Epigenetics in Precision Medicine

In recent years, knowledge of epigenetic mechanisms underlying disease onset and progression has proven crucial for the development of novel early diagnosis and prognosis biomarkers for patient stratification and precision medicine. Epigenetics in Precision Medicine, a new volume in the Translational Epigenetics series, provides a thorough discussion and overview of current developments in clinical epigenetics with special emphasis on epigenetic biomarkers that can be used for clinical diagnosis, prognosis, patient stratification, and treatment monitoring. Disease types discussed include cancer, metabolic disorders, neurodegenerative diseases, bone disease, and immune-related disorders. The book examines the challenges of advancing epigenetics research and translating findings to the clinic and drug discovery in each of these areas, as well as current solutions; chapter authors discuss how to leverage epigenomic technologies, applications, and tools, such as next-generation sequencing, to discover new epigenetic biomarkers in disease and drug studies.

Epigenetics in Precision Medicine focuses on complex epigenetic mechanisms in several pathologies, and explores how epigenetics can power the advance of precision medicine, not only by improving in vitro diagnostic and prognostic tools, but by providing new therapeutic approaches to treat human disease.

• Provides a thorough grounding in epigenetics-driven precision medicine, with emphasis on developing and implementing early diagnosis and prognosis biomarkers, and supporting patient stratification
• Empowers researchers and clinicians to incorporate epigenetics in new disease research, drug discovery, and clinical practice
• Features chapter contributions from international leaders in the field

• Language: English
• Publisher: Academic Press
• Release date: Nov 12, 2021
• ISBN: 9780323858052

Book preview

Preface

Although the Human Genome Project was completed in 2003 and large-scale genetic association analyses have been performed in many diseases in order to elucidate the mechanism of disease, the use of genetic markers alone is not sufficient to explain completely the disease onset and progression. Progress in the field of human epigenetics has been accelerated, thanks to recent technological advances and the comprehension of epigenetic dysregulation occurring in human diseases.

Since the NIH Roadmap Epigenomics Project, epigenetics has become a hot topic in biomedicine. Epigenetics controls myriad physiological and pathological states, toward mechanisms involving cell identity and gene expression in both health and disease. For a given pathogenic phenotype in a complex disease, there are multiple potential genes that could be genetically altered (i.e., mutations, copy number variations, and triplet expansion) and epigenetically controlled [i.e., DNA methylation, posttranslational modifications (PTMs) of histones and miRNAs and lncRNAs, etc.]. Therefore, the investigation of epigenetic mechanisms improves our understanding of the disease mechanisms contributing to the natural history of a disease. Moreover, epigenetics is demonstrating epigenetic marks and epigenetic mechanisms are useful as biomarkers to guide clinical decisions and improve personalized management of human diseases including the detection, diagnosis, deep phenotyping, risk stratification, and prognosis. Moreover, epigenetics may contribute to more accurate prediction of patients who will benefit from a specific treatment or prevention strategy for a particular disease, which has crucial implications for the health of the patient and the efficient use of healthcare system resources. In this regard, there are already some in vivo diagnostic (IVD) tests based on epigenetic biomarkers that are currently being commercialized and are described in this new volume Epigenetics in Precision Medicine. Importantly, it is expected that the number of epigenetic-based IVD tests will increase substantially in the coming years, after demonstrating the potential of epigenetic biomarkers to improve clinical settings.

Many methods, including next-generation sequencing-based technologies, are currently available to clinicians and researchers to analyze epigenetic biomarkers isolated from several biological sources. However, despite the spectacular progress of epigenetic research in human diseases, there are still some challenges to be solved such as the interpretation of existing and future data generated mainly by next-generation sequencing approaches in large cohorts of patients, the effect produced by epigenetic drugs or life style and dietary interventions in the epigenome, and the significance of epigenetic changes occurring in patients participating in clinical trials, just to mention a few. It is noteworthy that most studies addressing personalized medicine using epigenetic biomarkers and drugs have been performed in small exploratory cohorts and therefore had low statistical significance. So, the systematic inclusion of the analysis of epigenetic modifications (including DNA methylation, long noncoding RNAs, and microRNAs) in large-scale clinical trials, with well-selected patients, would greatly improve our capacity to define companion diagnostic biomarkers correlating with treatment response or even predict response before the start of the therapy.

Importantly, the integration of clinical information with data sets generated by high-throughput technologies will help decipher the complex interplay of the different epigenetic mechanisms and gene regulatory networks, thereby contributing to the identification of therapeutic targets, and demonstrate the pharmacological potential of new epigenetic drugs. Importantly, technical progress and bioinformatic methods will enable us to increase our capacity to analyze large cohorts of patients with high-dimensional multiomic data sets. So, it is conceivably an improvement of precision medicine, thanks to the incorporation of artificial intelligence and bioinformatics.

Finally, epigenetic drugs regulating epigenetic mechanisms are gaining particular interest for the treatment of several diseases. In the last decade, both the Food and Drug Administration (FDA) and the European Medicine Agency (EMA) have approved various epigenetic drugs used to treat a wide variety of diseases such as different cancers, hypertension, and even infections caused by viruses, among others. In particular, some epigenetic drugs such as DNA methyltransferase inhibitors, histone acetyltransferase modulators, histone deacetylase inhibitors, histone methyltransferase inhibitors, histone demethylase inhibitors, bromodomain inhibitors, and noncoding RNAs or antago-miRNAs are acquiring special relevance in the pharmaceutical industry.

Interestingly, life style and dietary regimes can also target epigenetic mechanisms, thereby impacting directly on the health status. In recent years, there is a growing interest in the exploration of the effect of dietary regimes on the epigenetic reprogramming. Therefore, in the coming years we will assist to an explosion of new therapeutic approaches based on changes in life style and nutrition in order to intervene in specific epigenetic mechanisms affecting health and disease status.

This book is focused on complex epigenetic mechanisms in several pathologies and explores how epigenetics can fuel precision medicine not only improving IVD and prognostic tools but also providing new efficient epigenetic-based therapeutic approaches to human diseases. The book focuses on recent progress in several areas of clinical epigenetics and discusses the advances in epigenomic technologies, including tools and applications based on next-generation sequencing to discover new epigenetic biomarkers that can help implement epigenetics into personalized medicine.

Furthermore, this new volume for Translational Epigenetic Series expands from the utility of epigenetic biomarkers for diagnosis and prognosis of several diseases to the use of epigenetic drugs for the treatment of some human diseases. The new volume focusses on the potential of epigenetics to improve the precision medicine in cancer (i.e., lung cancer, colorectal cancer, prostate cancer, gynecological cancers, bone cancer, pancreatic cancer, and brain cancer), cardiovascular and metabolic diseases, diabetes and obesity, inflammatory and autoimmune disorders, skin diseases, infertility, bone-related diseases, and neurodegenerative diseases, among others.

The book was written in a descriptive and comprehensive manner by outstanding experts in their corresponding fields for a broad target audience who are interested in this exciting field with the vocation of improving precision medicine.

José Luis García-Giménez, Valencia, Spain

Chapter 1: Perspectives and future directions of translational epigenetics in personalized and precision medicine

José Luis García-Giméneza,b,c; Jesús Beltrán-Garcíaa,b,c; Rebeca Osca-Verdegala,b,c; Federico V. Pallardóa,b,c;

Toshikazu Ushijimad;

Trygve O. Tollefsbole,f,g,h,i    a Department of Physiology, School of Medicine and Dentistry, University of Valencia, Valencia, Spain

b CIBER Enfermedades Raras, Center for Biomedical Network Research on Rare Diseases (CIBERER), Institute of Health Carlos III, Valencia, Spain

c INCLIVA Biomedical Research Institute, Valencia, Spain

d Division of Epigenomics, National Cancer Center Research Institute, Tokyo, Japan

e Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, United States

f Center for Aging, University of Alabama at Birmingham, Birmingham, AL, United States

g Comprehensive Diabetes Center, University of Alabama at Birmingham, Birmingham, AL, United States

h Nutrition Obesity Research Center, University of Alabama at Birmingham, Birmingham, AL, United States

i Cell Senescence Culture Facility, University of Alabama at Birmingham, Birmingham, AL, United States

Abstract

An epigenetic biomarker is any epigenetic mark or epigenetic mechanism which generally serves to evaluate health or disease status, progression, or treatment response. New discoveries in the field of epigenetics highlight the critical role of epigenetic mechanisms in health and diseases therefore generating new opportunities to identify epigenetic biomarkers for early detection, prognostication, and particularly personalized treatment of human diseases, the hallmark of precision medicine. Advances in precision medicine, including the development of new biomarkers and their translation to in vitro diagnostic (IVD) tests, the adoption of high-throughput technologies, such as next-generation sequencing and mass spectrometry into the clinical laboratory, together with the improvement of artificial intelligence applied to medical routine will substantially improve precision medicine in complex human diseases. In this chapter, we present the most advanced epigenetic IVD tests and epigenetic drugs which are currently being validated in clinical trials. In addition, we propose the basis of how epigenetic technology will contribute to the incorporation of precision medicine in clinical settings.

Keywords

cfDNA; Epigenetic biomarker; IVD; DNA methylation; miRNAs; Artificial intelligence

Abbreviations

AI 

artificial intelligence

ccfDNA 

cell-free circulating DNA

ChIP-Seq 

Chromatin Immunoprecipitation Sequencing

CRC 

colorectal cancer

DNMT 

DNA methyl transferase

DNMTi 

DNMT inhibitor

EGFR 

epidermal growth factor receptor

ENCODE 

ENCyclopedia of DNA elements

FDA 

Food and Drug Administration

FFEP 

formalin-fixed paraffin-embedded samples

HDAC 

histone deacetylase

HDACi 

histone deacetylase inhibitor

HEP 

Human Epigenome Project

IHEC 

International Human Epigenome Consortium

IVD 

in vitro diagnostic

slncRNA 

long non-coding RNAs

mAb 

monoclonal antibody

MS 

mass spectrometry

NGS 

next generation sequencing

PTMs 

post-translational modifications

RRBS 

Reduced Representation Bisulfite Sequencing

SAHA 

Suberoylanilide hydroxamic acid

WGBS 

Whole Genome Bisulfite Sequencing

Introduction

Precision medicine was defined by the National Research Council’s Toward Precision Medicine in 2008 as: The tailoring of medical treatment to the individual characteristics of each patient … to classify individuals into subpopulations that differ in their susceptibility to a particular disease or their response to a specific treatment. Preventive and therapeutic interventions can then be concentrated on those patients who will benefit, sparing expense and side effects for those who will not.¹ After the sequencing of the Human Genome, it was evident that genetics is not the only contributor to disease onset and progression and in some complex diseases genetics solely, or in combination with clinical data, cannot predict responses of patients to specific therapies. In fact, most human diseases are complex multifactorial pathologies, caused by genetic background and epigenetic mechanisms, which can modulate transcriptional programs and lead to adverse clinical outcomes. For a given pathogenic phenotype in a complex disease, there are multiple potential genes which could be genetically altered (i.e., mutations, copy number variations, and triplet expansions) and epigenetically controlled [i.e., DNA methylation, posttranslational modifications (PTMs) of histones, and miRNAs and lncRNAs].

In 2015, President Obama launched the Precision Medicine Initiative in the United States, which refers to individually tailored health care on the basis of a person’s genes, lifestyle and environmental effects.² In Europe, similar efforts were also initiated to implement personalized medicine, as recognized in the conclusions of the Council of the European Union at its meeting in December 2015.³

In this regard, epigenetics is a breakthrough science with the potential of improving precision medicine. Actually, epigenetic marks can act as bioarchives recruiting natural history of diseases and life-time exposures and explain biological changes that increase our susceptibility to diseases. Thus, epigenetics can be exploited for their diagnostic and prognostic value as biomarkers.⁴ Epigenetics and epigenome research provide more comprehensive analysis of human diseases. A number of initiatives have been launched during the past decade to map epigenomic and related (clinical and genetic) data, such as the International Human Epigenome Consortium (IHEC),⁵ the ENCyclopedia Of DNA Elements (ENCODE) project Consortium,⁶ the Human Epigenome Project (HEP) consortium,⁷ the NIH Roadmap Epigenomics Consortium⁸ and International Human Epigenome Project, EpiGeneSys (http://www.epigenesys.eu/en/) and more recently the 4D nucleosome project.⁹

In line with this, epigenetic biomarkers can help early diagnosis, disease progression monitoring, disease outcome prognostication, and prediction of future morbidities. Even more, epigenetic biomarkers can serve to select and stratify patients for specific treatments and to evaluate the positive or negative effects of therapeutic interventions in specific patient subsets.

Specifically, an epigenetic biomarker is defined as any epigenetic mark or altered epigenetic mechanism which generally serves to evaluate health or disease status and is particularly stable and reproducible during sample processing and analysis.¹⁰ An ideal biomarker can be measured in wide array of biospecimens such as body fluids (i.e., plasma, serum, saliva, semen, urine, and cerebrospinal fluid) or primary tissue samples [fresh tissue, cells, single cell isolated, fine-needle aspirates, formalin-fixed paraffin-embedded samples (FFPE), etc.].

Ideally, for precision medicine application in clinical settings, an epigenetic biomarker may cover at least one and preferably all of the following properties: (i) predict the risk of future disease development; (ii) detect a disease or pathological condition; (iii) provide information about the natural history of the disease; (iv) prognosticate outcome of disease; (v) become a companion diagnostic (i.e., the biomarker can respond to therapy); (vi) become a theragnostic biomarker by allowing simultaneous diagnosis and targeted therapy.¹¹

Knowledge regarding precision medicine in a wide array of diseases that continues to expand, however, for real implementation of precision medicine in clinical settings, it is essential to identify robust biomarkers using either -omic data alone, or in combination with clinical data which will allow identification of specific biomarkers,¹² performance of precision medicine-based clinical trials to deliver high-quality disease precision medicine, and incorporating artificial intelligence (AI) to manage properly -omic and clinical data to improve clinical settings.¹³ Epigenetics has experimentally advanced in the past decade, but growing evidence has demonstrated that epigenetic regulation is even more complex than genetics and not just a genetic complement. Therefore, although epigenetics can provide a lot of biomarker candidates, the current challenge is how to transform these candidates into a reliable biomarker.

In fact, although current research is generating a large number of biomarkers, the reality is that only few epigenetic biomarkers are integrated in clinical settings, mainly in cancer. Further efforts are being made in other biomedical fields such as cardiovascular diseases,¹⁴ psychiatric disorders,¹⁵,¹⁶ infertility,¹⁷ and others, but probably most of the new biomarkers discovered do not reach clinical applications in the next 5 years.

A major goal of biomedical research in epigenetics is to identify feasible biomarkers from the high number of candidates generated from Next-Generation Sequencing procedures [i.e., Whole Genome Bisulfite Sequencing (WGBS), Reduced Representation Bisulfite Sequencing (RRBS), Chromatin Immunoprecipitation Sequencing (ChIP-Seq) and Small RNA-seq, and long noncoding RNA-seq, among others]. This can include DNA methylation changes (e.g., CpG), histone modifications, noncoding RNAs expression, or chromatin structural changes (e.g., nucleosome positioning). However, the extraction of relevant biological data is time consuming and requires implementing bioinformatic and computational procedures to extract and analyze this amount of data. To address these challenges, an integrated approach combining data generation, data selection (including genetic, epigenetic, metabolomic, transcriptomic, and clinical data), and AI analysis on such data sets is needed.¹⁸

Genomic and epigenomic (and also other -omics) studies are usually performed independently, therefore contributing to the fact that the molecular, biological and physiological relationships between genetics and epigenetics remain almost unknown in most diseases. This situation supposes a disadvantage to develop precision medicine; therefore, the integrated understanding of genetic variation and epigenetic deregulation appears to now be more critical than ever before.¹⁹ Importantly, the current progress of AI technologies, such as machine learning and deep learning, is remarkable and enables multimodal analyses of big omics data. In this regard, it is important to develop platforms and automated systems (if possible) that can conduct multimodal analysis of medical big data using AI as this may accelerate the implementation of precision medicine.¹⁹

Some packages and bioinformatic tools have been developed to facilitate computational epigenetics analysis and comprehension. Some examples are CancerLocator, CpGenie,²⁰ DeepCpG,²¹ DeepSEA,²² EpiExplorer,²³ MethMarker,²⁴ RnBeads.²⁵ AI technologies not only will facilitate our understanding of the genetic and epigenetic interpatient heterogeneity, for example, in multiple cancers subtypes, but also will help to identify new biomarkers and contextualize them into the complex landscape of human diseases and in turn to stimulate the discovery of drug candidates against epigenetic targets.²⁶,²⁷

New drugs designed to interact with a specific molecular target, and especially those associated with the use of biomarkers which can predict patient response, showed the highest relative improvement in response rate.²⁸ In terms of clinical trials incorporating epigenetic biomarkers, an essential element that should be factored it is the feasibility of the test analyzing these biomarkers and the turnaround time for obtaining test results, particularly in those biomarkers analyzed by using next-generation sequencing approaches (i.e., RRBS or WGBS and SmallRNA-seq). However, the use of single biomarkers or specific signatures assessed by procedures such as RT-PCR approaches may help to incorporate epigenetic biomarkers to identify subsets of patients who can benefit from specific therapies and also to monitor the effectiveness of the applied therapies. In this regard, epigenetic biomarkers may help to demonstrate effectiveness of new therapies during clinical trials, by predicting trial endpoints, such as functional ability, disease progression, survival, or death; and even inform about the subject responsiveness to therapeutic interventions.

In parallel to the discovery and validation of new potential epigenetic biomarkers which can act in some cases as companion diagnostics for emerging epigenetic therapies. In this regard, we previously described laboratory strategies to accelerate the adoption of epigenetic biomarkers in the clinical routine.¹⁰,²⁹,³⁰

While mutations, specifically oncogenic mutations, in human cells are fixed and stable, epigenetic alterations are potentially reversible, and this reversibility makes them targets for specific epigenetic drugs. The modification of epigenetic marks well established for homeostatic physiological programs result in altered gene expression which in turn drive to diseases onset and progression. In cancer, several therapies based on the flexibility of the epigenetic programs have been developed. In this regard, DNMT inhibitors (DNMTi) and HDAC inhibitors (HDACi) alone or in combination with cytotoxic agents and targeted therapies have been clinically tested for several cancers and malignancies.³¹–³³ The development and clinical validation of epigenetic drugs is a promising field despite future treatments that most likely will consist of combining epigenetic drugs with other epigenetic drugs or nonepigenetic drugs.³⁴

Diagnostic, prognostic, and therapeutic applications of epigenetics in precision medicine

In the particular case of epigenetic biomarkers, there is currently a low number of commercially available epigenetic tests despite the fact that there is a considerable number of clinically validated biomarkers. There are a number of possible reasons for the limited number of commercially available test: (i) the scarce clinical trials performed; (ii) limited access of laboratories to technologies needed to measure them [including histone posttranslational modifications (PTMs) by mass spectrometry (MS), complex miRNA and DNA methylation profile signatures using next-generation sequencing (NGS)]; (iii) the arduous work of implementation of the regulatory roadmap during the development of such biomarkers; (iv) the scarce number of realistic studies of cost-effectiveness for the new biomarkers; (v) and the limited interest of the pharma industry to contribute to biomarker development unless such biomarkers would become a companion diagnostic tool.

Among the different properties described above for epigenetic biomarkers, some of the most valuable characteristics for such biomarkers are that several epigenetic markers (i.e., DNA methylation at specific CpGs, microRNAs, lnc-RNAs and posttranslational modifications in histones) can be detected in many body fluids including serum/plasma, nipple fluid aspirate, vaginal fluid, semen, saliva, crevicular fluid, and urine, therefore facilitating the incorporation of these potential epigenetic biomarkers in clinical settings.¹⁰,³⁵ Of special interest are cell-free circulating DNA (ccfDNA) and circulating microRNAs which have already provided commercially available IVD tests.

Recently, we published a compilation of IVD tests based on epigenetic biomarkers that are currently being commercialized for in vitro diagnostic in cancer. In Table 1.1, we present an IVD epigenetic test that is FDA-approved or CE-mark compliant, which helps to improve precision medicine. For colorectal cancers, several options are available, based on stool DNA (i.e., Cologuard and EarlyTect), liquid biopsy (Epi ProColon, EarlyTect, and Nu.Q), and FFPE tissue (miRPredX-31-3p). Some of them such as Epi ProColon and EarlyTect are being currently validated in large observational clinical trials (ClinicalTrials.gov Identifier: NCT03218423 for Epi ProColon and ClinicalTrials.gov Identifier: NCT03146520 for EarlyTect). Importantly, among the epigenetic IVD tests for colorectal cancers, miRpredX-31-3p predicts the potential clinical benefits associated with first-line anti-EGFR (epidermal growth factor receptor) therapy compared with anti-VEGF (vascular endothelial growth factor receptor)-based therapy or when second or further lines of treatment with anti-EGFR mAB therapy is more beneficial versus chemotherapy alone for multiple patient outcomes.³⁶–³⁸ In fact, in an interventional randomized clinical trial performed in 1808 subjects (ClinicalTrials.gov, Trial Registration ID: NCT03362684), the predictive potential of the miR-31-3p expression level was assessed to evaluate the clinical outcomes of patients treated with anti-EGFR therapy (cetuximab) plus FOLFOX-4 vs. FOLFOX-4 only in stage III CRC patients (the patients enrolled in the PETACC-8 Study).

Table 1.1

Other tests, such as the Therascreen MGMT Pyro kit for glioblastoma, can be used in, the DNA obtained from blood as well as FFPE tissues. The use of this test may allow personalized therapy in glioblastoma patients with temozolomide.⁶⁴,⁶⁵ For lung cancer, the test EpiproLung BL Reflex Assay measuring the methylation status of SHOX2 and PTGER4 by methyl-specific PCR from cfDNA in blood samples can be assessed. A recent study has demonstrated the potential use of the analysis of methylation status of SHOX2 in the therapeutic effect assessment and prognosis prediction of stage IV lung cancer patients undergoing first-line standard chemotherapy, combined radio- and chemotherapy or tyrosine kinase inhibitor (TKI)-based targeted therapy.⁶⁶

In cervical cancer, DNA methylation of ZNF582 gene can be tested using Cervi-M assay Methyl-specific PCR in epithelial cells obtained from cervical brush. Other CE-marked test is QIAsure methylation test which identifies cancer-specific epigenetic changes in the FAM19A4 and mir124-2 loci in cervical cells and enables the physician to assess whether a woman of 30 years or older with an HPV infection is progressing toward cervical cancer (CIN3 +) in need of treatment.⁶⁷,⁶⁸ For breast cancer, the Therascreen PITX2 DNA methylation RGQ PCR kit, which measures the methylation in the promoter 2 of the pituitary homeobox transcription factor 2, can be performed in both DNA from blood and FFPE samples. The Therascreen PITX2 DNA methylation test can be used to identify breast cancer patients who will benefit of the anthracycline-based chemotherapy.⁶⁹ For cancers of unknown origin, EPICUP assay based on DNA methylation profile assessed in FFPE tissue can be performed. Detailed information can be found in our recent review.¹¹

Epigenetic-based therapies

In the past decade both, the FDA and the EMA, have approved various epigenetic drugs used to treat a wide variety of diseases such as different cancers, hypertension, and even infections caused by viruses, among others. These drugs are usually classified into DNMTi or HDACi, although miRNA-based therapies (miR-therapies) are gaining popularity nowadays and some miR-therapies are currently in the last phase of clinical trials (Tables 1.2–1.4). In addition, some epigenetic drugs based on the regulation of histone posttranslational modifications, such as histone acetylase inhibitors (HATi) and histone methyltransferase inhibitors (HMTi), are already entering into the final clinical phases (Tables 1.2–1.4). Regarding the three FDA-approved DNMTis, two of them, 5-aza-2′-deoxycytidine and 5-azacytidine, are used to treat myelodysplastic syndromes,⁷⁰ although the potential of these drugs is being evaluated in different clinical trials for other cancers such as leukemia,²³,⁷¹,⁷² breast cancer⁷³,⁷⁴ and lung cancer,⁷⁵,⁷⁶ among others. The third, hydralazine, is in the preclinical evaluation to assess its potential to treat cervical cancer⁷⁶ and breast cancer.⁷⁷

Table 1.2

MDS, myelodysplastic syndrome; AML, acute myeloid leukemia; CMML, chronic myelomonocytic leukemia; MPN, myeloproliferative neoplasm.

Table 1.3

HDAC, histone deacetylase; EZH2, enhancer of zeste homolog 2; DOT1L, DOT1-like histone H3K79 methyltransferase; BET, bromodomain and extra-terminal motif; DLBCL, diffuse large B cell lymphoma; HL, Hodgkin’s lymphoma; NHL, non-Hodgkin’s lymphoma; AML, acute myeloid leukemia; MLL, mixed-lineage leukemia; MDS, myelodysplastic síndrome; MPN, myeloproliferative neoplasm; CLL, chronic lymphocytic leukemia.

Table 1.4

Note: ALS, amyotrophic lateral sclerosis; CHC, chronic hepatitis C; HCV, hepatitis C virus; MPM, malignant pleural mesothelioma; NASH, nonalcoholic steatohepatitis; NSCLC, nonsmall-cell lung carcinoma.

Regarding HDACis, suberoylanilide hydroxamic acid (SAHA), and romidepsin are approved by the FDA for the treatment of cutaneous T-cell lymphoma, and its efficacy against other types of cancer, such as urothelial carcinoma and colorectal carcinoma are currently being studied.⁷⁸,⁷⁹ Similarly, Panobinostat is proposed for the treatment of multiple myeloma, and is currently in preclinical studies to analyze its efficacy against chronic myeloid leukemia,⁸⁰ epithelioid sarcoma,⁸¹ endometrial cancer,⁸² and prostate cancer.⁸⁰ Finally, Belinostat is used to treat peripheral T-cell lymphoma, and it is in different clinical stages for the treatment of lung cancer⁸³ and multiple myeloma⁸² (Table 1.2). However, in addition to FDA-approved epigenetic drugs, numerous epigenetic-based therapies have been developed including natural compound derivatives or known drugs with new potential epigenetic applications [e.g., trichostatin A, resveratrol, curcumin, sulphoraphane, sinefungin, daminozide (N-(dimethylamino) succinamic acid)].³⁴,⁸⁴

Finally, miRNA-based therapies have not yet obtained FDA approval for medical intervention, although there are some candidate therapies in clinical development or in phase 1 and phase 2 clinical trials (Table 1.3). Among several miRNA mimics and antagomiR candidates being evaluated in clinical trials, Miravirsen (produced by Roche/Santaris), designed to treat hepatitis C, is considered the flagship product of this class of future drugs.⁸⁵

Conclusions and perspective

Although the Human Genome Project was completed in 2003 and large-scale genetic association analyses have been performed in a wide array of diseases, to explain the mechanism of disease onset and progression only using genetic markers is neither easy nor sufficient. Epigenetics has contributed to providing new avenues in the comprehension of events that occur in complex diseases, thereby providing new mechanisms based on epigenetic dysregulation that contribute to disease outcome. Future medicine advances toward predictive medicine, more precise and personalized practice and theragnosis to directly inform clinical decision-making and providing therapeutic solution in an specific and timely manner. Epigenetic biomarkers may further contribute to this purpose and to reduce the escalating healthcare expenditure worldwide.

There exists some barriers to effectively implementing precision medicine in the clinical routine such as the selection of the appropriate molecular test, the time required for results delivery, the tools available to interpret the results obtained from the selected epigenetic test, and also financial concerns. From a technical and technological point of view, to implement new epigenetic IVD tests into clinical settings, the new epigenetic tests developed should be easy-to-use, inexpensive and performed in widely extended analytical methods at clinical laboratories, as for example those based on RT-qPCR and microarrays for both DNA methylation and miRNA analyses. Next-generation sequencing is being completely implemented into clinical routine also for epigenetic analysis in part due to the fact that the prices for high throughput analysis are decreasing in recent years. An example of this kind of epigenetic test is the UroMark 150 biomarker assay for bladder cancer detection which was developed using Infinium 450K Human Methylation array (Illumina).⁸⁶ Recently, UroMark test was validated by analyzing 150 loci by means a microdroplet-based PCR application followed by NGS of amplification of target loci using RainDance Technology, and analyzing the results using a random forest bioinformatic analysis.⁸⁷ Besides the feasibility of the incorporation of NGS technologies in clinical laboratories, another aspect to consider is the price of such epigenetic tests. Collinson defined a series of interesting questions for evaluating the scientific and clinical evidence and cost-effectiveness for the clinical use of new potential biomarkers.⁸⁸ In addition, we further described laboratory strategies to accelerate the adoption of epigenetic biomarkers in clinical settings.¹⁰ Adoption of new technologies, such as NGS and MS, into the clinical laboratory, joined to the efforts led by the research and medical community, pharmaceutical industry, IVD, and medical device manufacturers, will substantially contribute to the development and introduction of new IVD tests based on epigenetic biomarkers. Moreover, serious efforts should be made to identify biomarkers in noninvasive biospecimens, preferably using biofluids, which will contribute to anticipating disease diagnosis and to also increase patient compliance with screening campaigns for cancer detection, as occurs, for example, with Epi proColon 2.0 test and EarlyTect colorectal cancer assay.

Moreover, the discovery of new epigenetic biomarkers not only may allow the implementation of epigenetic IVD test into the clinical routine but also may contribute to the development of a large number of epigenetic drugs that are expected to treat diseases in a personalized medicine manner. In this regard, epigenetic IVD test may increase the ability to adapt treatments to different stages of disease and even to different individual characteristics of patients, improving the clinical responses of as many patients as possible. Interestingly, as is being observed by the large number of clinical trials with epigenetic drugs, they are capable of modulating specific targets involved in a wide variety of diseases which allows the same drug can be postulated as a potential candidate for the treatment of different diseases.

Importantly, the potential of epigenetics in clinical settings, for both biomarker identification and epigenetic targets characterization, will be improved by adapting AI and machine learning procedures to epigenetic biomarkers analyses which in turn will contribute to definitely providing robust biomarkers for precision medicine.¹⁸ One important scenario is that the cost of specific treatments and the appropriate use of targeted therapies guided by epigenetic biomarkers are expected to streamline the high cost required to receive personalized therapies, as occurs in cancer. We definitely need to explore the possibility that more patients can benefit from precision medicine, to identify the correct treatment for the appropriate patient supported by appropriate biomarkers. In conclusion, epigenetics applied to precision medicine will provide more information than ever before which will improve patient care.

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Chapter 2: Translational epigenetics in precision medicine of colorectal cancer

Jesús Beltrán-Garcíaa,b,c; Rebeca Osca-Verdegala,b,c; Salvador Mena-Mollád,e; Marta Seco-Cerveraa,b; Lorena Peiró-Chovab,e; José Luis García-Giméneza,b,c; Pierre Laurent-Puigf,g; Andrés Cervantesb,h,i,j    a CIBER Enfermedades Raras, Center for Biomedical Network Research on Rare Diseases (CIBERER), Institute of Health Carlos III, Valencia, Spain

b INCLIVA Biomedical Research Institute, Valencia, Spain

c Department of Physiology, School of Medicine and Dentistry, University of Valencia, Valencia, Spain

d Department of Physiology, Faculty of Pharmacy, University of Valencia, Valencia, Spain

e EpiDisease S.L. Parc Científic de la Universitat de València Biomedical Research Institute INCLIVA, Spin-Off of CIBERER (ISCIII), Valencia, Spain

f Cordeliers Research Center, INSERM, CNRS SNC 5096, Sorbonne University, University of Paris, Paris, France

g Institute of Cancer Paris CARPEM, APHP, Department of Biology, European Hospital Georges Pompidou, Paris, France

h Medical Oncology Department, Hospital Clínico Universitario de Valencia, Valencia, Spain

i Center for Biomedical Network Research on Oncology (CIBERONC), Institute of Health Carlos III, Valencia, Spain

j Department of Medicine, School of Medicine and Dentistry, University of Valencia, Valencia, Spain

Abstract

Epigenetic alterations play a key role in the initiation, progression, and metastasis of cancer. Therefore, epigenetic marks and mechanisms are potential biomarkers for precision medicine in cancer. Considering the substantial role of the epigenetic alterations in DNA methylation, miRNA expression, and posttranslational modifications in histones in colorectal cancer (CRC) initiation and progression, worldwide research has identified new epigenetic biomarker for CRC diagnosis, prognosis, and prediction of treatment response, of which some have been approved and are currently being commercialized. In this chapter, we provide an overview of the most promising epigenetic biomarkers and describe commercially available epigenetic-based in vitro diagnostic tests for CRC that can actually be implemented into clinical practice.

Keywords

Precision medicine; Epigenetic biomarker; IVD; DNA methylation; miRNA; ctDNA; Circulating nucleosomes

Abbreviations

CIN 

chromosomal instability

CMS 

consensus molecular subtypes, consisting transcriptome-based classification of CIMP

CIMP 

CpG island methylator phenotype

CRC 

colorectal cancer

ctDNA 

circulating tumor DNA

EMT 

epidermal-mesenchymal transition

FIT 

fecal immunochemical test

FOBT 

fecal occult blood test

5-FU 

5-fluorouracil

MMR 

mismatch repair

MSI 

microsatellite instability

OS 

overall survival

TNM 

tumor-node-metastasis

Conflict of interest

PLP is a co-inventor of patent on the role of Mir31-3p in the prediction of response to anti-EGFR therapy. The other authors declare that this work was conducted in the absence of any commercial or financial relationships with the companies commercializing these tests that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by AES2016 (ISCIII) with grant number PI19/00994 and Proyectos de Desarrollo Tecnológico en Salud with grant number DTS21/00193 AES2017 and Plataformas ISCIII de apoyo a la I+D+i en Biomedicina y Ciencias de la Salud with grant number PT20/00029, co-financed by the European Regional Development Fund (ERDF), Instituto de Salud Carlos III and and CIBERer (Biomedical Network Research Center for Rare Diseases and INGENIO2010). J.B.G is supported by a grant Contratos i-PFIS (IFI18/00015) and co-financed by the European Social Fund. R.O.V. is supported by the grant PFIS FI20/00202 Contratos predoctorales de formación en investigación en salud Instituto de Salud Carlos III; R.O.V. is supported by the Grant Contract PFIS grant (FI20/00202). This study was supported by grants from the Instituto de Salud Carlos III [grant number PI18/01909] to A.C.

Introduction

Colorectal cancer (CRC, MIM 114500) encompasses two types of highly aggressive and common types of cancers, namely, colon cancer (the fourth most common malignancy) and rectal cancer (the eight most common one).¹ The prevalence of CRC cases varies geographically around the world but collectively, CRCs present the third most commonly diagnosed form of cancer worldwide in both sexes, accounting for 11% of all diagnosed cancer cases¹ and about the 9% of all cancer-related deaths.² In this context, 5-year survival rates range from more than 90% for stage I to less than 10% for stage IV CRC.³ To CRC, onset and progression contribute genetic mutations, copy number variations, epigenetic alterations, microsatellite instability (MSI), and altered transcription programs.⁴ These molecular inputs contribute to acquire increasingly dysplastic features in precursor lesions (adenomas and serrated lesions), which can sometimes progress to an adenocarcinoma. In particular, changes in DNA methylation and miRNAs signatures contribute to pathways dysregulation involved in various cellular mechanisms such as cell cycle, transcription, autophagy, apoptosis, inflammatory and immune response, and angiogenesis as well as invasion and metastasis. CRC is a complex disease because its heterogeneity in terms of its biological behavior, prognosis, and response to therapies. So, the early detection of precursor lesions and early-onset CRC in asymptomatic individuals during screening is essential for the CRC prevention and importantly, to improve the outcome of patients.

Colonoscopy is considered the gold standard for CRC screening because it has the potential to both detect and remove precursor lesions. However, colonoscopy is an invasive and expensive procedure, which is hampered by complications such as hemorrhage and perforation. Moreover, colonoscopy is associated with low compliance rates, so delaying diagnosis of CRC. In this context, the identification of new biomarkers in noninvasive or semiinvasive biospecimens is further recommended.⁵ So, the fecal occult blood test (FOBT) and fecal immunochemical test (FIT) were developed and are currently the most commonly used noninvasive screening tests in Europe and other Western countries. However, FOBT and FIT tests have lower sensitivity and specificity compared with colonoscopy, at least for precursor lesions such as adenomas.⁵ Moreover, both tests, FOBT and FIT, have limited sensitivity for detecting proximal compared to distal CRC⁶,⁷ and relatively low in detecting early stage I CRC (53%) and advanced adenomas (≥ 1.0 cm) (27%).⁸ Because the potential for reducing the burden of CRC by early detection is significant, several efforts are currently being made to develop, preferably noninvasive or semiinvasive [i.e., circulating tumor DNA (ctDNA)], CRC screening tests and to improve the adherence rates of participation for screening because people are reticent to be screened by current available methods.⁹ Besides the early identification of precursor lesions and early-stage CRC, it is also important to molecularly classify tumors to stratify patients properly. In this regard, the current tumor-node-metastasis (TNM) classification system for CRC staging is inadequate for prognostication.¹⁰ Moreover, the feasibility of TNM classification system for clinical decision-making is limited, particularly for intermediate stages.¹⁰ From a molecular perspective, CRC has been historically classified into subgroups on the basis of three pathophysiological pathways of carcinogenesis consisting on chromosomal instability (CIN), MSI, and CpG island methylator phenotype (CIMP).¹¹ CRC subgrouped as CIN, accounts for 80%–85% of CRCs and refers to a high level of alterations in chromosomes or large portions of chromosomes with duplications or deletions, which results in activation of cellular growth-promoting pathways and/or inhibition of apoptotic pathways.¹² These tumors came from adenomatous polyps on the basis of bi-allelic inactivating mutation of APC, SMAD4, TP53 genes and activating KRAS gene mutation.¹²,¹³ MSI subgroup is characterized by mutations in MLH1 gene and/or epigenetic silencing of the MLH1 gene promoter by hypermethylation, which accounts for the 80% of the sporadic cases of CRC.¹⁴,¹⁵ Notably, the likelihood of MSI in CRC varies according to the stage of CRC, with a higher incidence in the early stages (approximately 20% in stages I and II and 12% in stage III) and a lower incidence in the metastatic phase (4%–5%). CIMP subtype is characterized by epigenetic instability and widespread hypermethylation of promoter CpG island loci, resulting in the inactivation of several tumor suppressor genes or tumor-related genes (i.e., CACNAG1, SOCS1, RUNX3, NEUROG1, and MLH1). Several gene panels have been proposed to characterize CIMP + CRCs. The result of these analyses revealed that CIMP tumors have distinct etiology, molecular features, and epigenetic landscape.¹⁶ To overcome the shortcomings associated with tumor heterogeneity, even within the aforementioned CIN, MSI, and CIMP subgroups, Guinney and colleagues used a large-scale data sharing and analytics approach across six international expert teams to identify four gene expression-based consensus molecular subtypes (CMS1–4) of CRC.¹⁷ Generally, CMS1 tumors display a higher percentage of MSI, hypermethylation, and high level of mutations, and are associated with better survival. CMS2 subtype contributes to larger subset of subtypes accounting for almost 37% of all tumor subtypes. CMS2 tumors have SCNA high, microsatellite stable, activated WNT and Myc pathway and elevated EGFR with mutated TP53 gene. CMS3 tumors are characterized by high mutation rate in KRAS gene and epithelial characteristics. CMS4 has a high CpG methylator phenotype with strong stromal infiltration, upregulation of genes involved in epidermal-mesenchymal transition (EMT), upregulated angiogenic features, and hyperactivated TGFb pathway.¹⁶ Particularly, based on this recent consensus for molecular subtypes classification of CRCs, MSI is associated with CMS1 (MSI immune subtype), which is characterized by a CIMP + status.¹⁸,¹⁹ Concomitantly, the presence of a BRAF V600E mutation, is another common feature of CMS1 which can be found in about 30% of dMMR cases and is limited to sporadic MSI. Importantly, it is possible to test for the BRAF V600E mutation and MLH1 promoter methylation to differentiate sporadic tumors from Lynch syndrome-associated tumors in this setting.

The selection of appropriate therapies for CRC patients is also a clinical need. Among the therapies proposed for CRC, anti-EGFR mAb (epidermal growth factor receptor) therapy is not indicated for carriers of RAS mutations (~ 50% of patients with metastatic CRC) because the mutations in the RAS gene (mainly in exons 2, 3, and 4 of KRAS and NRAS) make metastatic CRC patients nonresponders to anti-EGFRs mAB treatment.²⁰ Beside genetic mutations, other epigenetic mechanisms should be considered. For example, Sun et al. found miR-31 represses the expression of RAS p21 GTPase activating protein (RASA1), and therefor activates CRC cell growth and tumorigenesis.²¹ These findings identify miR-31 as a modulator of this critical pathway and therefore affects patient responses to anti-EGFR therapy. Because aberrant DNA methylation, altered histone posttranslational modifications marks and the disruption of microRNAs programs play an important role in CRC, it is obvious the mechanisms underlying CRC that can be positively influenced by changing lifestyle are directly connected with epigenetics.²² Epigenetics, is a discipline contributing to spectacular advances in biomedicine, which aims to improve precision medicine by discovering new epigenetic mechanisms and providing new biomarkers, epigenetic targets, and epigenetic drugs with potential uses in cancer therapy. Epigenetic biomarkers can help in CRC screening, staging, disease follow-up, and therapy selection thanks to their ability for early diagnosis, disease progression monitoring, disease outcome prediction, selection and stratification of patients by risk, and even the evaluation of the positive or negative effects of therapeutic interventions in specific patient subsets.²³,²⁴ So, the identification of new biomarkers to allow clinicians to select those patients who can benefit from the established therapies is a current clinical need in CRC.

Potential epigenetic biomarkers for CRC

Epigenetic biomarkers for CRC based on DNA methylation

In CRC, aberrant hypomethylation and hypermethylation have been identified in the promoter regions of important tumor-suppressor genes,²⁵–²⁷ including CDKN2A (at the promoters of each of its two encoded distinct cell cycle-regulatory proteins), p16INK4A²⁸,²⁹ and p14ARF,³⁰ in the mismatch repair gene MLH1³¹ and the adenomatous polyposis coli (APC).³²

Particularly, MLH1 promoter methylation in CRCs is being used as epigenetic biomarker in CRCs that exhibit loss of MLH1 and/or PMS2 protein expression. In this regard, the most frequent cause of MLH1 inactivation is through somatic inactivation due to bi-allelic promoter hypermethylation.¹⁴ Thus, the analysis of MLH1 hypermethylation analysis in patients with CRC with loss of MLH1 expression is currently being implemented in clinical laboratories for differentiating between Lynch syndrome and sporadic CRCs with mismatch repair (MMR) deficiency. In addition, the MLH1 hypermethylation, together with BRAF mutations, are associated with serrated polyps and serrated adenocarcinomas.³³ The analysis of the methylation of MLH1 for the diagnosis of CRC has been tested in blood samples in different studies and found that sensitivity ranged from 18.4% to 42.9% and specificity 97.6%–100%.³⁴–³⁶ Guinney et al. identified four gene expression-based consensus molecular subtypes (CMS) to classify CRC (CMS1–4). Among them, CMS1 subgroup is defined by a high degree of promoter hypermethylation leading to MSI, including MLH1, and high mutational profile.¹⁷ Importantly, patients with MMR deficiency and high MSI status (dMMR-MSI-H) define a subgroup of CRC patients who have a favorable prognosis but who do not benefit from 5-fluorouracil (5-FU) treatment.³⁷,³⁸ Interestingly, dMMR-MSI-H CRC tumors are feasible to be treated with anti-PD-1 antibody (pembrolizumab) because these tumors have a strong lymphogenic antitumor immune response through the expression of programmed cell death 1 ligand 1 (PD-L1).³⁹,⁴⁰ Methylation of SFRP2 gene (secreted frizzled-related protein 2), which is a modulator of WNT signaling, allows to detect precancerous lesions (adenoma) in both blood/plasma and stool samples. In this context, SFRP2 methylation for adenoma detection gives rise a sensitivity ranging from 6.4% to 81.1% in plasma samples, with corresponding specificities ranging from 73% to 100%.⁴¹,⁴² In the case of stool-based SFRP2 methylation the sensitivity for adenoma detection ranged from 27.8% to 76% and specificity was 55%.⁴³,⁴⁴ The values of sensitivity for CRC detection were better than those obtained for the detection of precancerous lesions providing sensitivity ranging 56.3%–94.2% with specificity of 77%–96.8% in stool⁴³–⁴⁶;