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	J Clin Exp Oncol. Case report
Genshu Tate, Takuma Tajiria, Koji Kishimoto, Toshiyuki Mitsuya 

Title 25 words

Heterozygous loss of ten-eleven translocation 2 gene in a malignant melanoma with the BRAFV600E mutation

Department of Pathology, Showa University Fujigaoka Hospital, Yokohama, Japan
aPresent address: Department of Pathology, Tokai University Hachioji Hospital, Tokyo, Japan
*Address for correspondence: Genshu Tate, M.D., Department of Pathology, Showa University Fujigaoka Hospital, Fujigaoka 1-30, Aoba-ku, Yokohama 227-8501, Japan. Phone: +81-45-974-6383; Fax: +81-45-972-6242; E-mail:  HYPERLINK "mailto:gentate@yahoo.co.jp" gentate@yahoo.co.jp 

Abstract 300 words
Epigenetic gene silencing, including CpG island hypermethylation, can alter the expression of tumor-suppressor genes, and an inactivation of tumor-suppressor genes is involved in tumorigenic processes in many cancers. Genomic DNA methylation occurs predominantly on the 5 position of cytosine (5-methylcytosine, 5mC). The DNA methyltransferases that catalyze DNA methylation have been thoroughly investigated, whereas little is known regarding the enzymatic mechanisms of DNA demethylation. The ten-eleven translocation 2 (TET) protein converts 5mC to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine and finally to 5-carboxylcytosine (5caC). Subsequent decarboxylation of 5caC is carried out by either thymine-DNA glycosylase, and eventually TET-mediated reactions lead to DNA demethylation. The loss of 5hmc is an epigenetic characteristic in melanoma, and the expressions of all three TET genes is significantly lower in melanomas than in nevi, with the most dramatic decrease in the expression of TET2. The aim of the present study is to address whether the decreased level of 5hmc is caused by loss of heterozygosity (LOH) of the TET2 gene. Patient is a 92 year-old (yo) Japanese female who suffered from basal cell carcinoma (BCC) and malignant melanoma at 87 yo and 91 yo, respectively. Immunohistochemistery showed the significantly reduced level of 5hmc in malignant melanoma, but not in BCC. Both specimens of malignant melanoma and BCC were applied for a molecular analysis of SNPs in TET2 gene. The LOH of the TET2 gene was detected in malignant melanoma with a BRAFV600E mutation. This study suggests that heterozygous loss of the TET2 gene is likely one of the mechanisms underlying the low 5hmc level in the present case of malignant melanoma. 

Key words: Basal cell carcinoma; Heterozygous loss; Melanoma; Ten-eleven translocation 2; 

Introduction
     Epigenetic gene silencing, including CpG island hypermethylation, can alter the expression of tumor-suppressor genes, and an inactivation of tumor-suppressor genes is involved in tumorigenic processes in many cancers [1].  Genomic DNA methylation occurs predominantly on the 5 position of cytosine (5-methylcytosine, 5mC) in the context of CpG islands. The DNA methyltransferases that catalyze DNA methylation have been thoroughly investigated, whereas little is known about the enzymatic mechanisms of DNA demethylation. Recently, however, it was discovered that TET (ten-eleven translocation) protein, an alpha-ketoglutarate (alpha-KG)- and Fe (II)-dependent dioxygenase, has the capacity to catalyze sequential oxidation reactions [2, 3]. First, the TET protein converts 5mC to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine and finally to 5-carboxylcytosine (5caC). Subsequent decarboxylation of 5caC is carried out by either thymine-DNA glycosylase or other DNA repair enzymes, and eventually TET-mediated reactions lead to DNA demethylation [4]. The levels of 5hmC are dramatically reduced in human breast, liver, lung, pancreatic and prostate cancers. Decreased 5hmC levels are associated with reduced expressions of TET genes [5]. Three TET genes, TET1, TET2 and TET3, are expressed in humans. TET2 is mutationally inactivated in approximately 15% of myeloid cancers, including 22% of acute myeloid leukemia (AML) [6, 7].
     In the context of melanocytic tumors, the 5hmC level is high in mature melanocytes but lost in melanoma, and the loss of 5hmC is correlated with melanoma progression [8]. The expressions of all three TET genes are significantly lower in melanomas than nevi, with the most dramatic decrease in the TET2, thus suggesting that a diminished expression of TET family genes in melanomas is a molecular mechanism underlying the global loss of 5hmC, which is a new epigenetic hallmark of melanoma [8]. The present study was conducted to determine how the TET gene expression is reduced or silenced in melanoma. We analyzed the patient with malignant melanoma associated with basal cell carcinoma (BCC). Immunohistochemistery (IHC) showed the significantly reduced level of 5hmc in malignant melanoma, but not in BCC. The LOH of the TET2 gene was detected in malignant melanoma with a BRAFV600E mutation, implying that the one of the plausible reason for the reduced level of 5hmc in malignant melanoma seems to be LOH of TET2 in the present case. 

Patients and methods 
Patient is a 92 year-old (yo) Japanese female. Basal cell carcinoma, 3 mm in diameter was developed in her arm when she was 87 yo, and malignant melanoma, nodular type with 1 cm in diameter was found in the right dorsum of her foot at 91 yo. The tumor thickness of malignant melanoma was 4 mm and the metastasis of sentinel lymph node was revealed. Local injection of interferon was performed ten times. However, the metastasis, 7 mm in diameter was found in her right thigh at 92 yo.
     Genomic DNA extracted from formalin-fixed, paraffin-embedded tissues was used for the BRAF somatic mutation and SNP analyses [9]. SNPs of rs2285720, rs2647257, rs7679673, rs1541374, rs1391442 and rs949681 were used for the analysis of the heterozygosity of the TET2 gene located in 4q24 (Fig. 1).  Normal skin tissue was used for the germline analysis. The primers used for amplification of the genomic DNA and PCR conditions are listed in Table 1. Amplified DNA fragments were recovered from a low melting temperature agarose gel (2%) and subjected to a direct sequencing analysis using an automated DNA sequencing system (Model 377, Applied Biosystems, Foster City, CA, USA). 
     Immunohistochemistry (IHC) was performed as previously described [10]. 
IHC using primary antibodies against 5-Hydroxymethylcytosine (X2000; Active Motif, 
Carlsbad, CA, USA) was performed on formalin-fixed, paraffin-embedded tissue sections at 
4�C overnight. For detection, the DAKO LSAB+Kit was used. 

Results 
Hematoxylin eosin (HE) stain shows malignant melanoma adjacent to epidermis (Figure 2a). An inset in Figure 2a shows a nodular melanoma with 1cm in diameter. HE stain indicates BCC with the peripheral palisading (Figure 2b). IHC exhibits the significantly reduced level of 5-hmC in malignant melanoma (Figure 2c) but not in BCC (Figure 2d).  As shown in Figure 3a, V600E in BRAF was observed in malignant melanoma. Representative LOH analyses were indicated in Figures 3b-d.  SNP at rs949681 was observed in the germline sequence (Fig. 3b), whereas malignant melanoma (Fig. 3c) and BCC (Fig. 3d) showed LOH. SNPs at rs2285720 and rs1391442 showed LOH in malignant melanoma (data not shown), indicating the hetelozygous loss of the TET2 gene spanning approximately 25 Kb that contains one exon (Fig. 1). Heterozygosity at rs 2285720 was retained in BCC (data not shown). SNP and LOH analyses were summarized in Table 1. 
Discussion
     The BRAFT1799A mutation is a frequent genetic alteration in melanoma, detected in 50-70% and approximately 30% of Caucasian and Japanese cases of melanoma, respectively. This mutation results in the constitutively activated BRAFV600E kinase, which then promotes cell proliferation via the mitogen-activated protein kinase signaling pathway. BRAFV600E signaling causes widespread alterations in gene methylation in melanoma cells and BRAFV600E is a therapeutic target of melanoma [11-13]. 
     Lian et al. reported that the loss of 5hmC is an epigenetic hallmark and that downregulation of TET family enzymes is likely one of the mechanisms underlying 5hmC loss in melanoma [8]. Our immunohistochemical analysis, which also revealed a reduced expression of 5hmC in melanoma, together with the lack of reports in PubMed showing a link between malignant melanoma and TET2 loss of heterozygosity prompted us to address the question whether the BRAFV600E mutation is associated with LOH of TET2. 
     Tellez et al. analyzed 16 melanoma cell lines for aberrant methylations and concluded that aberrant hypermethylation is frequent in melanoma cell lines and occurs independently of BRAF and NRAS mutations [14]. In contrast, the BRAF mutation is frequently seen in sessile serrated adenoma and sporadic colorectal cancers exhibiting high levels of microsatellite instability, both of which are associated with DNA methylation [15]. The number of cases examined in the present study was limited; thus, further analyses are necessary to achieve a better understanding of the relationship between BRAFV600E and LOH of TET2.
     Investigations of oncogenes and tumor suppressor genes have provided molecular evidence for the concept of multistep carcinogenesis [16, 17]. The molecular pathogenesis of malignant melanoma appears to be sequential as well, and several genes are thought to be involved in disease progression [18, 19]. The present study was not in the early stage, as lymph node metastasis was observed. Hence, the question of whether BRAFV600E or LOH of TET2 is an earlier molecular alteration remains unresolved. The BRAF, NRAS, PTEN, RAC1 and PPP6C genes are responsible for the molecular tumorigenesis of malignant melanoma, and frequent genetic alterations are observed in these genes [18, 19]. However, in contrast to the molecular basis of adenoma carcinoma sequences observed in colorectal cancer, further studies are required to clarify the molecular pathogenesis of the initiation and progression of melanoma. Although LOH of TET2 is not fully examined, such phenomena have potential applicability for demethylation-based epigenetic treatment in melanoma patients with these genetic alterations. 
     LOH at rs949681 was observed not only in malignant melanoma but also in BCC, but the level of 5hmC is reduced in melanoma but not in BCC. The reason of this discrepancy may be the length of LOH because LOH spans approximately 25 Kb including one exon in malignant melanoma, but rs2285720 is retained in BCC. Thus it is possible that the region of LOH is short in BCC that dose not influence the translation of TET2.  
     In conclusion, the current study reports the case of a malignant melanoma patient whose tumor possessed the decreased level of 5hmC and heterozygous loss of TET2, suggesting that LOH of TET2 seems to be one of the possible mechanisms of the decreased 5hmC in human malignant melanoma.

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Figure legends
Figure 1.
An integrated germline map of the TET2 gene in the patient. SNPs examined in the present study are indicated by arrows. Boxes and vertical lines denote exons.

Figure 2. Histopathology and immunohistochemistry of malignant melanoma (a, c) and basal cell carcinoma (b, d). (a) Histology of malignant melanoma. E and M denote epidermis and malignant melanoma, respectively. The nest of malignant melanoma cells proliferates adjacent to the epidermis. The inset shows a nodular melanoma. (b) Histology of basal cell carcinoma exhibiting the peripheral palisading of the nuclei. (c) Immunohistochemistry of 5-hmC in malignant melanoma exhibiting the significantly reduced level (M) compared to that in epidermis (E). (d) Immunohistochemistry of 5-hmC in basal cell carcinoma (arrow) indicating the retained level of 5-hmC.  

Figure 3.
(a) DNA sequencing of BRAF in malignant melanoma indicating V600E. An arrow indicates the mutation position. The wild type sequence in codon 600 is T whereas the mutated nucleotide is A. (b, c) Representative sequences for the analysis of the heterozygous loss in the TET2 gene. Germline sequence of rs949681 in the TET2 gene shows C and T (b).  The arrow indicates the position of rs949681. However, only C is observed in malignant melanoma (c) and basal cell carcinoma (d), implying LOH in TET2. 
















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