DNA methylation: its role in transcriptional regulation and association with lung cancer

DNA methylation in transcriptional regulation

The term “Epigenetics” is used to define the heritable changes in phenotype or gene expression caused by mechanisms not involving primary DNA sequence alterations.¹ These mechanisms include DNA methylation, histone tail modifications, nucleosomal rearrangements/remodeling, and noncoding RNAs (ncRNAs). These mechanisms interact with each other and follow a precise molecular orchestration in order to maintain or change epigenetic marks throughout the genome; this crosstalk seems to be pivotal for the maintenance of cell differentiation, growth, and homeostasis.^2,3 There are many enzymes mediating this crosstalk. For instance, MeCP2, a methyl-binding domain-containing protein, decks on methylated DNA and recruits co-repressor complexes such as histone methyltransferases and histone deacetylases; thus, establishing an inactive chromatin state that hinders transcription factor accessibility and leads to loss of gene expression.^4,5 It has been shown that DNA methylation status can determine nucleosome occupancy and further influence gene expression.⁶ Recently, it has been shown that long ncRNAs also regulate the interaction between chromatin and remodeling complexes, such as PRC2,⁷ by bringing them to specific genomic loci and shifting their position when necessary.⁸

DNA methylation is the most studied epigenetic modification in humans. It designates the addition of a methyl group (CH₃) at position 5′ of the amino base cytosine within CpG dinucleotides,⁹ leading to the formation of 5-methylcytosine. This reaction is catalyzed by DNA methyltransferases (DNMTs), which can act de novo (mainly DNMT3a and DNMT3b) or maintain the existing methylation patterns (mainly DNMT1)^10–12 assisted by accessory proteins such as UHRF1. Demethylation can be passive when the maintenance mechanism fails to copy the pattern to the newly synthesized strand following DNA replication, or an active process that occurs via a 5-hydroxymethyl cytosine intermediate catalyzed by TET proteins and encompasses the action of a number of accessory enzymes.^11–17 It seems that the first oxidation of 5-methylcytosine to 5-hydroxymethyl cytosine is carried out by TET1¹³ while several ways of thereafter transforming 5-hydroxymethyl cytosine to cytosine have been suggested and involve both nuclear and base excision repair pathways.^15,16

DNA methylation is implicated in various physiological processes, such as cell differentiation, X-chromosome inactivation, gene imprinting, and genomic stability. DNA methylation alterations in human disease are well established to date,¹⁸ although the list of epigenetic-driven diseases is growing fast. Global hypomethylation¹⁹ and loci-specific hypermethylation²⁰ have been frequently reported in cancer.²¹ Originally, changes in DNA methylation were studied in gene promoters, where hypermethylation was associated with downregulation of tumor suppressor genes and hypomethylation triggers upregulation of oncogenes.²² More recently, taking advantage of the development of genome-wide analysis techniques, intra- and intergenic methylation changes have also been described. Alterations of DNA methylation within the gene body can affect alternative intragenic promoters and enhancers, ncRNA expression, transposable elements mobility, alternative splicing, or polyadenylation.^23,24 Intergenic methylation changes can also affect enhancers or insulators; therefore, leading to gene silencing or activation, respectively,²⁵ or alter the expression of intergenic ncRNAs.^23,24 The involvement of DNA methylation in transcriptional regulation is based on the chromatin conformation changes that are associated with it. As mentioned DNA methylation occurs in combination with other epigenetic mechanisms and determines whether a particular stretch of DNA is loose and accessible to the transcriptional machinery or is tightly wrapped around nucleosomes in a dense chromatin form prohibitive of transcription factor binding.²³ The most exciting point is probably that this relationship is bidirectional; it is now known that certain transcription factors can place epigenetic marks upon binding to DNA and can therefore alter DNA methylation.²⁶

In biomarker studies, both DNA methylation and messenger RNA (mRNA) expression compete for a place in molecular diagnostics. The main disadvantage of detecting DNA methylation only is that it does not indicate the level of expression deregulation of the affected gene(s). One would argue that expression deregulation ultimate proof is only demonstrated by protein assays; however, determining mRNA expression is providing good preliminary evidence. In the case of DNA methylation, there is no such evidence and one can hypothesize the outcome on RNA.

On the other hand, DNA methylation analysis has significant analytical and biological advantages. DNA methylation changes require at least one cell cycle, which makes this modification robust following sampling; RNA expression profiles deteriorate quickly upon removal of the sample from the body. In addition, DNA is a much more stable chemical entity than RNA; therefore, DNA methylation assays do not need special sampling requirements and are applicable to archival material, where mRNA analysis can be particularly challenging.

Lung cancer biomarkers

Lung cancer is one of the most frequently diagnosed cancers in Europe²⁷ and the US,²⁸ accounting for 13% of all new cases of cancer in the world.²⁹ Lung cancer is divided into two main types: small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). NSCLC represents approximately 85% of lung tumors with adenocarcinoma, squamous cell carcinoma, and large cell carcinoma representing the main histological subtypes. The precise histomolecular classification of lung tumors plays a critical role in personalized medicine and effective treatment of patients.³⁰ Lung cancer still remains the leading cause of cancer-related deaths worldwide.²⁹ This is mainly due to very late diagnosis,³¹ and there are worldwide efforts from the lung cancer research community to introduce national screening strategies.³² There is a consensus that implementation of molecular markers can assist clinical management of lung cancer. Multiple studies focused on the development of biomarkers for early detection,^33–36 metastatic risk assessment,^37,38 and therapeutic stratification of patients.^39–41

According to the Biomarkers Definitions Working Group, the term “biomarker” is used to describe a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biomarker qualification involves four major steps: 1) discovery phase, 2) experimental validation, 3) preclinical validation, and 4) clinical validation.^31,42 Numerous studies describe new biomarkers and their potential use in the clinic;^43,44 however, very few of them manage to reach clinical validation.³⁵ The main obstacles are the poor reproducibility of the studies, sample heterogeneity, methodological biases, quality control implementation, and significant interlaboratory variability in experimental performance and data analysis.⁴⁵ To date, biomarkers for lung cancer are mainly used to define and monitor therapy (eg, EGFR, KRAS, or ALK mutations).⁴⁶ DNA-based biomarkers present practical advantages,⁴⁷ and the established evidence regarding the involvement of epigenetic deregulation in human cancer, including lung,⁴⁸ sets research of DNA methylation biomarkers at very high priority in lung cancer diagnostics.⁴⁹

DNA methylation detection techniques and methodologies

The need for detection of abnormally methylated DNA in tissue samples and body fluids inevitably leads to discussing methodology issues and the appropriateness of different approaches. A large number of DNA methylation detection methods and modifications exist to date, each having certain advantages and disadvantages.

There are three major approaches regarding the means of discrimination between 5′-methyl cytosine and cytosine:

1. Chromatin immunoprecipitation,⁵⁰ which utilizes anti-5′-methyl cytosine³⁴ or methyl-binding domain proteins.⁵⁰
2. Methylation-specific restriction enzyme⁵¹-based methods, which take advantage of the differential recognition of methylated DNA sequences over their unmethylated counterparts.⁵²
3. Bisulfite-based methods, which utilize the fact that bisulfite deaminates cytosine to uracil but does not affect 5′-methyl cytosine, effectively transforming this epigenetic modification into a sequence difference.⁵³

Regarding throughput, genome-wide or gene/locus-specific approaches are employed according to the needs. Microarrays and deep sequencing are mainly employed in the first type while dideoxy sequencing,⁵⁴ pyrosequencing,⁵⁵ and MALDI-TOF⁵⁶ are normally utilized for medium-throughput, locus-specific applications. Historically, a few other techniques have been used, such as single-strand conformation polymorphism analysis⁵⁷ and high-resolution melting.⁵⁸ A very frequent method also used for locus-specific applications is methylation-specific polymerase chain reaction (MSP)^53,59 and its quantitative real-time derivative qMSP⁶⁰ or MethyLight.⁶¹

Various particularities can be recognized in detecting DNA methylation in body fluids. These samples are known to carry a tiny load of abnormal nucleic acids in the presence of high amounts of “contaminating” normal DNA. Exception to this is the detection of DNA methylation in the peripheral leukocytes in leukemias or for monitoring therapeutic schemes with epigenetic drugs⁶² where the target DNA is ample.

The reliable detection of nucleic acids associated with minimal residual disease in samples such as plasma/serum, urine, sputum, bronchial washings (BWs), saliva, is demanding and devious. The most widely used approach has been bisulfite conversion of DNA followed by MSP or qMSP. The concept here is that methylation-specific primers bearing cytosines (forward) and guanines (reverse) preferably at the 3′ end of both primers will generate amplicons only from the methylated DNA copies of the target sequence under optimal conditions. The vast experience of the research community in polymerase chain reaction amplification techniques allows, in combination with the availability of new engineered hot-start Taq (Thermus Aquaticus) polymerases and reliable thermocycling hardware, to reach high levels of fidelity. Endpoint MSP is a method that revolutionized epigenetic research.⁵³ Its main disadvantage is the lack of quantitation efficiency as well as problems in sensitivity and specificity. The latter can be overcome by using methylation enrichment pyrosequencing, which employs pyrosequencing to confirm the status of an MSP amplicon.⁶³ The real-time version of MSP, referred to as qMSP⁶⁰ or MethyLight,⁶¹ overcomes many of the endpoint assay problems. It is highly sensitive (especially when using fluorescent probes) in visualizing minute amounts that would never been seen on gels. The use of fluorescent probes adds one more level of sequence specificity and allows for multicolor detection of internal controls to normalize for DNA input. What is currently missing from qMSP/MethyLight is an internal control of bisulfite conversion.

As mentioned, an important problem in body fluids is the low availability of DNA. To make the problem more challenging, bisulfite conversion reduces DNA quality while the subsequent cleanup limits significantly recovery of DNA quantities. It is widely accepted today in the epigenetic biomarker research community that it is unlikely to find the perfect single marker. Most studies point to the discovery of panels of biomarkers, and thus multiple assays. Therefore, the tiny amount of DNA recovered has to be split in different reactions. One way to overcome this problem is to multiplex four targets per reaction using probes with different fluorescent dyes.⁶⁴ Of course the level of optimization required to prove equal amplification factors over five concentration logs between the different amplicons is significant. The abundance of each target is different; thus, in the absence of such calibration, the high-abundance target (10⁻¹–10⁻² per genome) will probably consume resources impairing amplification of the rare (≤10−³) copies. A different approach is the post-bisulfite whole-genome (bisulfitome) amplification,⁶⁵ which demonstrates promising findings, but further research is required to prove the potential extent of its use.

A last important point, frequently bypassed, regarding methodology is the amount of DNA added in a qMSP reaction. qMSP users claim detection of targets in dilution with normal DNA as low as 10⁻⁴. In this case, one needs to ensure 10⁴ genome equivalents (32 ng) are added in the reaction to have one abnormal copy keeping in mind that MSP targets only one bisulfite converted strand. This is also an important issue for consideration in whole bisulfite amplification.

Overall, massive progress has been demonstrated in the last decade in methodological approaches for DNA methylation detection in body fluids. Many academic groups have involved themselves in this research, and the contribution of the biotechnical industry must also be acknowledged. Although there are still issues regarding the quality control of the used techniques, there should be no doubt that these will be overcome and a clinically useful method will be very soon available.

DNA methylation aberrations in lung cancer

The high frequency of DNA methylation aberrations in lung cancer has been shown in a large number of studies. Development of epigenetic biomarkers, which could reach clinically validated applications, would assist in personalized management of patients with lung cancer. Identification of markers for early detection (diagnosis), risk assessment (prognosis), and therapeutic stratification of patients is of utmost importance and has been the subject of numerous studies during the last decade.⁶⁶ Investigating the tumorous, dysplastic, or normal lung tissue is a more direct way to search for biomarkers when compared with surrogate tissues. The relevant literature is vast, and older studies have been previously reviewed.⁴⁹ In the present review, we are summarizing the most recent (post-2012) information and attempt to evaluate the progress of DNA methylation biomarkers in the clinical management of lung cancer. We searched for reports in PubMed using combinations of keywords such as “lung cancer”, “DNA methylation”, “biomarker”, “diagnosis”, “early detection”, “prognosis”, “body fluids”, “plasma”, “serum”, “sputum”, “bronchial lavage”, and “bronchial washings”.

The findings of recent DNA methylation studies in lung cancer tissue are summarized in Table 1. This table contains 131 genes that can be categorized according to the biological processes they are involved in (Figure 1). While the vast majority of the studies analyzed in Table 1 are mainly focused on the aberrant promoter DNA methylation, studies investigating whole genome as well as gene body methylation patterns are also included. For instance, Nelson et al⁶⁷ tried to identify new methylation key markers utilizing whole-genome and exonic regions of various lung cancer-related genes. Among the most thoroughly studied genes for DNA methylation in lung cancer are RASSF1, RARβ, p16 (CDKN2A), MGMT, SHOX2, APC, BRCA1, and DAPK, providing a fair list of candidates with biomarker potential. However, Do et al,⁶⁸ in an attempt to confirm previous studies showing tumor-specific methylation of DNA repair genes, suggest that methylation frequency of lung cancer-associated genes like ATM, BRCA1, MLH1, and XPC is likely to be overestimated in the literature. This raises the importance of diligent validation of previous results, especially if obtained using different methodologies.

Figure 1 Bar chart demonstrating the distribution of the most important groups describing the function of the genes with abnormal DNA methylation in lung cancer tissue. Notes: Gene ontology was performed based on biological function using The Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 (http://david.abcc.ncifcrf.gov/).

Table 1 List of recent (post-2012) studies identifying abnormal DNA methylation patterns in human lung cancer tissue Abbreviations: AC, adenocarcinoma; BGS, bisulfite genomic sequencing; BSP, bisulfite sequencing PCR; COBRA, combined bisulfite restriction analysis; COPD, chronic obstruction pulmonary disease; FFPE, formalin fixed paraffin embedded; GEO, Gene Expression Omnibus; IQR, interquartile range; lncRNA, long non coding RNA; mRNA, messenger RNA; MS-HRM, methylation-sensitive high-resolution melting; MSP, methylation specific PCR; NCBI, National Center for Biotechnology Information; NSCLC, non-small-cell lung cancer; PCR, polymerase chain reaction; qMSP, quantitative methylation-specific PCR; SCC, squamous cell carcinoma; SCLC, small-cell lung cancer; T, tumor-only samples; T/N, tumor-normal pairs.

Diagnostic markers

As mentioned earlier, despite the advances in medicine and cancer biology, lung cancer is largely diagnosed at late stages, leading to poor prognosis and high mortality rates.³¹ Still the only screening method is low-dose computerized tomography scan of the lungs that is applied to high-risk individuals but includes exposure to radiation, a high rate of potential false positives, and high cost.⁶⁹ Development of a biomarker-based test could boost early or more accurate diagnosis that is able to distinguish among the different types of lung cancers. Zhao et al⁷⁰ proposed a panel of four genes (MYF6, SIX6, BCL2, and RARβ) that could offer a sensitivity of 93.07% and a specificity of 83.33% in the diagnosis of stage I NSCLC. A similar study shows that the methylation status of NEUROG2, NID2, RASSF1A, APC, and HOXC9 can also serve in early diagnosis of NSCLC (sensitivity 91.26%, specificity 84.62%); in particular, NEUROG2 seems to be able to discriminate between adenocarcinomas and squamous cell carcinomas.⁷¹ The DNA methylation status of miRNAs could also serve as a diagnostic marker in NSCLC. Hypermethylation in NSCLC tissue was observed for hsa-miR-34a, hsa-miR-9-2, and hsa-miR-9-3³³ as well as miR-193.⁷² Furthermore, in order to empower the precision of the diagnostic outcome of each study, several meta-analyses have been conducted.^70,73–75 Taking into account the results of many individual studies, these meta-analyses confirmed that the methylation status of MGMT,⁷⁰ CDKN2A,⁷⁴ APC,⁷⁵ and RARβ⁷³ promoters is strongly related to lung cancer development.

Prognostic markers

Lung cancer is a highly heterogeneous malignancy. Subpopulations of patients exhibit different risk of metastatic relapse.⁷⁶ Therefore, markers of prognostic value enabling prediction of the disease aggression and potential course present a long-standing clinically unmet need. The methylation status of numerous genes has been associated with patients’ survival. Recent studies have associated methylation of CDKN2A,⁷⁷ BRCA1,⁷⁸ HOXA-2/10,⁷⁹ CHFR,⁸⁰ TFPI-2,⁸¹ miR-9-3,⁷² and miR-34b/c⁸² with poor survival of patients with NSCLC and miR-886-3p⁸³ in SCLC patients. On the other hand, hypermethylation of SHOX2, PITX2,⁸⁴ DKK1,⁸⁵ and CYP24A1⁸⁶ was related to better prognosis in NSCLC. In addition to hypermethylation, cancer-specific hypomethylated loci have been proposed as prognostic factors. More specifically, low methylation levels of SCUBE3, SYT2, KCNC3, KCNC4, GRIK3, CRB1, SOCS2, ACTA1, ZNF660, MDFI, ALDH1A3, and SRD5A2 in lung tumors was an indicator of poor survival in a recent study by Lokk et al.⁸⁷ Surprisingly, although RASSF1 hypermethylation was mainly associated with poor outcome,^88–90 a study by Morán et al,⁹¹ proposed its protective value.

Therapeutic stratification markers

Directly linked to prognostication is the need for biomarkers that can predict response to particular therapeutic regimens in order to facilitate personalized therapy. Currently, chemotherapy and/or radiotherapy are used based on classical histopathological criteria with very little to no input from molecular constitution of the tumor. However, as personalized medicine rapidly gains ground with the use of targeted therapies (EGFR, ALK, etc) it becomes profound that a more global genetic and epigenetic profiling of individual tumors is required. Many studies have shown that specific DNA methylation signatures can divide patients into subgroups depending on their response to specific anticancer therapy.^76,92,93 Hypermethylation of IGFBP3 has been associated with lower response of NSCLC patients to cisplatin therapy,⁹² while methylation of miR-503 seems to also play a role in this pathway.⁹⁴ Another recent study reports that unmethylated SFRP5 is a predictor of response to EGFR tyrosine kinase inhibitors therapy, independently of EGFR genotype.⁹³ Also, a phase 2 clinical trial investigated whether MGMT promoter methylation can predict response to Temozolomide treatment in advanced aerodigestive tract (including lung cancer) and colorectal cancers. Unfortunately, the number of lung cancer patients exhibiting MGMT methylation was too small to support the power of the study, leading to uninterpretable results.⁹⁵

DNA methylation detection in body fluids

The high frequency of DNA methylation detected in primary tumors led researchers to explore the feasibility of detecting these alterations in body fluids for diagnostic purposes. Body fluids represent an extremely attractive clinical material for the management of disease because they can be collected using noninvasive or minimally invasive procedures. Assays target cancer-related DNA methylation abnormalities that may be detected in circulating tumor cells or in cell-free DNA or both. However, the detection of cancer-specific DNA methylation in body fluids presents major challenges, mainly associated with low target abundance in an excess of normal DNA. Similarly to the “Lung cancer biomarkers” section, older reports have been adequately reviewed previously;⁴⁹ therefore, we present the most recent updates (post-2012) in this review (Table 2).

Table 2 List of recent (post-2012) studies involving detection of DNA methylation in body fluids from patients with lung cancer Abbreviations: BGS, bisulfite genomic sequencing; CO, Colorado; EBUS-TNA, endobronchial ultrasound with transbronchial needle aspiration; NM, New Mexico; NSCLC, non-small-cell lung cancer; PCR, polymerase chain reaction; qMSP, quantitative methylation-specific PCR; qPCR, quantitative PCR; T/N, tumor -normal pairs.

Bronchial washings

Although bronchoscopy does not qualify as a noninvasive process, it is a standard part of the clinical workup of patients suspected for lung cancer, and currently, diagnosis on this sample type is confined to cytological examination. Unfortunately, the diagnostic efficiency of this technique is relatively poor and nearly half of the cases are missed.^96,97 Therefore, it is imperative to develop molecular biomarkers capable of improving the diagnostic efficiency of lung cytology.

The feasibility detecting promoter hypermethylation in bronchial lavage was demonstrated 15 years ago,⁹⁸ and this was followed by a number of relevant publications (reviewed by Liloglou et al⁴⁹). More recently, the combined detection of RASSF1 methylation and KRAS mutation in BWs has been reported to aid in the detection of malignancy in false-negative or ambiguous cytological outcomes by correctly diagnosing as cancer 29% of the false-negative or doubtful cytological outcomes.⁹⁹ Furthermore, a panel of DNA methylation biomarkers has been described to show a substantial gain in sensitivity of detection over standalone cytology.⁶⁰ In this large retrospective case-control study, encompassing BWs from 655 individuals, a diagnostic algorithm based on the methylation profile of CDKN2A, TERT, WT1, and RASSF1 demonstrated 82% sensitivity, 92% specificity, and a diagnostic accuracy of 85.9%. Finally, DNA methylation of SHOX2 alone demonstrated a very high sensitivity in BWs (96%) but the specificity was less than optimal (78%).¹⁰⁰ However, a significant improvement in specificity has been shown for SHOX2 methylation when endobronchial ultrasound with transbronchial needle aspiration (EBUS-TBNA) samples were used; specificity reached 99% in this study while sensitivity was improved (82%–99%).¹⁰¹ Moreover, the negative predictive value also increased from 80% to 99%, which from a clinical point of view, might help in avoiding further unnecessary invasive procedures.

Sputum

Sputum is another type of body fluid that has been widely tested for lung cancer biomarker qualification. Its major advantage over BWs is that it is noninvasive and can be practiced in three consecutive day collections at home and sent by post. Its major disadvantage though is the high heterogeneity of the sample in lung cell content (a large percentage of samples are inadequate and contain mainly oral epithelial cells),⁴⁹ especially when it is noninduced. The methylation status of numerous genes has been recently studied (Table 2) in both spontaneous and induced sputum specimens; some of them alone¹⁰² and some of them grouped in panels.¹⁰³ DNA methylation in sputum has also been shown to improve the classification accuracy. Leng et al,¹⁰³ in a large case–control study using two different cohorts of patients, reported that seven-gene panels improved the prediction accuracy of standalone clinicopathological data models. More recently, RASSF1 methylation seemed to significantly aid cytological evaluation by improving sensitivity (from 22% to 52%) without diminishing specificity (94%).¹⁰²

Plasma/serum

The detection of cancer-indicative biomarkers in blood has been the holy grail of cancer molecular diagnostics. Blood is not only an easily accessible and minimally invasive specimen but also highly standardized in the clinical setting across the globe. The two main problems lie with the high dilution of the potential target but also the lack of localized origin. In other words, abnormal DNA in the bloodstream could have originated anywhere in the body. Nevertheless both plasma and serum specimens from lung cancer patients have been used to detect DNA methylation aberrations in numerous genes;⁴⁹ the most recent studies are listed in Table 2. Although most of these alterations seem to be highly specific, they do not reach the sensitivity levels demanded in clinical diagnostics. For instance, SEPT9 promoter hypermethylation demonstrated 44.3% sensitivity and 92.3% specificity,¹⁰⁴ while BRMS1 exhibited 47.9% sensitivity and 100% specificity.¹⁰⁵ So far, RASSF1A and RARβ seem to be the most promising candidates since different studies have shown that both markers combined can achieve 75%–85% sensitivity and 75%–100% specificity.^106,107 Beyond diagnosis, blood has also been recently used to evaluate significance of gene promoter hypermethylation in plasma of NSCLC patients for prognosis and therapeutic monitoring.^105,107 Ponomaryova et al¹⁰⁷ report that the methylation index for RASSF1A and RARβ in plasma from lung cancer patients decreased after neoadjuvant chemotherapy and the methylation levels were comparable to those observed in healthy individuals after surgery. Furthermore, all five patients who had a relapse showed an increase in the methylation level of either gene within 9 months after surgery. More recently, the presence of promoter hypermethylation of BRMS1 in NSCLC patients’ plasma was associated with a lower overall survival for stage I–III and stage IV tumors.¹⁰⁵

Despite the increasing number of relevant studies addressing DNA promoter methylation as a promising biomarker in body fluids, the preclinical research context and lack of clinical validation in follow-up studies in different cohorts has hampered the development of clinically useful diagnostic markers. In this regard, so far the EpiProLung BL Reflex Assay (Epigenomics, Berlin, Germany) is the only clinically validated and Conformité Européene (CE)-labeled DNA methylation biomarker for diagnosing lung cancer. The assay, which determines the methylation level of SHOX2,³⁵ has been thoroughly validated in bronchial aspirates showing 78% sensitivity and 96% specificity.¹⁰⁰ In addition to bronchial aspirates, SHOX2 methylation has also been analyzed in other body fluids such as plasma, pleural effusions, and transbronchial aspirates.^101,108,109 Ilse et al¹⁰⁹ noted that the detection of malignancy in pleural effusions improved when cytological examination was combined with SHOX2 assessment and that SHOX2 methylation was not only found in malignant effusions from lung cancer patients but also in effusions from malignant tumors of other origins.

Concluding summary

DNA methylation is a critical epigenetic modification affecting chromatin conformation and transcriptional activity, subsequently influencing globally the biology of the mammalian cell. Inability to maintain correct DNA methylation patterns inevitably leads to disease. The list of relevant human diseases is growing and ranges from developmental disorders to autoimmune conditions and cancer. Lung cancer is the most lethal form of human neoplasia, and epigenetic deregulation has been extensively shown in lung neoplastic tissues utilizing a wide range of methodological approaches. The feasibility of identifying abnormally methylated DNA in body fluids has been established. The focus now is moving to identifying the correct targets and improving the methodologies to reach clinical standards. Despite the significant volume of relevant literature, currently there is not enough published work to include sufficient clinical validation to allow moving into a randomized clinical trial. The latter will be the ultimate test proving the patient benefit, since early detection of lung cancer will certainly lead to a stage shift and thereafter improvement of survival of lung cancer patients.

Acknowledgments

Paschalia Pantazi and Alexandra Papaioannou are recipients of IKY Fellowships of Excellence for Post Graduate Studies in Greece – Siemens program. Amelia Acha-Sagredo is a recipient of a postdoctoral fellowship from the Basque Government. Georgios Nikolaidis is supported by the EC program “Translational Potential” (TransPOT; EC Contract 285948). Triantafillos Liloglou’s research is supported by the Roy Castle Lung Cancer Foundation, UK. This article is dedicated to our dearest friend, Lefteris Konstandinidis, who died from lung cancer on September 2, 2014.

Disclosure

The authors report no conflicts of interest in this work.

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