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Biofluids

Introduction

Recent progress in the field of epigenetics has provided crucial insights into the mechanisms of tumorigenesis. Cancers originate from an accumulation of both genetic and epigenetic alterations. Researchers have documented that epigenetic changes occur in the initiation, progression, and metastasis in a number of tumor types. 

There are two major areas of interest in the clinical use of epigenetics: therapeutics and biomarker identification. Because epigenetic modifications are reversible, they have gained attention as a target of preventive and therapeutic medicine. For example, DNMT inhibitors and HDAC inhibitors in blood cancers have shown positive clinical impact (Hatzimichael, 2013).

In addition, epigenetic modifications hold promise as biomarkers for early cancer detection, prediction, screening, and prognosis.  

Liquid biopsy

Liquid biopsy is advantageously non-invasive, simply using body fluids such as blood, stool, urine, sputum, and saliva to serve as an excellent surrogate tissue for studying tumors. Primary tumors release tumor material into body fluids, mainly as circulating tumor cells, cell-free nucleic acids (DNA and RNA), and extracellular vesicles. Epigenetic alterations found in liquid biopsies have shown utility as biomarkers for early detection, prognosis, monitoring, and evaluation of therapeutic response in many types of cancer patients. In fact, changes in DNA methylation patterns, alterations of histone modifiers (such as methylation of H3K9me and acetylation of H3K27ac) and ncRNA expression observed in liquid biopsy samples are well documented in various cancers including colorectal cancer (Rodriguez-Casanova 2021).

Cell-free nucleic acids

During tumor development, tumor cells release DNA, mRNA, and microRNA into the blood circulation. This process occurs by apoptotic and necrotic cell deaths, characteristic of tumors. 

Cancer patients, unlike non-cancer patients, have a higher level of cfDNA in plasma or serum, and epigenetic alterations can be detected in these samples. For example, cFDNAs from a wide range of cancers have shown K-ras and p53 mutations (Zhuang 2017). In addition, Septin 9 methylation observed in cfDNAs may be a sensitive biomarker for early colorectal cancer detection. High frequency of aberrant hypermethylation of specific genes have been also detected in cfDNA from sputum (bronchial secretions common in smokers). Another study analyzed the methylation of specific genes in urine sediments from patients with prostate cancer and showed the sensitivity of DNA methylation biomarkers in distinguishing early and late prostate cancer (Rodriguez-Casanova 2021).

Additionally, numerous non-coding RNAs (ncRNAs) can be detected in liquid biopsy. Several studies have highlighted the role of ncRNAs in cell-to-cell communication through the promotion of differential gene expression in tumor cells and stroma. The disruption of ncRNA expression in cancer cells may also alter histone PTMs and DNA methylation levels (Church 2014).

cEVs

Exosomes, microvesicles, and apoptotic bodies can be found in different body fluids at high concentrations. These vesicles play an important role in cancer as they promote cell proliferation and invasion. These bodies can be isolated by ultracentrifugation, immunoaffinity, or precipitation. Molecular alterations have been described in exosomal DNA and exosomal ncRNAs from cancer patients (Liu 2020).

Circulating Tumor Cells (CTCs)

The analysis of DNA methylation has also been explored in circulating tumor cells isolated from blood. Data demonstrated that methylation profiling of CTCs represents a promising non-invasive approach for tumor detection.



Methodologies: Methods for Detecting Epigenetic Marks in Liquid Biopsy

DNA Modifications: Methylation and Hydroxymethylation

The detection of DNA methylation patterns is based on methods that either depend on sodium bisulfite conversion or on immunoprecipitation and methyl-sensitive restriction enzymes. 

Sodium Bisulfite Methods:

Genome-wide bisulfite-based approaches based on NGS allow to evaluate the whole methylome in liquid biopsy allowing discovery of the whole methylation landscape of cfDNA from cancer patients.

  • MCTA-seq (methylated CpG tandem amplification and sequencing) it is possible to analyze the methylation status of CpG Islands in cfDNA . 
  • CpG-targeted bisulfite sequencing for the analysis of methylation in cfDNA. After bisulfite treatment of plasma cfDNA, regions of interest are pulled down and sequenced, and the results are analyzed in combination with machine learning. 
  • Genome-wide level in liquid biopsy using methylation microarrays. This methodology utilizes bisulfite-converted DNA and has been applied to the study of cfDNA and CTCs from cancer patients. Approaches based on methylation microarrays and NGS have been used to identify epigenetic biomarkers in cfDNA for cancer detection. For instance, the analysis of ~850,000 CpGs in pooled cfDNA samples by MethylationEPIC array highlighted 1,384 differentially methylated CpG sites that discriminate CRC patients from healthy controls. (Gaillardo-Gomez, 2018)
  • cfDNA reduced representation bisulfite sequencing (cf-RRBS) is a cost-effective way to analyze the cfDNA methylome in liquid biopsy. In cf-RRBS, cfDNA is dephosphorylated prior to enzymatic digestion by the methylation-insensitive restriction enzyme MspI and sequenced. 
  • Genome-wide hydroxymethylation profiles can be obtained from cfDNA of cancer patients by 5hmC methods combined with NGS (Li et al., 2017).

Immunoprecipitation methods

  • cfMeDIP-seq This is a genome-wide method based on cell-free methylated DNA immunoprecipitation and high-throughput sequencing . Particularly, cfMeDIP-seq is a region-based method that reveals methylation status of genomic regions of at least 100 bp in length. cfMeDIP-seq is a low-input and sensitive approach that can be used for both early- and late-stage detection of multiple tumor types. (Shen et al., 2019). 
  • Methyl-CpG binding (MBD) proteins coupled with ddPCR (MBD–ddPCR) This locus-specific method is based on  immunoprecipitation using a methyl-binding domain and allows the detection of methylation sites in cfDNA.

The significance of NGS

The combination of NGS with machine learning has enabled the development of a test based on ~1 million CpG sites capable of detecting and localizing more than 50 tumor types. (Liu M. C. et al., 2020). Another research group designed a targeted NGS assay based on 9,223 hypermethylated CpG sites obtained from The Cancer Genome Atlas (TCGA), which proved useful for identifying tumor types (Liu et al., 2018). The PanSeer assay, which considers 10,613 CpG sites, allowed the detection of five cancer types regardless of the tissue of origin. Importantly, this assay enabled the detection of the presence of cancer in asymptomatic individuals years before standard diagnosis (Chen et al., 2020). 

Histone Modifications and Nucleosome Positioning 

Histone modifications in blood-circulating nucleosomes have revealed that-they can also contribute to cancer detection. For example, low levels of H3K9me3, H4K20me3, and H3K27me3 have been proposed as biomarkers for the diagnosis of colorectal cancer (Gezer et al. 2015). Different post-translational modifications detected in circulating nucleosomes have also proven useful for screening (Rodriguez-Casanova 2021). In addition, high concentrations of circulating nucleosomes in CRC patients have been associated with disease progression, poor therapy response, and reduced survival. Levels of nucleosomes in cancer patients are dynamic and thus can be useful to indicate the response to therapy in real time (Holdenrieder et al., 2001).

Detection methods:

  • ChIP-seq of cell-free nucleosomes (cfChIP-seq). This method enables the capture of circulating nucleosomes with different active chromatin marks that maintain the cell-of-origin genomic distribution of modifications and expression patterns. Other similar approaches have been used to quantify the level of histone marks associated with circulating cell-free nucleosomes in plasma of cancer patients (Vad-Nielsen et al., 2020). 
  • ChIP-qPCR of circulating nucleosomes can detect histone modifications in individual genes 
  • Measuring cfDNA fragmentation. Several studies showed that all cells may actively release DNA fragments. In multiple cancers, plasma cfDNA fragments have been shown to exhibit different sizes between healthy individuals and cancer patients (Mouliere et al., 2018). Researchers have proposed cfDNA fragmentation patterns as epigenetic biomarkers for early cancer detection. In addition, the analysis of cfDNA fragmentation patterns has proved useful for early detection and to predict the response of patients to immunochemotherapy. The different length of the DNA fragments present in stool has also been used as good tool to discriminate cancer patients from healthy ones. These approaches require a very low input of cfDNA from different types of fluids, allowing  early detection of cancer (Mouliere et al., 2018). Other genome-wide fragmentation methods, such as DNA evaluation of fragments for early interception (DELFI), have been recently proposed (Cristiano et al., 2019). 

Non-coding RNAs

Studies have identified circulating miRNA signatures for early detection cancer with high sensitivity and specificity. The levels of different miRNAs in blood can also provide information about the patient's disease stage, and represent a valuable tool for early detection of recurrence and the evaluation of response to therapy.  For instance, the overexpression of miR-21, is associated with the activation of relevant specific pathways that promote tumor pathogenesis in colorectal cancer (Xiong et al., 2013). In addition, recent transcriptomic analyses have revealed cancer-specific expression signatures of miRNAs, lncRNAs, and other ncRNAs (Song et al., 2020).

  • Microarrays and NGS have been used in liquid biopsy for the detection of several types of ncRNAs, including miRNAs, lncRNAs, and circRNAs
  • RT-qPCR and ddPCR, allow to quantify the expression levels of specific ncRNA transcripts in liquid biopsy with high sensitivity. 

Link to DNA methylation: DNA methylation may control the expression levels of ncRNAs in colorectal tumor cells. Exosomal miRNAs (e.g., miR-21 and miR-139-3p) analysis represents a valuable tool for diagnosis and prognosis. The analysis of miRNAs in circulating exosomes has revealed important strategies for the identification of treatment-resistant patients.

Opportunities - From research to translational biomarkers

The translation of circulating epigenetic biomarkers into the clinical setting will require large multicenter studies to demonstrate the clinical benefit of their use. 

  1. Choosing the right biomarkers:Currently a plethora of DNA methylation biomarkers in biofluids have been identified. Researchers must determine which should be chosen for further validation and introduced into screening. As with any biomarker research strategy, the ideal biomarker would be highly sensitive and specific in different populations, regardless of different etiologies,  patient age, gender or tumor stage. 
  2. Biomarker panels:Panel of biomarkers instead of a sole biomarker could increase the sensitivity and specificity of early detection, diagnosis, prevention or prediction of cancer. 
  3. Standardizing protocols -  Sample collection, processing, and storage must be improved for reproducibility. Efforts should be made to harmonize pre-analytical and analytical protocols according to the epigenetic alteration, circulating element, or biofluid. It is important to choose the best biofluid - what is the right surrogate tissue? The detection of some epigenetic biomarkers can be more sensitive in stool than plasma. For example, SEPT9 methylation, which was evaluated in stool and plasma from patients with better results from stool.
  1. Validation- The Early Detection Research Network (EDRN, USA) guidelines, biomarker panel should follow several steps before “in-the-field” including a preclinical investigation phase, establishment of clinical assay and its validation, assessment of the performance of the biomarker for early disease detection, screening assessment of the biomarker, and impact of screening. Guidelines have already been proposed for gene expression microarrays, proteomics, and immunology approaches for early cancer detection and screening. These guidelines will help further the development of novel cancer-related DNA methylation biomarkers.
  2. Cost Many technologies involve NGS-based approaches, which include library preparation and bioinformatic interpretation. However, in the future, these strategies should be associated with more adjusted costs and user-friendly bioinformatic solutions.



REFERENCES

Chen, X., Gole, J., Gore, A., He, Q., Lu, M., Min, J., et al. (2020). Non-invasive early detection of cancer four years before conventional diagnosis using a blood test. Nat. Commun. 11:3475. doi: 10.1038/s41467-020-17316-z

Church, T. R., Wandell, M., Lofton-Day, C., Mongin, S. J., Burger, M., Payne, S. R., et al. (2014). Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer. Gut 63, 317–325. doi: 10.1136/gutjnl-2012-304149epigenetic marks in liquid biopsy.

Cristiano, S., Leal, A., Phallen, J., Fiksel, J., Adleff, V., Bruhm, D. C., et al. (2019). Genome-wide cell-free DNA fragmentation in patients with cancer. Nature 570, 385–389. doi: 10.1038/s41586-019-1272-6

Eleftheria Hatzimichael, Tim Crook, "Cancer Epigenetics: New Therapies and New Challenges", Journal of Drug Delivery, vol. 2013, Article ID 529312, 9 pages, 2013. https://doi.org/10.1155/2013/529312

Gallardo-Gómez, M., Moran, S., Páez de la Cadena, M. et al. A new approach to epigenome-wide discovery of non-invasive methylation biomarkers for colorectal cancer screening in circulating cell-free DNA using pooled samples. Clin Epigenet 10, 53 (2018). https://doi.org/10.1186/s13148-018-0487-y

Gezer, U., Ustek, D., Yoruker, E. E., Cakiris, A., Abaci, N., Leszinski, G., et al. (2013). Characterization of H3K9me3- and H4K20me3-associated circulating nucleosomal DNA by high-throughput sequencing in colorectal cancer. Tumour Biol. 34, 329–336. doi: 10.1007/s13277-012-0554-5

Holdenrieder, S., Stieber, P., Bodenmuller, H., Busch, M., Fertig, G., Furst, H., et al. (2001). Nucleosomes in serum of patients with benign and malignant diseases. Int. J. Cancer 95, 114–120. doi: 10.1002/1097-0215(20010320)95:2<114::aid-ijc1020>3.0.co

Li, W., Zhang, X., Lu, X., You, L., Song, Y., Luo, Z., et al. (2017). 5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers. Cell Res. 27, 1243–1257. doi: 10.1038/cr.2017.121

Liu, W., Yang, D., Chen, L., Liu, Q., Wang, W., Yang, Z., et al. (2020). Plasma exosomal miRNA-139-3p is a novel biomarker of colorectal cancer. J. Cancer 11, 4899–4906. doi: 10.7150/jca.45548

Mouliere, F., Chandrananda, D., Piskorz, A. M., Moore, E. K., Morris, J., Ahlborn, L. B., et al. (2018). Enhanced detection of circulating tumor DNA by fragment size analysis. Sci. Transl. Med. 10:eaat4921. doi: 10.1126/scitranslmed.aat4921 

Rodriguez-Casanova 2021. Front. Cell Dev. Biol., 05 February 2021 | https://doi.org/10.3389/fcell.2021.622459 Rongyuan Zhuang, Song Li, Qian Li, Xi Guo, Feng Shen, Hong Sun, Tianshu Liu The prognostic value of KRAS mutation by cell-free DNA in cancer patients: A systematic review and meta-analysis PLoS One. 2017; 12(8): e0182562. Published online 2017 Aug 10. doi: 10.1371/journal.pone.0182562 

Shen, S. Y., Burgener, J. M., Bratman, S. V., and De Carvalho, D. D. (2019). Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA. Nat. Protoc. 14, 2749–2780. doi: 10.1038/s41596-019-0202-2

Song, W., Ren, J., Wang, C., Ge, Y., and Fu, T. (2020). Analysis of circular RNA-related competing endogenous RNA identifies the immune-related risk signature for colorectal cancer. Front. Genet. 11:505. doi: 10.3389/fgene.2020.00505

Vad-Nielsen, J., Meldgaard, P., Sorensen, B. S., and Nielsen, A. L. (2020). Cell-free Chromatin Immunoprecipitation (cfChIP) from blood plasma can determine gene-expression in tumors from non-small-cell lung cancer patients. Lung Cancer 147, 244–251. doi: 10.1016/j.lungcan.2020.07.023

Xiong, B., Cheng, Y., Ma, L., and Zhang, C. (2013). MiR-21 regulates biological behavior through the PTEN/PI-3 K/Akt signaling pathway in human colorectal cancer cells. Int. J. Oncol 42, 219–228. doi: 10.3892/ijo.2012.1707




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