Skip to main content

Hydroxymethylation and tumors: can 5-hydroxymethylation be used as a marker for tumor diagnosis and treatment?

Abstract

5-Methylcytosine (5mC) is considered as a common epigenetic modification that plays an important role in the regulation of gene expression. At the same time, 5-hydroxymethylcytosine (5hmC) has been found as an emerging modification of cytosine bases of recent years. Unlike 5mC, global 5hmC levels vary from tissues that have differential distribution both in mammalian tissues and in the genome. DNA hydroxymethylation is the process that 5mC oxidates into 5hmC with the catalysis of TET (ten-eleven translocation) enzymes. It is an essential option of DNA demethylation, which modulates gene expression by adjusting the DNA methylation level. Various factors can regulate the demethylation of DNA, such as environmental toxins and mental stress. In this review, we summarize the progress in the formation of 5hmC, and obtaining 5hmC in a cell-free DNA sample presents multiple advantages and challenges for the subject. Furthermore, the clinical potential for 5hmC modification in dealing with cancer early diagnosis, prognostic evaluation, and prediction of therapeutic effect is also mentioned.

Background

In the process of carcinogenesis, changes in epigenome as early events are generally the same as changes in the genome, which can inactivate tumor suppressor genes and activate proto-oncogenes and play an essential role in the occurrence, development, invasion, and metastasis of tumor [1]. For the past few years, researchers have extended the studies on tumors to the field of epigenetics. During tumor malignancy, distinct epigenetic modifications such as histone modification, DNA methylation, and demethylation, non-coding RNA may become effective biomarkers for high-risk assessment, early diagnosis, prognosis, and reference for drug treatment [2]. In mammals, TET dioxygenase mediates the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Then, replication-dependent 5mC dilution or thymine DNA glycosylase (TDG)-dependent base excision repair (BER) performed. Thus, 5-methylcytosine (5mC) can be reverted to unmodified cytosine (C) [3]. In a series of oxidation reactions, 5-hydroxymethylcytosine (5hmC) is the first product. 5-Methylcytosine (5mC) and α-ketoglutarate (α-KG) formed 5hmC with iron and oxygen as cofactors under the catalysis of ten-eleven translocation (TET) family proteins (Fig. 1). TET enzymes can further oxidize 5hmC to produce 5-formylcytosine and 5-carboxylcytosine [4, 5]. Consistent with their relatively low content in the genome, 5fC and 5caC have poor stability [4,5,6].

Fig. 1
figure 1

Chemical structures of 5-methylcytosine (5mC) and its oxidized modifications. Cytosine is converted to 5-methylcytosine by both DNMT1 and DNMT3. 5-Methylcytosine is oxidized by TET1, TET2, or TET3 to yield 5-hydroxymethylcytosine (5hmC). By a similar reaction, using the same enzymes, 5-hydroxymethylcytosine is further oxidized to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). Both 5-formylcytosine and 5-carboxycytosine are depyrimidated by the thymine DNA glycosylase (TDG) and processed by the base excision repair (BER) pathway. All these pathways could result in unmodified cytosine. ɑ-KG = ɑ-ketoglutaric acid; SA = succinic acid

There are two mechanisms of DNA demethylation: active demethylation and passive demethylation [7]. Active demethylation is that TET protein oxidized 5-mC to generate 5-hmC, 5-fC, and 5-caC. 5-fC and 5-caC can directly start base excision repair (BER) to generate cytosine under thymine glycosylase (TDG). Passive demethylation is also called the deamination pathway. In the reaction, 5-mC is converted into thymine under the deaminase, then initiate base excision repair with DNA thymine glycosylase and finally generate cytosine. The main methods of passive demethylation are the following [8]: Oxidize the methyl group to carboxyl group and complete demethylation by decarboxylation. Remove 5-mC by BER pathway and replace with unmodified cytosine. Specific deaminase recognizes and converts 5-mC to C. The 5-mC was converted to C through deamination to form a mismatch. Then, BER was started under TDG. Demethylation through hydrolysis. 5-hmC plays an essential role in both active demethylation and passive demethylation and is closely related to gene expression regulation. Different reaction processes regulate dynamic DNA demethylation. The availability and content of substrates and cofactors, post-transcription, and post-translation modifications of TET proteins are all factors. Previous studies that focused on structure and biochemical functions have elucidated how TET proteins and TDG mediate active DNA demethylation [8, 9]. Among them, TET proteins may play a different role than the traditional catalytic enzyme. Further analysis is needed to clarify the function of TET proteins themselves and their role in DNA demethylation. Besides, with the continuous development of mapping and tracking technology of 5mC oxidation products, the functions of oxidation products 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) can be studied in depth. Different from 5mC, the content of 5hmC in different tissue has better stability and stronger robustness, which presents tissue specificity, especially showing the differences between cancerous tissue and healthy tissue [10]. Since 5hmC is enriched in promoters, enhancers, and other transcriptional regulatory elements, it is more closely related to gene expression [11, 12]. This leads us to believe that it may have the potential of applying in the clinical diagnosis of tumors, like some vital regulatory molecules of epigenetics, and could be used to predict the metastasis, recurrence, and prognosis. Several surveys indicate that the potency and clinical convenience of tumor biomarkers are essential for the successful delivery of certain drugs and better clinical care for cancer patients. Due to the long incubation period of cancer and symptoms only start to emerge at an advanced stage, effective and timely treatment may not be available. Hence, efficient screening and early detection are particularly crucial to improve patient survival and quality of life [11, 13].

With the rapid development of liquid biopsy technique, cell-free DNA (cfDNA) originating from different tissues and entering blood circulation has become available research in recent years in the field of clinical diagnosis [14]. It only requires blood samples from cancer patients, with advantages of high efficiency, low cost, and smooth operation. From the perspective of patients, compliance is better and dynamic monitoring can be achieved. Most importantly, it makes up for the lack of inadequate acquisition due to the low content of 5hmC in tumor tissues [15, 16]. In this review, we list the diagnostic function of 5hmC in cfDNA in early detection as well as its advantages in anticancer drug selection and efficacy monitoring. Meanwhile, in a variety of different types of cancer, the specific mechanisms by which epigenetic pattern changes identify oncogene mutations and dysregulations remain to be elucidated, and the problem of standardization requirements for all kinds of samples is yet to be solved.

Advantages and disadvantages of ctDNA as 5hmC research sample

Liquid biopsy is a diagnostic analysis of body fluid samples, such as circulating tumor cells (CTC), circulating tumor DNA (ctDNA), and exosome in the blood [17]. In recent years, ctDNA and cfDNA detection have been drawing much attention in diagnosis and treatment and become the research hotspot, as they have advantages of convenience, minimally invasive, convenient real-time monitoring, and easier to reflect the information of genome-wide tumorigenesis [17]. The methods used in ctDNA and cfDNA detection are known as polymerase chain reaction (PCR) technology (such as ARMS-PCR (amplification refractory mutation system) and digital PCR) and the next-generation sequencing technology (NGS) [18]. Although some ctDNA tests have been proved to have clinical validity and feasibility in analyzing some advanced cancer species, such as colorectal cancer, most of the ctDNA tests lack sufficient evidence to support their clinical practicability [19, 20]. At this point, useful clinical evidence of ctDNA tests in early tumor detection, treatment monitoring, or residual tumor surveillance is limited, and there is also a lack of clinical practicability evidence.

Given the rapid progress of ctDNA research and clinical tools and guidelines, it will be necessary to re-evaluate the pertinent literature so that standardized guidelines for various indexes of body fluid samples will be available soon [21]. There are many uncertain factors in the pre-processing steps of ctDNA test samples, and these variables may affect the sample purity, so as the subsequent test evaluation [22]. On the whole, many factors may affect the final result in the following steps, including blood drawing, collecting, processing, storage, transportation, and DNA extraction and purification. Also, biological factors related to patients may affect the release of cell-free DNA through the bloodstream, such as smoking, anemia, non-malignant diseases like inflammation, and autoimmune diseases (Fig. 2). However, the technology of sample control is still limited and many problems remain to be solved [21, 23]. To determine whether ctDNA can be used as an active marker in clinical practice, it is necessary to comprehensively evaluate its accuracy, sensitivity, specificity, and repeatability [24, 25]. At present, the method for assessing the accuracy and uniqueness of the plasma cfDNA test is mainly through methodological comparison with paired tissue samples [26]. Studies have shown that there may be differences between plasma ctDNA tests and biopsy results, which may be caused either by the performance of analysis (such as the analysis sensitivity) or by other biological properties. Multiple factors such as cancer type, tumor stage, temporal and spatial heterogeneity of tumor, and different sampling time between tissue and plasma can cause differences. Due to the low content of ctDNA in plasma, it is significant to explore the lower detection limit [27]. Considering the impact of biological property and nonbiological factors above, it is more objective and recommended to verify the detectability near the detection limit by incorporating cell lines or artificially constructing standardized samples [28]. The minimum analytical sensitivity and specificity required for ctDNA tests should be established on specific intended uses to facilitate its clinical application while emphasizing that mutation results must be combined with other clinical information to adjust the therapeutic regimen. The proportion of ctDNA in plasma cell-free DNA (also known as purity) varies from person to person. Similarly, the percentage of ctDNA in total cell-free DNA in different patients also has significant differences [29, 30]. This explains why we need to study further the prognostic significance of mutation abundance in ctDNA tests.

Fig. 2
figure 2

Detecting 5hmC biomarkers in cfDNA of human cancers. The workflow of 5hmC-Seal profiling from cfDNA is shown. Purified cfDNA is ligated with standard sequencing adapters. 5hmC-containing cfDNA fragments are selectively labeled and captured, followed by PCR amplification and next-generation sequencing (NGS)

As epigenetic changes in cell-free DNA (cfDNA) have been widely observed to show high sensitivity and specificity in disease detection and classification, including 5-methylcytosine (5mC) and 5-methylcytosine (5hmC), it is vital to collect and integrate the bioinformation of cfDNA for future clinical application. However, due to the lack of effective collection, standardized quality control, and analysis procedures, the clinical utilization of these data cannot be effectively realized. CFEA (http://www.bio-data.cn/ CFEA) is a cell-free epigenome database used to involve 5mC, 5hmC, and NP (nucleosomal localization) of 27 kinds of human diseases. Bioinformation was quality controlled and standardized in the database. Besides, relevant clinical information was collected to facilitate the search and download of cell-free epigenome data. Users can better view and compare the development of the cfDNA epigenome in different stages of the tumor. The comprehensive and timely CFEA database has excellent potential for the development of liquid biopsy biomarkers for various human diseases [16].

The value of 5hmC in tumor diagnosis

Produced under the active demethylation mediated by the TET (ten-eleven transposition) dioxygenase family, 5mC is modified and oxidized to 5-hydroxymethyl cytosine (5hmC) and further oxidized to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) [10]. As a relatively stable DNA marker, 5hmC plays a role as a gene expression marker in the whole genome sequencing map of mammalian cells and tissues [31]. Since 5hmC is mainly distributed in the active transcriptional region, accompanied by open chromatin, and allowed histone modification, it is considered to be closely related to gene expression, which can be an ideal candidate biomarker with high chemical stability for cancer diagnosis [14, 32]. When 5mC in cfDNA was sequenced with bisulfite as a high background level inhibitory marker, the results were severely disturbed by DNA degradation [33]. However, unlike 5mC, 5hmC can be mapped with a low level of DNA and highly sensitive selective chemical markers, and the identified 5hmC has cancer type specificity [34,35,36]. Changes in 5hmC rich in enhancers, gene bodies, and promoters are closely related to changes in gene expression levels [37]. They can reproduce changes in gene expression in different cell states. Therefore, it can be combined with 5mC in cfDNA of liquid biopsy for non-invasive diagnosis and prognosis. Sensitive detection of cytosine modification patterns is helpful to identify cancer-specific biomarkers, and dynamic monitoring 5hmC needs to be put into clinical use because of the lower cost of obtaining blood samples, higher patient compliance, and clinical convenience [38].

Further, researchers found that in lung cancer patients, the content of 5hmC in cfDNA decreased gradually, while in the liver and pancreatic cancer patients, cell-free hydroxymethylome had their disease-specific changes [14, 26, 39]. However, for HCC (hepatocellular carcinoma), after calculating the HCC scores of postoperative and recurrent HCC samples, the researchers learned that HCC scores could accurately follow up on the treatment and understand the recurrence status of patients [40, 41]. In combination with the HCC score, cell-free 5hmC sequencing provides an opportunity to detect HCC, monitor treatment outcomes, and monitor recurrence status [42]. The unbiased gene-level analysis of tSNE (t-distributed stochastic neighbor embedding) showed that HCC patients could be isolated from HBV-infected patients and healthy individuals by comparing the cell-free 5hmC pattern [43]. HCC-specific differentially expressed genes (q < 0.001, fold change > 1.41, 1006 genes) could isolate HCC from healthy populations and most HBV samples [44].

Researchers have reported that an improved hMe-seal method makes the quantitative analysis of low levels of 5hmC in cfDNA possible, which has significant advantages over traditional methods [45, 46]. Firstly, hMe-seal does not further degrade cfDNA in samples, unlike the bisulfite method used for cell-free 5mC sequencing. Secondly, compared to genome-wide sequencing methods including mutation sequencing, the concentration of 5hmC not only improves the cost-effectiveness (10–20 million reads,~ 0.5-fold human genome coverage) but more importantly enables the quantitative determination of 5hmC cfDNA from low-frequency tissue sources, such as blood cell samples [33, 46]. In addition to data of the gene body, 5hmC in non-coding regions can also be used as potential biomarkers to predict cancer types [47]. What is noteworthy is that different types of cancer exhibit specific patterns of cell-free hydromethylome, which leads us to think that we can use specific cell-free 5hmC characteristics to predict cancer types with high precision. In the process of step by step clinical application, this will comprehensively analyze and summarize the genetic and epigenetic changes of various tumor states and further enhance the level of individualized diagnosis and precision medicine (Fig 3).

Fig. 3
figure 3

Advances in genome-wide 5-hydroxymethylcytosine spectrum in tumor studies. Genome-wide 5-hydroxymethylcytosine spectrum could advance the field of cancer research. 5hmc-based analysis can help understand the underlying mechanisms associated with cancer progression, identify new diagnostic tools, and provide more effective drug regimens and efficacy monitoring for cancer treatment

The potential value of 5hmC in different types of cancer

The traditional view is that a tumor is due to gene mutation and amplification caused by carcinogenic factors, which could lead to the disorder of cell proliferation and differentiation [48]. However, with further understanding of cancer in recent years, researchers found that regulatory mechanisms of the non-coding region perform more critical roles in the occurrence and progression of the tumor [49,50,51]. For example, DNA methylation, histone modification, and chromatin structure mutation are all found to alter in multiple types of tumors [52, 53]. Researchers found 5mC correlated with the initiation, progression, histological grade, and poor prognosis of human cancers. However, the biological significance of 5hmC in human cancer remains elusive [54]. Discovery of the mechanism for ten-eleven translocation enzymes (TET1, TET2, and TET3), which are capable of the oxidation 5mC to 5hmC and gene regulation, shows that cytosine methylation is essential in mammalian genomic DNA and transcriptional regulation [55, 56]. Global loss of 5hmC, associated with TET downregulation and alteration of TET functions, points to a link between cancer epigenetics and immunoregulation [57, 58].

If the usual way of DNA methylation which is mediated through the coordinated actions of several DNA methyltransferases (DNMTs) that transfer a methyl group from S-adenosyl methionine (SAM) to the carbon-5 position of cytosine does not occur, DNA becomes progressively demethylated through a passive replication-dependent mechanism [59, 60]. This dysregulation happens in both hematological and solid tumors, for example, colon, liver, lung, stomach, skin (melanoma), esophageal squamous cell carcinoma, prostate, blood, and breast tumors [61,62,63]. The most important thing is that the reduction of TET1 expression appears to be a tumor suppressor gene that can promote the growth and metastasis of cancer [64,65,66]. Thus, investigating the underlying molecular mechanisms between DNMTs and cancer is of great importance for therapeutic strategies.

5hmC and malignant melanoma

Melanoma is a common and aggressive form of cancer, which makes the diagnosis very difficult. Even in the same pathology, the outcomes can vary significantly for lesions [67, 68]. For now, there is only one panel of 31 RNA-based prognostic biomarkers known as Decision Dx-Melanoma that claims to improve prognostic predictions, which is only offered by a single laboratory in the USA [69, 70]. The decreased expression of 5hmC in malignant tissue has been shown consistently in a wide range of different cancers, including melanoma [71,72,73]. Also, the loss of 5hmC in melanoma has been reported to lead to reduced survival, the decrease of 5hmC was positively correlated with the prognosis of malignant melanoma [74]. At the same time, TET2 reduced the expression in different pathological stages of malignant melanoma [75]. Along with the TET2 reducing, the tumor became more malignant [76, 77].

Clinically, the detection of 5hmC in the skin section of malignant melanoma guides the early diagnosis and prognosis [78]. Besides, 5hmC showed a similarly low level, whether it was from an exposed part [79]. It suggests that the low content of 5hmC may not be correlated with ultraviolet light [80]. These all suggest that 5hmC may be a viable prognostic biomarker in melanoma [81, 82].

5hmC and breast cancer

Breast cancer (BC) is a very heterogeneous disease characterized by different molecular and histopathological features; responses to treatment of its subtypes differ based on estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) status [83, 84]. The complexity has been a big challenge for researchers and clinicians to stratify breast tumors. By evaluating a total of 15 pairs of normal and carcinoma samples in human breast tissue, the results show the levels of 5hmC were dramatically reduced in cancer group compared with the healthy breast tissues [85]. 5hmC of stem cells in breast cancer patients has reduced [86]. The expression of 5hmC was observed by immunohistochemical staining [87]. Researchers found that the nucleus of the unmutated breast cancer cells was 5hmC-positive, while the 5hmC in TET2-mutated tissue was negative [88]. In order to determine the prognostic role of TET family proteins and DNA glycosylase (TDG) in patients with early breast cancer, the expression of mRNAs encoding TET1-3 and TDG in 162 breast cancer tissues was quantified. The result that TET1 mRNA was significantly related to overall survival [89], as well as TET3 and TDG mRNAs were independent prognostic factors for patients, suggests the relationship between DNA methylation and central signaling pathways involved in cancer [90]. There was more lymphocyte infiltration near the tumor cells. The expression of 5hmC in breast cancer of mutated TET2 was decreased [91, 92]. That is to say, abnormal regulation of TET2 may lead the hydroxymethylation of DNA in the wrong way. Furthermore, these all have contributed to finding the epigenetic medication, which can enhance the antitumor immune response.

5hmC and bladder cancer

Despite significant advances in surgical techniques and adjuvant therapies, bladder cancer remains a highly prevalent and lethal malignancy [93]. Previous categories of tumor markers include proteomic markers [94, 95], genomic markers, and epigenetic markers [96, 97], and epigenetics is a field that combines genomics and proteomics [98]. The DNA methylation is the most common epigenetic change investigated in bladder tumor markers [99]. 5hmC level has seemed like an independent marker of bladder cancer gradually. In a study of 57 bladder cancer patients and 20 control individuals, by methylation-specific PCR, promoter methylation of E-cadherin, p16, p14, and RASSF1A in DNA isolated from exfoliated cells was evaluated [100].

Consistent with recent findings in different types of cancers, healthy bladder tissues showed intense nuclear 5hmC staining. However, a partial or complete loss of 5hmC was shown in bladder cancer. Patients with higher 5hmC levels had lived longer than the ones with lower levels; meanwhile, the lower 5hmC level, the higher stage and metastasis of the tumor. 5hmC is found to appear in gene-rich regions of the bladder in the normal genome and is correspondingly low in the cancer genome. The loss of 5hmC occurred in different cancer-related genes during bladder carcinogenesis. As the expression of TET2, L2HGDH, and vitamin C transporters (SVCT1 and SVCT2) was decreased in all bladder cancer cell lines, vitamin C is found to be capable of increasing 5hmC as a cofactor of TET proteins [101]. The researchers then treated several bladder cancer cell lines with vitamin C at various concentrations and periods. It turned out that vitamin C increased the 5hmC levels in T24 cells, which depends on time and concentration. Moreover, similar effects were observed that 5hmC levels increased with vitamin C, which inhibited cell growth in renal cancer [102]. It is also indicated that vitamin C can increase 5hmC levels in bladder cancer cells. Importantly, researchers observed that 5hmC promotes the activity of TET enzymes rather than increase the expression levels directly. It was also reported that high-dose vitamin C could restrain cancer cells by producing H2O2 [4], unlike low-dose vitamin C, which suppressed cancer growth in an H2O2-independent way that included 5hmC restoration.

At present, there has been much research on 5hmC in the tumor. However, their specific function and mechanism in various cancers are not clear. Therefore, analyzing cancer-related genome-wide hydroxymethylated regions and binding sites of TET proteins will help to figure out their importance in cancer.

Discussion

DNA methylation is a process in which cytosine (C) is catalyzed by DNA methyltransferase to produce 5mC, and the product is called the “fifth base” of DNA. DNA hydromethylation refers to the oxidation of 5mC by TET to produce 5hmC, the “sixth base” of DNA [103]. It is believed that methylation can inactive some genes, leading to gene silencing. However, demethylation can cause gene reactivation and expression, and the content of DNA methylation in tumors of different systems is still controversial. Studies have shown that the distribution of 5hmC is tissue-specific, and there are differences in the distribution of 5hmC in different organs and tissues. Also, the content of 5hmC in tissues and organs is affected by complex factors such as environmental toxins and the internal environment of the human body.

Because of the convenience of obtaining cfDNA and the smaller trauma to the human body, the study on 5hmC in cfDNA has attracted extensive attention from researchers. The acquisition and analysis of 5hmC in cfDNA provide a new perspective for the early diagnosis, effective treatment, and prognosis evaluation of tumors. With the development of biological information technology and high-throughput sequencing technology, the quantitative techniques of hydroxymethylation analysis such as hMeDIP-seq and TAB-Seq are becoming more and more mature, providing an advantageous method for DNA hydroxymethylation modification research in the tumor. The exploration of tumor-related genome-wide methylation regions is conducive to the in-depth understanding of the role of DNA hydromethylation modification in the development of solid tumors and hematological tumors, to find new tumor molecular markers and therapeutic targets, which may become a research hotspot in the next stage of methylation modified tumors.

In recent years, researches on hydromethylation mainly focus on tumors and psychiatric diseases [104, 105]. Studies have shown that during organ maturation and drug-induced response, 5hmC analysis can be used as an indicator of cell status and contribute to the identification of toxic carcinogen exposure [106, 107]. Dynamic enrichment patterns of methylation and hydromethylation markers may enable us to identify target genes for epigenetic modifications associated with prostate cancer [108]. Through a comprehensive analysis of global DNA methylation and hydroxymethylation in tumors and healthy tissues, colorectal cancer-related genes can be screened [109]. By changing the DNA methylation state, 5hmC can prevent the inactivation of some tumor suppressor genes and apoptotic genes. The decreased level of 5hmC leads to the lack of gene protection and the occurrence of tumors.

Although studies have suggested that methylation modification is associated with tumorigenesis and progression, studies on hydromethylation in ctDNA are still lacking. Moreover, the mechanism by which DNA methylation and hydroxymethylation affect oncogenesis and progression in different systems has not been clearly studied, and the specific role of epigenetic modification needs to be further explored. However, due to the complexity of biopsy required for the diagnosis of most tumors and cfDNA content in peripheral blood samples is susceptible to many external factors, it remains to be further studied whether other methods can be used to replace puncture biopsy in the future. What is the preference of hydromethylation in tumor-related genes for CpG sites? Which TET enzyme is involved in hydroxymethylation modification? How does hydromethylation status of specific gene promoters change after treatment? These problems need to be further explored.

Conclusions

As the intermediate product of cytosine modification, 5hmC has offered a broad perspective of the epigenetic regulation process. 5hmC is a prospective marker, which correlates to different stages of cancer. Future research may be inclined to explain the connection between 5hmC and intractable diseases, especially in early detection of tumors and the prediction of chemotherapeutic resistance. As far as the environment is concerned, its variety influences epigenetic modification, so as environmental toxins. After understanding the changes in 5hmC, we will get more details about abnormal reactions caused by DNA hydroxymethylation. The relationship between 5hmC and the human body is expected to be a current issue in future research.

Availability of data and materials

The datasets generated/analyzed during the current study are available.

References

  1. Kelly AD, Issa JJ. The promise of epigenetic therapy: reprogramming the cancer epigenome. Curr Opin Genet Dev. 2017;42:68–77.

    Article  CAS  PubMed  Google Scholar 

  2. Kanwal R, Gupta S. Epigenetic modifications in cancer. Clin Genet. 2012;81(4):303–11.

    Article  CAS  PubMed  Google Scholar 

  3. Shen L, Zhang Y. Enzymatic analysis of Tet proteins: key enzymes in the metabolism of DNA methylation. Methods Enzymol. 2012;512:93–105.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA, et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011;333(6047):1300–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pfaffeneder T, Hackner B, Truss M, Munzel M, Muller M, Deiml CA, et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew Chem Int Ed Engl. 2011;50(31):7008–12.

    Article  CAS  PubMed  Google Scholar 

  6. He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q, et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science. 2011;333(6047):1303–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kroeze LI, van der Reijden BA, Jansen JH. 5-Hydroxymethylcytosine: an epigenetic mark frequently deregulated in cancer. Biochim Biophys Acta. 2015;1855(2):144–54.

    CAS  PubMed  Google Scholar 

  8. Zhu JK. Active DNA demethylation mediated by DNA glycosylases. Annu Rev Genet. 2009;43:143–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Huang H, Jiang X, Li Z, Li Y, Song CX, He C, et al. TET1 plays an essential oncogenic role in MLL-rearranged leukemia. Proc Natl Acad Sci U S A. 2013;110(29):11994–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Klungland A, Robertson AB. Oxidized C5-methyl cytosine bases in DNA: 5-Hydroxymethylcytosine; 5-formylcytosine; and 5-carboxycytosine. Free Radic Biol Med. 2017;107:62–8.

    Article  CAS  PubMed  Google Scholar 

  11. Kranzhofer DK, Gilsbach R, Gruning BA, Backofen R, Nuhrenberg TG, Hein L. 5'-Hydroxymethylcytosine precedes loss of CpG methylation in enhancers and genes undergoing activation in cardiomyocyte maturation. PLoS One. 2016;11(11):e0166575.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Kim M, Costello J. DNA methylation: an epigenetic mark of cellular memory. Exp Mol Med. 2017;49(4):e322.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang J, Han X, Gao C, Xing Y, Qi Z, Liu R, et al. 5-Hydroxymethylome in circulating cell-free DNA as a potential biomarker for non-small-cell lung cancer. Genomics Proteomics Bioinformatics. 2018;16(3):187–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Song CX, Yin S, Ma L, Wheeler A, Chen Y, Zhang Y, et al. 5-Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages. Cell Res. 2017;27(10):1231–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Shahal T, Koren O, Shefer G, Stern N, Ebenstein Y. Hypersensitive quantification of global 5-hydroxymethylcytosine by chemoenzymatic tagging. Anal Chim Acta. 2018;1038:87–96.

    Article  CAS  PubMed  Google Scholar 

  16. Yu F, Li K, Li S, Liu J, Zhang Y, Zhou M, et al. CFEA: a cell-free epigenome atlas in human diseases. Nucleic Acids Res. 2020;48(D1):D40–d4.

    Article  PubMed  Google Scholar 

  17. Fernandes Marques J, Pereira Reis J, Fernandes G, Hespanhol V, Machado JC, Costa JL. Circulating tumor DNA: a step into the future of cancer management. Acta Cytol. 2019;63(6):456–65.

    Article  CAS  PubMed  Google Scholar 

  18. Shen Z, Wu A, Chen X. Current detection technologies for circulating tumor cells. Chem Soc Rev. 2017;46(8):2038–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tie J, Cohen JD, Wang Y, Christie M, Simons K, Lee M, et al. Circulating tumor DNA analyses as markers of recurrence risk and benefit of adjuvant therapy for stage III colon cancer. JAMA Oncol. 2019;5:1710.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Tie J, Wang Y, Tomasetti C, Li L, Springer S, Kinde I, et al. Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer. Sci Transl Med. 2016;8(346):346ra92.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Volckmar AL, Sultmann H, Riediger A, Fioretos T, Schirmacher P, Endris V, et al. A field guide for cancer diagnostics using cell-free DNA: from principles to practice and clinical applications. Genes Chromosomes Cancer. 2018;57(3):123–39.

    Article  CAS  PubMed  Google Scholar 

  22. Merker JD, Oxnard GR, Compton C, Diehn M, Hurley P, Lazar AJ, et al. Circulating tumor DNA analysis in patients with cancer: American Society of Clinical Oncology and College of American Pathologists joint review. J Clin Oncol. 2018;36(16):1631–41.

    Article  CAS  PubMed  Google Scholar 

  23. Cheng F, Su L, Qian C. Circulating tumor DNA: a promising biomarker in the liquid biopsy of cancer. Oncotarget. 2016;7(30):48832–41.

    PubMed  PubMed Central  Google Scholar 

  24. Vymetalkova V, Cervena K, Bartu L, Vodicka P. Circulating cell-free DNA and colorectal cancer: a systematic review. Int J Mol Sci. 2018;19(11):3356.

    Article  PubMed Central  CAS  Google Scholar 

  25. Cree IA, Uttley L, Buckley Woods H, Kikuchi H, Reiman A, Harnan S, et al. The evidence base for circulating tumour DNA blood-based biomarkers for the early detection of cancer: a systematic mapping review. BMC Cancer. 2017;17(1):697.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Li W, Zhang X, Lu X, You L, Song Y, Luo Z, et al. 5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers. Cell Res. 2017;27(10):1243–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chen J, Chen J, He F, Huang Y, Lu S, Fan H, et al. Design of a targeted sequencing assay to detect rare mutations in circulating tumor DNA. Genet Test Mol Biomarkers. 2019;23(4):264–9.

    Article  CAS  PubMed  Google Scholar 

  28. Nikolaev S, Lemmens L, Koessler T, Blouin JL, Nouspikel T. Circulating tumoral DNA: preanalytical validation and quality control in a diagnostic laboratory. Anal Biochem. 2018;542:34–9.

    Article  CAS  PubMed  Google Scholar 

  29. Wyatt AW, Annala M, Aggarwal R, Beja K, Feng F, Youngren J, et al. Concordance of circulating tumor DNA and matched metastatic tissue biopsy in prostate cancer. J Natl Cancer Inst. 2017;109(12).

  30. Barbany G, Arthur C, Lieden A, Nordenskjold M, Rosenquist R, Tesi B, et al. Cell-free tumour DNA testing for early detection of cancer - a potential future tool. J Intern Med. 2019;286(2):118–36.

    CAS  PubMed  Google Scholar 

  31. Nestor CE, Ottaviano R, Reddington J, Sproul D, Reinhardt D, Dunican D, et al. Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes. Genome Res. 2012;22(3):467–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Stroud H, Feng S, Morey Kinney S, Pradhan S, Jacobsen SE. 5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells. Genome Biol. 2011;12(6):R54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Liu Y, Siejka-Zielinska P, Velikova G, Bi Y, Yuan F, Tomkova M, et al. Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution. Nat Biotechnol. 2019;37(4):424–9.

    Article  CAS  PubMed  Google Scholar 

  34. Qing Y, Tian Z, Bi Y, Wang Y, Long J, Song CX, et al. Quantitation and mapping of the epigenetic marker 5-hydroxymethylcytosine. Bioessays. 2017;39(5):1700010.

    Article  CAS  Google Scholar 

  35. Nazor KL, Boland MJ, Bibikova M, Klotzle B, Yu M, Glenn-Pratola VL, et al. Application of a low cost array-based technique - TAB-Array - for quantifying and mapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells. Genomics. 2014;104(5):358–67.

    Article  CAS  PubMed  Google Scholar 

  36. Mooijman D, Dey SS, Boisset JC, Crosetto N, van Oudenaarden A. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables lineage reconstruction. Nat Biotechnol. 2016;34(8):852–6.

    Article  CAS  PubMed  Google Scholar 

  37. Mellen M, Ayata P, Dewell S, Kriaucionis S, Heintz N. MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. Cell. 2012;151(7):1417–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Yong E. Cancer biomarkers: written in blood. Nature. 2014;511(7511):524–6.

    Article  CAS  PubMed  Google Scholar 

  39. Li X, Liu Y, Salz T, Hansen KD, Feinberg A. Whole-genome analysis of the methylome and hydroxymethylome in normal and malignant lung and liver. Genome Res. 2016;26(12):1730–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Hlady RA, Sathyanarayan A, Thompson JJ, Zhou D, Wu Q, Pham K, et al. Integrating the epigenome to identify drivers of hepatocellular carcinoma. Hepatology. 2019;69(2):639–52.

    Article  CAS  PubMed  Google Scholar 

  41. Cai J, Chen L, Zhang Z, Zhang X, Lu X, Liu W, et al. Genome-wide mapping of 5-hydroxymethylcytosines in circulating cell-free DNA as a non-invasive approach for early detection of hepatocellular carcinoma. Gut. 2019;68(12):2195–205.

    Article  PubMed  Google Scholar 

  42. Dong ZR, Zhang C, Cai JB, Zhang PF, Shi GM, Gao DM, et al. Role of 5-hydroxymethylcytosine level in diagnosis and prognosis prediction of intrahepatic cholangiocarcinoma. Tumour Biol. 2015;36(4):2763–71.

    Article  CAS  PubMed  Google Scholar 

  43. Zhu X, Dresser K, Chen BJ. Loss of 5-hydroxymethylcytosine immunohistochemical expression is a useful diagnostic aid for distinguishing hepatocellular carcinoma in cytology fine needle aspiration specimens. Cytopathology. 2019;30(5):492–8.

    Article  PubMed  Google Scholar 

  44. Ye C, Tao R, Cao Q, Zhu D, Wang Y, Wang J, et al. Whole-genome DNA methylation and hydroxymethylation profiling for HBV-related hepatocellular carcinoma. Int J Oncol. 2016;49(2):589–602.

    Article  CAS  PubMed  Google Scholar 

  45. Yu M, Hon GC, Szulwach KE, Song CX, Zhang L, Kim A, et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell. 2012;149(6):1368–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Skvortsova K, Zotenko E, Luu PL, Gould CM, Nair SS, Clark SJ, et al. Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA. Epigenetics Chromatin. 2017;10:16.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014;6(224):224ra24.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Brison O. Gene amplification and tumor progression. Biochim Biophys Acta. 1993;1155(1):25–41.

    CAS  PubMed  Google Scholar 

  49. Nakagawa H, Fujita M. Whole genome sequencing analysis for cancer genomics and precision medicine. Cancer Sci. 2018;109(3):513–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Khurana E, Fu Y, Chakravarty D, Demichelis F, Rubin MA, Gerstein M. Role of non-coding sequence variants in cancer. Nat Rev Genet. 2016;17(2):93–108.

    Article  CAS  PubMed  Google Scholar 

  51. Diederichs S, Bartsch L, Berkmann JC, Frose K, Heitmann J, Hoppe C, et al. The dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding RNA and synonymous mutations. EMBO Mol Med. 2016;8(5):442–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Tai KY, Shiah SG, Shieh YS, Kao YR, Chi CY, Huang E, et al. DNA methylation and histone modification regulate silencing of epithelial cell adhesion molecule for tumor invasion and progression. Oncogene. 2007;26(27):3989–97.

    Article  CAS  PubMed  Google Scholar 

  53. Lu TY, Kao CF, Lin CT, Huang DY, Chiu CY, Huang YS, et al. DNA methylation and histone modification regulate silencing of OPG during tumor progression. J Cell Biochem. 2009;108(1):315–25.

    Article  CAS  PubMed  Google Scholar 

  54. Putiri EL, Tiedemann RL, Thompson JJ, Liu C, Ho T, Choi JH, et al. Distinct and overlapping control of 5-methylcytosine and 5-hydroxymethylcytosine by the TET proteins in human cancer cells. Genome Biol. 2014;15(6):R81.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Schubeler D. Function and information content of DNA methylation. Nature. 2015;517(7534):321–6.

    Article  CAS  PubMed  Google Scholar 

  56. Feng L, Lou J. DNA methylation analysis. Methods Mol Biol. 1894;2019:181–227.

    Google Scholar 

  57. Qiu L, Liu F, Yi S, Li X, Liu X, Xiao C, et al. Loss of 5-hydroxymethylcytosine is an epigenetic biomarker in cutaneous T-cell lymphoma. J Invest Dermatol. 2018;138(11):2388–97.

    Article  CAS  PubMed  Google Scholar 

  58. Lio CJ, Yuita H, Rao A. Dysregulation of the TET family of epigenetic regulators in lymphoid and myeloid malignancies. Blood. 2019;134(18):1487–97.

    Article  PubMed  PubMed Central  Google Scholar 

  59. van der Wijst MG, Venkiteswaran M, Chen H, Xu GL, Plosch T, Rots MG. Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase. Epigenetics. 2015;10(8):671–6.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Chen CC, Wang KY, Shen CK. The mammalian de novo DNA methyltransferases DNMT3A and DNMT3B are also DNA 5-hydroxymethylcytosine dehydroxymethylases. J Biol Chem. 2012;287(40):33116–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Kanazawa A, Tsukamoto T, Shimizu S, Yamamoto S, Murata A, Kubo S. Laparoscopic hepatectomy for liver cancer. Dig Dis. 2015;33(5):691–8.

    Article  PubMed  Google Scholar 

  62. Tekpli X, Urbanucci A, Hashim A, Vagbo CB, Lyle R, Kringen MK, et al. Changes of 5-hydroxymethylcytosine distribution during myeloid and lymphoid differentiation of CD34+ cells. Epigenetics Chromatin. 2016;9:21.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Tekpli X, Skaug V, Baera R, Phillips DH, Haugen A, Mollerup S. Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report. Cell Oncol (Dordr). 2016;39(6):583–9.

    Article  CAS  Google Scholar 

  64. Neri F, Incarnato D, Oliviero S. DNA methylation and demethylation dynamics. Oncotarget. 2015;6(33):34049–50.

    Article  PubMed  PubMed Central  Google Scholar 

  65. Neri F, Dettori D, Incarnato D, Krepelova A, Rapelli S, Maldotti M, et al. TET1 is a tumour suppressor that inhibits colon cancer growth by derepressing inhibitors of the WNT pathway. Oncogene. 2015;34(32):4168–76.

    Article  CAS  PubMed  Google Scholar 

  66. Kundaje A, Meuleman W, Ernst J, Bilenky M, Yen A, Heravi-Moussavi A, et al. Integrative analysis of 111 reference human epigenomes. Nature. 2015;518(7539):317–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Eggermont AMM, Blank CU, Mandala M, Long GV, Atkinson V, Dalle S, et al. Adjuvant pembrolizumab versus placebo in resected stage III melanoma. N Engl J Med. 2018;378(19):1789–801.

    Article  CAS  PubMed  Google Scholar 

  68. Gould Rothberg BE, Rimm DL. Biomarkers: the useful and the not so useful--an assessment of molecular prognostic markers for cutaneous melanoma. J Invest Dermatol. 2010;130(8):1971–87.

    Article  CAS  PubMed  Google Scholar 

  69. Yelamos O, Gerami P. Predicting the outcome of melanoma: can we tell the future of a patient’s melanoma? Melanoma Manag. 2015;2(3):217–24.

    Article  PubMed  PubMed Central  Google Scholar 

  70. Gerami P, Cook RW, Wilkinson J, Russell MC, Dhillon N, Amaria RN, et al. Development of a prognostic genetic signature to predict the metastatic risk associated with cutaneous melanoma. Clin Cancer Res. 2015;21(1):175–83.

    Article  CAS  PubMed  Google Scholar 

  71. Jin SG, Wu X, Li AX, Pfeifer GP. Genomic mapping of 5-hydroxymethylcytosine in the human brain. Nucleic Acids Res. 2011;39(12):5015–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Gu TP, Guo F, Yang H, Wu HP, Xu GF, Liu W, et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature. 2011;477(7366):606–10.

    Article  CAS  PubMed  Google Scholar 

  73. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;324(5929):930–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Cimmino L, Abdel-Wahab O, Levine RL, Aifantis I. TET family proteins and their role in stem cell differentiation and transformation. Cell Stem Cell. 2011;9(3):193–204.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Gambichler T, Sand M, Skrygan M. Loss of 5-hydroxymethylcytosine and ten-eleven translocation 2 protein expression in malignant melanoma. Melanoma Res. 2013;23(3):218–20.

    Article  CAS  PubMed  Google Scholar 

  76. Uchiyama R, Uhara H, Uchiyama A, Ogawa E, Takazawa Y, Ashida A, et al. 5-Hydroxymethylcytosine as a useful marker to differentiate between malignant melanomas and benign melanocytic nevi. J Dermatol Sci. 2014;73(2):161–3.

    Article  CAS  PubMed  Google Scholar 

  77. Mikoshiba Y, Ogawa E, Uchiyama R, Uchiyama A, Uhara H, Okuyama R. 5-Hydroxymethylcytosine is a useful marker to differentiate between dermatofibrosarcoma protuberans and dermatofibroma. J Eur Acad Dermatol Venereol. 2016;30(1):130–1.

    Article  CAS  PubMed  Google Scholar 

  78. Song F, Amos CI, Lee JE, Lian CG, Fang S, Liu H, et al. Identification of a melanoma susceptibility locus and somatic mutation in TET2. Carcinogenesis. 2014;35(9):2097–101.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Shibata T, Kokubu A, Miyamoto M, Sasajima Y, Yamazaki N. Mutant IDH1 confers an in vivo growth in a melanoma cell line with BRAF mutation. Am J Pathol. 2011;178(3):1395–402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Munari E, Chaux A, Vaghasia AM, Taheri D, Karram S, Bezerra SM, et al. Global 5-hydroxymethylcytosine levels are profoundly reduced in multiple genitourinary malignancies. PLoS One. 2016;11(1):e0146302.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  81. Ito S, D'Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature. 2010;466(7310):1129–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Abbas O, Miller DD, Bhawan J. Cutaneous malignant melanoma: update on diagnostic and prognostic biomarkers. Am J Dermatopathol. 2014;36(5):363–79.

    Article  PubMed  Google Scholar 

  83. Wu SG, He ZY, Li Q, Li FY, Lin Q, Lin HX, et al. Predictive value of breast cancer molecular subtypes in Chinese patients with four or more positive nodes after postmastectomy radiotherapy. Breast. 2012;21(5):657–61.

    Article  PubMed  Google Scholar 

  84. Arvold ND, Taghian AG, Niemierko A, Abi Raad RF, Sreedhara M, Nguyen PL, et al. Age, breast cancer subtype approximation, and local recurrence after breast-conserving therapy. J Clin Oncol. 2011;29(29):3885–91.

    Article  PubMed  PubMed Central  Google Scholar 

  85. Yang H, Liu Y, Bai F, Zhang JY, Ma SH, Liu J, et al. Tumor development is associated with decrease of TET gene expression and 5-methylcytosine hydroxylation. Oncogene. 2013;32(5):663–9.

    Article  CAS  PubMed  Google Scholar 

  86. Lorsbach RB, Moore J, Mathew S, Raimondi SC, Mukatira ST, Downing JR. TET1, a member of a novel protein family, is fused to MLL in acute myeloid leukemia containing the t(10;11)(q22;q23). Leukemia. 2003;17(3):637–41.

    Article  CAS  PubMed  Google Scholar 

  87. Tefferi A, Lim KH, Abdel-Wahab O, Lasho TL, Patel J, Patnaik MM, et al. Detection of mutant TET2 in myeloid malignancies other than myeloproliferative neoplasms: CMML, MDS, MDS/MPN and AML. Leukemia. 2009;23(7):1343–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Abdel-Wahab O, Mullally A, Hedvat C, Garcia-Manero G, Patel J, Wadleigh M, et al. Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies. Blood. 2009;114(1):144–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Liu C, Liu L, Chen X, Shen J, Shan J, Xu Y, et al. Decrease of 5-hydroxymethylcytosine is associated with progression of hepatocellular carcinoma through downregulation of TET1. PLoS One. 2013;8(5):e62828.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Yang L, Yu SJ, Hong Q, Yang Y, Shao ZM. Reduced expression of TET1, TET2, TET3 and TDG mRNAs are associated with poor prognosis of patients with early breast cancer. PLoS One. 2015;10(7):e0133896.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  91. Wu MZ, Chen SF, Nieh S, Benner C, Ger LP, Jan CI, et al. Hypoxia drives breast tumor malignancy through a TET-TNFalpha-p38-MAPK signaling axis. Cancer Res. 2015;75(18):3912–24.

    Article  CAS  PubMed  Google Scholar 

  92. Tefferi A, Levine RL, Lim KH, Abdel-Wahab O, Lasho TL, Patel J, et al. Frequent TET2 mutations in systemic mastocytosis: clinical, KITD816V and FIP1L1-PDGFRA correlates. Leukemia. 2009;23(5):900–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics, 2008. CA Cancer J Clin. 2008;58(2):71–96.

    Article  PubMed  Google Scholar 

  94. Petricoin EF, Zoon KC, Kohn EC, Barrett JC, Liotta LA. Clinical proteomics: translating benchside promise into bedside reality. Nat Rev Drug Discov. 2002;1(9):683–95.

    Article  CAS  PubMed  Google Scholar 

  95. Schiffer E, Mischak H, Theodorescu D, Vlahou A. Challenges of using mass spectrometry as a bladder cancer biomarker discovery platform. World J Urol. 2008;26(1):67–74.

    Article  CAS  PubMed  Google Scholar 

  96. Lodde M, Fradet Y. The detection of genetic markers of bladder cancer in urine and serum. Curr Opin Urol. 2008;18(5):499–503.

    Article  PubMed  Google Scholar 

  97. Vrooman OP, Witjes JA. Molecular markers for detection, surveillance and prognostication of bladder cancer. Int J Urol. 2009;16(3):234–43.

    Article  CAS  PubMed  Google Scholar 

  98. Taylor SE, Li YH, Smeriglio P, Rath M, Wong WH, Bhutani N. Stable 5-hydroxymethylcytosine (5hmC) acquisition marks gene activation during chondrogenic differentiation. J Bone Miner Res. 2016;31(3):524–34.

    Article  CAS  PubMed  Google Scholar 

  99. Kim YK, Kim WJ. Epigenetic markers as promising prognosticators for bladder cancer. Int J Urol. 2009;16(1):17–22.

    Article  PubMed  Google Scholar 

  100. Lin HH, Ke HL, Huang SP, Wu WJ, Chen YK, Chang LL. Increase sensitivity in detecting superficial, low grade bladder cancer by combination analysis of hypermethylation of E-cadherin, p16, p14, RASSF1A genes in urine. Urol Oncol. 2010;28(6):597–602.

    Article  CAS  PubMed  Google Scholar 

  101. Jankowska AM, Szpurka H, Tiu RV, Makishima H, Afable M, Huh J, et al. Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms. Blood. 2009;113(25):6403–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Lian CG, Xu Y, Ceol C, Wu F, Larson A, Dresser K, et al. Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. Cell. 2012;150(6):1135–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Kraus TF, Guibourt V, Kretzschmar HA. 5-Hydroxymethylcytosine, the “Sixth Base”, during brain development and ageing. J Neural Transm (Vienna). 2015;122(7):1035–43.

    Article  CAS  Google Scholar 

  104. Rustad SR, Papale LA, Alisch RS. DNA methylation and hydroxymethylation and behavior. Curr Top Behav Neurosci. 2019;42:51–82.

    PubMed  Google Scholar 

  105. Hack LM, Dick ALW, Provencal N. Epigenetic mechanisms involved in the effects of stress exposure: focus on 5-hydroxymethylcytosine. Environ Epigenet. 2016;2(3):dvw016.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  106. Zhu T, Brown AP, Ji H. The emerging role of ten-eleven translocation 1 in epigenetic responses to environmental exposures. Epigenet Insights. 2020;13. https://doi.org/10.1177/2516865720910155.

  107. Thomson JP, Hunter JM, Lempiainen H, Muller A, Terranova R, Moggs JG, et al. Dynamic changes in 5-hydroxymethylation signatures underpin early and late events in drug exposed liver. Nucleic Acids Res. 2013;41(11):5639–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Kamdar SN, Ho LT, Kron KJ, Isserlin R, van der Kwast T, Zlotta AR, et al. Dynamic interplay between locus-specific DNA methylation and hydroxymethylation regulates distinct biological pathways in prostate carcinogenesis. Clin Epigenetics. 2016;8:32.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  109. Gilat N, Tabachnik T, Shwartz A, Shahal T, Torchinsky D, Michaeli Y, et al. Single-molecule quantification of 5-hydroxymethylcytosine for diagnosis of blood and colon cancers. Clin Epigenetics. 2017;9:70.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Key Research and Development Plan (2016YFC1302900), Jilin Province Science and Technology Funds (20180201032YY, 20150204007YY, and 20140204022YY), Jilin Province Development and Reform Commission Funds (2014G073,2016C046-2), and Education Department of Jilin Province Funds (JJKH20170804KJ).

Author information

Authors and Affiliations

Authors

Contributions

Haoyue Gao participated in the conception and design of the study and contributed to drafting the article. Tianmin Xu revised it critically for relevant intellectual content. All authors have read and approved the final submitted manuscript.

Corresponding author

Correspondence to Tianmin Xu.

Ethics declarations

Ethics approval and consent to participate

Not applicable

Consent for publication

Consent for publication was obtained from the participants.

Competing interests

The authors declare that there is no conflict of interest regarding the publication of this article.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, T., Gao, H. Hydroxymethylation and tumors: can 5-hydroxymethylation be used as a marker for tumor diagnosis and treatment?. Hum Genomics 14, 15 (2020). https://doi.org/10.1186/s40246-020-00265-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s40246-020-00265-5

Keywords