- Email: [email protected]
Giant Cell Lesions of the Jaws: Does the Level of Vascularity and Angiogenesis correlate With Behavior?
J Oral Maxillofac Surg 70:1860-1866, 2012
Giant Cell Lesions of the Jaws: Does the Level of Vascularity and Angiogenesis correlate With Behavior? Zachary S. Peacock, DMD, MD,* Richard C. K. Jordan, DDS, PhD, FRCPath,† and Brian L. Schmidt, DDS, MD, PhD‡ Purpose: To compare vascularity and angiogenic activity in aggressive and nonaggressive giant cell
lesions (GCLs) of the jaws. Materials and Methods: This is a retrospective study of 14 GCLs treated at the University of California,
San Francisco. Immunohistochemistry was used to determine of the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), CD34, and CD31. VEGF and bFGF expression in giant cells (GCs) and surrounding mononuclear stroma was classified into 1) high immunoreactivity (⬎50% staining) and 2) low immunoreactivity (⬍50% staining). CD31- and CD34-stained vessels were counted at 200⫻ magnification. Clinical and radiographic records were reviewed to classify lesions as aggressive or nonaggressive. Results: Of the lesions, 8 were aggressive and 6 were nonaggressive. High VEGF expression was found within the GCs in 4 of 8 aggressive lesions compared with 1 of 6 nonaggressive lesions. The stroma in both groups had low staining. High staining of the GCs for bFGF was found in 6 of 8 aggressive lesions compared with 3 of 6 nonaggressive lesions. The stroma of all aggressive cases showed high expression of bFGF compared with 3 of 6 nonaggressive cases. The aggressive group had a mean of 20.1 ⫾ 5.4 vessels/high-powered field (hpf) stained for CD31 compared with 11.5 ⫾ 5.6 vessels/hpf in the nonaggressive group. The aggressive group had 24.6 ⫾ 7.0 vessels/hpf stained with CD34 compared with 18.5 ⫾ 4.0 vessels/hpf in the nonaggressive group. Conclusions: The vascularity and level of angiogenesis within aggressive GCLs are higher than those in nonaggressive lesions. © 2012 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 70:1860-1866, 2012 The giant cell lesion (GCL) describes a group of intraosseous nonodontogenic benign tumors containing multinucleated giant cells (GCs). Several entities in the jaws can share this histology, including hyperparathyroidism, cherubism, and the central GC granuloma.1 Controversy remains over the biologic basis of GCLs of the jaws and thus the rationale for some therapies. GCLs of the jaws are most commonly found in children and young adults, with up to 75% of cases occurring before 30 years of age. The lesion affects *Currently, Instructor in Oral and Maxillofacial Surgery, Massachusetts General Hospital, Boston, MA; Formerly, Resident, Department of Oral and Maxillofacial Surgery, University of California, San Francisco, San Francisco, CA. †Professor of Oral Pathology and Pathology and Associate Dean for Research, University of California, San Francisco, San Francisco, CA. ‡Professor, Department of Oral and Maxillofacial Surgery, and Director, Bluestone Center for Clinical Research, New York University College of Dentistry, New York, NY.
female patients twice as often as male patients. Stromal fibroblasts are thought to produce cytokines, resulting in the recruitment of monocytes, which subsequently transform into multinucleated GCs.2 Immunohistochemical studies have shown the GCs to be osteoclasts phenotypically.3 The GCL of the jaw may be a unique lesion or may simply represent a continuum of the same disease process as GC tumors affecting the long bones.4 In this study the term “giant cell lesion” is used and includes lesions that may have Address correspondence and reprint requests to Dr Peacock: Massachusetts General Hospital, Warren 1201, 55 Fruit St, Boston, MA 02114; e-mail: [email protected] © 2012 American Association of Oral and Maxillofacial Surgeons
0278-2391/12/7008-0$36.00/0 doi:10.1016/j.joms.2011.08.020
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Table 1. CRITERIA FOR CLASSIFYING GCLS OF JAWS
Lesion size Rapid growth Root resorption Tooth displacement Cortical bone perforation Recurrence after curettage
Aggressive
Nonaggressive
ⱖ5 cm Yes Often Often Often
⬍5 cm No No No No
Yes
No
NOTE. Adapted from Chuong et al.5 Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
been historically considered central GC reparative granulomas or GC tumors. GCLs can be classified as aggressive or nonaggressive based on clinical and radiographic features (Table 1).5 The aggressive form has 3 of the following 5 criteria: rapid growth, root resorption, tooth displacement, cortical bone thinning, or perforation. In addition, a GCL greater than 5 cm and/or recurring after enucleation and curettage is classified as aggressive. The nonaggressive lesion is often asymptomatic, grows slowly, and has a low rate of recurrence. Currently, there are no reliable histologic or molecular methods to differentiate aggressive from nonaggressive lesions. Angiogenesis is the process by which capillaries form from pre-existing blood vessels. It occurs physiologically (eg, embryologic development and wound healing) and in pathologic processes such as proliferative retinopathies and facilitation of tumor growth.6,7 Numerous cytokines and growth factors modulate angiogenesis through paracrine and autocrine pathways.8,9 Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are thought to be the most potent inducers of angiogenesis.10 VEGF, a mitogen for endothelial cells, has been found to promote angiogenesis both in vitro and in vivo.8,11 It enhances vascular permeability and serves as a survival factor for newly formed blood vessels.12,13 bFGF is the prototype of a family of 13 structurally related heparin growth factors. It is also a potent inducer of angiogenesis and is thought to act synergistically with VEGF on endothelial cells.11 CD31 and CD34 are known endothelial cell markers in vascular beds of both normal tissue and neoplasms.14,15 CD31 is a 130-kDa transmembrane glycoprotein that is a member of the immunoglobulin superfamily. It is present on the surface of platelets, monocytes, macrophages, and neutrophils and is a constituent of the endothelial intercellular junction. Its vital role in angiogenesis has been recognized
recently,16-18 and it is one of the best-known immunohistochemical markers for benign and malignant vascular tumors.15 CD34 is a 110-kDa transmembrane glycoprotein present on leukemic, endothelial, and stem cells. Although its function is still unclear, it is frequently used to characterize vascular tumors, progenitor cells, and solitary fibrous tumors.15 Vered et al19 showed that angiogenesis does occur in GCLs of the jaws, albeit at low levels. They found that VEGF and bFGF immunoreactivity was lower in the endothelial cells of GCLs compared with other physiologic and pathologic processes of angiogenesis. The multinucleated GCs and mononuclear stromal cells stained positive for both VEGF and bFGF. This may explain the response of GCLs to antiangiogenic treatment.20 Dewsnup and colleagues33 assessed CD34 staining density in GCLs and found increased vascular density in aggressive lesions; increased CD34 staining density was then found to be associated with aggressive clinical behavior.22 The purpose of this study was to compare the immunoreactivity of VEGF and bFGF of multinucleated GCs and the mononuclear stroma in aggressive and nonaggressive GCLs of the jaws. In addition, vascular density was compared by use of immunohistochemical staining of CD31 and CD34.
Materials and Methods CASE SELECTION
This retrospective study examined the records and tissue from patients treated for GCLs of the jaws at the Department of Oral and Maxillofacial Surgery, University of California, San Francisco, from 1995 to 2004. The study was approved by the University of California, San Francisco Institutional Review Board (CHR Committee on Human Research No. H11557-0701922). GCLs from patients with hyperparathyroidism, cherubism, and Noonan syndrome were excluded. Cases were included if complete clinical, radiographic, and pathologic records were available (minimum follow-up of 6 months). Cases were excluded for incomplete or unavailable records and inadequate tissue for sectioning and staining. All specimens were verified by a board-certified oral and maxillofacial pathologist to be GCLs of the jaws (R.C.K.J.). CLINICAL DATA
Patient records were reviewed independent of the histologic findings. Clinical data collected included age, gender, presence or absence of pain, swelling, root resorption, cortical thinning, perforation, and recurrence. On the basis of the classification system formalized by Chuong and Kaban5 2 groups were defined: 1) aggressive GCLs and 2) nonaggressive GCLs.
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IMMUNOHISTOCHEMISTRY
bound complexes were visualized by the application of a 0.05% solution of 3-amino-9-ethylcarbazole (Sigma-Aldrich, St Louis, MO) containing 0.3% hydrogen peroxide as a substrate. After incubation, the sections were washed and then lightly counterstained in hematoxylin and coverslipped. A pyogenic granuloma served as positive control to verify binding of antibodies to VEGF, bFGF, and CD31. A section of colorectal cancer was an additional positive control for VEGF. For CD34, a solitary fibrous tumor was used. Slides were stained with omission of each primary antibody as a negative control.
Immunohistochemistry was performed as previously described to determine the expression levels of VEGF, bFGF, CD31, and CD34.23 In brief, 5-m sections were cut and mounted on adherent glass slides. Sections were dewaxed in xylene and rehydrated in graded ethanols. Endogenous peroxidase activity was blocked by immersion in 0.5% methanolic peroxide for 15 minutes, followed by 2 washes in phosphate-buffered saline solution (PBS) for 5 minutes each. Slides were immersed in deionized water and then rinsed in PBS. Before staining with VEGF and CD31, antigen retrieval techniques were used. For VEGF, Tris (hydroxymethyl)aminomethane/ ethylenediaminetetraacetic acid at pH 9.0 (Dako, Carpinteria, CA) was used with microwaving. CD31 required antigen retrieval with citrate at pH 6.0 (Dako). Sections were then incubated for 1 hour at room temperature with the respective primary antibodies: 1) VEGF–rabbit monoclonal anti-VEGF (Labvision, Fremont, CA) diluted to 1:25 in PBS, 2) bFGF– prediluted mouse monoclonal anti-bFGF (Biogenics, Napa, CA), 3) CD31–prediluted mouse monoclonal anti-CD31 (Dako), and 4) CD34 –prediluted mouse monoclonal anti-CD34 (Biogenics). This was followed by 2 washes in PBS, incubation with a biotinylated secondary antibody (Envision Plus; Dako), and 2 PBS rinses, followed by the application of preformed avidin-biotin complex (Dako) for 30 minutes.24 The
QUANTIFICATION OF IMMUNOHISTOCHEMISTRY
Immunostained sections were assessed and quantified by an investigator (Z.S.P.) blinded to the clinical data for each case. Immunohistochemical reactivity of the multinucleated GCs and surrounding stroma for VEGF and bFGF was scored and classified into 2 groups: 1) high immunoreactivity (⬎50% staining) and 2) low immunoreactivity (⬍50% staining). CD31 and CD34 expression was scored by randomly selecting 3 areas of lesional tissue and counting the number of positively stained vessels within a 1.25-mm eyepiece graticule at 200⫻ magnification. The mean of the 3 random high-powered fields (hpf) was calculated.
Table 2. DEMOGRAPHIC AND CLINICAL DATA OF STUDY POPULATION (14 GCLs)
Case Age No. (yr) Gender 1 2 3 4 5 6
26 9 16 9 7 46
F M M M M M
7 8 9
19 12 9
F M F
10
20
F
11
4
F
12
14
F
13 14
20 15
F M
Location Maxilla, anterior Mandible, anterior Mandible, anterior Mandible, anterior Mandible, anterior Mandible, body, ramus Mandible, anterior Mandible, body Mandible, body and ramus Mandible, body and ramus Mandible, body, molar region Mandible, anterior and bilateral body Mandible, anterior Mandible, premolar, molar
Rate of Root Cortical Size Pain Growth Swelling Resorption Thinning Recurrence (cm) Aggressive N N N N N N
Slow Slow Slow Slow Slow Slow
Y N Y Y Y Y
N N N N N N
N N N N N N
N N N N N N
2.6 2.4 2.9 4.2 2.6 4.3
N N N N N N
Y Y N
Rapid Rapid Slow
Y Y Y
N N Y
Y Y N
Y N N
3.1 7.2 6.9
Y Y Y
Y
Rapid
Y
N
Y
N
3.1
Y
Y
Rapid
Y
Y
Y
Y
5.6
Y
Y
Rapid
Y
Y
Y
Y
10.2
Y
Y N
Rapid Slow
Y Y
N N
Y Y
Y Y
3.6 2.1
Y Y
Abbreviations: F, female; M, male; N, no; Y, yes. Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
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multinucleated GCs was found in 4 of 8 aggressive lesions compared with 1 of 6 nonaggressive lesions. Within the stroma, 1 aggressive lesion and 1 nonaggressive lesion had high immunoreactivity. Evidence of staining for bFGF was present in 12 of 14 cases (Fig 2). High staining of the GCs was found in 6 of 8 aggressive cases compared with 3 of 6 nonaggressive lesions. Two of the nonaggressive lesions had no staining of the GCs. The stroma of the aggressive group was highly stained in all 8 cases compared with only 3 of 6 in the nonaggressive group. Endothelial cells within the lesions were stained by CD31 and CD34 (Fig 3). For CD31-stained aggressive lesions, a mean of 20.1 vessels/hpf were present compared with 11.5 vessels/hpf in the nonaggressive group. In the CD34-stained group, a mean of 24.6 vessels/hpf were present in the aggressive group compared with 18.5 vessels/hpf in the nonaggressive group. The pyogenic granuloma was positively stained for VEGF, bFGF, and CD34. The colorectal cancer was also positive for CD34, as was the solitary fibrous tumor for CD31. No staining was seen in any GCLs stained without the primary antibody.
Discussion
FIGURE 1. Aggressive versus nonaggressive immunohistochemical staining for VEGF. A, Photomicrograph at 200⫻ magnification showing high staining (⬎50%) of GCs and stroma within aggressive GCL. B, Photomicrograph at 200⫻ magnification showing low staining (⬍50%) of GCs and stroma within nonaggressive GCL. Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
STATISTICAL ANALYSIS
For categorical data, differences were assessed with the Fisher exact test. For continuous data, 95% confidence intervals were calculated and compared. All tests were 2 tailed.
Results We identified 14 subjects (7 male and 7 female patients) that met the inclusion criteria (Table 2). Of these 14 patients, 13 had mandibular GCLs. The GCLs were aggressive in 8 and nonaggressive in 6. The mean ages of the patients with aggressive and nonaggressive lesions were 14.1 years and 18.8 years, respectively. Of the 8 subjects with aggressive lesions, 6 were female patients. Immunoreactivity to VEGF was seen in all 14 specimens (Fig 1, Table 3). High reactivity within the
Controversy remains regarding the GCL and its pathogenesis. The lesion has been proposed to be a reactive inflammatory, vascular, endocrine, or neoplastic process. GCLs of the jaws can be characterized as aggressive or nonaggressive based on certain clinical behaviors.5 No reliable immunophenotypic or molecular marker exists, however, to differentiate between aggressive and nonaggressive GCLs. This study characterized the vascularity of GCLs of the jaws. The aggressive lesions had increased expression of angiogenic markers VEGF and bFGF within both the multinucleated GCs and the mononuclear fibroblastic stroma. In addition, the aggressive lesions had increased vascularity quantified by immunoreactivity to endothelial markers CD31 and CD34. This suggests that angiogenesis occurs within GCLs and both multinucleated GCs and mononuclear stromal cells may be a source of VEGF and bFGF. VEGF and bFGF levels may be increased in aggressive lesions, leading to the increased vascular density. In treating patients with GCLs of the jaws, it would be beneficial and efficacious to have directed therapy that is selected based on a proven biologic basis. Unfortunately, both the etiology and cell of origin are unknown at this time. The GCL has been proposed to be a proliferative vascular lesion and has been shown to respond to antiangiogenic therapy after enucleation.20,25 Proliferative vascular lesions are known to
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Table 3. IMMUNOHISTOCHEMICAL STAINING OF VEGF, BFGF, CD31, AND CD34
Case No. Nonaggressive group 1 2 3 4 5 6 Mean ⫾ 95% CI Aggressive group 7 8 9 10 11 12 13 14 Mean ⫾ 95% CI
Age (yr)
CD31Stained Vessels/hpf
CD34Stained Vessels/hpf
High High Low Low High Low
11 4 26 8 12 8 11.5 ⫾ 5.6
10 23 22 16 24 16 18.5 ⫾ 4.0
High High High High High High High High
23 27 13 12 11 31 17 27 20.1 ⫾ 5.4
31 8 37 34 17 31 20 19 24.6 ⫾ 7.0
Gender
VEGF GC
VEGF Stroma
bFGF GC
bFGF Stroma
26 9 16 9 7 46
F M M M M M
Low Low Low Low High Low
High Low Low Low Low Low
High High Low Low High Low
19 12 9 20 4 14 20 15
F M F F F F F M
Low High Low High High High Low Low
Low Low Low Low High Low Low Low
Low High High High High High High High
Abbreviations: CI, confidence interval; F, female; M, male. Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
have overexpression of angiogenic proteins VEGF and bFGF.9 This study supports the theory that GCLs of the jaws are proliferative vascular lesions. Characterizing the GCLs as strictly a proliferative vascular process may be overly simplistic. The multinucleated GCs within these lesions have been shown to be osteoclasts or osteoclast-like.2,3,26 Multiple studies have shown resolution with calcitonin, a known inhibitor of osteoclasts.27-29 As previously theorized, the multinucleated GCs and mononuclear cells within the stroma may stimulate angiogenesis, increasing the circulatory delivery of progenitors of these osteoclast-like GCs.19,30 This would increase the osteolytic capacity and, hence, the aggressiveness of the lesion and has been termed “osteoclastogenesis.”29 Given favorable clinical responses to inhibitors of both angiogenesis through interferon ␣2a and osteoclasts through calcitonin, both processes may be important in development and progression of the lesion.20,25,27,29,31,32 Further controlled studies are needed to determine the right combination of medical therapy and surgical management for aggressive GCLs. Previous analyses of angiogenesis within GCLs have had inconsistent results. Vered et al19 quantified the expression of VEGF and bFGF within the multinucleated GCs, mononuclear spindle cells, and endothelial cells within GCLs. They found low levels of staining of these angiogenic proteins and concluded that these lesions have low angiogenic activity, but no comparison was made between aggressive and nonaggressive
lesions. Our study found increased levels of both of these proteins in aggressive lesions within the multinucleated GCs and the mononuclear stroma as compared with nonaggressive lesions. Immunoreactivity of CD34 has been extensively studied in GCLs of the jaws. O’Malley et al33 saw no differences between aggressive and nonaggressive lesions through counting the percentage of CD34-positive cells in a high-powered field. Positively stained endothelial cells were found only in the periphery of the lesions. Dewsnup et al21 showed significantly increased CD34 staining density in aggressive GCLs compared with nonaggressive lesions through computerized histologic analysis. They went on to show that staining density greater than 2.5% is predictive of aggressive clinical behavior.21 Our study found increased vascularity as assessed by both CD34 and CD31. CD31 has not been previously used to assess vascularity within these lesions. Like CD34, it is a transmembrane glycoprotein found on the surface of endothelial cells. The results of this study support the theory that a proliferative vascular component within GCLs may be responsible for their clinical aggressiveness. Increased angiogenesis and vascularity may allow for increased recruitment and differentiation of hematopoietic cells to osteoclasts, leading to more rapid osteolysis and enlargement. Limitations exist within this study. First, we evaluated a small number of cases. Although there were obvious trends in the representations of vascularity
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within these lesions, we were not able to show statistically significant differences. Second, despite use of positive and negative controls, limitations exist within the immunohistochemistry technique, including variability in antigen retrieval, to permit assessment of protein levels within tissue.34 Further study of the genotypic signature and phenotypic expression of these lesions is needed to help develop a reliable method to characterize clinical behavior at the time of biopsy. The goal is to develop suitable and reliable directed medical therapy to prevent recurrence of aggressive lesions. Given the proposed inter-related nature of angiogenesis and osteoclastogenesis, perhaps a combination of current medical therapies will improve patient outcomes. The results of this study suggest an increased vascularity within GCLs that are clinically aggressive. Characterization of VEGF and bFGF, protein markers
FIGURE 3. CD31 and CD34 immunohistochemical staining. A, Photomicrograph at 200⫻ magnification showing vessels stained for CD31 in aggressive GCL. B, Photomicrograph at 200⫻ magnification showing vessels stained for CD34 in aggressive GCL. Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
of angiogenesis, as well as endothelial proteins CD31 and CD34 within these lesions, may be helpful to predict clinical behavior.
References
FIGURE 2. Aggressive versus nonaggressive immunohistochemical staining for bFGF. A, Photomicrograph at 200⫻ magnification showing high staining (⬎50%) of GCs and stroma within aggressive GCL. B, Photomicrograph at 200⫻ magnification showing low staining (⬍50%) of GCs and stroma within nonaggressive GCL. Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
1. Austin LT Jr, Dahlin DC, Royer RQ: Giant-cell reparative granuloma and related conditions affecting the jawbones. Oral Surg Oral Med Oral Pathol 12:1285, 1959 2. Itonaga I, Hussein I, Kudo O, et al: Cellular mechanisms of osteoclast formation and lacunar resorption in giant cell granuloma of the jaw. J Oral Pathol Med 32:224, 2003 3. Flanagan AM, Nui B, Tinkler SM, et al: The multinucleate cells in giant cell granulomas of the jaw are osteoclasts. Cancer 62:1139, 1988 4. Resnick CM, Margolis J, Susarla SM, et al: Maxillofacial and axial/appendicular giant cell lesions: Unique tumors or variants of the same disease?—A comparison of phenotypic, clinical, and radiographic characteristics. J Oral Maxillofac Surg 68:130, 2010 5. Chuong R, Kaban LB, Kozakewich H, et al: Central giant cell lesions of the jaws: A clinicopathologic study. J Oral Maxillofac Surg 44:708, 1986
1866 6. Folkman J, Klagsbrun M: Angiogenic factors. Science 235:442, 1987 7. Folkman J: The role of angiogenesis in tumor growth. Semin Cancer Biol 3:65, 1992 8. Leung DW, Cachianes G, Kuang WJ, et al: Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246: 1306, 1989 9. Seghezzi G, Patel S, Ren CJ, et al: Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: An autocrine mechanism contributing to angiogenesis. J Cell Biol 141:1659, 1998 10. Ferrara N: Vascular endothelial growth factor: Basic science and clinical progress. Endocr Rev 25:581, 2004 11. Pepper MS, Ferrara N, Orci L, et al: Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem Biophys Res Commun 189:824, 1992 12. Senger DR, Galli SJ, Dvorak AM, et al: Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219:983, 1983 13. Gerber HP, Hillan KJ, Ryan AM, et al: VEGF is required for growth and survival in neonatal mice. Development 126:1149, 1999 14. De Young BR, Frierson HF Jr, Ly MN, et al: CD31 immunoreactivity in carcinomas and mesotheliomas. Am J Clin Pathol 110:374, 1998 15. Pusztaszeri MP, Seelentag W, Bosman FT: Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues. J Histochem Cytochem 54:385, 2006 16. DeLisser HM, Christofidou-Solomidou M, Strieter RM, et al: Involvement of endothelial PECAM-1/CD31 in angiogenesis. Am J Pathol 151:671, 1997 17. Matsumura T, Wolff K, Petzelbauer P: Endothelial cell tube formation depends on cadherin 5 and CD31 interactions with filamentous actin. J Immunol 158:3408, 1997 18. Zhou Z, Christofidou-Solomidou M, Garlanda C, et al: Antibody against murine PECAM-1 inhibits tumor angiogenesis in mice. Angiogenesis 3:181, 1999 19. Vered M, Buchner A, Dayan D: Giant cell granuloma of the jawbones—A proliferative vascular lesion? Immunohistochemical study with vascular endothelial growth factor and basic fibroblast growth factor. J Oral Pathol Med 35:613, 2006 20. Kaban LB, Troulis MJ, Ebb D, et al: Antiangiogenic therapy with interferon alpha for giant cell lesions of the jaws. J Oral Maxillofac Surg 60:1103, 2002
GIANT CELL LESIONS OF THE JAWS 21. Dewsnup NC, Susarla SM, Abulikemu M, et al: Immunohistochemical evaluation of giant cell tumors of the jaws using CD34 density analysis. J Oral Maxillofac Surg 66:928, 2008 22. Susarla SM, August M, Dewsnup N, et al: CD34 staining density predicts giant cell tumor clinical behavior. J Oral Maxillofac Surg 67:951, 2009 23. Macabeo-Ong M, Shiboski CH, Silverman S, et al: Quantitative analysis of cathepsin L mRNA and protein expression during oral cancer progression. Oral Oncol 39:638, 2003 24. Ginzinger DG, Godfrey TE, Nigro J, et al: Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis. Cancer Res 60:5405, 2000 25. Kaban LB, Mulliken JB, Ezekowitz RA, et al: Antiangiogenic therapy of a recurrent giant cell tumor of the mandible with interferon alfa-2a. Pediatrics 103:1145, 1999 26. Tiffee JC, Aufdemorte TB: Markers for macrophage and osteoclast lineages in giant cell lesions of the oral cavity. J Oral Maxillofac Surg 55:1108, 1997 27. de Lange J, Rosenberg AJ, van den Akker HP, et al: Treatment of central giant cell granuloma of the jaw with calcitonin. Int J Oral Maxillofac Surg 28:372, 1999 28. Pogrel MA, Regezi JA, Harris ST, et al: Calcitonin treatment for central giant cell granulomas of the mandible: Report of two cases. J Oral Maxillofac Surg 57:848, 1999 29. Pogrel MA: Calcitonin therapy for central giant cell granuloma. J Oral Maxillofac Surg 61:649, 2003 30. Collin-Osdoby P, Rothe L, Bekker S, et al: Basic fibroblast growth factor stimulates osteoclast recruitment, development, and bone pit resorption in association with angiogenesis in vivo on the chick chorioallantoic membrane and activates isolated avian osteoclast resorption in vitro. J Bone Miner Res 17:1859, 2002 31. Collins A: Experience with anti-angiogenic therapy of giant cell granuloma of the facial bones. Ann R Australas Coll Dent Surg 15:170, 2000 32. de Lange J, van Rijn RR, van den Berg H, et al: Regression of central giant cell granuloma by a combination of imatinib and interferon: A case report. Br J Oral Maxillofac Surg 47:59, 2009 33. O’Malley M, Pogrel MA, Stewart JC, et al: Central giant cell granulomas of the jaws: Phenotype and proliferation-associated markers. J Oral Pathol Med 26:159, 1997 34. Walker RA: Quantification of immunohistochemistry—Issues concerning methods, utility and semiquantitative assessment I. Histopathology 49:406, 2006
Giant Cell Lesions of the Jaws: Does the Level of Vascularity and Angiogenesis correlate With Behavior? Zachary S. Peacock, DMD, MD,* Richard C. K. Jordan, DDS, PhD, FRCPath,† and Brian L. Schmidt, DDS, MD, PhD‡ Purpose: To compare vascularity and angiogenic activity in aggressive and nonaggressive giant cell
lesions (GCLs) of the jaws. Materials and Methods: This is a retrospective study of 14 GCLs treated at the University of California,
San Francisco. Immunohistochemistry was used to determine of the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), CD34, and CD31. VEGF and bFGF expression in giant cells (GCs) and surrounding mononuclear stroma was classified into 1) high immunoreactivity (⬎50% staining) and 2) low immunoreactivity (⬍50% staining). CD31- and CD34-stained vessels were counted at 200⫻ magnification. Clinical and radiographic records were reviewed to classify lesions as aggressive or nonaggressive. Results: Of the lesions, 8 were aggressive and 6 were nonaggressive. High VEGF expression was found within the GCs in 4 of 8 aggressive lesions compared with 1 of 6 nonaggressive lesions. The stroma in both groups had low staining. High staining of the GCs for bFGF was found in 6 of 8 aggressive lesions compared with 3 of 6 nonaggressive lesions. The stroma of all aggressive cases showed high expression of bFGF compared with 3 of 6 nonaggressive cases. The aggressive group had a mean of 20.1 ⫾ 5.4 vessels/high-powered field (hpf) stained for CD31 compared with 11.5 ⫾ 5.6 vessels/hpf in the nonaggressive group. The aggressive group had 24.6 ⫾ 7.0 vessels/hpf stained with CD34 compared with 18.5 ⫾ 4.0 vessels/hpf in the nonaggressive group. Conclusions: The vascularity and level of angiogenesis within aggressive GCLs are higher than those in nonaggressive lesions. © 2012 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 70:1860-1866, 2012 The giant cell lesion (GCL) describes a group of intraosseous nonodontogenic benign tumors containing multinucleated giant cells (GCs). Several entities in the jaws can share this histology, including hyperparathyroidism, cherubism, and the central GC granuloma.1 Controversy remains over the biologic basis of GCLs of the jaws and thus the rationale for some therapies. GCLs of the jaws are most commonly found in children and young adults, with up to 75% of cases occurring before 30 years of age. The lesion affects *Currently, Instructor in Oral and Maxillofacial Surgery, Massachusetts General Hospital, Boston, MA; Formerly, Resident, Department of Oral and Maxillofacial Surgery, University of California, San Francisco, San Francisco, CA. †Professor of Oral Pathology and Pathology and Associate Dean for Research, University of California, San Francisco, San Francisco, CA. ‡Professor, Department of Oral and Maxillofacial Surgery, and Director, Bluestone Center for Clinical Research, New York University College of Dentistry, New York, NY.
female patients twice as often as male patients. Stromal fibroblasts are thought to produce cytokines, resulting in the recruitment of monocytes, which subsequently transform into multinucleated GCs.2 Immunohistochemical studies have shown the GCs to be osteoclasts phenotypically.3 The GCL of the jaw may be a unique lesion or may simply represent a continuum of the same disease process as GC tumors affecting the long bones.4 In this study the term “giant cell lesion” is used and includes lesions that may have Address correspondence and reprint requests to Dr Peacock: Massachusetts General Hospital, Warren 1201, 55 Fruit St, Boston, MA 02114; e-mail: [email protected] © 2012 American Association of Oral and Maxillofacial Surgeons
0278-2391/12/7008-0$36.00/0 doi:10.1016/j.joms.2011.08.020
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Table 1. CRITERIA FOR CLASSIFYING GCLS OF JAWS
Lesion size Rapid growth Root resorption Tooth displacement Cortical bone perforation Recurrence after curettage
Aggressive
Nonaggressive
ⱖ5 cm Yes Often Often Often
⬍5 cm No No No No
Yes
No
NOTE. Adapted from Chuong et al.5 Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
been historically considered central GC reparative granulomas or GC tumors. GCLs can be classified as aggressive or nonaggressive based on clinical and radiographic features (Table 1).5 The aggressive form has 3 of the following 5 criteria: rapid growth, root resorption, tooth displacement, cortical bone thinning, or perforation. In addition, a GCL greater than 5 cm and/or recurring after enucleation and curettage is classified as aggressive. The nonaggressive lesion is often asymptomatic, grows slowly, and has a low rate of recurrence. Currently, there are no reliable histologic or molecular methods to differentiate aggressive from nonaggressive lesions. Angiogenesis is the process by which capillaries form from pre-existing blood vessels. It occurs physiologically (eg, embryologic development and wound healing) and in pathologic processes such as proliferative retinopathies and facilitation of tumor growth.6,7 Numerous cytokines and growth factors modulate angiogenesis through paracrine and autocrine pathways.8,9 Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are thought to be the most potent inducers of angiogenesis.10 VEGF, a mitogen for endothelial cells, has been found to promote angiogenesis both in vitro and in vivo.8,11 It enhances vascular permeability and serves as a survival factor for newly formed blood vessels.12,13 bFGF is the prototype of a family of 13 structurally related heparin growth factors. It is also a potent inducer of angiogenesis and is thought to act synergistically with VEGF on endothelial cells.11 CD31 and CD34 are known endothelial cell markers in vascular beds of both normal tissue and neoplasms.14,15 CD31 is a 130-kDa transmembrane glycoprotein that is a member of the immunoglobulin superfamily. It is present on the surface of platelets, monocytes, macrophages, and neutrophils and is a constituent of the endothelial intercellular junction. Its vital role in angiogenesis has been recognized
recently,16-18 and it is one of the best-known immunohistochemical markers for benign and malignant vascular tumors.15 CD34 is a 110-kDa transmembrane glycoprotein present on leukemic, endothelial, and stem cells. Although its function is still unclear, it is frequently used to characterize vascular tumors, progenitor cells, and solitary fibrous tumors.15 Vered et al19 showed that angiogenesis does occur in GCLs of the jaws, albeit at low levels. They found that VEGF and bFGF immunoreactivity was lower in the endothelial cells of GCLs compared with other physiologic and pathologic processes of angiogenesis. The multinucleated GCs and mononuclear stromal cells stained positive for both VEGF and bFGF. This may explain the response of GCLs to antiangiogenic treatment.20 Dewsnup and colleagues33 assessed CD34 staining density in GCLs and found increased vascular density in aggressive lesions; increased CD34 staining density was then found to be associated with aggressive clinical behavior.22 The purpose of this study was to compare the immunoreactivity of VEGF and bFGF of multinucleated GCs and the mononuclear stroma in aggressive and nonaggressive GCLs of the jaws. In addition, vascular density was compared by use of immunohistochemical staining of CD31 and CD34.
Materials and Methods CASE SELECTION
This retrospective study examined the records and tissue from patients treated for GCLs of the jaws at the Department of Oral and Maxillofacial Surgery, University of California, San Francisco, from 1995 to 2004. The study was approved by the University of California, San Francisco Institutional Review Board (CHR Committee on Human Research No. H11557-0701922). GCLs from patients with hyperparathyroidism, cherubism, and Noonan syndrome were excluded. Cases were included if complete clinical, radiographic, and pathologic records were available (minimum follow-up of 6 months). Cases were excluded for incomplete or unavailable records and inadequate tissue for sectioning and staining. All specimens were verified by a board-certified oral and maxillofacial pathologist to be GCLs of the jaws (R.C.K.J.). CLINICAL DATA
Patient records were reviewed independent of the histologic findings. Clinical data collected included age, gender, presence or absence of pain, swelling, root resorption, cortical thinning, perforation, and recurrence. On the basis of the classification system formalized by Chuong and Kaban5 2 groups were defined: 1) aggressive GCLs and 2) nonaggressive GCLs.
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IMMUNOHISTOCHEMISTRY
bound complexes were visualized by the application of a 0.05% solution of 3-amino-9-ethylcarbazole (Sigma-Aldrich, St Louis, MO) containing 0.3% hydrogen peroxide as a substrate. After incubation, the sections were washed and then lightly counterstained in hematoxylin and coverslipped. A pyogenic granuloma served as positive control to verify binding of antibodies to VEGF, bFGF, and CD31. A section of colorectal cancer was an additional positive control for VEGF. For CD34, a solitary fibrous tumor was used. Slides were stained with omission of each primary antibody as a negative control.
Immunohistochemistry was performed as previously described to determine the expression levels of VEGF, bFGF, CD31, and CD34.23 In brief, 5-m sections were cut and mounted on adherent glass slides. Sections were dewaxed in xylene and rehydrated in graded ethanols. Endogenous peroxidase activity was blocked by immersion in 0.5% methanolic peroxide for 15 minutes, followed by 2 washes in phosphate-buffered saline solution (PBS) for 5 minutes each. Slides were immersed in deionized water and then rinsed in PBS. Before staining with VEGF and CD31, antigen retrieval techniques were used. For VEGF, Tris (hydroxymethyl)aminomethane/ ethylenediaminetetraacetic acid at pH 9.0 (Dako, Carpinteria, CA) was used with microwaving. CD31 required antigen retrieval with citrate at pH 6.0 (Dako). Sections were then incubated for 1 hour at room temperature with the respective primary antibodies: 1) VEGF–rabbit monoclonal anti-VEGF (Labvision, Fremont, CA) diluted to 1:25 in PBS, 2) bFGF– prediluted mouse monoclonal anti-bFGF (Biogenics, Napa, CA), 3) CD31–prediluted mouse monoclonal anti-CD31 (Dako), and 4) CD34 –prediluted mouse monoclonal anti-CD34 (Biogenics). This was followed by 2 washes in PBS, incubation with a biotinylated secondary antibody (Envision Plus; Dako), and 2 PBS rinses, followed by the application of preformed avidin-biotin complex (Dako) for 30 minutes.24 The
QUANTIFICATION OF IMMUNOHISTOCHEMISTRY
Immunostained sections were assessed and quantified by an investigator (Z.S.P.) blinded to the clinical data for each case. Immunohistochemical reactivity of the multinucleated GCs and surrounding stroma for VEGF and bFGF was scored and classified into 2 groups: 1) high immunoreactivity (⬎50% staining) and 2) low immunoreactivity (⬍50% staining). CD31 and CD34 expression was scored by randomly selecting 3 areas of lesional tissue and counting the number of positively stained vessels within a 1.25-mm eyepiece graticule at 200⫻ magnification. The mean of the 3 random high-powered fields (hpf) was calculated.
Table 2. DEMOGRAPHIC AND CLINICAL DATA OF STUDY POPULATION (14 GCLs)
Case Age No. (yr) Gender 1 2 3 4 5 6
26 9 16 9 7 46
F M M M M M
7 8 9
19 12 9
F M F
10
20
F
11
4
F
12
14
F
13 14
20 15
F M
Location Maxilla, anterior Mandible, anterior Mandible, anterior Mandible, anterior Mandible, anterior Mandible, body, ramus Mandible, anterior Mandible, body Mandible, body and ramus Mandible, body and ramus Mandible, body, molar region Mandible, anterior and bilateral body Mandible, anterior Mandible, premolar, molar
Rate of Root Cortical Size Pain Growth Swelling Resorption Thinning Recurrence (cm) Aggressive N N N N N N
Slow Slow Slow Slow Slow Slow
Y N Y Y Y Y
N N N N N N
N N N N N N
N N N N N N
2.6 2.4 2.9 4.2 2.6 4.3
N N N N N N
Y Y N
Rapid Rapid Slow
Y Y Y
N N Y
Y Y N
Y N N
3.1 7.2 6.9
Y Y Y
Y
Rapid
Y
N
Y
N
3.1
Y
Y
Rapid
Y
Y
Y
Y
5.6
Y
Y
Rapid
Y
Y
Y
Y
10.2
Y
Y N
Rapid Slow
Y Y
N N
Y Y
Y Y
3.6 2.1
Y Y
Abbreviations: F, female; M, male; N, no; Y, yes. Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
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multinucleated GCs was found in 4 of 8 aggressive lesions compared with 1 of 6 nonaggressive lesions. Within the stroma, 1 aggressive lesion and 1 nonaggressive lesion had high immunoreactivity. Evidence of staining for bFGF was present in 12 of 14 cases (Fig 2). High staining of the GCs was found in 6 of 8 aggressive cases compared with 3 of 6 nonaggressive lesions. Two of the nonaggressive lesions had no staining of the GCs. The stroma of the aggressive group was highly stained in all 8 cases compared with only 3 of 6 in the nonaggressive group. Endothelial cells within the lesions were stained by CD31 and CD34 (Fig 3). For CD31-stained aggressive lesions, a mean of 20.1 vessels/hpf were present compared with 11.5 vessels/hpf in the nonaggressive group. In the CD34-stained group, a mean of 24.6 vessels/hpf were present in the aggressive group compared with 18.5 vessels/hpf in the nonaggressive group. The pyogenic granuloma was positively stained for VEGF, bFGF, and CD34. The colorectal cancer was also positive for CD34, as was the solitary fibrous tumor for CD31. No staining was seen in any GCLs stained without the primary antibody.
Discussion
FIGURE 1. Aggressive versus nonaggressive immunohistochemical staining for VEGF. A, Photomicrograph at 200⫻ magnification showing high staining (⬎50%) of GCs and stroma within aggressive GCL. B, Photomicrograph at 200⫻ magnification showing low staining (⬍50%) of GCs and stroma within nonaggressive GCL. Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
STATISTICAL ANALYSIS
For categorical data, differences were assessed with the Fisher exact test. For continuous data, 95% confidence intervals were calculated and compared. All tests were 2 tailed.
Results We identified 14 subjects (7 male and 7 female patients) that met the inclusion criteria (Table 2). Of these 14 patients, 13 had mandibular GCLs. The GCLs were aggressive in 8 and nonaggressive in 6. The mean ages of the patients with aggressive and nonaggressive lesions were 14.1 years and 18.8 years, respectively. Of the 8 subjects with aggressive lesions, 6 were female patients. Immunoreactivity to VEGF was seen in all 14 specimens (Fig 1, Table 3). High reactivity within the
Controversy remains regarding the GCL and its pathogenesis. The lesion has been proposed to be a reactive inflammatory, vascular, endocrine, or neoplastic process. GCLs of the jaws can be characterized as aggressive or nonaggressive based on certain clinical behaviors.5 No reliable immunophenotypic or molecular marker exists, however, to differentiate between aggressive and nonaggressive GCLs. This study characterized the vascularity of GCLs of the jaws. The aggressive lesions had increased expression of angiogenic markers VEGF and bFGF within both the multinucleated GCs and the mononuclear fibroblastic stroma. In addition, the aggressive lesions had increased vascularity quantified by immunoreactivity to endothelial markers CD31 and CD34. This suggests that angiogenesis occurs within GCLs and both multinucleated GCs and mononuclear stromal cells may be a source of VEGF and bFGF. VEGF and bFGF levels may be increased in aggressive lesions, leading to the increased vascular density. In treating patients with GCLs of the jaws, it would be beneficial and efficacious to have directed therapy that is selected based on a proven biologic basis. Unfortunately, both the etiology and cell of origin are unknown at this time. The GCL has been proposed to be a proliferative vascular lesion and has been shown to respond to antiangiogenic therapy after enucleation.20,25 Proliferative vascular lesions are known to
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Table 3. IMMUNOHISTOCHEMICAL STAINING OF VEGF, BFGF, CD31, AND CD34
Case No. Nonaggressive group 1 2 3 4 5 6 Mean ⫾ 95% CI Aggressive group 7 8 9 10 11 12 13 14 Mean ⫾ 95% CI
Age (yr)
CD31Stained Vessels/hpf
CD34Stained Vessels/hpf
High High Low Low High Low
11 4 26 8 12 8 11.5 ⫾ 5.6
10 23 22 16 24 16 18.5 ⫾ 4.0
High High High High High High High High
23 27 13 12 11 31 17 27 20.1 ⫾ 5.4
31 8 37 34 17 31 20 19 24.6 ⫾ 7.0
Gender
VEGF GC
VEGF Stroma
bFGF GC
bFGF Stroma
26 9 16 9 7 46
F M M M M M
Low Low Low Low High Low
High Low Low Low Low Low
High High Low Low High Low
19 12 9 20 4 14 20 15
F M F F F F F M
Low High Low High High High Low Low
Low Low Low Low High Low Low Low
Low High High High High High High High
Abbreviations: CI, confidence interval; F, female; M, male. Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
have overexpression of angiogenic proteins VEGF and bFGF.9 This study supports the theory that GCLs of the jaws are proliferative vascular lesions. Characterizing the GCLs as strictly a proliferative vascular process may be overly simplistic. The multinucleated GCs within these lesions have been shown to be osteoclasts or osteoclast-like.2,3,26 Multiple studies have shown resolution with calcitonin, a known inhibitor of osteoclasts.27-29 As previously theorized, the multinucleated GCs and mononuclear cells within the stroma may stimulate angiogenesis, increasing the circulatory delivery of progenitors of these osteoclast-like GCs.19,30 This would increase the osteolytic capacity and, hence, the aggressiveness of the lesion and has been termed “osteoclastogenesis.”29 Given favorable clinical responses to inhibitors of both angiogenesis through interferon ␣2a and osteoclasts through calcitonin, both processes may be important in development and progression of the lesion.20,25,27,29,31,32 Further controlled studies are needed to determine the right combination of medical therapy and surgical management for aggressive GCLs. Previous analyses of angiogenesis within GCLs have had inconsistent results. Vered et al19 quantified the expression of VEGF and bFGF within the multinucleated GCs, mononuclear spindle cells, and endothelial cells within GCLs. They found low levels of staining of these angiogenic proteins and concluded that these lesions have low angiogenic activity, but no comparison was made between aggressive and nonaggressive
lesions. Our study found increased levels of both of these proteins in aggressive lesions within the multinucleated GCs and the mononuclear stroma as compared with nonaggressive lesions. Immunoreactivity of CD34 has been extensively studied in GCLs of the jaws. O’Malley et al33 saw no differences between aggressive and nonaggressive lesions through counting the percentage of CD34-positive cells in a high-powered field. Positively stained endothelial cells were found only in the periphery of the lesions. Dewsnup et al21 showed significantly increased CD34 staining density in aggressive GCLs compared with nonaggressive lesions through computerized histologic analysis. They went on to show that staining density greater than 2.5% is predictive of aggressive clinical behavior.21 Our study found increased vascularity as assessed by both CD34 and CD31. CD31 has not been previously used to assess vascularity within these lesions. Like CD34, it is a transmembrane glycoprotein found on the surface of endothelial cells. The results of this study support the theory that a proliferative vascular component within GCLs may be responsible for their clinical aggressiveness. Increased angiogenesis and vascularity may allow for increased recruitment and differentiation of hematopoietic cells to osteoclasts, leading to more rapid osteolysis and enlargement. Limitations exist within this study. First, we evaluated a small number of cases. Although there were obvious trends in the representations of vascularity
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within these lesions, we were not able to show statistically significant differences. Second, despite use of positive and negative controls, limitations exist within the immunohistochemistry technique, including variability in antigen retrieval, to permit assessment of protein levels within tissue.34 Further study of the genotypic signature and phenotypic expression of these lesions is needed to help develop a reliable method to characterize clinical behavior at the time of biopsy. The goal is to develop suitable and reliable directed medical therapy to prevent recurrence of aggressive lesions. Given the proposed inter-related nature of angiogenesis and osteoclastogenesis, perhaps a combination of current medical therapies will improve patient outcomes. The results of this study suggest an increased vascularity within GCLs that are clinically aggressive. Characterization of VEGF and bFGF, protein markers
FIGURE 3. CD31 and CD34 immunohistochemical staining. A, Photomicrograph at 200⫻ magnification showing vessels stained for CD31 in aggressive GCL. B, Photomicrograph at 200⫻ magnification showing vessels stained for CD34 in aggressive GCL. Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
of angiogenesis, as well as endothelial proteins CD31 and CD34 within these lesions, may be helpful to predict clinical behavior.
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FIGURE 2. Aggressive versus nonaggressive immunohistochemical staining for bFGF. A, Photomicrograph at 200⫻ magnification showing high staining (⬎50%) of GCs and stroma within aggressive GCL. B, Photomicrograph at 200⫻ magnification showing low staining (⬍50%) of GCs and stroma within nonaggressive GCL. Peacock, Jordan, and Schmidt. Giant Cell Lesions of the Jaws. J Oral Maxillofac Surg 2012.
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GIANT CELL LESIONS OF THE JAWS 21. Dewsnup NC, Susarla SM, Abulikemu M, et al: Immunohistochemical evaluation of giant cell tumors of the jaws using CD34 density analysis. J Oral Maxillofac Surg 66:928, 2008 22. Susarla SM, August M, Dewsnup N, et al: CD34 staining density predicts giant cell tumor clinical behavior. J Oral Maxillofac Surg 67:951, 2009 23. Macabeo-Ong M, Shiboski CH, Silverman S, et al: Quantitative analysis of cathepsin L mRNA and protein expression during oral cancer progression. Oral Oncol 39:638, 2003 24. Ginzinger DG, Godfrey TE, Nigro J, et al: Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis. Cancer Res 60:5405, 2000 25. Kaban LB, Mulliken JB, Ezekowitz RA, et al: Antiangiogenic therapy of a recurrent giant cell tumor of the mandible with interferon alfa-2a. Pediatrics 103:1145, 1999 26. Tiffee JC, Aufdemorte TB: Markers for macrophage and osteoclast lineages in giant cell lesions of the oral cavity. J Oral Maxillofac Surg 55:1108, 1997 27. de Lange J, Rosenberg AJ, van den Akker HP, et al: Treatment of central giant cell granuloma of the jaw with calcitonin. Int J Oral Maxillofac Surg 28:372, 1999 28. Pogrel MA, Regezi JA, Harris ST, et al: Calcitonin treatment for central giant cell granulomas of the mandible: Report of two cases. J Oral Maxillofac Surg 57:848, 1999 29. Pogrel MA: Calcitonin therapy for central giant cell granuloma. J Oral Maxillofac Surg 61:649, 2003 30. Collin-Osdoby P, Rothe L, Bekker S, et al: Basic fibroblast growth factor stimulates osteoclast recruitment, development, and bone pit resorption in association with angiogenesis in vivo on the chick chorioallantoic membrane and activates isolated avian osteoclast resorption in vitro. J Bone Miner Res 17:1859, 2002 31. Collins A: Experience with anti-angiogenic therapy of giant cell granuloma of the facial bones. Ann R Australas Coll Dent Surg 15:170, 2000 32. de Lange J, van Rijn RR, van den Berg H, et al: Regression of central giant cell granuloma by a combination of imatinib and interferon: A case report. Br J Oral Maxillofac Surg 47:59, 2009 33. O’Malley M, Pogrel MA, Stewart JC, et al: Central giant cell granulomas of the jaws: Phenotype and proliferation-associated markers. J Oral Pathol Med 26:159, 1997 34. Walker RA: Quantification of immunohistochemistry—Issues concerning methods, utility and semiquantitative assessment I. Histopathology 49:406, 2006