- Email: [email protected]
Review of the molecular pathogenesis of the odontogenic keratocyst
Oral Oncology 45 (2009) 1011–1014
Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Review
Review of the molecular pathogenesis of the odontogenic keratocyst Carolina Cavaliéri Gomes, Marina Gonçalves Diniz, Ricardo Santiago Gomez * Department of Oral Surgery and Pathology, School of Dentistry, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
a r t i c l e
i n f o
Article history: Received 13 July 2009 Received in revised form 1 August 2009 Accepted 17 August 2009 Available online 30 September 2009 Keywords: Odontogenic keratocyst Keratocystic odontogenic tumour PTCH Genetics Epigenetics Review
s u m m a r y The odontogenic keratocyst (keratocystic odontogenic tumour) (OKC) is one of the most prevalent odontogenic tumours. Since its initial description, a number of studies have focused on different aspects of this lesion, attempting to explain its distinctive biological behaviour. In this review the authors address the main genetic and epigenetic alterations reported on this tumour. Although most of the knowledge on this field is not being used in the clinical practice, some perspectives of translational studies are discussed. Ó 2009 Elsevier Ltd. All rights reserved.
Introduction The odontogenic keratocyst (OKC) (keratocystic odontogenic tumour) is an aggressive cystic lesion most frequently present in the second, third and fourth decades of life at the posterior mandible of male patients. Histologically, the OKC is characterized by a uniform, usually corrugated parakeratinized epithelium, 8–12 cells thick presenting a flat basal surface lining the fibrous wall.1 The columnar basal cells showing reversed nuclear polarity is an important feature and satellite cysts may be found in some cases. The aggressive behaviour and high recurrence rate of the OKC suggests a true neoplastic potential and prompted the World Health Organization Working Group to classify the OKC as a benign tumour with odontogenic epithelium and mature, fibrous stroma without odontogenic ectomesenchyme.2 The proliferative activity in the OKC was studied by various investigators and it was compared to other odontogenic cysts and tumours.3 Most of the researchers have found increased immunohistochemical expression of the proliferation markers Ki67 and PCNA in the OKC compared to other odontogenic lesions.4–12 It is important to mention that proliferative status is not always directly associated with biological behaviour or a true neoplastic nature. Non-neoplastic proliferative lesions may also demonstrate high expression of some proliferative markers.13 An-
* Corresponding author. Address: Department of Oral Surgery and Pathology, School of Dentistry, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627 Belo Horizonte, MG, CEP 31270 901, Brazil. Tel.: +55 31 34092477; fax: +55 31 34092430. E-mail address: [email protected] (R.S. Gomez). 1368-8375/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2009.08.003
other example is the Ki-67 expression in low grade mucoepidermoid carcinoma, a malignant neoplasm, which is significantly lower than that in glandular odontogenic cysts, an odontogenic cyst.14 Therefore, the understanding of the biology of a tumour should go beyond cell proliferation, revealing the aspects that influence the fine regulation of the cell cycle. Recently, some studies have focused on the molecular aspects of OKC. In this review we attempted to address the main genetic and epigenetic alterations reported in the OKC. The use of new molecular tools and their potential contribution to the understanding of the pathogenesis and treatment of the lesion are also discussed.
Genetic alterations The neplasic concept of OKC is supported by molecular studies that demonstrated loss of heterozygosity.15–17 The authors of these studies found evidence of allelic loss mainly in the p16, p53, PTCH, MCC, TSLC1, LTAS2 and FHIT genes. Taking into consideration that all of these genes are tumour-suppressor genes associated with different types of human neoplasia, these findings give further support to explain the aggressive behaviour of the OKC. In addition, observations have been made that daughter cysts are associated with a higher frequency of allelic loss.15 P53 is a tumour-suppressor gene that is commonly mutated in many malignant neoplasms, including oral squamous cell carcinoma.18 As the p53 protein has a short half-life, it cannot usually be detected by immunohistochemical methods. However, the primary antibody used, the sensitivity of the detection system, and fixation conditions of the tissue, among other factors, may lead
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to false-positive or false-negative results. Some studies have shown positive p53 labelling in epithelial cells of the OKC,9,19–21 but it was not followed by p53 gene alteration using stranded conformation polymorphism (PCR–SSCP).20 Additionally, positive immunohistochemical labelling for p53 was also found in dentigerous cysts and radicular cysts and p53 mutation was detected in only one OKC (12.9% of the cases).17 Taken together, it seems that p53 mutation is not a relevant event to OKC pathogenesis. The genomic aberrations in the OKC were also studied by Heikinheimo et al.22 using cDNA-expression arrays and array-comparative genomic hybridization. The authors found that several genes were overexpressed or underexpressed and exhibited deletions in 3q13.1, 5p14.3 and 7q31.3. They concluded that overexpressed and amplified genes in 12q13, including cytokeratin 6B (KRT6B), epidermal growth factor receptor ERBB3 and glioma-associated oncogene homologue 1 (GLI1), may contribute to the persistent growth characteristics of the OKC. As the authors suggested, the detachment of epithelium from the stroma, one of the microscopic hallmarks of the OKC, may be explained by the several gene aberrations related to cell adhesion observed in their study. The most important genetic alteration reported in the OKC is in the Drosophila Patched gene (PTCH1).23–27 The PTCH1 encodes a transmembrane receptor for Sonic Hedgehog (SHH) and other Hedgehog proteins. Normally, the PTCH1 represses the functions of the signalling effector Smoothened (SMO). The binding of HH on the PTCH1-SMO complex releases SMO, leading to the activation of target genes by the GLI family of transcription factors.28 The discovery that germline mutations of PTCH1 are the underlying cause of Nevoid Basal Cell Carcinoma Syndrome (NBCCS), prompted several authors to investigate the role of the HH pathway in the pathogenesis of a number of tumours associated with this syndrome, including the OKC. Mutations of PTCH1 in OKC associated with NBCCS were first described by Lench and colleagues.23 Subsequently, PTCH1 gene mutation was reported in the epithelial lining of sporadic cases of human OKC.24–27 Recently, Pan and Li29 demonstrated that Ki-67 labelling index in the epithelium of OKCs with PTCH1 mutation was significantly higher than in cases with no PTCH1 mutation. The authors concluded that PTCH1 mutations are associated with a subgroup of OKCs showing increased proliferative activity and thus may be related to a phenotype of higher recurrence tendency. An additional study showed that Gli1, which is a downstream signalling molecule of the SHH/PTCH pathway, is overexpressed in OKC.27 The fact that activation of SMO, like inactivation of PTCH1, upregulates the transcription of hedgehog target genes, was the basis for the investigation of activating mutations in the SMO gene in OKC, but the authors failed to demonstrate any alterations. Although the mutations detected in the PTCH1 were predicted to result in premature termination of PTCH1 protein and a high frequency of these mutations were clustered into the two large extracellular loops where hedgehog ligand binding occurs, no apparent genotype–phenotype correlations could be established yet.30 After PTCH1 mutation, the development of OKC may follow the ‘two-hit’ or the haploinsufficiency theory (Fig. 1). According to the former, the OKC associated with NBCCS would arise from precursor cells containing a hereditary ‘first hit’ followed by allelic loss of the second normal allele, while the sporadic OKC would arise from susceptible cells in which two somatic ‘hits’ have occurred. According to the haploinsufficiency model, the OKC would develop after the loss of only one PTCH allele, leading to reduction of gene dosage. The presence of normal PTCH protein in OKC cases with PTCH nonsense mutation (predicted to result in a truncated protein) suggests the occurrence of retention of one wild PTCH allele and supports the haploinsufficiency model.31 Alternative expression of mRNAs generated by a single gene is an important mechanism to produce different proteins and to reg-
Figure 1 Possible molecular mechanisms related to PTCH1 inactivation. One or more of these genetic or epigenetic alterations may act in cooperation causing PTCH inactivation, leading to Sonic Hedgehog pathway activation.
ulate genes in a time-dependent manner.32 There are several PTCH1 isoforms generated by splice variants of the first exon. These isoforms have different expression profiles, function and transcriptional regulation.33–37 The most prevalent isoforms of PTCH1 are PTCHb, PTCHd and PTCHe, which are generated by the first exons 1b, 1d and 1e, respectively. Although all of these isoforms can inhibit the HH protein activity, only 1b has an additional ability to completely inhibit SMO activity.33,34 Furthermore, transcripts of 1b were found to be exclusively upregulated in nodular basal cell carcinoma.33 Recently, our group studied the expression of PTCH1 first exons in 40 OKC tumours by RT-PCR.38 While none of the dental follicles expressed the exon 1b, it was detected in 90% of the OKC samples. The pattern of exon 1 expression observed in oral mucosa adjacent to the OKC was similar to the lesion. We concluded that OKC lining together with the oral adjacent mucosa presented overactivity of the SHH pathway. An interesting finding was that some of the lesions that were marsupialized lost the 1b expression, which, in turn, indicates that this procedure could alter PTCH1 variant profiles in some OKC. The elucidation of the molecular aspects that predict good response to marsupialization are important goals to be achieved. Epigenetic alterations Epigenetic alterations are considered important events in the tumorigenesis of benign and malignant tumours of the head and neck.39 The genes can be modified through this mechanism, without having their DNA sequences changed.40,41 DNA methylation is an epigenetic event characterized by the addition of a methyl group in cytosines within CpG islands, remodelling the chromatin and selectively activating or inactivating genes. The PTCH1 methylation has been suggested as an alternative to mutational causes of the PTCH pathway deregulation in tumours associated with NBCCS syndrome, such as meduloblastoma and basocellular carcinoma.42,43 Although none of these tumours presented methylation of the PTCH promoter, significantly higher methylation of the PTCH promoter region was correlated with low PTCH expression in breast cancer samples without PTCH mutation.44 Until now, the status of PTCH methylation in OKC not harbouring PTCH mutations remains unknown. Future directions, including examination of distal regions of the PTCH promoter, as well as CpG island containing other alternative exon variants may shed light on epigenetic mechanisms by which the PTCH pathway may be inactivated. The summary of the possible genetic and epigenetic mechanisms related to PTCH inactivation is demonstrated in Fig. 1. Our group studied the presence of methylation in P16, P21, P27, P53 and RB1 genes in OKC tumours by MSP (methylation specific
C.C. Gomes et al. / Oral Oncology 45 (2009) 1011–1014
PCR).45 In contrast to dental follicles, OKC samples presented methylation of the P21 gene. Epigenetic alteration of the RB1 gene was detected in some samples of dental follicles but in none of the OKC samples. This data revealed differences on the profile of genes methylated in an odontogenic tumour compared to normal odontogenic tissues. Whether epigenetics is really related to odontogenic tumours development is a question still not answered. Future molecular approaches MicroRNAs (miRNAs) are small non-coding RNAs which mediate gene expression at the post-transcriptional level by degrading or repressing target messenger RNAs (mRNA). It consists of a product about 22 nucleotides long and regulates mRNA translation by base pairing to partially complementary sites, predominantly in the 30 untranslated region (30 UTR) of mRNA.46,47 Overexpression of oncogenic miRNA may reduce protein products of tumour-suppressor genes. On the other hand, the loss of tumour-suppressor miRNA expression may cause elevated levels of oncogenic proteins. Although altered expression of miRNAs are important events in human cancers, to date there is no published study of tumour-suppressor or oncogenic miRNA in odontogenic cysts or tumours. Conclusion While some OKC tumours do not recur after marsupialization or other conservative approaches, some lesions show an aggressive behaviour. A number of clinical and surgical aspects influence such response, but up to now oral surgeons do not have reliable instruments to predict which lesion will recur or not. Studies involving the clinic-pathological, as well as the molecular aspects of this entity, will help to establish new parameters that could help clinicians to choose the most appropriate treatment for a specific tumour. Therefore, translational research joining the experience of different institutions is crucial. As some of the molecular pathways involved in the OKC are also deregulated in different types of human neoplasias (ex: PTCH gene in OKC and basal cell carcinoma), advances in one area could improve the overall current knowledge related to different tumours. It is the beginning of a new time. Conflict of Interest Statement None declared. Acknowledgements This study was supported by Grants from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and from Milênio/ Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. Dr. R.S. Gomez is research fellow of CNPq. References 1. Odell EW, Morgan PR. Biopsy pathology of the oral tissues. London: Chapman & Hall; 1998. 2. Barnes L, Eveson JW, Reichart P, Sidransky D. World Health Organization classification of tumours. Pathology & genetics. Head and neck tumours. Lyon: IARC Press; 2005. 3. Shear M. The aggressive nature of the odontogenic keratocyst: is it a benign cystic neoplasm? Part 1. Clinical and early experimental evidence of aggressive behaviour. Oral Oncol 2002;38:219–26. 4. Li TJ, Browne RM, Matthews JB. Quantification of PCNA positive cells within odontogenic jaw cyst epithelium. J Oral Pathol Med 1994;23:184–9. 5. Slootweg PJ. P53 protein and Ki-67 reactivity in epithelial odontogenic lesions. An immunohistochemical study. J Oral Pathol Med 1995;24:393–7.
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6. Li T-J, Browne RM, Matthews JB. Epithelial cell proliferations in odontogenic keratocysts: a comparative immunocytochemical study of Ki67 in simple, recurrent and basal cell naevus syndrome (BCNS)-associated lesions. J Oral Pathol Med 1995;24:221–6. 7. El Murtadi AE, Grehan D, Toner M, McCartan BE. Proliferating cell nuclear antigen staining in syndrome and nonsyndrome odontogenic keratocysts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;81:217–20. 8. Piattelli A, Fioroni M, Santinelli A, Rubini C. Expression of proliferating cell nuclear antigen in ameloblastomas and odontogenic cysts. Oral Oncol 1998;34:408–12. 9. Lo Muzio L, Staibano S, Pannone G, Bucci P, Nocini PF, Bucci E, et al. Expression of cell cycle and apoptosis-related proteins in sporadic odontogenic keratocysts and odontogenic keratocysts associated with nevoid basal cell carcinoma syndrome. J Dent Res 1999;78:1345–53. 10. Kimi K, Kumamoto H, Ooya K, Motegi K. Immunohistochemical analysis of cellcycle and apoptosis-related factor in lining epithelium of odontogenic keratocysts. J Oral Pathol Med 2001;30:434–42. 11. Saraçoglu U, Kurt B, Günhan O, Güven O. MIB-1 expression in odontogenic epithelial rests, epithelium of healthy oral mucosa and epithelium of selected odontogenic cysts. An immunohistochemical study. Int J Oral Maxillofac Surg 2005;34:432–5. 12. Kolár Z, Geierová M, Bouchal J, Pazdera J, Zboril V, Tyrdy´ P. Immunohistochemical analysis of the biological potential of odontogenic keratocysts. J Oral Pathol Med 2006;35:75–80. 13. Souza PEA, Mesquita RA, Gomez RS. Evaluation of p53, PCNA, Ki-67, MDM-2 and AgNOR in oral peripheral and central giant cell lesions. Oral Diseases 2000;6:35–9. 14. Kaplan I, Anavi Y, Manor R, Sulkes J, Calderon S. The use of molecular markers as an aid in the diagnosis of glandular odontogenic cyst. Oral Oncol 2005;41:895–902. 15. Agaram NP, Collins B, Barnes L, Lomago D, Aldeeb D, Swalsky P, et al. Molecular analysis to demonstrate that odontogenic keratocyst are neoplastic. Arch Pathol Lab Med 2004;128:313–7. 16. Henley J, Summerlin D-J, Tomich C, Zhang S, Cheng L. Molecular evidence supporting the neoplastic nature of odontogenic keratocyst: a laser capture microdissection study of 15 cases. Histopathology 2005;47:582–6. 17. Malcic´ A, Jukic´ S, Anic´ I, Pavelic´ B, Kapitanovic´ S, Kruslin B, et al. Alterations of FHIT and P53 genes in keratocystic odontogenic tumour, dentigerous cyst and radicular cyst. J Oral Pathol Med 2008;37:294–301. 18. Partridge M, Costea DE, Huang X. The changing face of p53 in head and neck cancer. Int J Oral Maxillofac Surg 2007;36:1123–38. 19. Lombardi T, Odell EW, Morgan PR. P53 immunohistochemistry of odontogenic keratocyst in relation to recurrence, basal-cell budding and basal-cell naevus syndrome. Archs Oral Biol 1995;40:1081–4. 20. Li TJ, Browne RM, Prime SS, Paterson IC, Matthews JB. P53 expression in odontogenic keratocyst epithelium. J Oral Pathol Med 1996;25:249–55. 21. Clark P, Marker P, Bastian HL, Krogdahl A. Expression of p53, Ki-67, and EGFR in odontogenic keratocysts before and after decompression. J Oral Pathol Med 2006;35:568–72. 22. Heikinheimo K, Jee KJ, Morgan PR, Nagy B, Knuutila S, Leiva I. Genetic changes in sporadic keratocystic odontogenic tumors (Odontogenic Keratocysts). J Dent Res 2007;86:544–9. 23. Lench NJ, Telford EA, High AS, Markham AF, Wicking C, Wainwright BJ. Characterisation of human patched germ line mutations in naevoid basal cell carcinoma syndrome. Hum Genet 1997;100:497–502. 24. Barreto DC, Gomez RS, Bale AE, Boson WL, De Marco L. PTCH gene mutations in odontogenic keratocysts. J Dent Res 2000;79:1418–22. 25. Ohki K, Kumamoto H, Ichinohasama R, Sato T, Takahashi N, Ooya K, et al. PTC, SMO, and GLI-1 in odontogenic keratocysts. Int J Oral Maxillofac Surg 2004;33:584–92. 26. Gu XM, Zhao HS, Sun LS, Li TJ. PTCH mutations in sporadic and Gorlinsyndrome-related odontogenic keratocysts. J Dent Res 2006;85:859–63. 27. Sun LS, Li XF, Li TJ. PTCH1 and SMO gene alterations in keratocystic odontogenic tumors. J Dent Res 2008;87:575–9. 28. Hooper JE, Scott MP. Communicating with Hedgehogs. Nat Rev Mol Cell Biol 2005;6:306–17. 29. Pan S, Li T-J. PTCH1 mutations in odontogenic keratocysts: are they related to epithelial cell proliferation? Oral Oncol. doi:10.1016/ j.oraloncology.2009.02.003. 30. Lindström E, Shimokawa T, Toftgård R, Zaphiropoulos PG. PTCH mutations: distribution and analyses. Hum Mutat 2006;27:215–9. 31. Barreto DC, Bale AE, De Marco L, Gomez RS. Immunolocalization of PTCH protein in odontogenic cysts and tumors. J Dent Res 2002;81:757–60. 32. Ayoubi TA, Van De Ven WJ. Regulation of gene expression by alternative promoters. FASEB J 1996;10:453–60. 33. Kogerman P, Krause D, Rahnama F, Kogerman L, Undén AB, Zaphiropoulos PG, et al. Alternative first exons of PTCH1 are differentially regulated in vivo and may confer different functions to the PTCH1 protein. Oncogene 2002;21:6007–16. 34. Shimokawa T, Rahnama F, Zaphiropoulos PG. A novel first exon of the Patched1 gene is upregulated by Hedgehog signaling resulting in a protein with pathway inhibitory functions. FEBS Lett 2004;578:157–62. 35. Agren M, Kogerman P, Kleman MI, Wessling M, Toftgård R. Expression of the PTCH1 tumour suppressor gene is regulated by alternative promoters and a single functional Gli-binding site. Gene 2004;330:101–14.
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36. Nagao K, Toyoda M, Takeuchi-Inoue K, Fujii K, Yamada M, Miyashita T. Identification and characterization of multiple isoforms of a murine and human tumour suppressor, patched, having distinct first exons. Genomics 2005;85:462–71. 37. Shimokawa T, Svärd J, Heby-Henricson K, Teglund S, Toftgård R, Zaphiropoulos PG. Distinct roles of first exon variants of the tumour-suppressor Patched1 in Hedgehog signaling. Oncogene 2007;26:4889–96. 38. Diniz MG, Borges ER, Guimarães ALS, Moreira PR, Brito JAR, Gomez MV, et al. PTCH1 isoforms in odontogenic keratocysts. Oral Oncol 2009;45:291–5. 39. Weber A, Wittekind C, Tannapfel A. Genetic and epigenetic alterations of 9p21 gene products in benign and malignant tumors of head and neck. Pathol Res Pract 2003;199:391–7. 40. Robertson KD, Wolffe AP. DNA methylation in health and disease. Nat Rev Genet 2000;1:11–9. 41. Narlikar GJ, Fan HY, Kingston RE. Cooperation between complexes that regulate chromatin structure and transcription. Cell 2002;108:475–87.
42. Cretnik M, Musani V, Oreskovic S, Leovic D, Levanat S. The Patched gene is epigenetically regulated in ovarian dermoids and fibromas, but not in basocellular carcinomas. Int J Mol Med 2007;19:875–83. 43. Pritchard JE, Olson JM. Methylation of PTCH1, the Patched 1 gene, in a panel of medulloblastoma. Cancer Genet Cytogenet 2008;180:47–50. 44. Wolf I, Bose S, Desmond JC, Lin BT, Williamson EA, Karlan BY, et al. Unmasking of epigenetically silenced genes reveals DNA promoter methylation and reduced expression of PTCH in breast cancer. Breast Cancer Res Treat 2007;105:139–55. 45. Rocha PR, Guimarães MM, Guimarães ALS, Diniz MG, Gomes CC, Brito JAR, et al. Methylation of P16, P21, P27, RB1 and P53 genes in odontogenic keratocysts. J Oral Pathol Med 2009;38:99–103. 46. Chen CZ. MicroRNAs as oncogenes and tumour suppressors. N Engl J Med 2005;353:1768–71. 47. Gomes CC, Gomez RS. MicroRNA and oral cancer: future perspectives. Oral Oncol 2008;44:910–4.
Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Review
Review of the molecular pathogenesis of the odontogenic keratocyst Carolina Cavaliéri Gomes, Marina Gonçalves Diniz, Ricardo Santiago Gomez * Department of Oral Surgery and Pathology, School of Dentistry, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
a r t i c l e
i n f o
Article history: Received 13 July 2009 Received in revised form 1 August 2009 Accepted 17 August 2009 Available online 30 September 2009 Keywords: Odontogenic keratocyst Keratocystic odontogenic tumour PTCH Genetics Epigenetics Review
s u m m a r y The odontogenic keratocyst (keratocystic odontogenic tumour) (OKC) is one of the most prevalent odontogenic tumours. Since its initial description, a number of studies have focused on different aspects of this lesion, attempting to explain its distinctive biological behaviour. In this review the authors address the main genetic and epigenetic alterations reported on this tumour. Although most of the knowledge on this field is not being used in the clinical practice, some perspectives of translational studies are discussed. Ó 2009 Elsevier Ltd. All rights reserved.
Introduction The odontogenic keratocyst (OKC) (keratocystic odontogenic tumour) is an aggressive cystic lesion most frequently present in the second, third and fourth decades of life at the posterior mandible of male patients. Histologically, the OKC is characterized by a uniform, usually corrugated parakeratinized epithelium, 8–12 cells thick presenting a flat basal surface lining the fibrous wall.1 The columnar basal cells showing reversed nuclear polarity is an important feature and satellite cysts may be found in some cases. The aggressive behaviour and high recurrence rate of the OKC suggests a true neoplastic potential and prompted the World Health Organization Working Group to classify the OKC as a benign tumour with odontogenic epithelium and mature, fibrous stroma without odontogenic ectomesenchyme.2 The proliferative activity in the OKC was studied by various investigators and it was compared to other odontogenic cysts and tumours.3 Most of the researchers have found increased immunohistochemical expression of the proliferation markers Ki67 and PCNA in the OKC compared to other odontogenic lesions.4–12 It is important to mention that proliferative status is not always directly associated with biological behaviour or a true neoplastic nature. Non-neoplastic proliferative lesions may also demonstrate high expression of some proliferative markers.13 An-
* Corresponding author. Address: Department of Oral Surgery and Pathology, School of Dentistry, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627 Belo Horizonte, MG, CEP 31270 901, Brazil. Tel.: +55 31 34092477; fax: +55 31 34092430. E-mail address: [email protected] (R.S. Gomez). 1368-8375/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2009.08.003
other example is the Ki-67 expression in low grade mucoepidermoid carcinoma, a malignant neoplasm, which is significantly lower than that in glandular odontogenic cysts, an odontogenic cyst.14 Therefore, the understanding of the biology of a tumour should go beyond cell proliferation, revealing the aspects that influence the fine regulation of the cell cycle. Recently, some studies have focused on the molecular aspects of OKC. In this review we attempted to address the main genetic and epigenetic alterations reported in the OKC. The use of new molecular tools and their potential contribution to the understanding of the pathogenesis and treatment of the lesion are also discussed.
Genetic alterations The neplasic concept of OKC is supported by molecular studies that demonstrated loss of heterozygosity.15–17 The authors of these studies found evidence of allelic loss mainly in the p16, p53, PTCH, MCC, TSLC1, LTAS2 and FHIT genes. Taking into consideration that all of these genes are tumour-suppressor genes associated with different types of human neoplasia, these findings give further support to explain the aggressive behaviour of the OKC. In addition, observations have been made that daughter cysts are associated with a higher frequency of allelic loss.15 P53 is a tumour-suppressor gene that is commonly mutated in many malignant neoplasms, including oral squamous cell carcinoma.18 As the p53 protein has a short half-life, it cannot usually be detected by immunohistochemical methods. However, the primary antibody used, the sensitivity of the detection system, and fixation conditions of the tissue, among other factors, may lead
e1012
C.C. Gomes et al. / Oral Oncology 45 (2009) 1011–1014
to false-positive or false-negative results. Some studies have shown positive p53 labelling in epithelial cells of the OKC,9,19–21 but it was not followed by p53 gene alteration using stranded conformation polymorphism (PCR–SSCP).20 Additionally, positive immunohistochemical labelling for p53 was also found in dentigerous cysts and radicular cysts and p53 mutation was detected in only one OKC (12.9% of the cases).17 Taken together, it seems that p53 mutation is not a relevant event to OKC pathogenesis. The genomic aberrations in the OKC were also studied by Heikinheimo et al.22 using cDNA-expression arrays and array-comparative genomic hybridization. The authors found that several genes were overexpressed or underexpressed and exhibited deletions in 3q13.1, 5p14.3 and 7q31.3. They concluded that overexpressed and amplified genes in 12q13, including cytokeratin 6B (KRT6B), epidermal growth factor receptor ERBB3 and glioma-associated oncogene homologue 1 (GLI1), may contribute to the persistent growth characteristics of the OKC. As the authors suggested, the detachment of epithelium from the stroma, one of the microscopic hallmarks of the OKC, may be explained by the several gene aberrations related to cell adhesion observed in their study. The most important genetic alteration reported in the OKC is in the Drosophila Patched gene (PTCH1).23–27 The PTCH1 encodes a transmembrane receptor for Sonic Hedgehog (SHH) and other Hedgehog proteins. Normally, the PTCH1 represses the functions of the signalling effector Smoothened (SMO). The binding of HH on the PTCH1-SMO complex releases SMO, leading to the activation of target genes by the GLI family of transcription factors.28 The discovery that germline mutations of PTCH1 are the underlying cause of Nevoid Basal Cell Carcinoma Syndrome (NBCCS), prompted several authors to investigate the role of the HH pathway in the pathogenesis of a number of tumours associated with this syndrome, including the OKC. Mutations of PTCH1 in OKC associated with NBCCS were first described by Lench and colleagues.23 Subsequently, PTCH1 gene mutation was reported in the epithelial lining of sporadic cases of human OKC.24–27 Recently, Pan and Li29 demonstrated that Ki-67 labelling index in the epithelium of OKCs with PTCH1 mutation was significantly higher than in cases with no PTCH1 mutation. The authors concluded that PTCH1 mutations are associated with a subgroup of OKCs showing increased proliferative activity and thus may be related to a phenotype of higher recurrence tendency. An additional study showed that Gli1, which is a downstream signalling molecule of the SHH/PTCH pathway, is overexpressed in OKC.27 The fact that activation of SMO, like inactivation of PTCH1, upregulates the transcription of hedgehog target genes, was the basis for the investigation of activating mutations in the SMO gene in OKC, but the authors failed to demonstrate any alterations. Although the mutations detected in the PTCH1 were predicted to result in premature termination of PTCH1 protein and a high frequency of these mutations were clustered into the two large extracellular loops where hedgehog ligand binding occurs, no apparent genotype–phenotype correlations could be established yet.30 After PTCH1 mutation, the development of OKC may follow the ‘two-hit’ or the haploinsufficiency theory (Fig. 1). According to the former, the OKC associated with NBCCS would arise from precursor cells containing a hereditary ‘first hit’ followed by allelic loss of the second normal allele, while the sporadic OKC would arise from susceptible cells in which two somatic ‘hits’ have occurred. According to the haploinsufficiency model, the OKC would develop after the loss of only one PTCH allele, leading to reduction of gene dosage. The presence of normal PTCH protein in OKC cases with PTCH nonsense mutation (predicted to result in a truncated protein) suggests the occurrence of retention of one wild PTCH allele and supports the haploinsufficiency model.31 Alternative expression of mRNAs generated by a single gene is an important mechanism to produce different proteins and to reg-
Figure 1 Possible molecular mechanisms related to PTCH1 inactivation. One or more of these genetic or epigenetic alterations may act in cooperation causing PTCH inactivation, leading to Sonic Hedgehog pathway activation.
ulate genes in a time-dependent manner.32 There are several PTCH1 isoforms generated by splice variants of the first exon. These isoforms have different expression profiles, function and transcriptional regulation.33–37 The most prevalent isoforms of PTCH1 are PTCHb, PTCHd and PTCHe, which are generated by the first exons 1b, 1d and 1e, respectively. Although all of these isoforms can inhibit the HH protein activity, only 1b has an additional ability to completely inhibit SMO activity.33,34 Furthermore, transcripts of 1b were found to be exclusively upregulated in nodular basal cell carcinoma.33 Recently, our group studied the expression of PTCH1 first exons in 40 OKC tumours by RT-PCR.38 While none of the dental follicles expressed the exon 1b, it was detected in 90% of the OKC samples. The pattern of exon 1 expression observed in oral mucosa adjacent to the OKC was similar to the lesion. We concluded that OKC lining together with the oral adjacent mucosa presented overactivity of the SHH pathway. An interesting finding was that some of the lesions that were marsupialized lost the 1b expression, which, in turn, indicates that this procedure could alter PTCH1 variant profiles in some OKC. The elucidation of the molecular aspects that predict good response to marsupialization are important goals to be achieved. Epigenetic alterations Epigenetic alterations are considered important events in the tumorigenesis of benign and malignant tumours of the head and neck.39 The genes can be modified through this mechanism, without having their DNA sequences changed.40,41 DNA methylation is an epigenetic event characterized by the addition of a methyl group in cytosines within CpG islands, remodelling the chromatin and selectively activating or inactivating genes. The PTCH1 methylation has been suggested as an alternative to mutational causes of the PTCH pathway deregulation in tumours associated with NBCCS syndrome, such as meduloblastoma and basocellular carcinoma.42,43 Although none of these tumours presented methylation of the PTCH promoter, significantly higher methylation of the PTCH promoter region was correlated with low PTCH expression in breast cancer samples without PTCH mutation.44 Until now, the status of PTCH methylation in OKC not harbouring PTCH mutations remains unknown. Future directions, including examination of distal regions of the PTCH promoter, as well as CpG island containing other alternative exon variants may shed light on epigenetic mechanisms by which the PTCH pathway may be inactivated. The summary of the possible genetic and epigenetic mechanisms related to PTCH inactivation is demonstrated in Fig. 1. Our group studied the presence of methylation in P16, P21, P27, P53 and RB1 genes in OKC tumours by MSP (methylation specific
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PCR).45 In contrast to dental follicles, OKC samples presented methylation of the P21 gene. Epigenetic alteration of the RB1 gene was detected in some samples of dental follicles but in none of the OKC samples. This data revealed differences on the profile of genes methylated in an odontogenic tumour compared to normal odontogenic tissues. Whether epigenetics is really related to odontogenic tumours development is a question still not answered. Future molecular approaches MicroRNAs (miRNAs) are small non-coding RNAs which mediate gene expression at the post-transcriptional level by degrading or repressing target messenger RNAs (mRNA). It consists of a product about 22 nucleotides long and regulates mRNA translation by base pairing to partially complementary sites, predominantly in the 30 untranslated region (30 UTR) of mRNA.46,47 Overexpression of oncogenic miRNA may reduce protein products of tumour-suppressor genes. On the other hand, the loss of tumour-suppressor miRNA expression may cause elevated levels of oncogenic proteins. Although altered expression of miRNAs are important events in human cancers, to date there is no published study of tumour-suppressor or oncogenic miRNA in odontogenic cysts or tumours. Conclusion While some OKC tumours do not recur after marsupialization or other conservative approaches, some lesions show an aggressive behaviour. A number of clinical and surgical aspects influence such response, but up to now oral surgeons do not have reliable instruments to predict which lesion will recur or not. Studies involving the clinic-pathological, as well as the molecular aspects of this entity, will help to establish new parameters that could help clinicians to choose the most appropriate treatment for a specific tumour. Therefore, translational research joining the experience of different institutions is crucial. As some of the molecular pathways involved in the OKC are also deregulated in different types of human neoplasias (ex: PTCH gene in OKC and basal cell carcinoma), advances in one area could improve the overall current knowledge related to different tumours. It is the beginning of a new time. Conflict of Interest Statement None declared. Acknowledgements This study was supported by Grants from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and from Milênio/ Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. Dr. R.S. Gomez is research fellow of CNPq. References 1. Odell EW, Morgan PR. Biopsy pathology of the oral tissues. London: Chapman & Hall; 1998. 2. Barnes L, Eveson JW, Reichart P, Sidransky D. World Health Organization classification of tumours. Pathology & genetics. Head and neck tumours. Lyon: IARC Press; 2005. 3. Shear M. The aggressive nature of the odontogenic keratocyst: is it a benign cystic neoplasm? Part 1. Clinical and early experimental evidence of aggressive behaviour. Oral Oncol 2002;38:219–26. 4. Li TJ, Browne RM, Matthews JB. Quantification of PCNA positive cells within odontogenic jaw cyst epithelium. J Oral Pathol Med 1994;23:184–9. 5. Slootweg PJ. P53 protein and Ki-67 reactivity in epithelial odontogenic lesions. An immunohistochemical study. J Oral Pathol Med 1995;24:393–7.
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