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Address for correspondence and reprint requests to Prof Onatolu Odukoya, Department of Oral Biology and Oral Pathology, College of Medicine University ofLagos/Lagos UniversityTeaching Hospital, Nigeria Email address: odukoyatolu2007@yahoo.com ©2013 Soyeli OO et al. Licensee Narain Publishers Pvt. Ltd. (NPPL) Submitted: October 21, 2013; Accepted: November 20, 2013 Published: December 4, 2013

Case report Open Access

Masson’s Trichrome and AgNOR Study of Fibrous dysplasia and Ossifying fibroma in Lagos University Teaching Hospital Patients

1Olujide Oladele Soyele, 2Olajumoke Ajibola Effiom, 2Onatolu Odukoya

1Department of Oral Biology and Oral Pathology, Lagos University Teaching Hospital, Nigeria.

2Department of Oral Biology and Oral Pathology, College of Medicine University of Lagos/Lagos University Teaching Hospital, Nigeria.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction: Fibrous dysplasia (FD) and ossifying fibroma (OF) pose diagnostic challenges. This study attempts to differentiate between the two lesions, using AgNOR and Masson’s Trichrome staining techniques. Study design: A retrospective study on FD and OF. Materials and methods: Forty cases each of fibrous dysplasia and ossifying fibroma were randomly selected from the oral biopsy archives of Lagos University Teaching Hospital. Three 5 microns paraffin sections per case were stained with Hematoxylin and Eosin, Masson’s Trichrome and AgNOR respectively. Proportions of bone trabeculae type and mean AgNOR scores for both lesions were analyzed for χ² and t statistics respectively using the Epi-info statistical package. Results: Proportions of Lamella bone trabeculae type in ossifying fibroma(52.5%) was significantly higher than in fibrous dysplasia (30.0%); proportions of mixed bone trabeculae type in fibrous dysplasia (70.0%) was significantly higher than in ossifying fibroma (50.0%) (p< 0.05). Proportions of woven bone trabeculae type in fibrous dysplasia (60.0%) was not significantly higher than in ossifying fibroma (55.0%) (p> 0.05). Mean AgNOR score for ossifying fibroma (130.4 ± 32.3/ 100 cells) was significantly higher than for fibrous dysplasia (92.3 ± 27.4/ 100 cells) (p < 0.05). Conclusion: Proportions of Lamella and mixed bone trabeculae types and mean AgNOR scores are important features to distinguish between fibrous dysplasia and ossifying fibroma, while proportions of woven bone trabeculae type is a poor distinguishing feature. This is the first demonstration of employing mixed bone trabeculae proportions to distinguish between fibrous dysplasia and ossifying fibroma. Key words: fibroosseous lesions: jaws: histochemistry

Introduction

Fibro-osseous lesions of the jaws which include fibrous dysplasia (FD) and ossifying fibroma (OF), are poorly defined heterogeneous group of lesions that affect the jaws and other craniofacial bones. They are characterized by the replacement of bone with a benign

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connective tissue matrix containing varying degrees of mineralization in the form of woven/lamellar bone and or sometimes, cementum-like, round, acellular, intensely basophilic structures [1,2]. The World Health Organization currently defines fibrous dysplasia as a genetically based sporadic disease of the bone which may affect either single or multiple bones (monostotic or polyostotic types respectively). When it occurs in different craniofacial bones, it is regarded as craniofacial fibrous dysplasia. Central ossifying fibroma however is a benign neoplasm which often presents with well-demarcated borders. It is histologically composed of fibrocellular stroma and variable amounts of mineralized material [3].

Fibrous dysplasia has been reported to occur with a global prevalence of 2.5% of all bone tumors and 7.5% of all benign bone neoplasms [4,5]. It has also been reported to account for 74.0% of fibroosseous lesions in Africa [6]. In contrast, the precise global frequency of ossifying fibroma is yet to be determined due to dearth of reported epidemiological studies and varying terminologies that have been used for its description [3, 7]. For example, cementoossifying fibroma and cementifying fibroma which were initially regarded as types of fibroosseous lesions like ossifying fibroma, have basically been grouped as ‘ossifying fibroma’ by the World Health Organization, while psammomatoid and trabecular juvenile aggressive ossifying fibromas have been grouped as variants of ossifying fibroma [3].

Despite the overwhelming remarkable similarities that exist between fibrous dysplasia and ossifying fibroma, both lesions still present with some differences in age, gender, jaw location [8,9], racial predilection [7,8,9,10] and radiological presentation [11,12,13].

Histologically, fibrous dysplasia often presents with a monotonous distribution of bone and fibrous connective tissue stroma with a characteristic lesional margin that blends with its surrounding adjacent tissue, w h i l e ossifying

fibroma presents with a storiform distribution of fibrous connective tissue of a tumor mass which is clearly demarcated from normal adjacent tissue [14, 15]. This difference in tumor growth patterns results in different treatment procedures for these lesions. While curettage is the treatment of choice for ossifying fibroma due to the fact that it can be easily shelled out, fibrous dysplasia may have to be surgically resected, if necessary, especially in cases of huge disfiguring lesions [16-19].

Nucleolar organizer regions (NORs) represent loops of DNA that code for ribosomal RNA. They are considered important in protein synthesis [20, 21]. NORs can be selectively stained by silver nitrate via a simple technique called the AgNOR technique. With the aid of light microscopy these are visualized as black or brown intra-nuclear AgNOR dots [22]. Previous studies have employed the AgNOR technique to compare the proliferating activities of ossifying fibroma and fibrous dysplasia [23,24] and Masson’s Trichrome histochemical staining technique to histologically distinguish between fibrous dysplasia and ossifying fibroma based on the proportions of mineralized content of woven and lamellar bone within the lesions [25,26]. To the best of our knowledge, there has been no report of any study done which has employed the use of both histochemical techniques (Masson’s Trichrome and AgNOR) together within the same study, using a fairly large pool of cases to distinguish between fibrous dysplasia and ossifying fibroma. The present study therefore aimed to distinguish between fibrous dysplasia and ossifying fibroma using Masson’s Trichrome and AgNOR histochemical stains.

Patients and Methods

From the period 1970 to 2009, all cases of fibroosseous lesions diagnosed in the Department of Oral Pathology and Oral Biology, Lagos University Teaching Hospital, were retrieved from the oral biopsy archives. Cases previously diagnosed as ‘cementifying

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Figure 1: Woven bone in a lesion of fibrous dysplasia (Masson’s Trichrome stain. Magnification x 100)

Figure 2: Histogram showing the distribution of woven bone trabeculae type in fibrous dysplasia and ossifying fibroma

fibroma’, ‘cement-ossifying fibroma’ and ‘ossifying fibroma’ were all broadly re-categorized as ossifying fibroma according to World Health Organization classification [3].

From the archival reports, a total of 157 cases retrieved were diagnosed as ossifying fibroma, while a total of 134 cases were diagnosed as fibrous dysplasia. For each lesion (i.e. fibrous dysplasia and ossifying fibroma), 40 cases were randomly selected (i.e. 40 cases of ossifying fibroma and 40 cases of fibrous dysplasia). Three 5 micron paraffin embedded tissue sections (group 1, group 2, group 3) were cut for each case, and stained with hematoxylin and eosin (group1), Masson’s Trichrome (group2) and AgNOR stains (group3) respectively. All hematoxylin and eosin stained slides (group1) were viewed with a light microscope to re-confirm diagnosis of the specific type of fibroosseous lesion.

Masson’s Trichrome staining was conducted using the multi steps method of Asonova and Milgalkin [27] which was modified by Gulati et al [26] and Ralis & Watkins [28]. All sections stained with Masson’s Trichrome (group2) were viewed with a light microscope using the 40 magnification objective (x40) to observe the

proportions of type of bone (i . e . whether woven, lamella or mixed) trabeculae per case. The proportion of the type of bone trabeculae per case was determined by examining 240 bone trabeculae for each case and recording the number of each type of bone trabeculae observed /case. The number of type of bone trabeculae related to the total number of trabeculae examined (240 per case) was subsequently computed as proportion of the type of bone trabeculae in each case. This proportion was expressed as a percentage.

AgNOR staining was conducted using the Ploton et al [29] technique. The AgNOR stained slides (group3) were viewed with light microscope with x 100 objective to identify positive reactions viewed as black intra-nuclear dots (NORs) in the fibroblasts. The NOR scores per case were evaluated as number of NORs observed in 100 fibroblast cells. The mean score for each lesion was subsequently computed.

Statistical analysis

Epi- info statistical package was used to analyze results obtained in the present study. Proportions of bone trabeculae type of 30.0% or below were considered as low, while proportions higher than the 30.0% were considered as high proportions. Proportions of bone trabeculae types for fibrous dysplasia and ossifying fibroma were compared using χ2

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Table 1: Distribution of Woven Bone Trabeculae at Low and High Proportions in FD & OF

Proportion

Type of Lesion

FD OF TOTAL

Freq % Freq % Freq %

0 – 30 16 40 18 45 34 42.5

31 – 90 24 60 22 55 46 57.5

TOTAL 40 100 40 100 80 100

P=0.41 (Fisher’s exact test) (not statistically significant) Table 2: Distribution of Lamellar Bone Trabeculae at Low and High Proportions in FD & OF

Proportion

Type of Lesion

FD OF TOTAL

Freq % Freq % Freq %

0 – 30 28 70 19 47.5 47 58.8.5

31 – 90 12 30 21 52.5 33 41.2

TOTAL 40 100 40 100 80 100

P=0.03 (Fisher’s exact test) (statistically significant)

Figure 3: Lamellar bone in lesion of ossifying fibroma (Masson’s Trichrome stain. (Magnification x 100)

Figure 4: Histogram showing the distribution of lamellar bone trabeculae type in fibrous dysplasia and ossifying fibroma

analysis and the mean AgNOR scores for fibrous dysplasia and ossifying fibroma were compared using the t-statistic.

Results

In comparing type of fibroosseous lesion with proportion of bone trabeculae, no association was established for woven bone in respect of FD (40.0% low proportion of woven bone and 60.0% high proportion of woven bone) and OF (45.0% low proportion of woven bone and 55.0% high proportion of woven bone (figures 1 and 2, table 1; p=0.41 fisher’s exact test). However, a statistically significant association

was established for lamella bone in respect of FD (70.0% low proportions of lamella bone trabeculae and 30.0% of high proportions of lamella bone) and OF (47.5% low proportions of lamella bone trabeculae and 52.5% of high lamella bone proportions) (figure 3 and 4 and table 2; p = 0.03 Fisher’s exact test). Furthermore, a statistically significant association was established for mixed bone trabeculae in respect of FD (30.0% low proportions of the mixed bone trabeculae and 70.0% high proportion of mixed bone trabeculae) and OF (50.0% low proportions of the mixed bone trabeculae and 50.0% had high proportions) (figure 5 and 6 and table 3; p = 0.05 Fisher’s

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Table 3: Distribution of Mixed Bone Trabeculae at Low and High Proportions in FD & OF

Proportion

Type of Lesion

FD OF TOTAL

Freq % Freq % Freq %

0 – 30 12 30 20 50 32 40

31 – 90 28 70 20 50 48 60

TOTAL 40 100 40 100 80 100

P=0.05 (Fisher’s exact test) (statistically significant) Table 4: Distribution of Mean AgNOR Scores/100 cells in FD and OF

Range Median Mode Mean

FD 47.1250-161.3750 89.50 77.0 92.3369 ± 27.4207

OD 88.1250-210.1525 125.50 88.1250 130.4069 ± 32.3392

FD vs OF: t=5.6787; df=78 (p< 0.001) (value is statistically significant)

Figure 5: Mixed bone trabeculae in a lesion of fibrous dysplasia (Masson’s Trichrome stain. Magnification x 100)

Figure 6: Histogram showing the distribution of mixed bone trabeculae type in fibrous dysplasia and ossifying fibroma

exact test). The mean AgNOR score of 130.4± 32.3/100 cells (1.3±0.3/cell) which was recorded for ossifying fibroma was significantly higher than the mean AgNOR score of 92.3±27.4/100 cells (0.9±0.3/cell) which was recorded for fibrous dysplasia (figure 7a and 7b and Table IV; p< 0.001Fisher’s exact test).

Discussion

Masson’s trichrome stain

Observation from our present study shows that determination of the proportion of woven bone trabeculae alone within the lesions may not be an adequate distinguishing feature, although

previous studies have stated that the presence of high proportions of woven bone suggests the diagnosis of fibrous dysplasia instead of ossifying fibroma [15,30] .

Relatively high proportions of lamella bone was more commonly observed with cases of ossifying fibroma when compared to those of fibrous dysplasia, thereby implying that high lamellar bone proportions may be an important feature to distinguish between both fibrous dysplasia and ossifying fibroma. Although previous studies have reported that ossifying fibroma consists of lamellar bone and woven bone in varying proportions [10, 25], our study implies that a lesion with high lamella bone proportion is more likely to be an ossifying fibroma than a fibrous dysplasia.

The relatively high proportions of mixed bone trabeculae observed more commonly in fibrous

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Figure 7(a): intra-nuclear black AgNOR dots in fibroblasts of fibrous dysplasia . (AgNOR stain. Magnification x 1000) (b): Relatively more intra-nuclear AgNOR dots in fibroblasts of ossifying fibroma (AgNOR stain. Magnification x 1000)

dysplasia than ossifying fibroma also suggests an important distinguishing role for this bone trabeculae type in the histological diagnosis of fibrous dysplasia. High proportion of mixed bone trabeculae that was observed to be associated with fibrous dysplasia may be attributed to the slow turnover of bone from pure woven bone to pure lamellar bone in fibrous dysplasia [31,32].To the best of our knowledge, this is the first study where mixed bone trabeculae quantitative analysis has been suggested as a distinguishing feature for fibrous dysplasia of the jaws.

AgNOR stain

AgNOR histochemical staining techniques have rarely been employed to distinguish fibrous dysplasia from ossifying fibroma of the jaws [23,24]. Mean AgNOR score of 92.3 ±27.4 per 100 cells (0.9/cell) recorded for fibrous dysplasia was significantly lower than the mean AgNOR score of 130.4 ± 32.3 per 100 cells (1.3/cell) for ossifying fibroma. This finding is similar to reports from a Japanese population (0.95/cell for fibrous dysplasia and 1.46/cell for ossifying fibroma) [23], and an Iranian population (0.73 for fibrous dysplasia and 1.75 for ossifying fibroma) [24]. Higher mean AgNOR scores recorded for ossifying fibroma indicates higher proliferative fibroblastic activity which further confirms its neoplastic nature. However, mutation of HRPT2 (parafibromin) which is a tumor suppressor gene does not appear to play a

role in distinguishing ossifying fibroma from fibrous dysplasia [33].

Update on distinguishing fibrous dysplasia from ossifying fibroma

Recent studies [34-36], have added to our knowledge of features that distinguish fibrous dysplasia from ossifying fibroma. Osteocalcin has been reported to be more expressed in stroma cells of lesions of fibrous dysplasia while its expression is more markedly expressed in bone trabeculae of ossifying fibroma [34]. GNAS gene mutation has also been reported to be a reliable adjunct to differentiate ossifying fibroma from fibrous dysplasia of the jaws, as the GNAS gene mutation was detected in fibrous dysplasia but not in ossifying fibroma [35]. Furthermore, peritrabecular clefting has been reported to occur in 86.5 % (32 from 68 cases) of FD while no case of peritrabecular clefting (from a series of 37 cases) was observed in OF [36].

Conclusion

In an attempt to distinguish between ossifying fibroma and fibrous dysplasia, the presence of higher proportions of lamella bone trabeculae is suggestive that a fibroosseous lesion i s ossifying fibroma, while the presence of higher proportions of mixed bone trabeculae is suggestive that a fibroosseous lesion is fibrous dysplasia. Woven bone trabeculae presence is not a reliable distinguishing feature to suggest that a fibroosseous lesion is either ossifying

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fibroma or fibrous dysplasia. The mean AgNOR score per cell which was observed to be significantly higher in ossifying fibroma than in fibrous dysplasia may serve as a reliable parameter to distinguish between the two lesions.

Authors' Contribution

OOS did the major laboratory work for the study as part of his project in partial fulfillment for the award of the Fellowship of the West African College of Surgeons. OAE supervised the project and assisted with final write up of the manuscript.

OO generated the idea for the project, took part in the supervision of the project and vetted the final write up of the manuscript.

Conflict of Interests

The authors declare that there are no conflicts of interests.

Ethical Considerations

The study was approved by the institute ethics committee.

Funding

None declared

Acknowledgement

We acknowledge with thanks all past and present consultant Oral Pathologists and Oral and Maxillofacial Surgeons who have contributed to the biopsy service record and clinical information used in generating data for this study. We are also indebted to Lagos University Teaching Hospital, the custodian of our biopsy and clinical records, for making it possible for us to carry out this study. Abstract and Oral poster presentation of manuscript were presented at the 16th

International congress of the International

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