Muna.doc J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 121 Immunohistochemical assessment of tumor suppressor gene Wwox in relation to proliferative marker KI67 proteins expression in giant cell lesions of the jaws and giant cell tumor of long bones Muna Salih Merza, B.D.S., M.Sc., Ph.D. (1) ABSTRACT Background: Peripheral giant cell lesion (PGCL) and central giant cell lesion (CGCL) of the jaws have a distinct clinical behavior.Giant cell tumour (GCT) is a benign locally aggressive neoplasm affects the long bones. Both lesions are characterized histologically by multinucleated giant cells in a background of ovoid to spindle-shaped mesenchymal cells. The WW domain-containing oxidoreductase (WWOX) gene is located at 16q23.1–16q23.2, a region that spans the second most common human fragile site, FRA16D, at 16q23.2.The Ki-67 antigen is a nuclear protein that is associated with and may be necessary for cellular proliferation.Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0). This study aimed to evaluate and compare immunohistochemical expression of tumor suppressor gene (WWOX) and proliferative marker (ki67) in giant cell lesions (GCLs) of the jaws and long bones. Materials and methods: Forty five retrospective paraffin embedded tissue blocks of giant cell lesions of the jaw and long bones were included in this study.Sections were stained immunohistochemically with anti WWOX and anti ki67 monoclonal antibodies. Results: Positive WWOX expression was found in 12 cases (80%), 14cases (93.3%)and12 (80%) of CGCG, PGCG and GCT studied cases respectively, with thehighest strong positive expression observed in PGCG.Positive Ki67 expression was found in 12 cases (80% ), 13cases ( 86.7 % )and10(66.7%) of CGCG,PGCG and GCT studied cases respectively with the high proliferative expression score has been recorded in PGCG .Statistically highly significant difference was found in the Ki67expression among different giant lesion types (p=0.006), whilenon-significant difference was found in WWOX expression. Non-significant correlation was found between expression of WWOXand Ki67 in CGCG, PGCG and GCT studied cases. Conclusions: Similar immunohistochemical expression of WWOX and Ki67 ingiant cell lesions of the jaw and GCT of long boneswith non-significant correlation between them in different studied lesionssuggests that those lesions may be the same disease but with different clinical behavior. Keywords: Wwox, KI67. (J Bagh Coll Dentistry 2015; 27(1):121-127). INTRODUCTION Central giant cell lesion (CGCL) and peripheral giant cell lesion (PGCL) are pathological conditions of the jaws that share the same microscopic features, but differ clinically in terms of their behavior (1). They are of unknown origin located more frequently in the mandible than maxilla, occurring in the 2nd and 3rd decades of life. Females are more frequently affected than males (2,3). Peripheral giant cell lesion (PGCL) is considered as a reactive process associated with a local irritating factor that shows low recurrence after treatment, especially if the irritating factor is eliminated. On the other hand, central giant cell lesion (CGCL) presents a variable clinical behavior ranging from slow and asymptomatic growth without recurrence to rapid, painful and recurrent growth (4). The GCT of long bones is a rare benign neoplasm, characterized by local aggressiveness, high recurrence rates and metastasis to the lung (5).It apparently arises from the mesenchymal cells of the connective tissue frame work. (1) Assistant Professor, Department of Oral Diagnosis, College of Dentistry, University of Baghdad These cells differentiate into fibroblast-like stromal components and multinucleated cells of osteoclastic type (6-9). The principal characteristic of this tumor is the unpredictable biological behavior (8). The WWOX gene (WW-domain containing oxidoreductase) is a candidate tumor suppressor gene located at 16q23.3-24.1, spanning the second most common fragile site, FRA16D (10). The WWOX protein contains two N-terminal WW domains and a central short chain oxidoreductase- like domain (11), that mediate protein–protein interactions. WW domains mediate complexes associated with signaling pathways implicated in a variety of cellular processes such as transcriptional regulation and protein stability (12). WWOX physically interacts via its first WW domain with the p53 homolog, p73 and induces cell apoptosis (13). Numerous studies have correlated loss of WWOX expression with cancer development, including some associating WWOX alteration with poor prognosis and outcome in various cancer types (11), suggesting a growth advantage for tumors with loss of WWOX. Ki-67 represents a nuclear protein forming part of DNA replicase complex that provides a simple, rapid J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 122 and reliable means of evaluating the growth fraction of neoplastic cell populations (14). Ki-67 has a short half-life; hence it can be used as a marker for actively proliferating cells. Since it is not expressed during the resting phase of a cell cycle, it functions as a specific indicator of cellular proliferation (15). MATERIALS AND METHODS Forty five formalin-fixed, paraffin-embedded tissue blocks (15 cases of CGCG, 15 cases of PGCG and 15 cases of GCT) were obtained randomly from the archives of the department of Oral & Maxillofacial Pathology/ College of Dentistry/ University of Baghdad and Al-Shaheed Ghazi Hospital/ Medical City / Baghdad during the period (1976-2012). The clinical data were obtained from surgical reports available with the tissue specimens .The diagnosis of each case was confirmed by examining the hematoxylin and eosin (H&E) sections by two experienced pathologists. Four µm thick sections were cut for immunoshitochemcial staining with anti WWOX and anti Ki67 monoclonal antibodies (Mabs) (AbcamUK). Abcam expose mouse and rabbit HRP/DAB immunohistochemical detection kit (Catalog No. ab80436, Cambridge, UK) was used for both primary antibodies (WWOX, Ki67).Negative and positive controls were included in each IHC run. Human colon carcinoma tissue blocks were used for WWOX and tonsil tissue blocks for Ki657 (according to antibodies manufacturer). For immunohistochemistry, the sections were mounted on positively charged slides. Slides were baked in hot air oven at 65°C overnight. Sections were sequentially dewaxed through a series of xylene, graded alcohol and water immersion steps. Antigen (Ag) retrieving was done for both WWOX and Ki67 Abs as recommended by the manufacturer. Then endogenous peroxidase activity was blocked followed by blocking the non- specific staining. Primary Abs (100 ml) was applied for each section. A dilution of (1:75) for WWOX, and (1:100) for Ki67 were used. After an overnight incubation and washing with phosphate buffered solution (PBS), secondary Abs were applied, incubated and rinsed with a stream of PBS. Primary Abs were visualized with 3, 3- diaminobenzidine (DAB) chromogen, then counterstained with Mayer's hematoxyline, dehydrated and mounted. Immunohistochemical expression recorded in percentage of stained stromal cells and multinucleated giant cells in the studied lesions was classified and scored as follows: - For WWOX, cytoplasmic or cytoplasmic/nuclearstaining pattern was considered positive for WWOX immunostaining. Immunoreactivity was classified as follows: (1) negative <10%, (2) weak positive 10-50% and (3) strong positive ≥ 50% (10). For Ki67 any brown nuclear staining, regardless of its intensity was considered to be positive (16,17). Immunoreactivity was classified as follows: (-) ≤ 5% negative, (+) 6-25 % low proliferation ,(++) 26-50% moderate proliferation and (+++) 51-100% high proliferation of the considered positive cells (18). All the data of the studied samples were subjected to computerized statistical analysis using SPSS version 19 computer program. Kruskall-Wallis H test was used to compare between the percentages of markers expression among lesion types. Mann-Whitney U test used after Kruskall- Wallis H test if it is significant to test any significant difference between each two lesions. Spearman’s Rank correlation test was used to test the relation between the markers in each lesion type. The positive sign of r value means there is direct relation and vice versa. RESULTS The results of present study revealed female predilection in both central and peripheral giant cell granulomma comprising (60%) of (9) cases in each of them. Similarly, in GCT, there was female predilection comprising (53.3 %) of 8 cases as shown in table (1 and 2). The age range of the patients was 8-52years with a mean of (25.93±13.5) yearsfor C.G.C.G. and age range of 3-75years with a mean of (41.6±23.5) yearsfor peripheral giant cell granuloma Whereas for patients with giant cell tumor the age range was 2-73 years with a mean of (32.5±17.5) years. As demonstrated in table (1 and 2). Considering site distribution, giant cell granuloma lesions were distributed between upper and lower jaw as follow: C.G.C.G. presented in maxilla in 20% (3 cases) and in the mandible80% (12 cases), P.G.C.G occurred in maxilla in 46.7% (7 cases) and in the mandible 53.3% (8 cases). Whereas, Giant cell tumor cases were distributed among various body regions beginning with head area andRadius bone 33.33% (5 cases) for each of them followed by Femur bone 26.66% (4 cases) and Tibia bone 6.7% (1 case), as shown in table (1 and 2). J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 123 Table 1: The demographic and clinical description of 15 patients with central giant cell granuloma and 15 patients with peripheral giant cell granuloma Lesion type Site Sex Age Mand. Maxilla Male Female Mean S.D. Min. Max. CGCG 12 (80%) 3 (20%) 6 (40%) 9 (60%) 25.93 13.6 8 52 PGCG 8 (53.3%) 7 (46.7%) 6 (40%) 9 (60%) 41.6 23.5 3 75 Table 2: The demographic and clinical description of 15patients with Giant cell Tumor Sex Age Site Male Female Mean SD Min. Max. Tibia Femur Radius Head 7 (46.7%) 8 (53.3 %) 32.5 17.5 2 73 1 (6.7%) 4 (26.7%) 5 (33.3%) 5 (33.3%) Assessment of the immunohistochemical expression of WWOX and KI67 Mabs Positive WWOX Immunostaining was detected as brown cytoplasmic or cytoplasmic with nuclear expression, mostly in multinucleated giant cells and some of mononuclear cells of CGCG, PGCG and GCT (Fig. 1-3). Analysis ofimmunohistochemicalexpression of WWOX Ab. in CGCG,PGCG and GCT revealed positive expression in 12 cases (80% )of CGCL , 14 cases (93.3 % )of PGCL and12(80%) of GCT studied cases .Results revealed predominance of score 3 i.e strong positive expression in bothCGCG and PGCG with 10 cases ( 66.7 %) of CGCG and 12 cases ( 80%) of PGCG respectively ,whereas in GCT , positive cases of WWOX expression revealed equal distribution between strong and weak expression scores with 6 cases (40 %) for each one as shown in the table(3). Table 3: Immunohistochemical expression of WWOX in all giant cell lesions Lesions I II III Total C.G.C.G. 3 (20%) 2 (13.3%) 10 (66.7%) 15(100%) P.G.C.G. 1 (6.7%) 2 (13.3%) 12 (80%) 15(100%) G.C.T. 3 (20%) 6 (40%) 6 (40%) 15(100%) Positive KI67 Immunostaining was detected as brown nuclear expression of stromal cells ofCGCG, PGCG and GCT (Fig. 4-6). Analysis of immunohistochemicalexpression of Ki67 Ab. in CGCG,PGCG and GCT revealed positive expression in 12 cases (80% ) of CGCL , 13cases ( 86.7 % )of PGCL and10(66.7%) of GCT studied cases .Positive casesrevealed predominance of high proliferative expression scorein bothCGCG and PGCG with 9 cases (60%) of CGCG and 10 cases (66.7%) of PGCG respectively ,whereas in GCT, results revealed low proliferation score in 5 cases (33.3 %), 3 cases (20%) with moderate proliferation score and the remaining 2 cases (13.3%) with strong expression scoreas shown in the table(4). Table 4: Immunohistochemical expression of KI67 in all giant cell lesions Lesions -ve I II III C.G.C.G. 3 (20%) 1 (6.7%) 2 (13.3%) 9 (60%) P.G.C.G. 2 (13.3%) 1 (6.7%) 2 (13.3%) 10 (66.7%) G.C.T. 5 (33.3%) 5 (33.3%) 3 (20%) 2(13.3%) J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 124 Figure 1: Positive WWOX immunostaining in CGCG(X40) Figure2: Positive WWOX immunostainig in PGCG(X40) Figure 3: Positive WWOX immunostaining in GCT(X40) Figure 4: Positive Ki67 immunostaining in CGCG (X40) Figure 5: Positive Ki67 immunostaining in PGCG (X40) Figure 6: Positive Ki67 immunostaining in GCT(X40) J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 125 Comparison the percentages of Ki67 and Wwox expression among different studied giant lesion types Statistical analysis using Kruskal Wallis and Mann Whitney tests revealed that, there is a highly significant difference in the expression of Ki67 between the high PGCG mean percentage (65%) &low GCT mean percentage (25.67%) withp value =0.006 .On the other hand ,non significant difference was found in WWOX expression amongthe studied giant lesions (p=0.060) (Table 5 and 6) Table 5: Comparison the percentages of Ki67 and Wwox markers among the lesions Markers Lesions Descriptive statistics Comparison N Median Mean S.D. Min. Max. Mean Rank Kruskall- Wallis H test d.f. p-value Ki67 CGC 15 60 51.67 33.31 0 90 24.27 10.39 2 0.006 (HS) PGC 15 80 65 33.49 0 95 29.97 GCT 15 20 25.67 25.06 0 75 14.77 Wwox CGC 15 80 60 33.91 0 90 24.37 5.64 2 0.060 (NS) PGC 15 80 69.33 24.92 0 90 27.83 GCT 15 50 45 31.11 0 85 16.80 Table 6: Mann-Whitney U test after Kruskall-Wallis H test for Ki67 Lesions Mean Rank Mann-Whitney U test Z-test p-value CGC 13.27 79 -1.39 0.162 (NS) PGC 17.73 CGC 19 60 -2.19 0.028 (S) GCT 12 PGC 20.23 41 -2.97 0.003 (HS) GCT 10.77 Correlation between thepercentages of Ki67 and WWox in the studied cell lesions: Results of present study and according to spearman’s test correlation revealed non- significant correlation between the expression of WWOXand KI67 in CGCG, PGCG and GCT studied cases (Table 7-9). Table7: Relation between the percentages of Ki67 and WWOX in CGCG Markers WWOX Ki67 R 0.488 p-value 0.065 (NS) Table 8: Relation between the percentages of Ki67 and WWOX in PGCG Markers WWOX Ki67 R 0.315 p-value 0.253 (NS) Table 9: Relation between the percentages of Ki67 and WWOX in GCT Markers WWOX Ki67 R 0.339 p-value 0.216 (NS) DISCUSSION Giant cell lesions of the jaw and GCT of long bones have a distinct clinical behavior. Both lesions are characterized histologically by multinucleated giant cells in a background of ovoid to spindle-shaped mesenchymal cells. There is a basic question whether both lesions are separate entities or variants of the same disease, (18) and if biological behavior differences are supported by a distinct pattern of certain markers proteins expression or not. Therefore this study was conducted in an attempt to assess the expression of WWOX tumor suppressor gene and proliferative marker Ki67 in giant cell lesions of the jaw bones in comparison to the giant cell tumor of long bones and to correlate their expression with each other in each studied lesion J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 126 to explain its possible role in the biological behavior of giant cell lesions and tumor. The WWOX gene is a recently cloned tumor suppressor gene that spans the FRA16D fragile region. WWOX protein contains two WW domains that are generally known to mediate protein-protein interaction (13). The subcellular localization of WWOX has been a controversial issue among the different research groups. Numerous immunohistochemical studies have shown that WWOX is a cytoplasmic protein both in normal and neoplastic tissues (20-25). Other laboratories have reported that WWOX localizes in mitochondria and nuclei of some cells (26). In the present study, both cytoplasmic and cytoplasmic/nuclear staining pattern was considered positive in both mononuclear and multinucleated giant cells. Results revealed strong positive WWOX expression in majority of PGCG and CGCG studied cases, mostly in the giant cells; this finding is supported by results of previous study (27). These findings support what previously reported that WWOX has an important role in apoptosis since it is mostly expressed in giant cells which have been shown to be the main source of this apoptotic event (26). Concerninig expression in GCT ,and up to my knowledge this is the first study that attempt to compare WWOX expression in giant cell lesions of the jaw bones and giant cell tumor of long bones.The present finding showed that the majority of GCT cases showed positive expressionwith non-significant difference obtained in WWOX expression amongCGCG, PGCG and GCT studied cases.This finding is in accordance with previous study conducted using other tumor suppressor gene (28) which supports the theory that these lesions are a spectrum of disease rather than different entities. On other hand the present findings indicate that WWOXcould not be used to explain the differences between the giant cell lesions of the jaws and GCT of the long bones. The Ki-67 antigen is a human nuclear protein used as a marker for cellular proliferation (15) Ki- 67 antigen is expressed during the G1, S, G2 and M phases of the cell cycle within the nucleus but is not expressed during the G0 (resting) phase, and thus it is a widely accepted proliferation marker and is useful in predicting the development of human neoplasm (14). Although the biological behavior is not only reflected by the proliferation index by tumor cells, but still it represents a clue on tumor activity .The results of previous study showed greaterKi-67 immunoreactivity in PGCL compared to CGCL(29). Similar results obtained in the present study. Additionally, CGCG and PGCG had a higher proliferative activity than GCT with a highly significant difference in Ki67 expression was found between CGG, PGCG and giant cell tumor. Similar results obtained in previous studies (29-31). 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Maria do Socorro Aragão; Marta RabelloPiva; Cassiano Francisco WeegeNonaka; Roseana de Almeida Freitas; Lélia Batista de Souza; Leão Pereira PintoCentral giant cell granuloma of the jaws and giant cell tumor of long bones - an immunohistochemical comparative study. J Appl Oral Sci 2007;15:4. الخالصة ان ورم الخالیا العمالقة ھومن االورام الحمیدة المحددة .ان آفة الخالیا العمالقةالطرفیةوآفة الخالیا العمالقة المركزیة للفكین لھاسلوكسریري واضح :الخلفیة من الخالیا البیضویة الى مغزلیة الشكل من خالیا اللحمة العدوانیة یصیب العظام الطویلة وتتمیز كال االفات تشریحیا من الخالیا العمالقة المتعددة النوى في خلفیة ھو ) Ki-67(ان بروتین .وھي المنطقة التي تضم الموقع البشري الھش االكثر شیوعا) 16q23.2–16q23.1(یقع ضمن منطقة) WWOX(ان جین .المتوسطة ھدفت .ثناء كل االطوار الفعالة من دورة الخلیة ولكنھ غائب عن الخالیا الساكنةان ھذا البروتین موجود ا.بروتین نووي مقترن بعملیة تكاثر الخالیا وضروري لھا في آفات الخالیا العمالقة للفكین ) ki67(ومعلم التكاثر ) WWOX(ھذه الدراسة الى تقییم ومقارنة االظھار الكیمیائي النسیجي المناعي للجین المثبط لالورام .والعظام الطویلة تم صبغ .تم تضمین خمس واربعین عینة نسیجیة استرجاعیةمطمورة بالبارافین من آفات الخالیا العمالقة للفكین والعظام الطویلة في ھذه الدراسة : المواد والطرق ).ki67(وال) WWOX(المقاطع النسیجیة لھذه العینات باستخدام الصبغات النسیجیة المناعیة بمضادات ال (,)CGCG(من الحاالت المدروسة من ال %) 80( حالة 12و%) 93.3(حالة 14,%) 80(حالة 12في ) WWOX(ل وجد االظھار االیجابي ل:النتائج PGCG (و)GCT (ووجد االظھار االیجابي لل .على التوالي)Ki67 ( حالة 12في)حاالت من الحاالت المدروسة من 10و %) 86.7(حالة 13,%) 80 )CGCG ( ,)PGCG(و)GCT (یل مؤشر عالي للتكاثر في على التوالي مع تسج)PGCG.( وجد فرق كبیر للغایة بین الحاالت المدروسة في اظھار ال )Ki67 ( مع عدم وجود فرق بینھا في درجة اظھار ال)WWOX.( تم العثور على عالقة غیر كبیرة بین ال)WWOX ( و)Ki67 ( في مختلف آفات الخالیا .العمالقة المدروسة في آفات الخالیا العمالقة في الفكین والعظام الطویلة مع وجود عالقة غیر معنویة ) Ki67(و) WWOX(الكیمیائي النسیجي المناعي للتشابھ الظھور :االستنتاجات .بینھما في مختلف الحاالت المدروسة االمر الذي یدل على ان ھذه اآلفات قد تكوننفس المرض ولكن بحاالت سریریة مختلفة