Methylation in cell-free DNA for early cancer detection
Authors: F. Fece de la Cruz, R.B. Corcoran
Journal: Annals of Oncology (2018)

Liquid biopsies and, in particular, analysis of cell-free DNA (cfDNA), have emerged as a promising and potentially transformative non-invasive diagnostic approach in oncology [1.Haber D.A. Velculescu V.E. Blood-based analyses of cancer: circulating tumor cells and circulating tumor DNA.Cancer Discov. 2014; 4: 650-661Crossref PubMed Scopus (490) Google Scholar, 2.Siravegna G. Marsoni S. Siena S. Bardelli A. Integrating liquid biopsies into the management of cancer.Nat Rev Clin Oncol. 2017; 14: 531-548Crossref PubMed Scopus (1105) Google Scholar]. cfDNA is composed of fragmented DNA released by cells into the circulation, typically as a result of cell death. cfDNA found in the plasma of healthy patients is composed of germline DNA released by normal cells. In cancer patients, some component of the overall cfDNA is composed of DNA released by tumor cells, often termed circulating tumor DNA (ctDNA). The fraction of ctDNA amidst the background of overall cfDNA is highly variable and often low, particularly in patients with early stage cancers [3.Bettegowda C. Sausen M. Leary R.J. et al.Detection of circulating tumor DNA in early- and late-stage human malignancies.Sci Transl Med. 2014; 6: 224ra224Crossref Scopus (2958) Google Scholar]. Thus, the detection and analysis of tumor-derived cfDNA poses several challenges and has required the development of specialized technologies with high analytical sensitivity and specificity. While recent studies have demonstrated the clinical potential of cfDNA for tumor genotyping and blood-based tracking of therapeutic response and resistance [4.Forshew T. Murtaza M. Parkinson C. et al.Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA.Sci Transl Med. 2012; 4: 136ra68Crossref PubMed Scopus (921) Google Scholar, 5.Murtaza M. Dawson S.J. Tsui D.W. et al.Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA.Nature. 2013; 497: 108-112Crossref PubMed Scopus (1308) Google Scholar, 6.Lanman R.B. Mortimer S.A. Zill O.A. et al.Analytical and clinical validation of a digital sequencing panel for quantitative, highly accurate evaluation of cell-free circulating tumor DNA.PLoS One. 2015; 10: e0140712Crossref PubMed Scopus (460) Google Scholar, 7.Schrock A.B. Pavlick D.C. Klempner S.J. et al.Hybrid capture-based genomic profiling of circulating tumor DNA from patients with advanced cancers of the gastrointestinal tract or anus.Clin Cancer Res. 2018; Crossref PubMed Scopus (35) Google Scholar], one of the most transformative potential applications of cfDNA analysis is to detect the presence of cancer in patients without clinical evidence of disease. This potential has been demonstrated most clearly through the detection of residual disease following curative intent cancer surgery [8.Diehl F. 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By screening post-operative cfDNA for the presence of specific mutations identified in the patient's resected tumor, detection of these tumor-specific mutations using highly sensitive techniques (often capable of detecting mutant alleles present at a frequency of 0.01%–0.1% or less) can accurately identify those patients who will eventually recur. Similarly, the potential to detect nascent cancers in asymptomatic individuals with a simple screening blood test when they are still curable could revolutionize cancer medicine. Most studies to date utilizing cfDNA for cancer detection have focused on the detection of mutations in cancer-related genes. However, this approach for early cancer detection poses several key challenges. First, cfDNA levels in patients with early-stage cancers are often much lower than with advanced disease [3.Bettegowda C. Sausen M. Leary R.J. et al.Detection of circulating tumor DNA in early- and late-stage human malignancies.Sci Transl Med. 2014; 6: 224ra224Crossref Scopus (2958) Google Scholar]. Second, unlike the residual disease setting discussed above, there is no prior knowledge of what specific mutations might be present in an individual patient's tumor. Third, benign lesions may harbor some of the same mutations commonly seen in certain tumors, leading to potential false positives. For example, BRAF V600 mutations, which are present in nearly 7% of advances cancers, are often observed in benign nevi [12.Shain A.H. Yeh I. Kovalyshyn I. et al.The genetic evolution of melanoma from precursor lesions.N Engl J Med. 2015; 373: 1926-1936Crossref PubMed Scopus (622) Google Scholar]. Many mutations detectable in cfDNA can also originate from the bone marrow through a process known as clonal hematopoiesis of indeterminate potential (CHIP), which increases exponentially with age [13.Jaiswal S. Fontanillas P. Flannick J. et al.Age-related clonal hematopoiesis associated with adverse outcomes.N Engl J Med. 2014; 371: 2488-2498Crossref PubMed Scopus (2506) Google Scholar]. Indeed, evidence of CHIP is observed in 10%–15% of patients over the age of 70 years. Finally, since many cancers share common mutations in genes such as TP53 and KRAS, which are mutated in ∼50% and ∼20% of all cancers, respectively, localizing an early cancer to a specific organ site following the detection of mutations in cfDNA poses a significant challenge. In this study, Liu et al. evaluate an alternative approach for early cancer detection based on assessing the methylation status of thousands of CpG sites in cfDNA [14.Liu L. Toung J.M. Jassowicz A.F. et al.Targeted methylation sequencing of plasma cell-free DNA for cancer detection and classification.Ann Oncol. 2018; 29: 1445-1453Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]. Indeed, widespread methylation changes are commonly observed across multiple cancer types, with tumors of certain tissue origin displaying specific methylation patterns [15.Leygo C. Williams M. Jin H.C. et al.DNA methylation as a noninvasive epigenetic biomarker for the detection of cancer.Dis Markers. 2017; 2017: 3726595Crossref PubMed Scopus (75) Google Scholar]. Thus, there are several potential advantages of assessing methylation. As mutation detection in cfDNA focuses on changes in a finite number of genes, these techniques are limited not just by analytical sensitivity and specificity, but also by the absolute number of cancer genomes present in a single tube of blood. If no DNA fragment from a specific mutated locus is present in single blood draw, no technique, no matter how perfect, can identify the presence of cancer. However, given the widespread methylation changes present in most cancers, assessing thousands of CpG sites increases the chances that tumor-derived DNA may be detectable in a given blood sample. Another key advantage is that methylation patterns often reflect the epigenetic origin of specific cancers and have been used to unmask the tissue of origin for cancers of unknown primary [16.Moran S. Martinez-Cardus A. Sayols S. et al.Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis.Lancet Oncol. 2016; 17: 1386-1395Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar]. Thus, if evidence of cancer is detected, this approach offers the potential to interpolate tumor origin from these data to guide clinical efforts to localize and intervene. To develop this approach, the authors mined the Cancer Genome Atlas database to identify 10 888 CpG sites frequently found to be hypermethylated in 32 tumor types. CpG sites also methylated in control cfDNA isolated from healthy individuals were excluded, resulting in 9322 individual CpG sites for analysis. In brief, whole-genome amplification was carried out on bisulfite-treated cfDNA before hybrid capture to preserve molecular diversity from the limited amount of cfDNA input. Sequencing data across CpG sites was then analyzed with a novel algorithm to determine a single methylation score for each sample (Figure 1). The authors evaluated this pan-cancer methylation assay in 68 patients with advanced cancer (including colorectal, breast, melanoma and non-small-cell lung cancer) who were not receiving systemic therapy at the time of the blood draw, as well as 66 healthy control individuals. The methylation score correctly detected the presence of cancer in 86.8% of cancer patients and 0% of the 25 healthy patients from the validation set. Importantly, using a tissue classification algorithm based on methylation patterns, the authors correctly identified the tissue origin of each cancer in 76.3% of patients. The ability to infer tissue origin from cfDNA highlights the potential advantages of incorporating methylation analysis into blood-based early detection strategies. A key limitation of this study is that it involved patients with advanced cancers, rather than patients with early stage and potentially curable cancers, which is the point at which screening tests would need to be effective in order to provide curative treatment options. Further evaluation will be necessary in larger numbers of patients with early cancers and ultimately in asymptomatic individuals to better assess the potential of this approach. Still, this study is an important proof-of-concept for how incorporating DNA methylation changes into early detection strategies may be beneficial. Recently, two blood-based early detection studies were reported that highlight the potential benefit of looking beyond mutation detection alone in cfDNA. Chan et al. used detection of Epstein–Barr Virus DNA in cfDNA from 20 174 Chinese patients to screen for nasopharyngeal carcinoma, with a sensitivity of 97.1% and a specificity of 98.6% [17.Chan K.C.A. Woo J.K.S. King A. et al.Analysis of plasma Epstein–Barr virus DNA to screen for nasopharyngeal cancer.N Engl J Med. 2017; 377: 513-522Crossref PubMed Scopus (387) Google Scholar]. The CancerSEEK assay assessed secreted tumor-related protein biomarkers in addition to detection of key cancer mutations in cfDNA in 1005 patients with non-metastatic cancer and 812 healthy controls, with sensitivities ranging from 69% to 98% by tumor type, and a sensitivity of 99% [18.Cohen J.D. Li L. Wang Y. et al.Detection and localization of surgically resectable cancers with a multi-analyte blood test.Science. 2018; 359: 926-930Crossref PubMed Scopus (1332) Google Scholar]. This present study suggests that DNA methylation could also be a useful tool that should be further evaluated, either alone, or in conjunction with mutation detection, in cfDNA for improved early cancer detection strategies. NIH/NCI (R01CA208437), NIH/NCI Gastrointestinal Cancer SPORE (P50CA127003), and a Stand Up To Cancer Colorectal Dream Team Award (all to RBC).