Detection of Cell Types Contributing to Cancer From Circulating, Cell-Free Methylated DNA

Abstract

Detection of cellular changes in tissue biopsies has been the basis for cancer diagnostics. However, tissue biopsies are invasive and limited by inaccuracies due to sampling locations, restricted sampling frequency, and poor representation of tissue heterogeneity. Liquid biopsies are emerging as a complementary approach to traditional tissue biopsies to detect dynamic changes in specific cell populations. Cell-free DNA (cfDNA) fragments released into the circulation from dying cells can be traced back to the tissues and cell types they originated from using DNA methylation, an epigenetic regulatory mechanism that is highly cell-type specific. Decoding changes in the cellular origins of cfDNA over time can reveal altered host tissue homeostasis due to local cancer invasion and metastatic spread to distant organs as well as treatment responses. In addition to host-derived cfDNA, changes in cancer cells can be detected from cell-free, circulating tumor DNA (ctDNA) by monitoring DNA mutations carried by cancer cells. Here, we will discuss computational approaches to identify and validate robust biomarkers of changed tissue homeostasis using cell-free, methylated DNA in the circulation. We highlight studies performing genome-wide profiling of cfDNA methylation and those that combine genetic and epigenetic markers to further identify cell-type specific signatures. Finally, we discuss opportunities and current limitations of these approaches for implementation in clinical oncology.

Keywords: Cell-free DNA (cfDNA); cellular damage; circulating tumor DNA (ctDNA); deconvolution; liquid biopsy; tissue-of-origin; tumor microenvironment.

FIGURE 1

Complementary role of tissue and liquid biopsies in oncology. Localized solid tissue biopsies are invasive and provide a snapshot of limited representational heterogeneity based on the small piece of tissue that is excised. In comparison, liquid biopsies are minimally invasive and allow for serial sampling to provide systemic information about the primary tumor as well as distant metastatic sites indicated in different colors. Thus, liquid biopsies complement tissue biopsies and increase representation of heterogeneity supporting the tracking of clonal evolution over time.

FIGURE 2

Factors contributing to probability of signal detection. (A) Tumors release mutant genomic and epigenomic cfDNA into the circulation. Normal cell types from the surrounding microenvironment and other somatic cells also release cfDNA into the circulation that can be identified through cell-type specific epigenetic markers. Combining tumor cell- and normal host cell-derived signals can increase target abundance in the circulation to increase sensitivity, while maintaining specificity. (B) Target abundance, sequencing depth, and breadth of genomic regions assayed are factors that determine signal detection probability in the circulation of cancer patients. (C) Relative abundance of cfDNA populations and associated specificity.

FIGURE 3

Applications for detection and localization of metastasis and Cancer of Unknown Primary (CUP). (A) CfDNA in healthy individuals is mostly of hematopoietic origin. (B) The composition of cell-free DNA changes with disease. In this example, primary lung cancer results in increased levels of ctDNA identified by tumor-specific genomic and epigenomic markers, as well as increased levels of cfDNA from the surrounding lung microenvironment identified by normal cell-specific epigenetic markers. (C) Genomic mutations occur independently in primary and metastatic tumor sites. Liquid biopsies are capable of capturing this heterogeneity; however, mutations alone cannot localize these clonal populations to their tissue origins at the primary tumor site and distant metastatic site. As a complementary approach, normal tissue- and cell-type epigenetic markers can be used for detection and localization of metastasis and Cancers of Unknown Primary (CUP).

FIGURE 4

DNA methylation technologies and platforms for signal detection. (A) Scale representation of DNA methylation technologies from targeted First- and Second-Generation toward comprehensive Third-Generation applications in liquid biopsies. (B) Methods for detection of DNA methylation. The same DNA sequence is found in all non-tumor cells, and simple sequence analysis cannot be used to distinguish its cell-type identity. However, these methods can be used to detect DNA methylation (5mc) and DNA hydroxymethylation (5hmc) levels in tumor and non-tumor cells. MeDIP-seq, Methylated DNA immunoprecipitation sequencing; MBD, methyl-CpG-binding domain sequencing; WGBS, Whole Genome Bisulfite Sequencing; BSAS, Bisulfite Amplicon Sequencing; RRBS, Reduced Representation Bisulfite Sequencing; MCTA-seq, methylated CpG tandem amplification and sequencing; MSP, Methylation Specific PCR; MRE-seq, methylation-sensitive restriction enzyme sequencing; HELP, Hpall-tiny fragment enrichment by ligation-mediated PCR; MSCC, Methyl-sensitive Cut Counting; EM-seq, Enzymatic Methyl-Sequencing; TAPS, TET-assisted pyridine borane sequencing; TAB-seq, TET-assisted bisulfite sequencing; ACE-seq, APOBEC-coupled epigenetic sequencing; hmc-CATCH, chemical-assistant C-to-T conversion of 5hmC sequencing; oxBS-seq, oxidative bisulfite sequencing.

FIGURE 5

Tissue-of-origin deconvolution analysis. CfDNA is a mixture of fragments released from healthy and diseased cells in different tissue types throughout the human body into the circulation. DNA methylation is highly cell-type specific and can be used to identify the cellular origins of cfDNA at specific markers. Tissue-of-origin (TOO) analysis traces cfDNA molecules back to the tissues and cell types they originated from and use changing tissue proportions to reveal altered tissue homeostasis in diseased states or during therapy.

FIGURE 6

Predicting treatment response and therapy-related toxicities from combined genetic and epigenetic analyses of cfDNA. The minimally invasive nature of liquid biopsies allows for serial sampling to monitor changes over time, especially under selective pressures from ongoing therapy. CtDNA can be used to track clonal heterogeneity over time to assess treatment response and detect treatment-resistant clones. Normal cell-specific cfDNA methylation patterns can be used in combination with ctDNA to assess the impact of treatment to the surrounding tumor microenvironment and to monitor for therapy-related toxicities in somatic cell-types. Acronyms: ctDNA, (circulating tumor DNA); cme-DNA, (circulating methylated cell-free DNA).