Analysis of the neoplastic nature and biological potential of sporadic and nevoid basal cell carcinoma syndrome-associated keratocystic odontogenic tumor

BACKGROUND: Keratocystic odontogenic tumor (KCOT), also known as odontogenic keratocyst, is a benign cystic neoplasm, which may be associated with nevoid basal cell carcinoma syndrome (NBCCS) and if it does, will occur as multiple cystic lesions. KCOT is locally destructive despite its bland histological features. However, the neoplastic nature of KCOT is not well established. Heparanase is an endo-d-glucuronidase enzyme that specifically cleaves heparan sulfate (HS) and the increase of its level in tumors promotes invasion, angiogenesis, and metastasis. METHODS: To investigate the neoplastic character of KCOT, we studied the localization patterns of heparanase in KCOT, focusing on the differences between sporadic and NBCCS-associated KCOTs, by immunohistochemistry and in situ hybridization. To compare the expression pattern of these cysts with non-tumorous odontogenic developmental cyst, dentigerous cyst was included. RESULTS: All the odontogenic cysts showed positive immunoreaction for heparanase protein in various intensities. The expression pattern of heparanase gene corresponded to that of protein expression. Interestingly, intense gene and protein expressions were observed in KCOT associated with NBCCS compared with sporadic ones and dentigerous cyst. CONCLUSIONS: The results implied that heparanase expression may be correlated with the neoplastic properties of KCOT, particularly in NBCCS-associated cases.     

PCR-SSCP和DNA测序检测牙源性角化囊肿中PTCH基因的突变

目的 检测牙源性角化囊肿（OKC）中PTCH基因突变的特点。方法 采用PCR-SSCP筛查与DNA直接测序的方法对12例OKC进行PTCH基因突变的检测, 其中2例为痣样基底细胞癌综合征（NBCCS）相关OKC,10例为散发OKC。 结果 在4例OKC中发现了4处突变,其中2处生殖细胞突变发生在2例NBCCS相关OKC,2处体细胞突变发生在2例散发OKC。另外,还在10例OKC中检测到了8处PTCH基因多态性。结论 NBCCS相关OKC和散发OKC均可发生PTCH基因突变,但突变水平不同,PTCH基因的突变在二者的发病中可能均起重要作用。     

PTCH gene mutations in odontogenic keratocysts

An odontogenic keratocyst (OKC) is a benign cystic lesion of the jaws that occurs sporadically or in association with nevoid basal cell carcinoma syndrome (NBCCS). Recently, the gene for NBCCS was cloned and shown to be the human homologue of the Drosophila segment polarity gene Patched (PTCH), a tumor suppressor gene. The PTCH gene encodes a transmembrane protein that acts in opposition to the Hedgehog signaling protein, controlling cell fates, patterning, and growth in numerous tissues, including tooth. We investigated three cases of sporadic odontogenic keratocysts and three other cases associated with NBCCS, looking for mutations of the PTCH gene. Non-radioactive single-strand conformational polymorphism and direct sequencing of PCR products revealed a deletion of 5 base pairs (bp) in exon 3 (518delAAGCG) in one sporadic cyst as well as mutations in two cysts associated with NBCCS, a nonsense (C2760A) and a missense (G3499A) alteration. This report is the first to describe a somatic mutation of PTCH in sporadic odontogenic keratocysts as well as two novel mutations in cysts associated with NBCCS, indicating a similar pathogenesis in a subset of sporadic keratocysts.     

牙源性角化囊肿SMO基因突变检测

目的检测牙源性角化囊肿(odontogenic keratocyst,OKC)是否存在SMO基因突变,进一步完善对OKC发病机制的认识。方法收集2012年9月至2017年6月就诊于北京大学口腔医学院·口腔医院口腔颌面外科的OKC患者,10例为痣样基底细胞癌综合征性OKC(女性4例,男性6例),20例为散发性OKC(女性7例,男性13例)。采集患者的病变组织,分离衬里上皮和纤维间质,采用Sanger测序法分别检测上皮与间质DNA中SMO基因突变情况。结果检测发现3个SMO基因突变位点,即1例综合征性OKC携带c.2081C>G(p.P694R)突变,2例散发性OKC分别携带c.907C>T(p.L303F)突变和c.1247_1248delinsAA(p.G416E)突变,前2例突变为未被报道过的SMO新突变,且2例散发性OKC均不伴PTCH1突变。结论除PTCH1突变外,OKC还存在SMO基因突变,可能与OKC的发病机制有关。     

牙源性角化囊肿中PTCH基因的突变检测

目的检测牙源性角化囊肿（OKC）中PTCH基因突变的发生频率、类型及分布特点，分析散发OKC与伴发痣样基底细胞癌综合征（NBCCS）OKC之间的分子病理联系。方法收集8例OKC新鲜病变组织（4例散发，4例伴发NBCCS），提取DNA，采用PCR直接测序法检测OKC病变组织中的PTCH基因突变。结果分别于4例NBCCS—OKC和2例散发OKC中检测到6处PTCH基因突变，2例为错义突变，引起1个氨基酸的改变；其余4例突变分别为1～7个碱基插入或缺失，其中3例引起读码框的改变（移码突变），并导致蛋白质的提前截断，1例导致了2个氨基酸的插入。结论PTCH基因突变不仅常见于NBCCS—OKC，部分散发OKC病变也可以发生该基因的异常。     

Contributions of PTCH gene variants to isolated cleft lip and palate.

OBJECTIVE: Mutations in patched (PTCH) cause the nevoid basal cell carcinoma syndrome (NBCCS), or Gorlin syndrome. Nevoid basal cell carcinoma syndrome may present with developmental anomalies, including rib and craniofacial abnormalities, and predisposes to several tumor types, including basal cell carcinoma and medulloblastoma. Cleft palate is found in 4% of individuals with nevoid basal cell carcinoma syndrome. Because there might be specific sequence alterations in PTCH that limit expression to orofacial clefting, a genetic study of PTCH was undertaken in cases with cleft lip and/or palate (CL/P) known not to have nevoid basal cell carcinoma syndrome. RESULTS: Seven new normal variants spread along the entire gene and three missense mutations were found among cases with cleft lip and/or palate. One of these variants (P295S) was not found in any of 1188 control samples. A second variant was found in a case and also in 1 of 1119 controls. The third missense (S827G) was found in 5 of 1369 cases and in 5 of 1104 controls and is likely a rare normal variant. Linkage and linkage desequilibrium also was assessed using normal variants in and adjacent to the PTCH gene in 220 families (1776 individuals), each with two or more individuals with isolated clefting. Although no statistically significant evidence of linkage (multipoint HLOD peak = 2.36) was uncovered, there was borderline evidence of significant transmission distortion for one haplotype of two single nucleotide polymorphisms located within the PTCH gene (p = .08). CONCLUSION: Missense mutations in PTCH may be rare causes of isolated cleft lip and/or palate. An as yet unidentified variant near PTCH may act as a modifier of cleft lip and/or palate.     

One germline mutation of PTCH gene in a Chinese family with non-syndromic keratocystic odontogenic tumours

Keratocystic odontogenic tumours (KOCTs) are common benign cystic tumours that arise sporadically or associated with nevoid basal cell carcinoma syndrome (NBCCS). PTCH mutation can be found in sporadically or NBCCS associated KOCTs. Few PTCH mutations in families with non-syndromic KOCTs have been reported. Through PCR and gene sequence analysis, the authors discovered one missense mutation c.3277G>C in exon 19 of PTCH gene in a Chinese family with non-syndromic KOCTs. This mutation causes one highly conserved glycine residue transit to arginine on the 10th transmembrane region of PTCH protein. This work revealed that the missense mutation of PTCH is the causative and dominant gene of KOCTs in this family.     

PTCH germline mutations in Chinese nevoid basal cell carcinoma syndrome patients

OBJECTIVES: PTCH, the human homologue of the Drosophila segment polarity gene, patched, has been identified as the gene responsible for nevoid basal cell carcinoma syndrome. The aim of this study was to investigate PTCH gene mutation in Chinese patients with nevoid basal cell carcinoma syndrome. MATERIALS AND METHODS: DNA was isolated from both odontogenic keratocyst tissue and peripheral blood of five patients with syndrome and one patient with only multiple odontogenic keratocysts, and mutational analysis of the PTCH gene performed by direct sequencing after amplification of all 23 exons by polymerase chain reaction (PCR). RESULTS: A previously reported germline mutation (c.2619C>A) was identified in two familial cases involving the mother and the daughter, with the mother also carrying a novel somatic mutation (c.361_362insGAGC). Three novel germline PTCH mutations (c.1338_1339insGCG, c.331delG and c.1939A>T) were detected in three unrelated patients with syndrome. The patient with multiple odontogenic keratocysts who failed to fulfill the diagnostic criteria of the syndrome also carried a novel germline mutation (c.317T>G). CONCLUSION: The frequent germline PTCH mutations detected in our series provide further evidence for the crucial role of PTCH in the pathogenesis of nevoid basal cell carcinoma syndrome in Chinese.     

PTCH mutations in sporadic and Gorlin-syndrome-related odontogenic keratocysts

Odontogenic keratocysts are relatively common lesions that may occur in isolation or in association with nevoid basal cell carcinoma syndrome (or Gorlin syndrome). The PTCH gene has been reported to be associated with Gorlin syndrome. We investigated 10 cases of non-syndromic keratocysts and two other cases associated with Gorlin syndrome, looking for PTCH mutations. Four novel and 1 known PTCH mutations were identified in five individual patients. Of the 5 mutations identified, 2 were germ-line mutations (2619C>A; 1338_1339insGCG) in 2 cysts associated with Gorlin syndrome, and 3 were somatic mutations (3124_3129dupGTGTGC; 1361_1364delGTCT; 3913G>T) in 3 non-syndromic cysts. This report describes PTCH mutations in both non-syndromic and Gorlin-syndrome-related odontogenic keratocysts in Chinese patients, and suggests that defects of PTCH are associated with the pathogenesis of syndromic as well as a subset of non-syndromic keratocysts.     

Familial multiple odontogenic keratocysts

Multiple odontogenic keratocysts (OKCs) are principle features of nevoid basal cell carcinoma syndrome (NBCCS; Gorlin-Goltz syndrome). NBCCS is a genetic disorder transmitted by an autosomal dominant gene with variable expressivity, which is important to recognize when a patient has multiple OKCs. The cysts of the jaws are among the most common findings. Another feature is a certain appearance of the face, such as: large calvaria, high-arched eyebrows, broad nasal root, and mild hypertelorism. Before-therapy diagnosis is, therefore, as important as after-therapy diagnosis. Genetic counseling and examination may also be indicated. The purpose of this paper was to present a family case report of nevoid basal cell carcinoma syndrome with multiple odontogenic keratocysts. The features identified by these combined clinical, imaging, and histologic findings are described, along with a brief mention of the family history and a review of the literature.     

Keratocystic odontogenic tumour: reclassification of the odontogenic keratocyst from cyst to tumour

The purpose of this paper is to review the features and behaviour of the odontogenic keratocyst (OKC), now officially known as the keratocystic odontogenic tumour (KCOT); to analyze a series of histologically confirmed KCOT cases; and to review and discuss the redesignation of KCOT and the implications for treatment. Redesignation of the OKC as the KCOT by the World Health Organization (WHO) is based on the well-known aggressive behaviour of this lesion, its histology and new information regarding its genetics. Abnormal function of PTCH, a tumour suppressor gene, is noted to be involved in both nevoid basal cell carcinoma syndrome and sporadic KCOTs. Normally, PTCH forms a receptor complex with the oncogene SMO for the SHH ligand. PTCH binding to SMO inhibits growth-signal transduction. SHH binding to PTCH releases inhibition of the signal transduction pathway. If normal functioning of PTCH is lost, the proliferation-stimulating effects of SMO are permitted to predominate. A review of the literature was conducted and results were tabulated to determine whether treatment modality is related to recurrence rate. More aggressive treatment - resection or enucleation supplemented with Carnoy"s solution with or without peripheral ostectomy - results in a lower recurrence rate than enucleation alone or marsupialization. Notably, the recurrence rate after marsupialization followed by enucleation is not significantly higher than that following the so-called aggressive modalities. Our case series consists of 21 patients treated for KCOTs. Results were organized to demonstrate recurrence as it relates to size of lesion and time since treatment and incidence as it relates to patient age and location in the jaws. In our series, the average KCOT surface area measured radiographically was 14 cm². Most lesions were within the 0-15 cm² range and lesions in this range resulted in the greatest number and proportion of recurrences. The recurrence rate of 29% in our case series was consistent with previously established data; all recurrences occurred within 2 years post-intervention. The incidence of primary lesions was highest in the age group 70-79 years; most lesions occurred in the posterior mandible. WHO"s reclassification of the OKC as the KCOT based on behaviour, histology and genetics underscores the aggressive nature of the lesion and should motivate clinicians to manage the disease in a correspondingly aggressive manner. The most effective interventions for the KCOT are either enucleation with Carnoy"s solution, or marsupialization with later cystectomy. Future treatment may involve molecular-based modalities, which may reduce or eliminate the need for aggressive surgical management.     

Investigation of chromosome 9q22.3-q31 DNA marker loss in odontogenic keratocysts

Multiple basal cell carcinomas and odontogenic keratocysts of the jaws are a feature of the inherited naevoid basal cell carcinoma syndrome (NBCCS), although both occur more commonly as single, sporadic cases. The NBCCS gene has been mapped to chromosome 9q22.3-q31 and loss of heterozygosity for DNA markers from this region has been observed in familial and sporadic basal cell carcinomas. Based on these observations, we undertook a pilot study to determine if a similar pattern of chromosome loss occurs in odontogenic keratocysts. DNA extracted from microdissected odontogenic keratocyst epithelium was examined for loss of heterozygosity for six polymorphic DNA markers mapping to human chromosome 9q22.3-q31. Allelotype loss was detected in epithelium from three, single, sporadic odontogenic keratocysts. These results implicate homozygous inactivation of the NBCCS gene in the initiation and progression of the odontogenic keratocyst.     

New mutation of the patched homologue 1 gene in a Chinese family with naevoid basal cell carcinoma syndrome

Naevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome, is inherited in an autosomal dominant mode characterised by a combination of developmental anomalies and a predisposition to form tumours. Our aim was to search for patched homologue 1 (PTHC1) mutations in a Chinese family with NBCCS. Mutation analysis of PTCH1 was done of all 10 members of this family by amplified polymerase chain reaction and direct sequencing. Two novel PTCH1 mutations (3146A→T, 1686C→T) were identified in all five affected members. The mutation, 3146A→T in exon 17, is predicted to lead to different PTCH protein translations. 1686C→T mutation in exon 11 is a nonsense mutation. These mutations were not found in any unaffected members of this family or in 100 unrelated healthy Chinese people. Our findings suggest that the 3146A→T mutation in the PTCH gene may be the cause of NBCCS by affecting the conformation and function of the PTCH protein.     

痣样基底细胞癌综合征伴先天性左眼缺失1例

痣样基底细胞癌综合征（NBCCS）又称基底细胞痣综合征或Goltz-Gorlin综合征，是一种复杂且罕见的常染色体显性遗传疾病，大量研究文献证实PTCH1基因与NBCCS有关。本文报道1例NBCCS伴先天性左眼缺失的病例，并结合相关文献探讨以进一步了解该综合征。     

Keratocystic odontogenic tumor: a retrospective study of 183 cases

In 2005, the WHO Working Group considered odontogenic keratocyst (OKC) to be a tumor and recommended the term keratocystic odontogenic tumor (KCOT), separating the lesion from the orthokeratinizing variant, which is now considered an odontogenic cyst. We analyzed the clinicopathological features of KCOTs encountered over a period of 28 years at Meikai University Hospital. The diagnosis was confirmed by reevaluation of hematoxylin and eosin-stained slides on the basis of the 2005 WHO Classification. Clinical history was also taken into consideration. A total of 183 KCOTs were found, and the two genders were affected almost evenly (51.3% male; 48.7% female; male to female ratio 1.05 to 1). Patient age at the time of diagnosis ranged from 6 to 78 years, with a peak in the third decade of life (mean age: 32.8 years). The mandible was the site of occurrence of 70.5% of tumors; 16.4% occurred in the maxilla and 13.1% in both. Association with the nevoid basal cell carcinoma syndrome (NBCCS) was found in 6.0% of all tumors, and recurrence was found in 13.1% of patients. We found that tumors that initially appeared in the maxilla alone had a higher recurrence rate than those that first appeared in the mandible alone. Pathological examination of KCOT is important to avoid misdiagnosis and provide appropriate treatment and follow-up.     

Nevoid basal cell carcinoma syndrome: molecular biology and new hypotheses.

To understand the molecular biology of the nevoid basal cell carcinoma syndrome, the hedgehog signaling network is explained. The syndrome is caused by mutations in patched, a tumor suppressor gene. A single point mutation in one patched allele may be responsible for the malformations found in the syndrome. Inactivation of both patched alleles results in the formation of tumors and cysts (basal cell carcinomas, odontogenic keratocysts, and medulloblastomas). Several new hypotheses are proposed to account for a minority of nevoid basal cell carcinoma syndrome patients. Evidence is provided to suggest that these hypotheses deserve to be investigated.     

Imaging modality correlations of an odontogenic keratocyst in the nevoid basal cell carcinoma syndrome: a family case report

Multiple maxillary and mandibular cysts are principle features of nevoid basal cell carcinoma syndrome (NBCCS; Gorlin-Goltz syndrome). We present a family case report of NBCCS with odontogenic keratocyst where the findings on plain films, CT, clinical, and histopathologic examinations are compared and analyzed. The systemic manifestations included frontal bossing, odontogenic keratocyst, ectopic calcification in 1 patient, and bifid rib in 1 patient. CT examination displayed aspects of bone morphology not visible on the plain films. Odontogenic keratocyst diagnosis was confirmed by histopathological examination. The features identified by these combined clinical, imaging, and histologic findings are helpful in identifying an NBCCS patient, distinguishing keratocyst from others cysts or neoplasic lesions, and can therefore influence surgical management. NBCCS is a rare autosomal dominant cancer predisposition syndrome, which is important to recognize when a patient has multiple odontogenic keratocysts, because lifelong monitoring is essential for patient management.     

Expression of cell cycle and apoptosis-related proteins in sporadic odontogenic keratocysts and odontogenic keratocysts associated with the nevoid basal cell carcinoma syndrome.

Odontogenic keratocysts are occasionally (4-5%) associated with the nevoid basal cell carcinoma syndrome, a pleiotropic, autosomal disorder presenting a spectrum of developmental abnormalities and a predisposition for the development of different neoplasms. The aim of this study was to establish whether keratocysts showing clinically aggressive behavior associated with nevoid basal cell carcinoma syndrome reflect differences in cellular proliferation rate and/or in the expression of oncoproteins and tumor suppressor genes. For this reason, formalin-fixed paraffin-embedded sections of odontogenic keratocysts associated with the nevoid basal cell carcinoma syndrome (16 cases) and sporadic odontogenic keratocysts (16 cases) were compared for expression of proliferating cell nuclear antigen (PCNA) and p53, bcl-2, and bcl-1 (cyclin D1) onco-proteins. Most of the epithelial lining of odontogenic keratocysts associated with the nevoid basal cell carcinoma syndrome showed nuclear immunopositivity for p53 protein and overexpression of cyclin D1 with various degrees of staining intensity. All sporadic odontogenic keratocysts were negative for p53 and cyclin D1. The expressions of bcl-2 oncoprotein were found to be substantially similar between the two groups of lesions, with a cytoplasmic immunopositivity localized only in the resting reserve basal layer of the epithelium. PCNA expression showed no statistically significant difference between the two groups of lesions. In conclusion, the finding of cyclin D1 and p53 overexpression in odontogenic keratocysts associated with the nevoid basal cell carcinoma syndrome could be considered a hallmark of a mutated cellular phenotype, thus leading to the hypothesis that their aggressive clinical behavior could be due to a dysregulation of the expression of cyclin D1 and p53 proteins, involved in a check-point control of cellular proliferation.     

MMP‐13 expression in keratocyst odontogenic tumour associated with NBCCS and sporadic keratocysts

Oral Diseases (2010) 16 , 795–800 Objective: To investigate the matrix metalloproteinase (MMP)‐13 expression in associated and non‐nevoid basal cell carcinoma syndrome (NBCCS) Odontogenic Keratocysts (OCKs) in order to contribute to a better understanding of the differences in the growth pattern between them. Materials and methods: Thirty‐nine paraffin‐embedded blocks of OCKs, 26 sporadic OCKs and 11 NBCCS‐associated KCOTs were studied by immunohistochemistry to evaluate MMP‐13 expression both in epithelial and stromal layers. A semi‐quantitative scale was used to evaluate immunostaining. Obtained data were compared between the two groups, using Fischer’s exact test and the chi‐square test. Results: Only 13 of 26 sporadic OCKs showed a positive immunostaining, whilst 11 KCOTs resulted in positive labelling for MMP‐13 expression. Moreover, syndromic cysts displayed a more intense and diffuse MMP‐13 labelling of the stromal tissue. Instead, in non‐syndromic forms, the staining pattern of MMP‐13 in stromal tissue was completely absent. Fisher's exact test showed a statistically significant greater prevalence of KCOTs‐immunolabelled cysts with respect to sporadic OCKs. Conclusions: Results from this study point out that the biological behaviour of these cysts could be related not only to the epithelial layer but also to stromal tissue in that... MMP‐13 overexpression in stromal tissue of NBCCS‐associated KCOTs could clarify the higher aggressiveness of these cysts.     

一个中国汉族痣样基底细胞癌综合征家系致病基因的定位研究

目的定位一个中国汉族痣样基底细胞癌综合征(NBCCS)家系的致病基因.方法选择SHH信号系统的基因作为该NBCCS家系致病基因的候选基因,用微卫星遗传标记在候选基因染色体区域定位致病基因.结果连锁分析结果发现,PTCH2基因所在染色体区域微卫星遗传标记D1 s2797 LODZMAX为1.31(平均遗传距离73.81)、D1 s2802 LODZMAX为1.26(平均遗传距离73.81),支持连锁.构建单体型发现家系内所有病变表型的个体D1s2797和D1 s2802的基因型一致,无交换重组现象.而SHH,PTCH,SMO基因染色体区域微卫星遗传标记连锁分析结果不支持连锁.结论这个中国汉族NBCCS家系的致病基因定位在PTCH2基因染色体区域1p32.3,遗传距离为1.85cM.