Ibtehal Final.doc J Bagh College Dentistry Vol. 26(2), June 2014 Immunohistochemical Oral Diagnosis 74 Immunohistochemical expression of HOXA1, and Ki-67 proteins of oral squamous cell carcinoma Ibtehal Qhtan Al-Etbi, B.D.S., M.Sc. (1) Riyadh Othman Al-Kaisi, B.D.S., M.Sc., Ph.D. (2) ABSTRACT Background: Oral squamous cell carcinoma (OSCC) is the most prevalent malignant neoplasm of the oral cavity and constitutes a major health problem in developing. In the last 30 years, the 5-year survival rate of patients with oral SCC has not improved despite advance in diagnostic techniques. To improve early diagnosis for this deadly disease, new biological markers are needed. HOX genes encode homeodomain-containing transcription factors involved in the regulation of cellular proliferation and differentiation during embryogenesis. HOX gene expression has been described in several adult tissues, where they performed important roles in maintaining homeostasis. Few studies have suggested that HOXA1 plays a role in tumorigenesis. Besides being overexpressed in several tumors, HOXA1 influences numerous cellular processes including proliferation, apoptosis and epithelialmesenchymal transition (EMT), and HOXA1 overexpression is sufficient for malignant transformation ofnontumorigenic epithelial cells. Ki-67 is a specific marker of proliferation and the expression of which is strictly associated with cell proliferation and is widely used in pathology as a proliferation marker to measure the growth fraction of cells in human tumors.The aims of this study were to evaluate the immunohistochemical expression of HOXA1 & Ki-67 in OSCC & to correlate the expression of the studied markers with the clinicopathological findings and with each other Materials and Methods: Thirty formalin-fixed, paraffin- embedded tissue blocks of oral squamous cell carcinoma were included in this study. H&E stain was done for each block for reassessment of histological examination. An immunohistochimical stain was performed using anti HOXA1 and anti Ki-67 poly clonal antibodies. Results:The expression of HOXA1 and Ki-67were positive in all oral squamous cell carcinoma cases & in all layers (100%), while the expression was restricted to the basal and supra basal layer in normal oral mucosa. Statistically non-significant correlation observed between each marker with clinico-pathological parameters. While a statistically significant association was found between the expressions of two markers, (p-value= 0.027). Conclusion: The statistically significant association observed between expressions of HOXA1 with the specific marker of proliferation Ki-67. This suggested important role in oral SCC development and progression. Keywords: OSCC, HOXA1, Ki-67. (J Bagh Coll Dentistry 2014; 26(2): 74-78). الخالصة :الخلفیة الطبیة التشخیصیة إال إن وبالرغم من تطور التقنیات . تجویف الفمي ویمثل المشكلة الرئیسیة المؤدیة للوفاة في بلدان العالم الثالثالسرطان الخالیا الحرشفیة ھو السرطان السائد في إن الكشف المبكر لسرطان الخالیا الحرشفیة الفموي مھم جدا للحد من خطورتھ . مستوى سنوات البقاء الخمسة المعتمدة في علم األورام لم یتطور بشكل مفید في السنوات الثالثین االخیرة المحفزة للنمو والتمایز اثناء النمو Homeoboxالتابعة لجینات HOXوھو احد افراد عائلة جین HOXA1ولذلك تم التركیز على إیجاد واسمات بیولوجیة جدیدة ومنھا جین وقد اثبتت دراسات حدیثة دوره المسرطنالفعال في العدید من االمراض السرطانیة حیث یظھر بشكل غیر . الجنیني والتكوین العضوي وقد یظھر في األنسجة البالغة عند الحاجة ایضا مبكر لسرطان ھو مؤشر التكاثر الرئیسي في النواة وھو المساعد في كشف وجود اي انقسامات في األنسجة وبالتالي فلھ فائدة عظیمة في التنبؤ والكشف ال Ki-67 .في األنسجةمتوازن وكذلك ربط ظھور كل منھما مع . Ki-67 لجین بمؤشر التكاثرفي السرطان الحرشفي للفم وربط ظھور ا HOXA1 تھدف الدراسة الحالیة الى التحري والتحقق من ظھور جین. الفم . المعطیات السریریة المرضیة لسرطان الفم الحرشفي مع ین والمطمورة بشتضمنت ھذه الدراسة ثالثین عینة استرجاعیة ألشخاص مصابین بسرطان الفم الحرشفي والتي استخرجت من المقاطع النسیجیة المثبتة بالفورمال: المواد والطرق بعد لك اجریت الصبغات الكیمیائیة النسیجیة المناعیة باستخدام . البارافین وجرى صبغ كل عینة بصبغتي الھیماتوكسلین واالیوسین إلعادة تقییمھا لغرض الفحص النسیجي المرضي . على شرائح نسیجیة دقیقة من العینات Ki-67 ومضاد HOXA1 مضاد اما نسبة إصابة الذكور %) . 70( ت ھذا السرطان تقع في االعمار التي تفوق الخمسین عامًا وأن معظم تلك الحاالت تركزت في الذكور وبنسبة أظھرت الدراسة أن اكثر حاال: النتائج %) 73,3(ت سریریا بشكل اورا م ومعظمھا ظھر%) 36,7(كذلك اظھرت الدراسة ان معظم الحاالت كانت في اللسان ). 1:2:(الى االناث فقد اظھرت ھذه الدراسة إلى انھا تساوي من النوع الواضح التمایز لسرطان الفم %) 40,0(من الحاالت السرطانیة ھي من النوع المتوسط التمایز و%) 43,3( اما الفحوصات النسیجیة المرضیة لھذه الدراسة فقد أظھرت ان . كان ایجابیًا في الطبقة السفلى فقط من النسیج المخاطي الفموي الطبیعي، بینما كان ایجابیًا في كل Ki-67ومؤشر التكاثر HOXA1 اظھرت ھذه الدراسة ایضًا ان تعبیر. الحرشفي HOXA1 مما یدل على الدور المھم لجین Ki-67 ومؤشر التكاثر HOXA1 كذلك اظھرت ھذه الدراسة وجود عالقة واضحة بین ظھور جین. طبقات النسیج الحرشفي لسرطان الفم واخیرا بینت ھذه الدراسة عدم وجود ایة عالقة بین العاملین السابقین والمتغیرات السریریة المرضیة مسرطن وذلك بواسطة تحفیز انقسام الخالیا وبالتالي النمو السرطاني الفمويكعامل .االخرى المصاحبة لسرطان الفم الحرشفي في انقسام الخالیا والنمو السرطاني لذلك تقترح ھذه الدراسة اجراء دراسات HOXA1 داللة على دور جین Ki-67 مع مؤشر التكاثر HOXA1 تالزم وجود جین: االستنتاجات . او مع مؤشر تكاثر اخر Ki-67 مع مؤشر التكاثر HOX او عضو اخر لجین HOXA1 جدیدة بعینات اكثر عددا لمعرفھ الدور الحقیقي لجین INTRODUCTION Squamous cell carcinoma (SCC) accounts to more than 90% of malignant tumors of the oral cavity and oropharynx. It is often related to considerable mortality and morbidity rates, and presents a variable etiology related to alcohol and tobacco abuse associated with genetic factors (1,2). (1) Master student, Department of Oral Diagnosis, College of Dentistry, University of Baghdad. (2) Professor, Department of Oral Diagnosis, College of Dentistry, University of Baghdad. The homeobox genes, is a master regulators of morphogenesis and cell differentiation during embryogenesis, have emerged as potential candidates to be also involved with carcinogenesis. This important family of genes codes regulatory proteins that act as transcriptional factors controlling the development of several tissues including orofacial tissue (3,4). HOXA1 is a member of HOX genes family which issubgroup of homeobox genes have important role in OSCC development and J Bagh College Dentistry Vol. 26(2), June 2014 Immunohistochemical Oral Diagnosis 75 progression. Ki-67 is a cell cycle associated human nuclear protein present in peri- chromosomal region (5). The estimated half-life of Ki-67antigen is 60- 90 minutes, and the Ki-67antigen starts to be expressed in S phase, progressively increasing through S and G2 phase and reaching a plateau at mitosis. After cell division, the cell return to G1with a stock of Ki-67antigen, whose level decreases rapidly during this phase (6,7). This study aimed to: • Evaluate the immunohistochemical expression of HOXA1 and Ki-67 markers in oral squamous cell carcinoma. • Correlate the expression of either marker with each other and with the Clinico-pathological parameters (age, sex, tumor site, clinical presentation, and histopathological grades) of OSCC. MATERIALS AND METHODS A retrospective study was performed on thirty- formalin- fixed paraffin embedded blocks of OSCCwere collected from the archives of Oral Pathology laboratory, College of Dentistry, Baghdad University, Al-Kadhimiya teaching hospital, and Al-Shaheed Ghazi Hospital/ Medical City / Baghdad from (2010-2013).The diagnosis of each case was confirmed by examining the Hematoxylin and Eosin (H&E) sections by two experienced pathologists. Four micrometer thick sections were cut and mounted on positively charged slides and stained immunohistochemically with monoclonal antibodies using anti HOXA1 and anti Ki- 67polyclonal antibodies (Abcam UK). Abcam expose mouse and rabbit HRP/DAB immunohistochemical detection kit (Catalog No. ab80436, Cambridge, UK) was used. RESULTS Clinicopathological Findings of OSCC cases were designed as follows: Most of the cases 21 (70%) aged were above 50 years and the majority of the cases were males 21 (70 %). The most common site was the tongue 11 cases (36.7%) and most of the cases were presented as mass22 cases (73.3%). Histopathological examination showed that 13 cases of OSCC (43.3%) were moderately differentiated, followed by 12 cases (40%) well differentiated and 5 cases (16.7%) were poorly differentiatedas shown in table (1). Immunohistochemical staining with HOXA1primary antibody showed that HOXA1 expression was positive in all examined OSCC specimens andwas observed as a nuclear stain restricted to the basal and suprabasal layers in healthy mucosae figures (1), whereas a broad positivity with variable distribution and intensity was found in the OSCC samples as shown in figures (2,3). score +1 was found in 26.6% (8 cases), score +2 and score +3 both found in 36.7% (11 cases) table(2) . Immunohistochemical staining with Ki-67 primary antibody showed that Ki-67 expression was positive in all examined OSCC specimens. Ki-67immunostaining as shown in figure (4) was observed as a nuclear stain restricted to the basal layer in healthy mucosae, whereas a broad positivity with variable distribution and intensity was found in the OSCC samples as shown in figures (5,6). Half of the cases (15 cases) were moderately proliferated score (++) and other (15 cases) were highly proliferated score (+++). Regarding correlation of two markers with the clinico- pathological findings of OSCC cases reveal that There was no significant correlation of these two markerswith the clinico- pathological findings(age, sex, tumor site, clinical presentation and histopathological grades). Concerning Correlation between HOXA1 and Ki-67 expression score Table (3), result of present study revealed statistically significant positive correlation with P value = 0.027. Figure 1: HOXA1 Expression in normal oral mucosa (10X). Figure 2: Positive expression of HOXA1 in well differentiated OSCC (10X). J Bagh College Dentistry Vol. 26(2), June 2014 Immunohistochemical Oral Diagnosis 76 Table 1: Clinico-pathological characteristics of 30 OSCC cases Percent % Frequency Age 70 21 >50 30 9 24-50 Sex 70 21 Male 30 9 Female Tumor site 36.7 11 Tongue 23.2 7 Maxilla 20 6 Mandibul 6.7 2 floor of mouth 6.7 2 buccal mucosa 6.7 2 Lip Histological Grading 40 12 Well 43.3 13 Moderate 16.7 5 Poor Clinical Presentation 73.3 22 Mass 20 6 Ulcer 6.7 2 White lesion Table 2: HOXA1 expression in 30 cases of OSCC HOXA1 score Frequency Percent Score 1+ 8 26.6% Score 2+ 11 36.7% Score 3+ 11 36.7% Total 30 100% Figure 3: Positive expression of HOXA1 in moderate differentiated OSCC (10X). Table 3: Correlation between HOXA1and Ki-67expression in OSCC. HOXA1 score Ki-67 score Total X2 Sig. ++ +++ N % 1+ 5 3 8 26.6 7.227 0.027 (S) 2+ 8 3 11 36.7 3+ 2 9 11 36.7 Total 15 15 30 100 Figure 4: Ki-67 Expression in normal oral mucosa (10X). Figure 5: Positive expression of Ki-67 in well differentiated OSCC (10X). Figure 6: Positive expression of Ki-67 in moderately differentiated OSCC (10X). DISCUSSION Concerning the epidemiological parameters, including age, sex, site, clinical presentation, studies showed variable results; these inconsistent findings among different studies could be credit with the fact that the current study and some of the others are not an epidemiological type of studies, therefore the limited number and the random selection of the cases according to what is available preclude for definitive clinical findings. J Bagh College Dentistry Vol. 26(2), June 2014 Immunohistochemical Oral Diagnosis 77 Assessment of HOXA1 Immunohistochemistry Immunoreactivity for HOXA1was observed as nuclear stain. Positive HOXA1 expression was observed in all the studied cases of OSCC this finding was agreed with previous study (8) which was similar to this study.cytoplasmic staining was also observed in some cases with nuclear stain, this explain by interaction between HOXA1 with numerous protein and transcription factors which was present primarily in the cytoplasm and involved in critical developmental process and then upon activation translocate to the nucleus to perform their function this interaction improved by study (9). In normal oral mucosa, immuno staining was restricted to the basal and suprabasal layers only due to the fact that squamous epithelium keeps a continuous physiological regeneration in normal conditions, while broad positivity with variable distribution in OSCC sample, the intensity of HOXA1expression was found beyond basal localization suggests that a correlation between HOXA1 expression and tumor progression may exist. Few studies(8)concerned HOXA1expression in OSCC which may be due to the fact that recently more attention has been paid to study this genes andTo our knowledgethis study is the first study in Iraq which demonstrates the HOXA1 expression in OSCC particularly or other cancer. However, many other studies (9-12) show expression of various members of HOX gene family in OSCC. Furthermore, aberrant expression of numerous HOX genes has been reported in various malignancies such as hematological malignancies (10) and variety of other solid tumors (13-15) Regarding Correlation of HOXA1 expression with clinic-pathological parameters; this study revealed that HOXA1 expression was not correlated with age, sex, clinical presentation and location of tumor. This finding was in agreement with previous study concerning OSCC (8), a non- significant correlation also was found concerning HOXA1 expression and different tumor grades, opposite results were found by previous study (8) Assessment of Ki-67 immunohistochemistry: Cell proliferation is a biological process of vital importance and this control is lost in cancer (16). Therefore, the knowledge of cellular proteins that control cell proliferation is essential for understanding tumor biology (17,18). Ki-67 antigen is a specific marker of proliferating cells (19).The IHC reactivity for NF Kb p65 was evaluated on the basis of presence or absence of brown nuclear and cytoplasmic staining (20) This study showed positive nuclear staining of Ki-67 antigen in all OSCC cases and in all layers, whereas in normal oral mucosa positive Ki-67 immunoreactivity was seen in the basal cell layer only (5). In addition, half of the positive cases showed high expression score and other half showed moderate expression score. Regarding the correlation of Ki-67 positive expression with age the results of the present study showed statistically non-significant correlation in Ki-67 expression between the two age groups. This finding agreed with previous study (21) and disagreed with other (22). Regarding the sex and sit of tumor there was statistically non-significant correlation. This is in accordance with (20-23), Also non-significant correlation was found in Ki-67 expression with different histopathological grades this finding was agree with some studies (1,24,25) and disagree with others (16,22,26,27) . Correlations between HOXA1 and Ki-67 expression in OSCC Uncontrolled cell proliferation plays a critical role in the development of a wide variety of carcinomas. 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