Ⅰ. INTRODUCTION
Cemento-ossifying fibroma (COF) is a benign fibro-osseous lesion of the jaws1), which is closely related to other osseous lesions, i.e., fibrous dysplasia, cementifying periapical dysplasia, cemento-osseous florid dysplasia, etc, according to the 1992 classification of WHO2). It has been described as a demarcated or rarely encapsulated neoplasm consisting of fibrous tissue and varying amounts of mineralized materials resembling bone and cementum3). COF can arise from any part of the facial skeleton and skull but mostly fom jaws4). Mandible is more frequently involved than maxilla5), and it usually appears within the bone and rarely in gingival soft tissues6).
COF are supposed to be arisen from undifferentiated mesenchymal cells of periodontal ligament origin, which have potential capability to form bone, cementum and fibrous tissue in combination7). COF is slow-growing, and is frequently found in women between the third and fourth decades of life8). It has a female to male predilection of 2:19). Although the underlying exact cause is not known yet, a history of trauma in the area of the lesion has been issued in the literatures4). COF is generally asymptomatic until the growth produces a noticeable swelling and mild deformity, and displacement of teeth may be early clinical features5).
Radiographs are a primary tool in the diagnosis of COF, exhibiting radiolucent in 53%, sclerotic in 7% and mixed density in 40%7). Computed tomogram may provide information for diagnosis as well as treatment planning. The final diagnosis of COF should be confirmed histologically10). Treatment of COF is mainly surgical resection of the lesion, and followed by graft reconstruction. Prognosis is relatively excellent7), and recurrence following surgical resection is generally uncommon11).
In this study, we reported a case of COF in the mandible of a female patient in her fourth decade of life. In this case, the tumor tissue recently grew rapidly. It was suspected to be transformed into malignant tumor, but histological observation showed a benign lesion filled with dilated vasculatures. Therefore, we examined the aneurysmal tumor lesion of COF by immunohistochemical (IHC) array method, and investigated characteristic protein expressions for abnormal ossification and aneurysmal cystic changes of COF.
Ⅱ. CASE REPORT
A 31 years old female visited the dental hospital of Gangneung-Wonju National University. She had been suffered from a bony swelling in the right premolar region of the mandible for 12 years, recently grown rapidly. A fistula tract developed on the right anterior mandibular border, but the lesion was relatively asymptomatic. In the radiological examination, the tumor mass was radiolucent and localized at premolar region in 11 years ago, but recently the lesion was rapidly swollen bucco-lingually with irregular radiolucent and radiopaque areas, forming multiple cystic spaces (Fig. 1 A and B). Under the diagnosis of calcifying odontogenic cyst, the mandibular mass was surgically resected, and the removed specimen was examined pathologically. This case report has been approved by Institutional Review Board (GWNUDH-IRB2016–11)
After decalcification, the dissected tumor mass disclosed multiple small cystic spaces and calcifying fibrous tissue, mimicking calcifying odontogenic cyst or ameloblastoma. Dissected tumor mass was fixed in 10% neutral buffered formalin, processed routinely, and embedded with paraffin. Histological microsections in 4 μm thick were mounted on glass slides and stained with hematoxylin and eosin staining, and also followed by histochemical Masson-Trichrome staining and IHC staining using 30 different antisera.
The tumor was composed of fibro-osseous tissue replacing the central area of mandiblular body, and it gradually expanded and pushed out adjacent cortical bone. The tumor lesion involving whole mandibular body was relatively well demarcated from normal bone, but gradually grew with multiple cystic swellings, and resulted in a huge fibroosseous tumor covered with membranous cortical bones (Fig. 1C).
In the low magnification, microsections showed many calcifying cementoid materials and ossifying trabeculae. The multiple cystic spaces were closely associated with the fibro-osseous tumor, and greatly swollen and filled with plasma fluid (Fig. 2A1). In the Masson trichrome stain, the peripheral mesenchymal tissue of main tumor was mostly stained red and continuously distributed in the cyst wall (Fig. 2A2)
In the histological observation, the fibrous mesenchymal cells were proliferating diffusely, produced rudimentary bony spicules. The osteoid bones were usually irregular in shape and continuously deposited by woven bone to form abortive bony materials resembling globular cementum. The cystic spaces were turned out to be aberrant vascular channels lined by endothelial cells, which were separated from systemic circulation and underwent cystic changes by fluid accumulation (Fig. 2B). The stromal fibrous tissue was highly cellular and composed of numerous spindle or stellate mesenchymal cells producing osteoid materials (Fig. 2C).
In the high magnification, the inner lumen of cystic spaces was lined by thin endothelial cells, and the cystic fluid materials were degenerative and occasionally recruited many macrophages (Fig. 2D). In the Masson trichrome stain, the fibrous cyst wall was stained red, and the adjacent mesenchymal tissue of fibro-osseous tumor was also stained red (Fig. 2E). These findings indicated that the mesenchymal cells of cystic wall and the periphery of main tumor remained undifferentiated, and did not produce collagen bundles.
IHC array was performed to detect the expression of α -tubulin*, bone morphogenetic protein-2 (BMP-2), caspase-3*, caspase-9*, β-catenin*, capillary morphogenetic protein-2 (CMG-2)@, deoxyhypusine hydroxylase (DOHH)&, hepatocyte growth factor (HGF)*, hypoxia inducible factor 1α (HIF-1α)*, heat shock protein-70 (HSP-70)*, vascular endothelial growth factor-A (VEGF-A)*, matrix metalloproteinase-1 (MMP-1)*, MMP-3*, MMP-9*, MMP-10*, osteoprotegerin (OPG*), p53*, phosphorylated RAC-alpha serine/threonine-protein kinase 1/2/3 (pAKT1/2/3)*, poly [ADP-ribose] polymerase (PARP)*, proliferating cell nuclear antigen (PCNA)*, receptor activator of nuclear factor kappa- Β ligand (RANKL)* , runt-related transcription factor 2 (RUNX-2)*, S-100@, signal transducer and activator of transcription 3 (STAT-3)*, transglutaminase 2 (TGase 2)&, transforming growth factor β1 (TGF-β1)*, tumor necrosis factor α (TNFα)*, versican*, von Willebrand factor (vWF)* and Wnt-1* (*SantaCruz Inc. Biotech., USA; @ ZYMED, CA, USA; &Abcam, Cambridge, UK).
Immunohistochemistry reaction protocols varied according to the target antigen and manufacturers’ protocols. After deparaffinization and rehydration of the tissue sections in xylene followed by ethanol, sections were incubated with 0.5% hydrogen peroxide in phosphate-buffered saline (PBS) for 30 minutes. Primary anti-human polyclonal antibodies were diluted and added. Then, the tissues were incubated overnight at 4°C. Secondary biotinylated antibody was added for 30 min, followed by streptavidin peroxidase for 1 hour. The slides were visualized with diaminobenzidine (DAB). Sections were counterstained and then subjected to alcohol and xylene baths before being mounted for examination. Between each incubation steps all slides were rinsed twice in phosphate-buffered saline (PBS)12).
Histochemical stains were observed under ordinary light microscope, and particularly, microscopic images of different IHC staining were captured by a digital camera (DP-70, Olympus Co., Japan) and each representative images were analyzed by colorimetry method (Adobe Photoshop CS6, USA) to quantify the expression levels of different proteins examined in this study. Expressions of the same proteins from normal bony tissue found in the periphery of the COF tumor lesion were used as a control group (Fig. 3).
Colorimetric analysis of IHC staining showed strong positive reactions of BMP-2 (107.8%), CMG2 (108.4%), HSP-70 (110%), VEGF-A (109.6%), OPG (107.2%), RANKL (108.8%), and S-100 (107.6%), and slight positive reactions of DOHH (104.5%), HGF (104.6%), HIF-1α (105.2%), MMP-1 (106.3%), MMP-3 (105.5%), p53 (104.1%), RUNX2 (103.4%), TGase 2 (105.3%), TGF-β1, TNFα (105.7%), versican (104.3%), Wnt1 (104.3%), caspase 3 (103.8%), and caspase 9 (103.1%), while rare reactions of β-catenin, MMP-9, MMP-10, pAKT1/2/3, PARP, PCNA, STAT3, and vWF compared to the control obtained from the bony tissue in the periphery of COF lesion (Fig. 4).
These data indicated that the progressive tumor growth via PCNA signaling was regulated by p53 signaling, and subsequently underwent cellular apoptosis and angiogenesis. VEGF-A, a key cytokine for angiogenesis was activated by HIF-1α, which was stimulated by p53 and HGF. VEGF-A induced abortive de novo angiogenesis in COF, and resulted in aneurysmal cystic changes of vessels. On the other hand, the cellular apoptosis of COF was enhanced by p53, Wnt1/β -catenin, and TGase-2, but inhibited by STAT3, MMPs, and pAKT1/2/3. Increases of VEGF-A and TGase-2 expressions positively affected the osteoblastic RANKL/OPG ratio to induce active osteogenesis in COF.
Ⅲ. DISCUSSION
Although it was known that COF is a benign fibro-osseous lesion of the jaws, clinic-pathological behaviors are variable as solid, multiple13), cystic14), recurrent15), and giant16) tumor, and showed deregulation of key Wnt/β-catenin signaling pathway genes, which were known to be relevant to the molecular pathogenesis of COF.17) In this study, we reported a case of COF occurred in the mandible of female patient in her fourth decade of life. The tumor tissue was closely associated with dilated vasculatures, thereby, the tumor tissue of COF was examined by IHC array method, and investigated whether the dilated vasculature was relevant to the actively proliferating COF lesion.
The present COF showed a benign fibro-osseous lesion forming immature and rudimentary bony spicules and globular cementum, however, its stromal fibrous tissue was relatively abundant and filled with numerous spindle or stellate mesenchymal cells18). These mesenchymal cells were mainly neoplastic, and IHC array showed positive reactions of PCNA, p53, and HGF, while they were rarely positive for caspase 9 and PARP but strongly positive for cellular survival and regeneration proteins, pAKT1/2/3, MMP-1, and MMP-3. Therefore, it was presumed that the mesenchymal cells were proliferative in the lack of apoptosis, but they were regulated by p53 signaling, which subsequently activated HGF and HIF-1α in the downstream pathways.
IHC array revealed marked increases in the expression of BMP-2, RANKL, and OPG, but slight increase in the expression of RUNX2 in the tumor tissue of COF. BMP-2 is a key signaling component in bone formation19), and belongs to the transforming growth factor β (TGF-β) superfamily20). RUNX2 is an important bone- specific transcription factor which is essential for the differentiation of osteoblasts from mesenchymal precursors21). Recently, it has been shown that osteoblasts regulate differentiation of osteoclasts by two factors, the receptor activator of nuclear factor-κB ligand (RANKL), and the osteoprotegerin (OPG). RANKL is a transmembrane ligand expressed by osteoblasts and bone marrow stromal cells. Following binding to RANK, a receptor for osteoclast differentiation, activation, and survival, RANKL induces osteoclastogenesis. OPG, also produced by osteoblasts and marrow stromal cells, acts as a decoy receptor for RANKL, thus regulating bone metabolism22). In this case, IHC array revealed marked increases of BMP-2, RANKL, and OPG, slight increase in the expression of RUNX2, and concurrent increases in the expression of TGF-β1 and TNFα. These findings may indicate that ossification was activated but not progressive and resulted in abnormal bone or cementum. According to the IHC array, we assumed that the lower expression of RUNX2 than BMP-2, RANKL, and OPG might be relevant to the abnormal ossification of COF.
IHC array also revealed consistent increases in the expression of HIF-1α, VEGF-A and CMG2 in the tumor tissue of COF. It is well known that hypoxia is one of the basic features of the substantive tumor microenvironment, and anaerobic status induces the expression of a series of genes involved in angiogenesis23). Hypoxia increases expression of hypoxia inducible factor 1 (HIF-1), which serves as hypoxia sensor and activates compensatory and adaptive mechanisms24). HIF-1 is a heterodimeric factor consisting of an inducible oxygen-sensitive α subunit (HIF-1α) and a constitutive oxygen-insensitive β subunit (HIF-1β/ARNT)25). HIF-1α is a transcription factor that under hypoxic conditions, accumulates in endothelial cells and can bind to VEGF gene promoter and induce VEGF gene expression24). The overexpression of HIF-1α has been confirmed in human tumors as compared with that in the respective normal tissues25). Vascular endothelial growth factor (VEGF) is the most important angiogenic factor. A binding site in HIF-1α is found in the upstream VEGF promoter. HIF-1α participates in the transcription control of VEGF in tissues and improves the stability of VEGF mRNA, which results in angiogenesis23). Capillary morphogenesis gene-2 (CMG2) showed enhanced expression on endothelial cells undergoing blood vessel growth or angiogenesis in vitro or in vivo model systems. Its role in endothelial proliferation has been documented26). In this case, we assumed that the dilated vascular channels were produced by the compensatory hyperplasia due to growing tumor mass. IHC array revealed the increased expression of HIF-1α, VEGF-A and CMG2 in the tumor tissue of COF, therefore, it was also presumed that the consistent expression of de novo angiogenesis factors of HIF-1α, VEGF-A, and CMG2 might be relevant to the aneurysmal cystic changes of vessels in COF tumor tissue.
In order to understand well the nature of the aneurysmal cystic change in the tumor tissue of COF in this case, we reviewed the literature for such cases. We found that a few cases of aneurysmal bone cysts (ABCs) which were associated with COFs in the jaws have been reported in the literature27). However, the studying of the histological features of aneurysmal bone cysts and comparison with those of this case made us exclude the diagnosis of an aneurysmal bone cyst secondary to a cemento-ossifying fibroma. Microscopically, aneurysmal bone cysts show many blood-filled cystic spaces of varying sizes, which are lined by spindle/plump fibroblasts, which are admixed with few multinucleate giant cells. Intervening connective tissue and the solid areas are usually highly cellular and vascular, containing some bony trabeculae28). These features were not competent with the histological findings of this case. Therefore, we assumed that the aneurysmal cystic change and the atypically vascular channels were produced by the compensatory hyperplasia due to growing tumor mass. Based on the IHC array findings of this case, we presented a diagram illustrating simplified genetic signaling pathways to explain abnormal protein expressions in COF (Fig. 5), indicating that the lower expression of RUNX2, osteoblast differentiation factor, than BMP-2 RANKL, and OPG might be related to the abnormal ossification of COF, and also showed that the consistent expressions of angiogenesis factors, HIF-1α, VEGF-A, and CMG2 might be related to the aneurysmal cystic changes of vessels in COF.
In the clinico-pathological data including IHC array, the present COF showed cellular characteristics exhibiting progressive growth in the lack of apoptosis, continuous maintenance of ossification, and de novo angiogenesis followed by aneurysmal cystic change. Therefore, it was assumed that the recent overgrowth of present COF was not caused by the malignant transformation but by the increased pressure by aneurysmal cystic change, and also suggested that the mesenchymal tumor cells of present COF remained undifferentiated resembling odontogenic mesenchyme.
