole
`
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`Non Invasive Identification of
`Gliomas With IDH1/2 Mutation by
`Analysis of Circulating Plasmatic
`DNA, D-2-hydroxyglutarate
`Dosage in Biological Liquids and
`Detection by Brain SPEctro-MRI:
`Impact for Diagnosis and Follow-up
`
`A Pilot Study to Evaluate the
`Predictive Value of Circulating
`Tumor DNA for Clinical Outcome
`in Patients With Advanced Head
`and Neck and Lung Cancers
`
`Prognostic Value of Circulating
`Tumoral Free DNA Versus
`Circulating Tumoral Cells in
`Patients with Colorectal Cancer
`Stage II–III
`
`Circulating Cell-free DNA in
`Metastatic Melanoma Patient:
`Mutational Analyses in Consecutive
`Measurement Before and After
`Chemotherapy
`
`Development of a noninvasive
`diagnostic approach of IDH1
`mutated gliomas through
`combination of mutation detection
`from cfDNA, urine, and MRI
`analysis
`
`Glioma, IDH1/IDH2
`mutation
`
`Intervention Model:
`Single Group Assignment
`
`NCT02597335 Recruiting
`patients
`
`Masking: Open Label
`
`Primary Purpose:
`Diagnostic
`
`Evaluation of predictive value of
`cfDNA in patients with stage III–IV
`NSCLC in terms of response to
`treatment
`
`Stage IV head and neck
`cancer or stages III–IV
`nonsmall cell lung cancer
`
`Observational Model:
`Case-Only
`
`NCT02245100 Recruiting
`patients
`
`Carper and Claudio [365]
`
`Comparison of prognostic value of
`KRAS and RASSF2A methylation
`between cfDNA CTCs and
`assessment of prognostic value of
`cfDNA and CTCs in localized
`CRC patients
`
`Determination of mutation status in
`ctDNA in metastatic melanoma
`patients with the Sequenom Mass
`Array NGS technology and
`comparison of results before and
`after treatment with primary tumor
`genotype
`
`Colorectal neoplasms
`
`Intervention Model:
`Single Group Assignment
`
`NCT02556281 Recruiting
`patients
`
`Masking: Open Label
`
`Primary Purpose:
`Diagnostic
`
`Metastatic (stage IV)
`melanoma
`
`Intervention Model:
`Single Group Assignment
`
`NCT02133222 Recruiting
`patients
`
`Masking: Open Label
`
`Primary Purpose:
`Screening
`
`Continued
`
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`Table 1 Selected Randomized Trials Which Include ctDNA Analysis—cont’d
`
`Condition
`
`Melanoma
`
`Trial (Official Title)
`
`Description
`
`aFour individual trials as listed in
`Santiago-Walker et al. [68]
`
`Analysis of cfDNA data from four
`clinical studies of the BRAF
`inhibitor (BRAFi) dabrafenib or the
`MEK inhibitor (MEKi) trametinib
`for determination of association
`between BRAF mutation status in
`cfDNA and tumor tissue and the
`association of BRAF cfDNA
`mutation status with baseline factors
`and clinical outcome
`
`Study Design
`
`ClinicalTrials.
`gov Identifier
`
`Status
`
`References
`
`Masking: Open Label
`
`NCT01153763 Ongoing
`
`Primary Purpose:
`Treatment
`
`Randomized
`
`NCT01227889 Ongoing
`
`Santiago-Walker
`et al. [68]
`
`Santiago-Walker
`et al. [68]
`
`Masking: Open Label
`
`Primary Purpose:
`Treatment
`
`Nonrandomized
`
`Masking: Open Label
`
`Primary Purpose:
`Treatment
`
`NCT01266967 Completed Santiago-Walker
`et al. [68]
`
`Randomized
`
`NCT01245062 Ongoing
`
`Masking: Open Label
`
`Primary Purpose:
`Treatment
`
`Solid tumors
`
`Masking: Open Label
`
`NCT01283945 Ongoing
`
`Primary Purpose:
`Treatment
`
`Santiago-Walker
`et al. [68]
`
`Soria et al. [366] and
`Dienstmann et al. [367]
`
`An Open-Label, Dose-escalation,
`Phase I/IIa Study to Determine the
`Maximum Tolerated Dose,
`Recommended Dose, Efficacy,
`Pharmacokinetics and
`Pharmacodynamics of the Dual
`VEGFR-FGFR Tyrosine Kinase
`Inhibitor, E-3810, Given Orally as
`Single Agent to Patients With
`Advanced Solid Tumors
`
`Determination of maximum
`tolerated dose and recommended
`dose of lucitanib in patients with
`advanced solid tumors as well as
`pharmacokinetics via cfDNA
`analysis
`
`00012
`
`

`

`A Phase III Randomized Trial of
`MRI-Mapped Dose-Escalated
`Salvage Radiotherapy
`Postprostatectomy: The MAPS
`Trial
`
`A Phase III, multicenter,
`open-label, randomized trial of
`®
`) versus
`Erlotinib (Tarceva
`chemotherapy in patients with
`advanced NSCLC with mutations
`in the Tyrosine Kinase (TK) domain
`of the EGFR
`
`A Phase II, Double-blind,
`Randomized Study to Assess the
`Efficacy of AZD6244 in
`Combination With Dacarbazine
`Compared With Dacarbazine Alone
`in First Line Patients With BRAF
`Mutation-Positive Advanced
`Cutaneous or Unknown Primary
`Melanoma
`
`Determination of effect of radiation
`boost on MRI lesion on initial
`complete biochemical response and
`determination of incidence and
`relationship of circulating DNA and
`tumor cells to tissue biomarkers and
`initial complete biochemical
`response
`
`Assessment of safety and efficacy of
`erlotinib compared with standard
`chemotherapy for first-line
`treatment of European patients with
`advanced EGFR-mutation positive
`NSCLC and assessment of response
`rate, OS, and EGFR-mutation
`status in serum or plasma
`
`Analytical Validation of BRAF
`Mutation Testing from Circulating
`Free DNA Using the Amplification
`Refractory Mutation Testing
`System.
`Aung et al.
`
`Prostate cancer, prostate
`adenocarcinoma
`
`Randomized
`
`Masking: Open Label
`
`Primary Purpose:
`Treatment
`
`NCT01411345 Recruiting
`patients
`
`Nonsmall cell lung cancer Randomized
`
`Masking: Open Label
`
`Primary Purpose:
`Treatment
`
`NCT00446225 Completed Rosell et al. [368],
`Karachaliou et al. [369],
`and Costa et al. [370]
`
`Melanoma
`
`Randomized
`
`NCT00936221 Completed Robert et al. [371]
`
`Masking: Double Blind
`(Subject, Caregiver,
`Investigator, Outcomes
`Assessor)
`
`Primary Purpose:
`Treatment
`
`aA Phase II (BRF113710) Single-arm, Open-Label Study of GSK2118436 in BRAF Mutant Metastatic Melanoma. A Phase III Randomized, Open-Label Study Comparing GSK2118436 to Dacarbazine (DTIC) in Previously Untreated Subjects With
`BRAF Mutation-Positive Advanced (Stage III) or Metastatic (Stage IV) Melanoma. BRF113929: An Open-Label, Two-Cohort, Multicentre Study of GSK2118436 as a Single Agent in Treatment Naı¨ve and Previously Treated Subjects With BRAF
`Mutation-Positive Metastatic Melanoma to the Brain. A Phase III Randomized, Open-Label Study Comparing GSK1120212 to Chemotherapy in Subjects With Advanced or Metastatic BRAF V600E/K Mutation-Positive Melanoma.
`
`00013
`
`

`

`86
`
`Samantha Perakis et al.
`
`3. THE BIOLOGY OF CIRCULATING TUMOR DNA
`
`Compared to the number of studies addressing the clinical applica-
`bility of ctDNA, data regarding the actual origin, the kinetics, and the
`mechanisms of release and clearance are limited and often contradictory;
`therefore, the biology of cfDNA remains unclear.
`ctDNA is not only found in cancer patients but also in healthy individ-
`uals, although elevated levels have been reported in cancer patients [3,70],
`suggesting that the release of DNA is in principle a physiological process. In
`addition, increased levels of cfDNA can be found in patients with certain
`conditions like SLE, rheumatoid arthritis, trauma, myocardial infarction,
`and stroke [71–75]. Under physiological conditions, cfDNA is degraded
`by peripheral blood DNase activity, leading to relatively low levels of
`cfDNA in healthy individuals. The fact that cancer patients show elevated
`levels of cfDNA might be attributed to lower DNase activity in blood plasma
`of cancer patients [76,77] since it was reported that mean DNase I levels
`were found to be lower in cancer patients than in healthy controls [76].
`In addition, DNA of cancer patients could be resistant to DNase digestion
`as demonstrated by using bacterial DNase [78,79]. Moreover, it is thought
`that as the volume of the tumors increases, the number of apoptotic and dead
`cells also increases due to increased cellular turnover. This might induce a
`massive release of DNA fragments sometimes from both tumor and adjacent
`normal cells, leading to imbalances between cfDNA spread and clearance.
`Diaz et al. stated that tumors consisting of 50 million malignant cells release
`sufficient DNA for the detection of ctDNA in blood and that this is below
`the limit of resolution of radiology studies [80]. Nevertheless, cancer patients
`show a dramatic variability of absolute cfDNA levels and fractions of ctDNA
`ranging from less than 1 to more than 90%, depending on tumor burden,
`stage, vascularity, cellular turnover, and response to therapy [3,70]. There-
`fore, one of the major challenges in the analysis of plasma DNA is the dif-
`ferentiation of circulating DNA derived from the tumor from nontumor
`circulating DNA.
`
`3.1 The Nature of Circulating Tumor DNA
`cfDNA in blood is double-stranded [21] and forms a specific ladder pattern
`known from apoptotic cells, ranging from 60 to 1000 bp [81]. Therefore,
`apoptosis is considered to be the main driver for release of DNA. Consis-
`tent with an apoptotic origin and the length of DNA wrapped around a
`
`00014
`
`

`

`Advances in Circulating Tumor DNA Analysis
`
`87
`
`nucleosome including a linker region, the distribution of cfDNA fragment
`lengths has a mode near 166 bp. Several groups have shown that plasma
`DNA molecules showed a predictable fragmentation pattern reminiscent
`of nuclease-cleaved nucleosomes, which is typically observed from apo-
`ptotic cells [57,82]. Further evidence for apoptosis as the major source of
`cfDNA came from experiments using mice, which showed that the predom-
`inant fragments in plasma from xenografted animals were mononucleosome
`derived [83]. Yet, other mechanisms including necrosis and active secretion
`were shown to contribute to the release of cfDNA [47,84]. There is also
`evidence that ctDNA in plasma may participate in tumorigenesis and the
`development of metastases via transfection-like uptake of such nucleic acids
`by susceptible cells, a process called genometastasis [85].
`Since studies using cffDNA have suggested that the size of circulating
`DNA fragments is an important accessible parameter [82], Jiang et al. used
`massively parallel sequencing for size profiling of plasma DNA from
`90 patients with hepatocellular carcinoma, 67 with chronic hepatitis B,
`36 with hepatitis B-associated cirrhosis, and 32 healthy controls. Compared
`to other conditions, slightly different size distributions were observed in
`HCC patients, indicating that plasma DNA molecules released by tumors
`might be shorter and preferentially carry the tumor-associated copy number
`aberrations [86]. Similar observations were already made in 2011, when the
`Thierry group demonstrated the presence of a higher proportion of cfDNA
`fragments below 100 bp particularly in samples from cancer [87]. However,
`data
`from our
`group suggest
`that
`an inefficient
`clearance of
`nucleosomal-derived DNA may lead to multiples of mono-nucleosomal
`DNA which correlated with higher fractions of tumor DNA [57,88].
`
`3.2 Release and Clearance
`The release of ctDNA is thought to be a fluctuating, stochastic process rather
`than a continuous process and in a few studies it appeared to be related to
`in