BJD

British Journal of Dermatology

P H O T OB I O L O G Y

Altered global methylation and hydroxymethylation status in vulvar lichen sclerosus: further support for epigenetic mechanisms T. Gambichler, S. Terras, A. Kreuter and M. Skrygan Department of Dermatology, Ruhr-University Bochum, Gudrunstraße 56, 44791 Bochum, Germany

Summary Correspondence Thilo Gambichler. E-mail: [email protected]

Accepted for publication 20 October 2013

Funding sources None.

Conflicts of interest None declared DOI 10.1111/bjd.12702

Background Epigenetics refers to functionally relevant changes in the genome other than those of DNA sequence that can lead to changes in gene expression or cellular phenotype. There is evidence that epigenetics is relevant in the pathogenesis of autoimmune diseases such as vulvar lichen sclerosus (VLS), as well as in cancer, including cutaneous squamous cell carcinoma, which is frequently associated with VLS. Objectives To study the global methylation and hydroxymethylation status in healthy controls and VLS lesions before and after long-term ultraviolet (UV)A1 treatment. Methods We studied 12 controls and 10 patients with VLS who were treated with medium-dose UVA1 four times weekly for 3 months. Immunohistochemistry and mutation analyses (polymerase chain reaction) were performed for 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), isocitrate dehydrogenases (IDHs) and the ten-eleven translocation (TET)2 enzyme. Results After 3 months of treatment, 5mC was significantly increased in VLS compared with baseline and controls. However, compared with controls 5hmC levels were significantly reduced in baseline VLS, but normalized after UVA1 treatment. Compared with controls, IDH1 expression was significantly higher in both treated and baseline VLS. By contrast, IDH2 levels were significantly reduced in baseline VLS compared with controls and UVA1-treated VLS. However, gene sequencing of the IDH1, IDH2 and TET2 genes did not reveal evidence of mutations. Conclusions VLS is associated with altered expression of IDH enzymes and aberrant hydroxymethylation, indicating an epigenetic background for the pathogenesis of VLS. UVA1 phototherapy may cause normalization of 5hmC patterns, but also global DNA hypermethylation in VLS lesions, raising concerns with respect to an increased risk of photocarcinogenesis.

What’s already known about this topic?

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Vulvar lichen sclerosus (VLS) is an autoimmune-mediated skin disorder that is frequently associated with cutaneous squamous cell carcinoma. Ultraviolet (UV)A1 appears to be beneficial in lichen sclerosus but may also be associated with increased risk of photocarcinogenesis.

What does this study add?



© 2013 British Association of Dermatologists

Altered expression of isocitrate dehydrogenases and aberrant global methylation and hydroxymethylation patterns were observed in VLS, indicating an epigenetic background for the pathogenesis of this condition.

British Journal of Dermatology (2014) 170, pp687–693

687

688 Altered methylation and hydroxymethylation in VLS, T. Gambichler et al.

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Following long-term UVA1 phototherapy of VLS, normalization of 5-hydroxymethylation patterns and global DNA hypermethylation were observed. The latter may raise concerns regarding photocarcinogenesis.

Lichen sclerosus (LS) is an inflammatory skin disorder with an autoimmune background, affecting mainly the anogenital areas in both men and women, and has been associated with squamous cell carcinoma (SCC).1 The efficacy of ultraviolet (UV) A1 phototherapy in extragenital LS was first established by our study group.2 Recently, Beattie et al.3 suggested that UVA1 may be of benefit in the management of vulvar LS (VLS). Nevertheless, concerns may arise regarding UVA1 treatment of genital LS given the risk of tumorigenesis, which is increased anyway in the genital type of the disease.1,4,5 Apart from genetic factors, global epigenetic changes and gene promoterspecific methylation patterns have been observed in inflammatory conditions and many cancer types as playing an essential role in carcinogenesis. Epigenetic modifications define how genetic information is read and used by cells. Epigenetic modifications are influenced by environmental factors, some of which can induce epigenetic signalling that may contribute to biological processes such as ageing. Methylation is the principal epigenetic modification of DNA and the histones that package DNA into chromatin inside a cell. Epigenetic modifications influence gene expression and enable the differentiation of pluripotent stem cells into distinct cell types early in embryological development. Epigenetics includes the investigation of differences in phenotype, in the absence of variation in the genetic code.6 In the case of skin cancers, aberrant methylation of tumour suppressor gene promoters is associated with their transcriptional inactivation. Environmental carcinogens such as UV radiation can act through epigenetic mechanisms. Hypomethylation is associated with activation of systemic autoimmune diseases, such as systemic and cutaneous lupus erythematosus and scleroderma. This may be through a mechanism of immunological cross-reactivity with hypomethylated DNA from pathogenic bacteria. Epigenetic factors may also be relevant in the pathogenesis of psoriasis and other inflammatory skin diseases such as LS.6 Hypermethylation of genes such as TP53, KRAS, CDKN2A (p16) and DAPK1 has been reported in both genital LS and LS-associated SCCs.7–13 DNA methylation at the 5-position of cytosine is well recognized as an important epigenetic modification in human health and disease.10 Recent evidence has demonstrated that 5-methylcytosine (5mC) can be converted by teneleven translocation (TET) enzymes in association with isocitrate dehydrogenases (IDHs) to 5-hydroxymethylcytosine (5hmC). The latter has become a major focus of epigenetic research. TET and IDH enzymes are mutated in several types of cancer, affecting their activity and likely altering genomic 5hmC and 5mC patterns. Furthermore, oxidation of 5mC to British Journal of Dermatology (2014) 170, pp687–693

5hmC appears to be a step in several active DNA demethylation pathways, which may be important for normal processes, as well as in global hypomethylation during cancer development and progression.14,15 UV radiation also plays an important role in formation of cytosine derivatives.16 Hence, in order to find out (i) whether the pathogenesis of VLS has an epigenetic background, and (ii) whether long-term UVA1 treatment of VLS is associated with epigenetic changes that might suggest photocarcinogenicity, we aimed to assess cell-cycle regulators, the mutation status of several skincancer-associated genes, and global methylation and hydroxymethylation and their regulator enzymes before and after long-term UVA1 treatment of VLS.

Materials and methods Patients The present investigation comprised 10 women (mean  SD age 492  197 years) with clinically and histologically proven VLS, who also participated in a clinical randomized controlled trial recently published by our research group.17 We included only patients with active disease (duration of disease < 2 years) and without specific treatment for at least 4 weeks prior to the beginning of the study. For controls we recruited 12 women who had undergone plastic surgery within the genital region (mean  SD age 47  63 years). The protocol of the study was approved by the ethics review board of the Ruhr-University Bochum. The study was conducted according to the Declaration of Helsinki principles. Informed consent was obtained from every subject included. Ultraviolet A1 irradiation protocol and clinical assessment Punch biopsies of 4 mm were obtained from vulvar skin before and after 3-month UVA1 treatment. UVA1 was given four times per week using a specially designed phototherapy device for use in the anogenital region (Sellamed; Sellas, Gevelsberg, Germany). The irradiance of the UVA1 source was 24 mW cm 2 (25 cm distance to the UVA1 source). During the first five sessions, the UVA1 dose was increased in steps of 10 J cm 2 starting from 10 J cm 2 up to a maximum dose of 50 J cm 2, four times weekly. Clinical assessment of efficacy was performed at baseline and after 3 months of UVA1 treatment by means of the total clinician’s score (TCS). Using the TCS, hypopigmentation, sclerosis, atrophy, hyperkeratosis, erosions, oedema and erythema are each assigned a value from © 2013 British Association of Dermatologists

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0 to 3, with 0 being ‘absent’, 1 being ‘slight’, 2 being ‘moderate’ and 3 being ‘severe’. The total of these values was considered the TCS, with a maximum score of 21 points. Histology and immunohistochemistry Histology was performed using routine haematoxylin and eosin staining. Immunohistochemical staining was performed as previously described more in detail.18 We used the following commercially available antibodies: rabbit polyclonal 5hmC-specific antibody at 1 : 500 dilution (Active Motif, Carlsbad, CA, U.S.A.); mouse monoclonal 5mC-specific antibody at 1 : 1000 dilution (Active Motif); rabbit polyclonal TET2-specific antibody at 1 : 500 dilution (Abcam, Cambridge, U.K.); rabbit monoclonal IDH1-specific antibody at 1 : 200 dilution (Abcam); mouse monoclonal IDH2-specific antibody at 1 : 200 dilution (Abnova GmbH c/o EMBLEM, Heidelberg, Germany); mouse monoclonal p16INK4a-specific antibody at 1 : 40 dilution (Ventana Medical Systems, Basel, Switzerland) and mouse monoclonal p53-specific antibody at 1 : 50 dilution (Dako, Hamburg, Germany). No staining was shown when omitting the primary antibodies (negative controls). Histological and immunohistochemical specimens were independently analysed in a blinded manner by two observers (200–4009 magnification). The reduction of the dermal lymphocytic infiltrates was assessed in haematoxylin and eosinstained samples as measured semiquantitatively in five fields of view (0, no lymphocytes; 1, slight lymphocytic infiltrates; 2, moderate lymphocytic infiltrates; 3, strong lymphocytic infiltrates) before and after treatment. For immunohistology, the entire epidermis was evaluated using the H-score system obtained by multiplying the intensity of the stain (0, no staining; 1, weak staining; 2, moderate staining; 3, intense staining) by the percentage (0–100) of keratinocytes showing that staining intensity.15 Mutation analyses Mutation analyses were performed on formalin-fixed paraffinembedded lesional biopsy specimens of patients with VLS obtained at baseline and after 3 months of UVA1 treatment. Table 1 Immunohistochemistry dataa of healthy controls and patients with vulvar lichen sclerosus (VLS) before and after 3 months of ultraviolet (UV)A1 phototherapy

Polymerase chain reaction (PCR) was performed using commercially available primers for exon 4 of IDH1, exon 4 of IDH2, exons 3 and 11 of TET2, exons 4, 6 and 7 of TP53 and codon 12 (G34 and G35) of KRAS, exon 3 of HRAS and exons 2 and 3 of NRAS (SeqLab, G€ ottingen, Germany). PCR was performed as previously described in more detail.18 Statistics Data were analysed (MEDCALC Software, Mariakerke, Belgium) using the paired t-test and Kruskal–Wallis ANOVA. Correlation studies were performed using the Spearman coefficient of rank correlation. No a adjustment for multiple testing was performed, as this was an explorative study. P-values ≤ 005 were considered significant.

Results Treatment outcome Clinical outcome was assessed by TCS based on the absence or severity of seven clinical symptoms of LS, as well as a histology score, to evaluate changes in lymphocytic infiltrate. Compared with baseline (112  41) the TCS observed in patients with VLS was significantly decreased after UVA1 treatment (75  46; P = 00086). However, when compared with baseline (21  07), the histology score of dermal lymphocytic infiltrates was not significantly reduced after UVA1 treatment (219  08; P = 057). Hence, despite a moderate clinical improvement, inflammatory signs on histology remained unchanged after UVA1 treatment. Global methylation and hydroxymethylation The DNA methylation status was examined by immunohistochemistry using antibodies that recognize the methylated forms of cytosine, 5mC and 5hmC. As detailed in Table 1, immunohistochemistry demonstrated a significant (P = 0043) median increase of 5mC immunoreactivity scoring in UVA1irradiated VLS (median 225, range 60–285) compared with baseline (median 126, range 40–270) and normal controls

Parameter

Healthy controls (A)

Baseline VLS (B)

UVA1-treated VLS (C)

P-valueb

5mC 5hmC TET2 IDH1 IDH2 p53 p16

82 265 240 80 50 120 12

126 120 255 155 0 150 15

225 2475 265 180 125 150 75

0043; C vs. A and B 00038; B vs. A and C 023 00062; A vs. B and C 0012; B vs. A and C 047 052

(20–210) (110–295) (140–285) (10–140) (0–180) (6–210) (0–120)

(40–270) (0–270) (30–285) (80–270) (0–10) (10–200) (1–150)

(60–285) (60–285) (160–300) (80–280) (0–160) (10–270) (0–150)

5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine; TET2, ten-eleven translocation 2 enzyme; IDH, isocitrate dehydrogenase. aH-score expressed as median (range). bSignificant results are in bold.

© 2013 British Association of Dermatologists

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(a)

(b)

(c)

(d)

(e)

(f)

Fig 1. 5-Methylcytosine levels are increased after long-term ultraviolet (UV)A1 exposure of vulvar lichen sclerosus (VLS) (c) compared with healthy skin (a) and baseline VLS (b). 5-Hydroxymethylcytosine levels in baseline VLS (e) are decreased compared with healthy controls (d) and UVA1-treated VLS (f).

(median 82, range 20–210). As shown in Figure 1, median 5hmC levels were significantly (P = 00038) reduced in VLS at baseline (median 120, range 0–270) compared with healthy controls (median 265, range 110–295). However, 5hmC immunoreactivity in UVA1-treated skin (median 2475, range 60–285) was comparable with that found in healthy controls (P > 005), indicating normalization of hydroxymethylation status after phototherapy. Protein expression of tumour suppressor genes, isocitrate dehydrogenases 1 and 2 and ten-eleven translocation 2 enzyme In order to assess tumour suppressor genes and regulators of 5mC/5hmC levels, we studied by means of immunohistochemistry the protein expression of p53, p16INK4a, IDH1, IDH2 and TET2. Immunoreactivity scoring for TET2, p53 and p16INK4a did not demonstrate significant differences between the groups assessed (P = 023, P = 047 and P = 052, respectively). However, median IDH1 expression was significantly (P = 00062) higher in VLS at baseline (median 155, range 80–270) and post-UVA1 treatment (median 180, range 80– 280) compared with healthy controls (median 80, range 10– 140). As shown in Figure 2, median IDH2 immunoreactivity was significantly (P = 0012) reduced in baseline VLS (median 0, range 0–10) compared with healthy controls (median 50, range 0–180) and post-UVA1-irradiated VLS (median 125, range 0–160). Correlation studies did not reveal a significant British Journal of Dermatology (2014) 170, pp687–693

relationship between the markers assessed, except for 5hmC (r = 077, P = 00034) and 5mC (r = 080, P = 0002), which strongly correlated with TET2 expression in VLS at baseline. Moreover, baseline TET2 immunoreactivity in VLS strongly correlated (r = 072, P = 00082) with baseline IDH2 immunoreactivity. Mutation analyses Mutations in the tumour suppressor genes and IDH1, IDH2 and TET2 were assessed using PCR. Gene sequencing of TP53, NRAS, KRAS, HRAS, IDH1, IDH2 and TET2 revealed no evidence of mutations either at baseline or at the post-treatment assessment.

Discussion Previously, potential oncogenic mutations in genes such as TP53 and KRAS have been found in genital LS, LS-associated SCC and healthy genital skin.11,12 However, in our study cohort we could not detect mutations in the KRAS, NRAS or TP53 genes. Similarly to our previously reported data on short-standing VLS,18 we did not found altered p53 or p16INK4a protein expression in VLS at baseline, indicating that cell-cycle regulation is not substantially altered at the beginning of the disease. Although the median post-UVA1 expression of p16INK4a dropped by about 50% compared with baseline, there was no statistically significant difference, which © 2013 British Association of Dermatologists

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Fig 2. Expression of ten-eleven translocation 2 protein was shown to be strong in healthy controls (a), as well as in untreated (b) and ultraviolet (UV)A1-treated vulvar lichen sclerosus (VLS) (c). Isocitrate dehydrogenase (IDH)1 expression was very weak in baseline VLS (e) compared with healthy controls (d) and UVA1-treated VLS (f). Compared with healthy controls (g) and UVA1-treated VLS (i), IDH2 expression was markedly reduced in baseline VLS (h).

is likely due to the small sample size investigated. However, chronic broadband UVA irradiation of human keratinocytes can induce alterations in histone modification and sever promoter hypermethylation of p16INK4a, leading to substantial impairment of p16INK4a transcription.10 Guerrero et al.8 demonstrated that there was increasing hypermethylation of genes from isolated VLS associated with vulvar SCC. The genes were hypermethylated more frequently in vulvar SCCs associated with LS than in those not associated with LS.19 Epigenetic silencing of p16INK4a in vulvar SCC and precursor lesions including VLS was also observed by Aide et al.7 and Lerma et al.9 Recent data indicate that regulators © 2013 British Association of Dermatologists

of 5-hydroxymethylation, including IDH and TET enzymes, are involved in early tumour development and progression.14,15,19 In the present study, we showed for the first time that global 5hmC levels in the epidermis are significantly reduced in VLS compared with healthy controls, and that a 3-month course of UVA1 treatment leads to normalization of 5hmC levels. We hypothesize that VLS-related alterations in 5-hydroxymethylation may lead to aberrant expression of genes associated with biological functions related to autoimmune and inflammation response.20 We also observed a massive increase of the global 5mC level in UVA1-treated VLS compared with baseline VLS, as well as British Journal of Dermatology (2014) 170, pp687–693

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in healthy controls, possibly also indicating hypermethylation of skin cancer-associated tumour suppressor genes.10 It is well known that 5mC plays an even more important role in UVB-induced mutations in mammalian cells, and it is present almost exclusively at CpG dinucleotides of promoter regions.4 These sequences are known mutational hotspots in cancer-relevant genes such as TP53 and TP16. Mechanistically, the longer wavelength absorption of 5mC relative to cytosine most likely explains the preferential susceptibility of dipyrimidines containing 5mC for sunlight or UVB-induced cyclobutane pyrimidine dimer formation.4 UVAinduced mutations are characterized by statistically significant increases in G-to-T transversions and small tandem base deletions relative to mutations derived spontaneously.4,21,22 We observed that, compared with controls, IDH1 expression in VLS at baseline was significantly higher than in controls, whereas IDH2 expression was significantly reduced in VLS at baseline. IDH mutations explaining altered expressions levels could not be detected in the exons that we assessed. Notably, global 5mC and 5hmC levels, as well as IDH2 expression, were significantly increased after medium-dose UVA1 treatment, which was paralleled by moderate clinical improvement even though routine histology did not demonstrate improvement with regard to the inflammatory infiltrates. The increase of 5hmC levels may be explained by UVA1-induced enhancement of IDH2 expression catalysing the conversion of a-ketoglutarate, which is an essential factor for 5hydroxymethylation by TET enzymes. However, whether the increase of IDH2 and 5hmC levels represents an important mode of action of phototherapy in VLS cannot be concluded from our data. Jo et al.23 demonstrated that IDHs play a significant role in cellular defence against UV-induced oxidative injury, and may thus be upregulated in response to UV. Together, there is a wealth of evidence indicating that epigenetics is relevant in the pathogenesis of cancer and autoimmune conditions. With respect to common skin cancers, aberrant methylation of tumour suppressor gene promoters is associated with their transcriptional inactivation. Environmental carcinogens such as UV may act through epigenetic mechanisms. Interestingly, it has been reported that hypomethylation is associated with activation of autoimmune diseases such as lupus erythematosus and scleroderma.4,6,22 Nevertheless, our data indicate that VLS is more likely associated with hypohydroxymethylation. Further evidence for the significance of epigenetics in VLS was recently reported by Terlou et al.,24 who demonstrated an autoimmune phenotype in VLS, characterized by increased levels of T-helper 1-specific cytokines, a dense T-cell infiltrate and enhanced microRNA155 (BIC) expression. In conclusion, we have shown for the first time that VLS is associated with aberrant 5-hydroxymethylation and altered expression of IDH enzymes, providing evidence for an epigenetic background in the pathogenesis of VLS. Long-term UVA1 phototherapy of VLS is paralleled by an increase of 5hmC and IDH2 levels, indicating that UVA1 causes normalization of 5-hydroxymethylation patterns in VLS. By contrast, British Journal of Dermatology (2014) 170, pp687–693

the strong increase of post-UVA1 5mC levels likely associated with DNA hypermethylation may raise concerns with respect to an increased risk of photocarcinogenesis. However, a currently unanswered question remains as to whether, overall, a treatment such as UVA1 might add to cancer risks through direct UV-related effects or whether, by improving VLS, it might overall reduce risks. Moreover, further studies are warranted to assess these changes with UVA1 for other indications, and before and after other treatments for VLS to decide whether the differences that we found before and after UVA1 treatment all relate to UVA1 in general (and would be found in other diseases) or to VLS response (and would be found with any effective intervention).

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lichen sclerosus. A randomized controlled study. JAMA Dermatol 2014; in press. Gambichler T, Kammann S, Tigges C et al. Cell cycle regulation and proliferation in lichen sclerosus. Regul Pept 2011; 167:209–14. Dumitrescu RG. Epigenetic markers of early tumor development. Methods Mol Biol 2012; 863:3–14. Figueroa-Romero C, Hur J, Bender DE et al. Identification of epigenetically altered genes in sporadic amyotrophic lateral sclerosis. PLoS One 2012; 7:e52672. Kim SI, Jin SG, Pfeifer GP. Formation of cyclobutane pyrimidine dimers at dipyrimidines containing 5-hydroxymethylcytosine. Photochem Photobiol Sci 2013; 12:1409–15.

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22 Pfeifer GP, Kadam S, Jin SG. 5-Hydroxymethylcytosine and its potential roles in development and cancer. Epigenetics Chromatin 2013; 6:10. 23 Jo SH, Lee SH, Chun HS et al. Cellular defense against UVBinduced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase. Biochem Biophys Res Commun 2002; 292:542–9. 24 Terlou A, Santegoets LA, van der Meijden WI et al. An autoimmune phenotype in vulvar lichen sclerosus and lichen planus: a Th1 response and high levels of microRNA-155. J Invest Dermatol 2012; 132:658–66.

British Journal of Dermatology (2014) 170, pp687–693

Altered global methylation and hydroxymethylation status in vulvar lichen sclerosus: further support for epigenetic mechanisms.

Epigenetics refers to functionally relevant changes in the genome other than those of DNA sequence that can lead to changes in gene expression or cell...
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