**3. Odontogenic keratocysts**

Odontogenic keratocysts (OKCs) represent a rare form of odontogenic cysts which originate from dental lamina remnants or eventually from the basal layer of upper and lower jaw oral epithelium before the odontogenesis ended. Since it was first described in 1876, this form of cysts grabs scientific attention mostly because of its developmental variabilities, histological appearance, and genetic basis [2, 58]. In the past few years, the World Health Organization (WHO) made an attempt to create more appropriate classification of these cysts. Recently, they were considered as keratocystic odontogenic tumors for their aggressive behavior, high mitotic rate, and association with genetic and chromosomal abnormalities. The newest WHO classification reclassifies them again as odontogenic keratocysts because *PTCH1* gene mutations were detected, similarly to other developmental cysts such as dentigerous cyst [59–61]. Despite many classifications, pathologists and surgeons face difficulties in the establishment of proper diagnosis. This is because keratocysts cannot be clinically and radiographically distinguished from other odontogenic cysts. Moreover, it is still debatable what the optimal therapeutic approach is in the treatment of keratocysts in order to prevent recidive, which is a characteristic of this disease [2].

Odontogenic keratocyst is histologically characterized with stratified squamous epithelium, which is five to eight layers thick with palisaded hyperchromatic basal cell layer and "corrugated" parakeratotic epithelial cells on luminal surface [2, 62, 63].

#### **Figure 6.**

*Macroscopic view (A) and histological morphology (B) of odontogenic keratocyst. Inflamed fibrous wall lined with nonspecific stratified squamous epithelium (arrow) with focally corrugated surface (arrow head). The presence of satellite cysts and solid islands in the cystic wall is marked with a star.*

Parakeratinization of the surface layer is one of the histological features that predicts recurrence together with the higher level of cell proliferative activity in the epithelium, binding in the basal layer of the epithelium, supraepithelial split of the epithelial lining, presence of daughter cells or also called satellite cells (**Figure 6**) [64].

Considering clinical and histological features together with unclear etiology and therapeutic strategy odontogenic keratocyst still remains debatable. Controversies in nature of these lesions are reflection of limited knowledge about their origin. Recent molecular and genetic discoveries try to elucidate these pathological entities and thus approach closer to better therapy modalities.

#### **3.1 Molecular basis of odontogenic keratocysts**

Odontogenic keratocysts are locally aggressive and have a tendency to recur over time. Keratocysts can happen sporadically or in conjunction with Gorlin-Goltz syndrome (Gorlin syndrome, OMIM 109400) [62, 64]. Gorlin syndrome is an autosomally inherited disease with a germline mutation in *PTCH* gene, located on chromosome 9q22. The incidence is 1:60,000 newborns. Patients with this syndrome present with a spectrum of developmental abnormalities which affect skin, nervous system, eyes, endocrine system, and bones. Skin abnormalities include basal cell carcinoma, benign dermoid cysts and tumors, palmar and plantar keratosis, and dermal calcinosis. Dental and bone abnormalities include multiple keratocysts, mild mandibular prognathism, kyphoscoliosis, and other vertebral defects like bifurcation of ribs, spina bifida, and others. Eye abnormalities include hypertelorism, wide nasal bridge, congenital blindness and strabismus. Neurological abnormalities include mental retardation, calcification of dura mater, and others. For the diagnosis of Gorlin syndrome, two major or one major and two minor criteria are needed [62, 64, 65].

The major criteria are as follows: multiple basal cell carcinoma or one tumor diagnosed before 20 years of age, histologically proven odontogenic keratocyst, three or more palmar or plantar pits, bilamellar calcification of the falx cerebri, fused or markedly splayed ribs, and first-degree relatives with Gorlin syndrome. The minor criteria include macrocephaly; congenital cleft lip or palate, frontal bossing, coarse face, or hypertelorism; other skeletal abnormalities; radiological deformities like bridging of the sella turcica, vertebral abnormalities, and ovarian fibroma; and medulloblastoma.

**69**

is shown in **Figure 7**.

*smoothened (Smo) and disheveled (Dsh).*

**Figure 7.**

interesting [64].

*Oral Pathology: Gene Expression in Odontogenic Cysts DOI: http://dx.doi.org/10.5772/intechopen.80555*

The appearance of keratocysts in Gorlin syndrome patients can be seen in up to 92% of all patients. Even though it has been shown that the syndromic form of keratocysts contains much higher numbers of proliferating epithelial cells and satellite cysts within the cystic wall, pathological examination cannot differentiate between syndromic and sporadic forms of this disease. Similarly, it has been shown that the syndromic form has higher rate of recurrence than the sporadic form. However, the syndromic form has shown more aggressive behavior than the sporadic form. Thus, radiographic appearance of multiple keratocyst formations

*Schematic representation of PTCH molecular structure. (A) Domain structure of PTCH protein with 12 transmembrane domains (blue) and low complexity regions (pink). It contains 23 coding exons spanning 74 kb and encoding a 1447 amino acid receptor glycoprotein. (B) PTCH is a membrane-bound receptor for hedgehog protein (yellow). PTCH1 gene produces patched protein, which is a homolog of Drosophila segment polarity gene. This receptor is a part of the hedgehog signaling cascade, which include downstream proteins such as* 

*PTCH1* gene produces Patched protein, which is a homolog of *Drosophila* segment polarity gene. It contains 23 coding exons spanning 74 kb and encoding a 1447 amino acid receptor glycoprotein. This receptor is a part of the Hedgehog signaling cascade, which includes downstream proteins such as Smoothened (Smo) and Disheveled (Dsh). Molecular structure including schematic domain representation

*PTCH* is a tumor suppressor and as such requires both gene copies to be mutated

The hallmark of odontogenic keratocysts is mutation in *PTCH* gene, which is a receptor in Sonic hedgehog signaling (Shh) pathway. This pathway is important for proper differentiation of embryonic cells, and mutations in this pathway lead

in tumors. Thus, patients with Gorlin syndrome already have one inherited mutation and can acquire the second mutation easier than patients with sporadic form. It has been shown that mutations in this gene often occur as LOH (loss of heterozygosity), meaning that both copies are mutated through different genetic mechanisms such as deletions, mutations, gene silencing, and others. After PTCH mutation, keratocysts can acquire additional genetic alterations which accelerate tumor development. When LOH was analyzed in sporadic form, several additional tumor suppressor genes were affected such as *TP53*. The association between these mutations and appearance of satellite microcysts in the cystic wall is particularly

should raise suspicion of a possible Gorlin syndrome [63].

**3.2 Gene expression in odontogenic keratocysts**

*Oral Pathology: Gene Expression in Odontogenic Cysts DOI: http://dx.doi.org/10.5772/intechopen.80555*

#### **Figure 7.**

*Gene Expression and Control*

**Figure 6.**

Parakeratinization of the surface layer is one of the histological features that predicts recurrence together with the higher level of cell proliferative activity in the epithelium, binding in the basal layer of the epithelium, supraepithelial split of the epithelial lining, presence of daughter cells or also called satellite cells (**Figure 6**) [64].

*Macroscopic view (A) and histological morphology (B) of odontogenic keratocyst. Inflamed fibrous wall lined with nonspecific stratified squamous epithelium (arrow) with focally corrugated surface (arrow head). The* 

Considering clinical and histological features together with unclear etiology and therapeutic strategy odontogenic keratocyst still remains debatable. Controversies in nature of these lesions are reflection of limited knowledge about their origin. Recent molecular and genetic discoveries try to elucidate these pathological entities

Odontogenic keratocysts are locally aggressive and have a tendency to recur over time. Keratocysts can happen sporadically or in conjunction with Gorlin-Goltz syndrome (Gorlin syndrome, OMIM 109400) [62, 64]. Gorlin syndrome is an autosomally inherited disease with a germline mutation in *PTCH* gene, located on chromosome 9q22. The incidence is 1:60,000 newborns. Patients with this syndrome present with a spectrum of developmental abnormalities which affect skin, nervous system, eyes, endocrine system, and bones. Skin abnormalities include basal cell carcinoma, benign dermoid cysts and tumors, palmar and plantar keratosis, and dermal calcinosis. Dental and bone abnormalities include multiple keratocysts, mild mandibular prognathism, kyphoscoliosis, and other vertebral defects like bifurcation of ribs, spina bifida, and others. Eye abnormalities include hypertelorism, wide nasal bridge, congenital blindness and strabismus. Neurological abnormalities include mental retardation, calcification of dura mater, and others. For the diagnosis of Gorlin syndrome, two major or one major and two

The major criteria are as follows: multiple basal cell carcinoma or one tumor diagnosed before 20 years of age, histologically proven odontogenic keratocyst, three or more palmar or plantar pits, bilamellar calcification of the falx cerebri, fused or markedly splayed ribs, and first-degree relatives with Gorlin syndrome. The minor criteria include macrocephaly; congenital cleft lip or palate, frontal bossing, coarse face, or hypertelorism; other skeletal abnormalities; radiological deformities like bridging of the sella turcica, vertebral abnormalities, and ovarian

and thus approach closer to better therapy modalities.

*presence of satellite cysts and solid islands in the cystic wall is marked with a star.*

**3.1 Molecular basis of odontogenic keratocysts**

minor criteria are needed [62, 64, 65].

fibroma; and medulloblastoma.

**68**

*Schematic representation of PTCH molecular structure. (A) Domain structure of PTCH protein with 12 transmembrane domains (blue) and low complexity regions (pink). It contains 23 coding exons spanning 74 kb and encoding a 1447 amino acid receptor glycoprotein. (B) PTCH is a membrane-bound receptor for hedgehog protein (yellow). PTCH1 gene produces patched protein, which is a homolog of Drosophila segment polarity gene. This receptor is a part of the hedgehog signaling cascade, which include downstream proteins such as smoothened (Smo) and disheveled (Dsh).*

The appearance of keratocysts in Gorlin syndrome patients can be seen in up to 92% of all patients. Even though it has been shown that the syndromic form of keratocysts contains much higher numbers of proliferating epithelial cells and satellite cysts within the cystic wall, pathological examination cannot differentiate between syndromic and sporadic forms of this disease. Similarly, it has been shown that the syndromic form has higher rate of recurrence than the sporadic form. However, the syndromic form has shown more aggressive behavior than the sporadic form. Thus, radiographic appearance of multiple keratocyst formations should raise suspicion of a possible Gorlin syndrome [63].

*PTCH1* gene produces Patched protein, which is a homolog of *Drosophila* segment polarity gene. It contains 23 coding exons spanning 74 kb and encoding a 1447 amino acid receptor glycoprotein. This receptor is a part of the Hedgehog signaling cascade, which includes downstream proteins such as Smoothened (Smo) and Disheveled (Dsh). Molecular structure including schematic domain representation is shown in **Figure 7**.

*PTCH* is a tumor suppressor and as such requires both gene copies to be mutated in tumors. Thus, patients with Gorlin syndrome already have one inherited mutation and can acquire the second mutation easier than patients with sporadic form. It has been shown that mutations in this gene often occur as LOH (loss of heterozygosity), meaning that both copies are mutated through different genetic mechanisms such as deletions, mutations, gene silencing, and others. After PTCH mutation, keratocysts can acquire additional genetic alterations which accelerate tumor development. When LOH was analyzed in sporadic form, several additional tumor suppressor genes were affected such as *TP53*. The association between these mutations and appearance of satellite microcysts in the cystic wall is particularly interesting [64].

#### **3.2 Gene expression in odontogenic keratocysts**

The hallmark of odontogenic keratocysts is mutation in *PTCH* gene, which is a receptor in Sonic hedgehog signaling (Shh) pathway. This pathway is important for proper differentiation of embryonic cells, and mutations in this pathway lead

to abnormal cellular proliferation and differentiation, which is strongly associated with the development of certain tumors [66, 67]. This section will describe genes that are involved in pathogenesis of odontogenic keratocysts through Sonic hedgehog pathway (Shh). The members of this pathway include secreted SHH ligand, which binds to its receptor *PTCH*, a 12-pass transmembrane protein, as described previously (**Figure 7**). In the absence of SHH ligand, PTCH inhibits a transmembrane protein called Smoothened (SMO).

In odontogenic keratocysts, *PTCH* mutations include LOH, deletions, point mutations, and others. The consequence of *PTCH* mutations are the constitutive activation of Shh pathway. When *PTCH* is mutated and inactivated, it is expected that SMO would be disinhibited and thus activated. This is exactly what is found in odontogenic keratocysts, where Smo overexpression has been shown by transcriptional and immunohistochemical studies [67–69]. Besides overexpression of SMO and PTCH, other downstream genes such as *GLI1*, *CCND1*, and *BCL-2* have been shown to be overexpressed in odontogenic keratocysts, indicating that these SHH pathway genes contribute to the development of these lesions.

Another downstream target of Shh signaling is a transcription factor SOX-2 [70] that is expressed in progenitor cells in epithelial tissues. SOX-2 expression is associated with elements of tooth development, especially in the region of the third molars in the lower jaw, which is the place where OKCs are usually located [71, 72].

Besides studies on Shh pathway, a list of genes that are upregulated in odontogenic keratocysts is shown in **Table 2**. Bioinformatic analysis has shown that other genes, such as *TP53* and *PCNA*, appear as the leaders and initiators of gene expression that is important for the development of odontogenic keratocysts [61, 73, 74]. Their analysis has shown that genes related to cell cycle and apoptosis are often dysregulated in these cysts, implying the recurrence of these cysts. Studies have shown that *TP53*, *PCNA*, p63, and Ki-67 expression is higher in keratocysts than in other types of odontogenic cysts. *TP53* is a tumor suppressor gene with several different functions in the cell including apoptosis, cell cycle arrest, and DNA repair.

The second gene that is found to be associated with odontogenic keratocysts is proliferating cell nuclear antigen (*PCNA*), which encodes a protein located in the nucleus and associated with DNA polymerase delta. It acts as a homotrimer and is


#### **Table 2.**

*Genes that show high expression in odontogenic keratocysts, detected either at the RNA or protein level (modified from [63]).*

**71**

*Oral Pathology: Gene Expression in Odontogenic Cysts DOI: http://dx.doi.org/10.5772/intechopen.80555*

**4. Conclusion**

development of these lesions.

**Acknowledgements**

drawing **Figure 2**.

**Conflict of interest**

Authors declare no conflict of interest.

implicated in the leading strand synthesis during DNA replication. DNA damage induces ubiquitination of this protein through RAD6-dependent DNA repair pathway. Expression of both *TP53* and *PCNA* in different odontogenic lesions was higher in the suprabasal layer of keratocysts than in radicular cyst. Overexpression of *PCNA* in the suprabasal layer implies its neoplastic nature and a tendency toward recurrence.

This chapter summarizes gene expression profiles of radicular cysts as the most common member of odontogenic cysts and keratocysts, a specific entity of epithelial developmental cysts, in order to uncover possible mechanism of pathogenesis that would help in the timely diagnosis and discovery of novel therapeutic options for these two types of jaw cysts. Pathogenesis of radicular cysts is associated with differential expression of genes involved in bone metabolism (RANK-RANKL-OPG pathway) and inflammation (chemokines and their receptors). However, the most extensively studied genes in the pathogenesis of radicular cysts belong to the family

of matrix metalloproteinases (MMPs), which show increased expression.

Specific entities of odontogenic cysts are odontogenic keratocysts, which are prone to recidive. This trait of keratocysts to recur makes them similar to tumors, which can be also seen in their gene expression profiles. The hallmark of odontogenic keratocysts is mutation in *PTCH* gene, which is a receptor in Sonic hedgehog signaling (Shh) pathway. Mutations in PTCH gene lead to the constitutive activation of this pathway. Besides overexpression of PTCH, other downstream genes such as *SMO*, *GLI1*, *CCND1*, and *BCL-2* have been shown to be overexpressed in odontogenic keratocysts, indicating that these SHH pathway genes contribute to the

Authors would like to thank Armin Klančević, DMD, for his contribution by

implicated in the leading strand synthesis during DNA replication. DNA damage induces ubiquitination of this protein through RAD6-dependent DNA repair pathway. Expression of both *TP53* and *PCNA* in different odontogenic lesions was higher in the suprabasal layer of keratocysts than in radicular cyst. Overexpression of *PCNA* in the suprabasal layer implies its neoplastic nature and a tendency toward recurrence.
