doi: 10.1111/jop.12208
J Oral Pathol Med (2015) 44: 59–66 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
DNA methyltransferase immunohistochemical expression in odontogenic tumours Douglas Magno Guimar~ aes, Daniella Moraes Antunes, Carina Magalh~aes Esteves Duarte, Leonardo Borges Ferro, Fabio Daumas Nunes Department of Oral Pathology, School of Dentistry, University of S~ ao Paulo, S~ ao Paulo, Brazil
BACKGROUND: Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalysed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyses the expression of DNMTs in odontogenic tumours. METHODS: Formalin-fixed and paraffin-embedded tissue samples of 20 ameloblastomas, 10 calcifying cystic odontogenic tumours, 10 calcifying epithelial tumours, 10 adenomatoid odontogenic tumours, 10 keratocystic odontogenic tumours, five ameloblastic fibromas, two ameloblastic fibro-odontomas, four central odontogenic fibromas, seven peripheral odontogenic fibromas and 10 odontogenic myxomas were included. Immunohistochemical expression of DNMT1, 3A and 3B was assessed using a semi-quantitative analysis, and also a correlation with p21, p27 and E-cadherin immunoexpression was made. RESULTS: DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. DNMT1 expression was directly correlated with p27 expression in ameloblastomas. CONCLUSION: The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours. J Oral Pathol Med (2015) 44: 59–66 Keywords: DNA methyltransferases; epigenetics; methylation; odontogenic tumours
Correspondence: Fabio Daumas Nunes, DDS, PhD, Cidade Universitaria, Av. Professor Lineu Prestes 2227, Butant~a, Zip Code (CEP) 05508-000, S~ao Paulo, S~ao Paulo, Brazil. Tel: +55 11 3091 7902, Fax: +55 11 3091 7894, E-mail: [email protected] Accepted for publication April 21, 2014
Introduction DNA methylation, the covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalysed by DNA methyltransferases (DNMTs). These enzymes constitute a family with three members: DNMT1, DNMT3A and DNMT3B. DNMT1 is responsible for maintaining the methylation pattern of the template strand for the other strips that occurs after DNA duplication. DNMTs 3A and 3B are responsible for de novo methylation, as they are unable to distinguish methylated from unmethylated regions (1). Changes in the pattern of DNA methylation have been linked to the pathogenesis of various benign and malignant neoplasms including oral (2, 3), gastric (4), breast (5), ovary (6) and prostate tumours (7); however, the literature is still scarce on reporting epigenetic events in odontogenic tumours. Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. The World Health Organization (WHO) classifies these tumours according to their epithelial or ectomesenchymal compounds. Benign lesions of odontogenic epithelium with mature, fibrous stroma without the participation of ectomesenchyme are the most common. Important members of this group are ameloblastoma, calcifying epithelial odontogenic tumour (CEOT), adenomatoid odontogenic tumour (AOT) and keratocystic odontogenic tumour (KOT) (8). Although these lesions grow slowly, they are known to be locally aggressive and require special clinical attention. Some of them, such as ameloblastoma, AOT and KOT, have been previously reported to possess methylated genes (9–13). Mesenchyme participates, together with the epithelium, in odontogenesis and is frequently involved in oncogenesis. Calcifying cystic odontogenic tumour (CCOT), ameloblastic fibroma (AF) and ameloblastic fibro-odontoma (AFO) are classified in the subgroup odontogenic epithelium with odontogenic ectomesenchyme, with or without hard tissue formation. The most worrying aspect of the latter two lesions is their potential for malignant transformation. It is believed that one-third of ameloblastic fibrosarcomas originates from one of these two tumours (14, 15).
(6) (20) (40) (50) (40) (80) (25) (20)
KOT, keratocystic odontogenic tumour; AOT, adenomatoid odontogenic tumour; CEOT, calcifying epithelial odontogenic tumour; CCOT, calcifying cystic odontogenic tumour; AF, ameloblastic fibroma; AFO, ameloblastic fibro-odontoma; COF, central odontogenic fibroma; POF, peripheral odontogenic fibroma; OM, odontogenic myxoma. J Oral Pathol Med
(10) (10) (60)
(100) (100)
(75) (100) (90)
(86)
(10) (10)
(20) (40)
15 10 9 0 0 4 2 0 0 0 1 1 6 (25)
(50) (40)
(30)
(75) (14)
5 0 1 1 0 0 0 0 6 0 0 2 4 (100) (40) (30)
(25)
(60) (100)
0 0 0 3 0 0 0 3 1 0 5 4 0 0 0 0 6 10 0 0 1 0 10 4 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 (10) 0 2 (20) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (100)
(100) (90) (100) (80) (100) (100) (100) (100) (100) (100) (70) (10) (10)
(25)
(75)
20 9 10 8 10 4 2 4 7 10 0 0 10 (50) (40)
(30) (20) (40)
10 4 0 0 0 1 0 3 0 7 1 1 (50) (40) (20) (20) (10) (75) (50) (25) (100)
10 4 2 2 1 3 1 1 7 0 3 2 4
Score 2 (%)
DNMT3B
Score 0 (%) Score 3 (%) Score 2 (%)
DNMT3A
Score 1 (%) Score 0 (%) Score 3 (%)
DNMT, DNA methyltransferase; KOT, keratocystic odontogenic tumour; AOT, adenomatoid odontogenic tumour; CEOT, calcifying epithelial odontogenic tumour; CCOT, calcifying cystic odontogenic tumour; AF, ameloblastic fibroma; AFO, ameloblastic fibro-odontoma; COF, central odontogenic fibroma; POF, peripheral odontogenic fibroma; OM, odontogenic myxoma; DC, dentigerous cyst; RC, radicular cyst; NOM, normal oral mucosa.
1 2 4 5 4 0 0 4 1 2
(94) (80) (60) (50) (60) (100) (100) (20) (75) (80)
(20) (10) (60)
16 8 6 5 6 4 2 1 3 8
(48) (30) (60) (50) (50) (50) (50) (50) (43) (30)
(30) (40) (60)
9 3 6 5 5 2 1 2 3 3
(52) (70) (40) (50) (50) (50) (50) (50) (57) (70)
(50)
10 7 4 5 5 2 1 2 4 7
(16–71) (10–65) (11–40) (07–43) (11–51) (11–12) (11–28) (18–54) (19–51) (17–50)
(10)
33 29 16 32 29 11 19 38 34 26
(80) (80)
Ameloblastoma KOT AOT CEOT CCOT AF AFO COF POF OM
(20) (80)
Mx (%)
0 2 8 0 1 0 0 0 0 0 2 1 6
Md (%)
0 0 0 8 8 0 1 0 0 3 4 6 0
F (%)
Ameloblastoma KOT AOT CEOT CCOT AF AFO COF POF OM DC RC NOM
M (%)
Score 2 (%)
Site
Age (years)
DNMT1
Gender Tumour
Score 1 (%)
Table 1 Clinical information of the 87 cases studied
Table 2 Scores of the immunohistochemical evaluation to DNMT1, 3A and 3B in the odontogenic tumours studied
Materials and methods Formalin-fixed and paraffin-embedded (FFPE) tissue samples were selected from the Oral Pathology Service, School of Dentistry, University of S~ao Paulo. Only samples from 2005 to 2012 were included. Specimens with insufficient tissue, poorly fixed, from syndromic patients or recurrent lesions were discarded. Samples consisted of 20 ameloblastomas, 10 CCOT, 10 CEOT, 10 AOT, 10 KOT, five AF, two AFO, four COF, seven POF and 10 OM. For comparison, 20 non-neoplastic odontogenic tissues, 10 samples of both dentigerous and radicular cysts, and 10 normal oral parakeratinized epithelium tissues were included. One section of each specimen was subjected to H&E and analysed by two pathologists to confirm the initial diagnosis. The age, gender and localization of each tumour are represented in Table 1. The Ethics Committee of the School of Dentistry at University of S~ao Paulo approved this study. Immunohistochemistry was performed using the labelled polymer method. Sections 3 lm thick were
Score 1 (%)
Odontogenic tumours may also arise from a proliferating mesenchyme without epithelial participation. Central and peripheral odontogenic fibroma (COF and POF, respectively) and odontogenic myxoma (OM) are members of the subgroup mesenchyme and/or odontogenic ectomesenchyme with or without odontogenic epithelium. The latter, despite being considered benign, can display locally destructive growth, affecting large regions of bone with high recurrence rates (8, 16). Although some epigenetic studies have been conducted in odontogenic tumours, showing the methylation of cellcycle-associated genes, as p21 and p27 (12), a study with the three types of DNMT in several different members of this group is missing. As DNA methylation, catalysed by DNMTs, is regarded as a key player in epigenetic silencing of transcription, and gene silencing is important for tumour behaviour, the present study analyses the expression of DNMTs in the most representative benign odontogenic tumours according to WHO classification.
Score 0 (%)
60
Score 3 (%)
DNA methyltransferase expression in odontogenic tumours Guimar~ aes et al.
DNA methyltransferase expression in odontogenic tumours Guimar~ aes et al.
obtained from FFPE tissues and mounted on silanized slides. Slides were deparaffinized in xylene, rehydrated in a graded alcohol series and washed in tap water. For antigen retrieval, the sections were placed in a microwave with citric acid solution, pH 6.0, for 15 min. Endogenous peroxidase activity was blocked in methanol containing 0.3% hydrogen peroxide. DNMT1 (IMG-261A, 1:400 dilution; Imgenex, San Diego, CA, USA), DNMT3A (IMG-268A, 1:200 dilution; Imgenex) and DNMT3B (IMG-184A, 1:100 dilution; Imgenex) antibodies were incubated at 4°C overnight. Immunohistochemistry to p21, p27 and E-cadherin was also analysed in tumours with high DNMTs expression to address if there was an association between the expressions of these proteins. These reactions were performed as described above using anti-p21 (M0637, 1:100; Dako, Carpinteria, CA, USA), anti-27 (dcs72, 1:200 dilution; Biogenex Fremont, CA, USA) and anti-E-cadherin (3195, 1:800 dilution; Cell signalling Beverly, MA, USA). These procedures were followed by use of the ADVANCED (Dako) polymer conjugate and Dako Liquid DAB plus Substrate Chromogen System TM (3,30-diaminobenzidine) (Dako) for antigen– antibody complex visualization. All sections were counterstained with Mayer haematoxylin. Placenta sections were used as a positive control for the three antibodies. Two experienced pathologists independently examined multiple fields and made a descriptive analysis of each case. In addition, a semi-quantitative analysis was performed
where only nuclear staining was considered as positive, as nuclear-expressed DNMTs are the actual functional DNMT proteins. Samples were individually scored as grade 0 (≤25% positive cells), grade 1 (>25% and ≤50% positive cells), grade 2 (>50% and ≤75% positive cells) and grade 3 (>75%) (6, 17). For p27 and p21, a semi-quantitative analysis was performed following the score 0 (≤5% positive cells), score 1 (>5% and ≤25% positive cells), score 2 (>25% and ≤50% positive cells) and score 3 (>50%) (18). Also, for the previous antibodies, only the nuclear staining was considered as positive. E-cadherin membrane labelling was scored as grade 1 (≤25% positive cells), grade 2 (>25% and ≤50% positive cells), grade 3 (>50% and ≤75% positive cells) and grade 4 (>75%) (19). Statistical analysis with the nonparametric Pearson’s chi-square test with a significance level of 5% was performed to evaluate the difference between scores in each tumour.
61
Results The scores representing the percentage of positive cells with nuclear expression of DNMT1, 3A and 3B in each tumour are shown in Table 2. In ameloblastoma, DNMT1 was observed in more than 50% of tumour cells, in both peripheral and central cells (Fig. 1A). The expression of DNMT1 in KOT was predominantly nuclear in more than 50% of cells and mostly in the upper basal cell layer
A
B
C
D
E
F
G
H
I
J
K
L
Figure 1 Immunohistochemical expression of DNMT1. (A) The nuclear and cytoplasmic expression in peripheral and central cells of an ameloblastoma plexiform area. (B) Nuclear expression in keratocystic odontogenic tumour suprabasal layer cells. (C) Nuclear and cytoplasmic expression in adenomatoid odontogenic tumour rosette-like structures. (D) Absence of expression in calcifying epithelial odontogenic tumour and nuclear and cytoplasmic expression in clear cells compounds (insert). (E) Cytoplasmic expression in basal cells layer of calcifying cystic odontogenic tumour (insert). (F and G) Cytoplasmic and nuclear expression in epithelial islands and mesenchymal cells of ameloblastic fibroma and ameloblastic fibro-odontoma, respectively. (H and I) Nuclear and cytoplasmic expression in odontogenic epithelium and mesenchymal cells of central odontogenic fibroma and peripheral odontogenic fibroma, respectively. (J) Nuclear expression in odontogenic myxoma stellate and spindle-shaped cells. Original magnification, 9400. J Oral Pathol Med
DNA methyltransferase expression in odontogenic tumours Guimar~ aes et al.
62
(Fig. 1B). Nuclear expression of DNMT1 in AOT was observed in about 50% of cells, with expression being more evident in duct-like and rosette-like structures (Fig. 1C). Tumours showing less of these structures presented a lower expression. In CEOT, polyhedric tumoural cells showed no expression in 80% of cases (Fig. 1D). However, clear cells were positive when present (Fig. 1D insert). DNMT1 expression in CCOT was mainly cytoplasmic in the basal layer (Fig. 1E). In two cases (20%), nuclear expression was observed only in this layer (Fig. 1E insert). In the other mixed odontogenic tumours (AF and AFO), nuclear expression of DNMT1 was observed in more than 50% of epithelial cells and in stromal component cells (Fig. 1F,G). In mesenchyme-derived odontogenic tumours with or without odontogenic epithelium, nuclear expression of DNMT1 was observed in more than 50% of the cells, that is, COF and POF were positive in both odontogenic epithelium and mesenchymal cells (Fig. 1H,I). In OM, nuclear expression of DNMT1 was observed in more than 75% of stellate and spindle-shaped cells (Fig. 1J). In DC, the DNMT1 expression was lower, with 70% of cases expressing in
J Oral Pathol Med (2015) 44: 59–66 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
DNA methyltransferase immunohistochemical expression in odontogenic tumours Douglas Magno Guimar~ aes, Daniella Moraes Antunes, Carina Magalh~aes Esteves Duarte, Leonardo Borges Ferro, Fabio Daumas Nunes Department of Oral Pathology, School of Dentistry, University of S~ ao Paulo, S~ ao Paulo, Brazil
BACKGROUND: Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalysed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyses the expression of DNMTs in odontogenic tumours. METHODS: Formalin-fixed and paraffin-embedded tissue samples of 20 ameloblastomas, 10 calcifying cystic odontogenic tumours, 10 calcifying epithelial tumours, 10 adenomatoid odontogenic tumours, 10 keratocystic odontogenic tumours, five ameloblastic fibromas, two ameloblastic fibro-odontomas, four central odontogenic fibromas, seven peripheral odontogenic fibromas and 10 odontogenic myxomas were included. Immunohistochemical expression of DNMT1, 3A and 3B was assessed using a semi-quantitative analysis, and also a correlation with p21, p27 and E-cadherin immunoexpression was made. RESULTS: DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. DNMT1 expression was directly correlated with p27 expression in ameloblastomas. CONCLUSION: The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours. J Oral Pathol Med (2015) 44: 59–66 Keywords: DNA methyltransferases; epigenetics; methylation; odontogenic tumours
Correspondence: Fabio Daumas Nunes, DDS, PhD, Cidade Universitaria, Av. Professor Lineu Prestes 2227, Butant~a, Zip Code (CEP) 05508-000, S~ao Paulo, S~ao Paulo, Brazil. Tel: +55 11 3091 7902, Fax: +55 11 3091 7894, E-mail: [email protected] Accepted for publication April 21, 2014
Introduction DNA methylation, the covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalysed by DNA methyltransferases (DNMTs). These enzymes constitute a family with three members: DNMT1, DNMT3A and DNMT3B. DNMT1 is responsible for maintaining the methylation pattern of the template strand for the other strips that occurs after DNA duplication. DNMTs 3A and 3B are responsible for de novo methylation, as they are unable to distinguish methylated from unmethylated regions (1). Changes in the pattern of DNA methylation have been linked to the pathogenesis of various benign and malignant neoplasms including oral (2, 3), gastric (4), breast (5), ovary (6) and prostate tumours (7); however, the literature is still scarce on reporting epigenetic events in odontogenic tumours. Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. The World Health Organization (WHO) classifies these tumours according to their epithelial or ectomesenchymal compounds. Benign lesions of odontogenic epithelium with mature, fibrous stroma without the participation of ectomesenchyme are the most common. Important members of this group are ameloblastoma, calcifying epithelial odontogenic tumour (CEOT), adenomatoid odontogenic tumour (AOT) and keratocystic odontogenic tumour (KOT) (8). Although these lesions grow slowly, they are known to be locally aggressive and require special clinical attention. Some of them, such as ameloblastoma, AOT and KOT, have been previously reported to possess methylated genes (9–13). Mesenchyme participates, together with the epithelium, in odontogenesis and is frequently involved in oncogenesis. Calcifying cystic odontogenic tumour (CCOT), ameloblastic fibroma (AF) and ameloblastic fibro-odontoma (AFO) are classified in the subgroup odontogenic epithelium with odontogenic ectomesenchyme, with or without hard tissue formation. The most worrying aspect of the latter two lesions is their potential for malignant transformation. It is believed that one-third of ameloblastic fibrosarcomas originates from one of these two tumours (14, 15).
(6) (20) (40) (50) (40) (80) (25) (20)
KOT, keratocystic odontogenic tumour; AOT, adenomatoid odontogenic tumour; CEOT, calcifying epithelial odontogenic tumour; CCOT, calcifying cystic odontogenic tumour; AF, ameloblastic fibroma; AFO, ameloblastic fibro-odontoma; COF, central odontogenic fibroma; POF, peripheral odontogenic fibroma; OM, odontogenic myxoma. J Oral Pathol Med
(10) (10) (60)
(100) (100)
(75) (100) (90)
(86)
(10) (10)
(20) (40)
15 10 9 0 0 4 2 0 0 0 1 1 6 (25)
(50) (40)
(30)
(75) (14)
5 0 1 1 0 0 0 0 6 0 0 2 4 (100) (40) (30)
(25)
(60) (100)
0 0 0 3 0 0 0 3 1 0 5 4 0 0 0 0 6 10 0 0 1 0 10 4 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 (10) 0 2 (20) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (100)
(100) (90) (100) (80) (100) (100) (100) (100) (100) (100) (70) (10) (10)
(25)
(75)
20 9 10 8 10 4 2 4 7 10 0 0 10 (50) (40)
(30) (20) (40)
10 4 0 0 0 1 0 3 0 7 1 1 (50) (40) (20) (20) (10) (75) (50) (25) (100)
10 4 2 2 1 3 1 1 7 0 3 2 4
Score 2 (%)
DNMT3B
Score 0 (%) Score 3 (%) Score 2 (%)
DNMT3A
Score 1 (%) Score 0 (%) Score 3 (%)
DNMT, DNA methyltransferase; KOT, keratocystic odontogenic tumour; AOT, adenomatoid odontogenic tumour; CEOT, calcifying epithelial odontogenic tumour; CCOT, calcifying cystic odontogenic tumour; AF, ameloblastic fibroma; AFO, ameloblastic fibro-odontoma; COF, central odontogenic fibroma; POF, peripheral odontogenic fibroma; OM, odontogenic myxoma; DC, dentigerous cyst; RC, radicular cyst; NOM, normal oral mucosa.
1 2 4 5 4 0 0 4 1 2
(94) (80) (60) (50) (60) (100) (100) (20) (75) (80)
(20) (10) (60)
16 8 6 5 6 4 2 1 3 8
(48) (30) (60) (50) (50) (50) (50) (50) (43) (30)
(30) (40) (60)
9 3 6 5 5 2 1 2 3 3
(52) (70) (40) (50) (50) (50) (50) (50) (57) (70)
(50)
10 7 4 5 5 2 1 2 4 7
(16–71) (10–65) (11–40) (07–43) (11–51) (11–12) (11–28) (18–54) (19–51) (17–50)
(10)
33 29 16 32 29 11 19 38 34 26
(80) (80)
Ameloblastoma KOT AOT CEOT CCOT AF AFO COF POF OM
(20) (80)
Mx (%)
0 2 8 0 1 0 0 0 0 0 2 1 6
Md (%)
0 0 0 8 8 0 1 0 0 3 4 6 0
F (%)
Ameloblastoma KOT AOT CEOT CCOT AF AFO COF POF OM DC RC NOM
M (%)
Score 2 (%)
Site
Age (years)
DNMT1
Gender Tumour
Score 1 (%)
Table 1 Clinical information of the 87 cases studied
Table 2 Scores of the immunohistochemical evaluation to DNMT1, 3A and 3B in the odontogenic tumours studied
Materials and methods Formalin-fixed and paraffin-embedded (FFPE) tissue samples were selected from the Oral Pathology Service, School of Dentistry, University of S~ao Paulo. Only samples from 2005 to 2012 were included. Specimens with insufficient tissue, poorly fixed, from syndromic patients or recurrent lesions were discarded. Samples consisted of 20 ameloblastomas, 10 CCOT, 10 CEOT, 10 AOT, 10 KOT, five AF, two AFO, four COF, seven POF and 10 OM. For comparison, 20 non-neoplastic odontogenic tissues, 10 samples of both dentigerous and radicular cysts, and 10 normal oral parakeratinized epithelium tissues were included. One section of each specimen was subjected to H&E and analysed by two pathologists to confirm the initial diagnosis. The age, gender and localization of each tumour are represented in Table 1. The Ethics Committee of the School of Dentistry at University of S~ao Paulo approved this study. Immunohistochemistry was performed using the labelled polymer method. Sections 3 lm thick were
Score 1 (%)
Odontogenic tumours may also arise from a proliferating mesenchyme without epithelial participation. Central and peripheral odontogenic fibroma (COF and POF, respectively) and odontogenic myxoma (OM) are members of the subgroup mesenchyme and/or odontogenic ectomesenchyme with or without odontogenic epithelium. The latter, despite being considered benign, can display locally destructive growth, affecting large regions of bone with high recurrence rates (8, 16). Although some epigenetic studies have been conducted in odontogenic tumours, showing the methylation of cellcycle-associated genes, as p21 and p27 (12), a study with the three types of DNMT in several different members of this group is missing. As DNA methylation, catalysed by DNMTs, is regarded as a key player in epigenetic silencing of transcription, and gene silencing is important for tumour behaviour, the present study analyses the expression of DNMTs in the most representative benign odontogenic tumours according to WHO classification.
Score 0 (%)
60
Score 3 (%)
DNA methyltransferase expression in odontogenic tumours Guimar~ aes et al.
DNA methyltransferase expression in odontogenic tumours Guimar~ aes et al.
obtained from FFPE tissues and mounted on silanized slides. Slides were deparaffinized in xylene, rehydrated in a graded alcohol series and washed in tap water. For antigen retrieval, the sections were placed in a microwave with citric acid solution, pH 6.0, for 15 min. Endogenous peroxidase activity was blocked in methanol containing 0.3% hydrogen peroxide. DNMT1 (IMG-261A, 1:400 dilution; Imgenex, San Diego, CA, USA), DNMT3A (IMG-268A, 1:200 dilution; Imgenex) and DNMT3B (IMG-184A, 1:100 dilution; Imgenex) antibodies were incubated at 4°C overnight. Immunohistochemistry to p21, p27 and E-cadherin was also analysed in tumours with high DNMTs expression to address if there was an association between the expressions of these proteins. These reactions were performed as described above using anti-p21 (M0637, 1:100; Dako, Carpinteria, CA, USA), anti-27 (dcs72, 1:200 dilution; Biogenex Fremont, CA, USA) and anti-E-cadherin (3195, 1:800 dilution; Cell signalling Beverly, MA, USA). These procedures were followed by use of the ADVANCED (Dako) polymer conjugate and Dako Liquid DAB plus Substrate Chromogen System TM (3,30-diaminobenzidine) (Dako) for antigen– antibody complex visualization. All sections were counterstained with Mayer haematoxylin. Placenta sections were used as a positive control for the three antibodies. Two experienced pathologists independently examined multiple fields and made a descriptive analysis of each case. In addition, a semi-quantitative analysis was performed
where only nuclear staining was considered as positive, as nuclear-expressed DNMTs are the actual functional DNMT proteins. Samples were individually scored as grade 0 (≤25% positive cells), grade 1 (>25% and ≤50% positive cells), grade 2 (>50% and ≤75% positive cells) and grade 3 (>75%) (6, 17). For p27 and p21, a semi-quantitative analysis was performed following the score 0 (≤5% positive cells), score 1 (>5% and ≤25% positive cells), score 2 (>25% and ≤50% positive cells) and score 3 (>50%) (18). Also, for the previous antibodies, only the nuclear staining was considered as positive. E-cadherin membrane labelling was scored as grade 1 (≤25% positive cells), grade 2 (>25% and ≤50% positive cells), grade 3 (>50% and ≤75% positive cells) and grade 4 (>75%) (19). Statistical analysis with the nonparametric Pearson’s chi-square test with a significance level of 5% was performed to evaluate the difference between scores in each tumour.
61
Results The scores representing the percentage of positive cells with nuclear expression of DNMT1, 3A and 3B in each tumour are shown in Table 2. In ameloblastoma, DNMT1 was observed in more than 50% of tumour cells, in both peripheral and central cells (Fig. 1A). The expression of DNMT1 in KOT was predominantly nuclear in more than 50% of cells and mostly in the upper basal cell layer
A
B
C
D
E
F
G
H
I
J
K
L
Figure 1 Immunohistochemical expression of DNMT1. (A) The nuclear and cytoplasmic expression in peripheral and central cells of an ameloblastoma plexiform area. (B) Nuclear expression in keratocystic odontogenic tumour suprabasal layer cells. (C) Nuclear and cytoplasmic expression in adenomatoid odontogenic tumour rosette-like structures. (D) Absence of expression in calcifying epithelial odontogenic tumour and nuclear and cytoplasmic expression in clear cells compounds (insert). (E) Cytoplasmic expression in basal cells layer of calcifying cystic odontogenic tumour (insert). (F and G) Cytoplasmic and nuclear expression in epithelial islands and mesenchymal cells of ameloblastic fibroma and ameloblastic fibro-odontoma, respectively. (H and I) Nuclear and cytoplasmic expression in odontogenic epithelium and mesenchymal cells of central odontogenic fibroma and peripheral odontogenic fibroma, respectively. (J) Nuclear expression in odontogenic myxoma stellate and spindle-shaped cells. Original magnification, 9400. J Oral Pathol Med
DNA methyltransferase expression in odontogenic tumours Guimar~ aes et al.
62
(Fig. 1B). Nuclear expression of DNMT1 in AOT was observed in about 50% of cells, with expression being more evident in duct-like and rosette-like structures (Fig. 1C). Tumours showing less of these structures presented a lower expression. In CEOT, polyhedric tumoural cells showed no expression in 80% of cases (Fig. 1D). However, clear cells were positive when present (Fig. 1D insert). DNMT1 expression in CCOT was mainly cytoplasmic in the basal layer (Fig. 1E). In two cases (20%), nuclear expression was observed only in this layer (Fig. 1E insert). In the other mixed odontogenic tumours (AF and AFO), nuclear expression of DNMT1 was observed in more than 50% of epithelial cells and in stromal component cells (Fig. 1F,G). In mesenchyme-derived odontogenic tumours with or without odontogenic epithelium, nuclear expression of DNMT1 was observed in more than 50% of the cells, that is, COF and POF were positive in both odontogenic epithelium and mesenchymal cells (Fig. 1H,I). In OM, nuclear expression of DNMT1 was observed in more than 75% of stellate and spindle-shaped cells (Fig. 1J). In DC, the DNMT1 expression was lower, with 70% of cases expressing in
