Recurrent COF in a NBCCS patient: Case report and in vitro analysis

Abstract

Objective:

Central odontogenic fibromas (COF) are rare, benign tumors derived from dental mesenchymal tissue that may occur in the maxilla or mandible. This report describes primary and recurrent COF in the mandible of a Nevoid Basal Cell Carcinoma Syndrome (NBCCS) patient.

Method:

A 36-year-old African American male presented with a COF and its recurrence 17 months later. Tissue pieces were obtained from both occurrences with IRB-approved signed consent. Collected tissue pieces were dissected, one portion was formalin-fixed and paraffin-embedded, and the other was cultured for the isolation of cell populations from the primary (COdF-1) and recurrent (COdF-1a) tumors. qRT-PCR, immunohistochemistry, and DNA sequencing were used for gene and protein analysis of the primary tumor and cell populations.

Results:

Histopathological analysis of the tumor showed sparse odontogenic epithelial cords in fibrous connective tissue. qRT-PCR analysis of tumor and cell populations (COdF-1 and COdF-1a) detected VIM, CK14, CD34, CD99 and ALPL mRNA expression. Protein expression was confirmed by immunohistochemistry. CD34 expression in primary tissues was higher than tumor cells due to tumor vascularization. DNA sequencing indicated the patient had PTCH1 mutations.

Conclusions:

Histopathology, mRNA and protein expression indicate the rare occurrence of COF in a patient with mutated PTCH1 gene and NBCCS.

Introduction

Odontogenic fibromas (OF) are rare, benign tumors derived from mesenchymal tissue of dental origin ¹. In 2017, the World Health Organization (WHO) classified OFs as neoplasms of “mature fibrous connective tissue, with variable amounts of inactive-looking odontogenic epithelium.”^2-4 Previously accepted subtypes included simple or epithelium-poor type COF, which has sparse cells with thin collagen fibers and a substantial aggregate of ground substances that affords it a fibromyxoid matrix and minimal odontogenic epithelium islands and cords. ^{3, 5, 6} Contrastingly, complex or epithelial-rich type COFs were described as cellular fibrous connective tissue with comparatively more odontogenic epithelium islands and cords. COF tumors may also have occasional areas of calcifications.

OFs are defined based on location in relation to the jaw, extraosseous (peripheral, POF) and intraosseous (central, COF).⁷ COFs account for 0.1-1.5% of all odontogenic tumors.⁸ This tumor is a slow growing, asymptomatic lesion, that presents itself as cortical expansion of the jaw bone.⁹ Earlier data reported COFs primarily occurring in the mandible compared to maxilla, however it is now shown that COFs occur with equal frequency between the mandible and maxilla.^{7, 10, 11} Mandibular COFs are most prevalent in the posterior premolar/molar area, whereas maxillary COFs are most prevalent in the anterior area. Although infrequent, COFs have also been shown to be more aggressive than their peripheral counterpart, promote paresthesia of the mandibular inferior dental nerve, and induce painful swelling.^{9, 12} COFs have been reported in ages 4 years old to 80 years old with an average age of 31.6 years and has a predominance for the males (1.13:1, M:F). From the reported COFs, 32 cases reported the race of the patient: 75% (n=24) of the patients were Caucasian, 18.75% (n=6) were African American, and 6.25% (n=2) were Asian.⁵

Radiographically, COFs present as a unilocular or multilocular radiolucency with well-defined margins.^{9, 10, 12, 13} Multilocular COFs are more commonly associated with increased aggression, inclusion of tooth tissue, severe root resorption of adjacent teeth, and displacement of neighboring teeth.¹² Multilocular, radiolucent COFs present very similarly to other odontogenic tumors such as odontogenic keratocysts (OKC), ameloblastomas, and odontogenic myxomas, thus requiring further expert opinions on clinical, radiographic, and histopathological features for proper diagnosis.^{9, 13} In terms of treatment, the lesion responds well to total enucleation and curettage of the cavity. Potential for malignancy has not been reported to date. Recurrence is uncommon and has been reported in 5.6% of COF cases.⁵ Although recurrence and malignant potential are minimal, follow-up is essential, as there have been cases reported of recurrence up to 9 years after the initial surgery.^{7, 9, 12}

In this report, we describe a COF occurring in a patient with Nevoid Basal Cell Carcinoma Syndrome (NBCCS), also called Gorlin syndrome and Gorlin-Goltz syndrome. NBCCS is a rare, autosomal dominant disorder characterized by mutations on the Patched gene (PTCH1) located on chromosome 9q22-q31. NBCCS prevalence is estimated to be between 1/19,000 to 1/256,000 individuals.¹⁴ Identification of potential NBCCS patients uses family history in combination with clinical identification of various physical abnormalities resulting from the mutations. Some of the physical abnormalities include calcification of the falx cerebri, palmar/plantar pits, bifid ribs, basal cell carcinoma, hypertelorism, ovarian and cardiac fibromas, and, most notably, OKCs.^{14, 15} Shimada et al. reported 80% of patients with NBCCS had OKCs and 30% of those patients’ experienced recurrence.¹⁴ The patient reported here has had multiple OKCs removed surgically, including recurrences. COFs have not been associated previously with NBCSS; however, Gorlin syndrome patients are known to develop a variety of tumors and other forms of fibromas.

The mutation associated most with NBCCS is the tumor suppressor gene PTCH1, which affects the Hedgehog Signaling Pathway and has various embryological development responsibilities such as organogenesis, nervous cell type specification, skeletal development, and endochondral ossification.¹⁶ A Sonic Hedgehog (Shh) ligand dimer binds to and inhibits the 12-transmembrane protein Patched (Ptch1) protein, resulting in the activation of the 7-transmembrane protein, G-coupled protein receptor Smoothened (SMO). SMO activates proteins in the Gli family which then translocate to the nucleus and phosphorylate the transcription factor Gli1. If Shh is inhibited due to mutation in the protein or in precursors, Ptch1 will bind to and inhibit SMO, resulting in a downregulation of Gli proteins. However, a mutation in PTCH1 causes SMO to be constitutively active, thus allowing Gli proteins to activate transcription of target genes. This is noted to cause increased cellular proliferation and tumorigenesis as seen in NBCCS patients.¹⁷ Mutations in PTCH1 have been reported in NBCCS, rhabdomyosarcomas, medulloblastomas, meningiomas, OKCs, and other sporadic tumors.^{15, 18}

In this clinical case review, we report a COF and recurrence in a NBCCS patient, which carries a PTCH1 polymorphism. We collected tissue from both the primary and recurrent tumors for the establishment of primary cell cultures (COdF-1 and COdF-1a, respectively).

Materials and Methods

Patient sample and establishment of primary cell culture

A 36-year-old African American male with a right posterior mandible lesion agreed to participate in this study with informed consent. This study was conducted in accordance with The Code of Ethics of the World Medical Association in line with the Declaration of Helsinki and independently reviewed and approved by the University of Alabama at Birmingham Institutional Review Board. A tumor piece was collected following the surgical resection of the primary and recurrent tumors. Tissue pieces were dissected. Half of the collected tissue pieces were fixed in formalin and paraffin embedded (FFPE), and the remaining pieces were minced and placed in culture. Cells for the primary and recurring populations (COdF-1 and COdF-1a, respectively) were established as outgrowths in alpha-MEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (α-MEM/10%FBS/antibiotics).

Immunohistochemistry (IHC)

The formalin-fixed, paraffin-embedded tissue was sliced at 5 μm thick sections, deparaffinized, and rehydrated for immunohistochemistry (IHC). Citrate antigen retrieval was performed, samples blocked in hydrogen peroxide and Ultra V block (Thermo Fisher Scientific). COdF1 and COdF-1a cells were grown in α-MEM/10%FBS/antibiotics on 4-well chamber slides and then fixed with 10% formalin for immunocytochemistry. Samples were incubated with primary antibodies for specific staining and HRP polymer-conjugated secondary antibodies. Negative controls were incubated with PBST and secondary antibody without primary antibody. Commercially available antibodies used were the following: ALP, CK14 (Santa Cruz Biotechnology), VIM (Dako North America Inc.), CD34 (Ventana), and CD99 (Thermo Fisher Scientific). Diaminobenzidine tetrachloride was used as the chromogen (Biocare Medical), and hematoxylin was used for counterstaining. The samples were imaged using a Nikon Eclipse TE2000-E inverted microscope (Nikon Instruments).

Quantification Real Time PCR (qRT-PCR)

A Qiagen RNeasy Mini Kit was used to isolate total RNA from the COdF-1 and COdF-1a cells. For FFPE tissue RNA isolation, 10 μm thick tumor sections were deparaffinized using xylene and ethanol. The tissue pellets were washed with ethanol and dried at 55°C. Roche High Pure RNA paraffin kit (Roche Diagnostics) was used to isolate the RNA. All RNA was reverse transcribed into cDNA using the Bio-Rad iScript cDNA Synthesis Kit. qRT-PCR was performed using the RT²SYBRGreen/Rox qPCR master mix (SABiosciences) and relative gene transcriptional levels detected using the ABI Prism 7500 Sequence Detection System (Applied Biosystems). Primers for vimentin (VIM), cytokeratin 14 (CK14), CD34, CD99, and alkaline phosphatase (ALP) were obtained from RT² quantitative PCR primer assays (SABiosciences). Cycle threshold (Ct) values relative to transcriptional expression levels were obtained and normalized to the housekeeping gene GAPDH to determine the delta Ct (ΔCt) values.

DNA extraction

Using the Wizard genomic DNA extraction kit (Promega), the total genomic DNA was extracted from the COF cell populations from the primary tumor (COdF-1) and recurrent tumor (COdF-1a) per the manufacturer’s protocol.

PCR and direct DNA sequencing

The coding and exon-intron boundaries of the PTCH1 was amplified using gene-specific primers via PCR. The PCR product was checked on an agarose gel, and 5 μL PCR products were purified by ExoSap enzyme. Bigdye 3.0 by ABI 3730 sequencer was used for bi-directional sequencing of the PCR product. The sequencing results were compared against a PTCH1 reference sequence (NC 000009;98205264-98279247) using SeqScape2.5 (Applied Biosystems). The alterations were checked against databases to determine if the alterations are known single-nucleotide polymorphisms (SNPs).

Results

Clinical and Histopathological Findings

The patient is a 36-year-old African American male with NBCCS and has undergone multiple surgeries for recurrent OKCs of the mandible and the maxilla. In May 2011 he presented with a new lesion in his right posterior mandible that was radiographically characterized with a unilocular, radiolucent background and well-defined borders (COdF-1, Fig. 1 A-C). Subsequently, surgical enucleation, curettage, and cryotherapy were performed. Histological findings were consistent with those of an COF. The patient followed up six months later and had no complaints of discharge nor evidence of facial or oral edema. The patient was seen in October 2012 and presented with a swollen mass on the right posterior mandible and complained of intraoral drainage, thus indicating recurrence. The panorex confirmed a recurring, unilocular radiolucency under and around the roots of tooth #30 (COdF-1a, Fig. 1D). In December 2012, the patient underwent a more extensive surgery than the first COF resection. Enucleation, curettage, and extraction of #30 was performed, followed by peripheral ostectomy and bone grafting. Post-operative radiographs confirmed complete enucleation, extraction of tooth #30, and bone grafting. The histopathological features confirmed the recurrence of COF. Follow-ups in June 2013, January 2014, and February 2015 showed healthy bone growth and no recurrence. His last follow-up radiograph was in 2023 and there was no evidence of recurrence (E).

Figure 1:

Clinical and histopathological examination of the central odontogenic fibroma of the mandible. (A) Radiographic image showing a unilocular, radiolucency of the right anterior mandible. (B) Sagittal computed topography showing radiolucent lesion of the posterior right mandible. (C) 3D reconstruction sagittal view of the bone lesion at tumor location. (D) Radiographic image showing radiolucency created by tumor recurrence. (E) Radiographic image from follow-up visit showing no recurrence.

The COdF-1 and COdF-1a tumors were stained with hematoxylin and eosin (H&E; Fig. 2, primary tumor, A-H; recurrent tumor, I-L; available as eSlide VM07016-07030). The tumor histopathology depicted mature stroma with a myxoid background with areas of collagen hyalinization and odontogenic epithelial cords and islands.^{2, 4, 19}

Figure 2:

(A-L) Hematoxylin and eosin (H&E) staining of tumor sections (primary tumor, A-H; recurrent tumor, I-L) at multiple magnifications (A-H: 2, 4, 20, and 40x; I-L: 4, 10, 20, 40x, eSlide VM07016-07030). Neoplastic fibrous tissue with areas of collagen hyalinization and islands of inactive odontogenic epithelium. Scale bar indicates 50 μm.

Primary Cell Population Establishment and Characterization

The COdF-1 tumor samples (Fig. 3A, B) were used to establish explant cultures. The cellular outgrowths from the primary sample (COdF-1, Fig. 3C) and recurrent sample (COdF-1a, data not shown) were used to establish cell populations from the isolated cells.

Figure 3:

Establishment of the central odontogenic fibroma (COdF-1) and recurrent (COdF-1a) cell populations. (A-B) Photomicrographs of primary (A) and recurrent (B) tumor pieces used for ex vivo culture. (C) Phase contrast micrograph of COdF cell population outgrowths. (D) Gene expression profiles of cell type markers from the COdF-1 and COdF-1a cell populations and primary tumor from formalin-fixed, paraffin-embedded tissue (paraffin) determined by qRT-PCR. The housekeeping gene GAPDH was used to normalize the dataset. All experiments were performed in triplicate and, and each reaction, in duplicate. The error bars indicate the SD.

qRT-PCR was performed on the COdF-1 primary cells, COdF-1a recurrent cells, and primary tumor tissue isolated from paraffin-embedded tissue (Fig. 3E). VIM, CK14, CD34, CD99, and ALPL transcript levels were detected at varying levels of expression. VIM and CD99 were found to be the most highly expressed. CD34 was weakly expressed in cultured cells. The primary tissue showed higher expression of CD34, which is expected as the tumor was vascularized.

Immunohistochemistry

The primary tumor tissue and cells, as well as the recurrent samples, all demonstrated strong vimentin expression and lacked CK14 expression as detected by IHC (Fig. 4). The COdF-1 and COdF-1a tumor tissues were both positive for CD34 due to both tissues being highly vascular, however, the COdF-1 primary cells and COdF-1a primary recurrent cells were both negative, as expected due to the endothelial cells not expanding in vitro. CD99 staining was weakly positive for COdF-1, negative for COdF-1 cells, and positive for COdF-1a and COdF-1a cells. ALP was positive for all samples.

Figure 4:

Immunohistochemical characterization of the primary central odontogenic fibroma (COdF-1) and recurrent tumor (COdF-1a) and the established cell populations (COdF-1 cells and COdF-1a) (10x). Tumors and cells stained positive for vimentin and ALP and negative for cytokeratin 14 (CK14). Blood vessels in the tumors stained positive for the vascular marker CD34, while cells were negative. Tumors (COdF-1 and COdF-1a) and COdF-1a cells were weakly positive for CD99. Positively stained controls are provided for each antibody used. Negative staining control with no primary antibody was done for all experiments, representative image shown.

DNA Sequencing

Total DNA sequencing was performed on the COdF-1 and COdF-1a cells, and the same polymorphisms were found in exons 22 and 23 of PTCH1 for each cell population, as expected in syndromic cases with germline mutations. A threonine to serine SNP at 1195 (Thr1195Ser) was found on exon 22. Exon 23 had two SNPs: Pro1216Arg and Pro1315Leu. The primary cell sequence was compared against PTCH1 reference sequence (Table 1).

Table 1.

PTCH1 genetic alterations in COdF primary cells

Location	SNP	Mutation	Reference
Exon 3	c.526C>T	-	rs2066834
Exon 5	c.735A>G	-	rs1805154
Exon 14	c.1746A>G	-	rs16909898
Exon 14	c.1656C>T	-	rs2227970
Intron 15	c.1797+25T>C	-	rs62637628
Intron 18	c.2689+21A>G	-	rs2236406
Intron 19	c.2943T>G	-	rs2066835
Exon 22	c.3408C>T	-	rs138240178
Exon 22	c.3385A>T	p.Thr1195Ser	rs2236405
Intron 22	c.3607-9C>T	-	rs2236404
Exon 23	c.3845C>A	p.Pro1216Arg	rs2227968
Exon 23	c.3941C>T	p.Pro1315Leu	rs357564

Discussion

In this study, we established two primary COF cell populations (COdF-1 & COdF-1a) from a 36-year-old African American male diagnosed with NBCSS and a prior history of OKCs. COF is not commonly associated with NBCSS. And to our knowledge, COdF-1 and COdF-1a are the first patient-derived cell populations of this tumor type. The establishment of primary COF populations aids in elucidating cellular characteristics, which could lead to the determination of NBCCS tumor markers and the development of novel therapeutics.

COFs are benign, primary bone-forming tumors with a mesenchymal origin. The high expression of vimentin, and lack of CK14 expression, in COdF-1 and COdF-1a tissues suggests a primordial mesenchymal origin. Both tumor and cells have high ALP expression, which is known to be overexpressed in bone-involving tumors and may regulate bone mineralization.^{20, 21} COFs consist of a high composition of cellular fibroblastic connective tissue with vascularization.¹¹ Neovascularization is commonly associated with positive expression of CD34+.²² CD99 expression has been linked with the fibroblasts-like cells in the stroma of COF.²³ In our study, both COdF-1 and COdF-1a tissues exhibited high expression of CD34+ and all but COdF-1 cells stained positive for CD99.

The patient in our study was diagnosed with NBCSS.²⁴ PTCH1 mutations, associated with this syndrome, include those that cause loss of heterozygosity in the 9q22.3-31 region and affect hedgehog signaling. PTCH1 mutations were found in both COdF-1 and COdF-1a during DNA sequencing. Both had SNP polymorphisms in exon 22 (Thr1195Ser) and exon 23 (Pro1216Arg & Pro1315Leu) of PTCH1. Thr1195Ser and Pro1315Leu had been associated with tumors in ameloblastoma patients.²⁵ In previous findings, Pro1315Leu polymorphisms has been found in syndromic and non-syndromic patients with OKC, and from a calcifying odontogenic tumor.^{24, 26}

NBCCS is commonly associated with the development of OKC, COF has not been reported. However, 75% of females with NCBBS have been diagnosed with ovarian fibromas, and 5% of patients report cardiac fibromas.^{27, 28}

Conclusion

We report a COF and recurrence in a NBCCS patient, which carries a PTCH1 polymorphism, and the establishment of a primary cell cultures. However, recurrence is uncommon and has been reported only in low percentage of total COF cases.⁵ NBCCS patients are known to develop a variety of tumors and other forms of fibromas; however, COF is not commonly associated with NBCSS. We established two primary cell populations, COdF-1 and COdF-1a. These are the first patient-derived cell populations of this tumor type and are useful for molecular characterizatin of the tumor.

Statement of Clinical Relevance.

Central odontogenic fibromas (COF) are more aggressive than peripheral counterparts and can cause paresthesia of the mandibular inferior dental nerve and induce painful swelling. COF has not been associated with Nevoid Basal Cell Carcinoma Syndrome or carrying a Patched1 (PTCH1) mutation.