Thaer.doc J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 128 Immunohistochemical expression of MMP9, as a marker of local invasion in Hodgkin’s and Non-Hodgkin’s lymphoma of the head and neck region Thaer K. Ali, B.D.S., M.Sc. (1) Bashar H. Abdulla, B.D.S., M.Sc., Ph.D. (2) Khitam R. Kadhim, M.B.Ch.B., F.I.C.M.S. (3) ABSTARCT Background: Malignant lymphoma is the generic term given to tumors of the lymphoid system and specifically of lymphocytes and their precursor cells, While all lymphomas are malignant neoplasms, there is a wide spectrum of clinical behavior, with some following an indolent clinical course and others behaving in an aggressive manner (will causing death in a short time frame if left untreated). The metastatic process involves intravasation and extravasation of tumor cells, followed by reimplantation of tumor cells, formation of a new tumor stoma, Degradation of the extracellular matrix and components of the basement membrane by proteases facilitates the detachment of tumor cells, their crossing of tissue boundaries, and invasion into adjacent tissue compartments. The importance of tumor-associated proteases in invasion and metastasis has been demonstrated for a variety of solid malignant tumors. MMP family has been implicated in tumor cell invasion and metastasis. Material and methods: This study included 67 formalin-fixed, paraffin-embedded histopathologically diagnosed lymphoma blocks (head and neck lesions), 24 Hodgkin’s Lymphoma and 43 Non Hodgkin’s Lymphoma. Immunohistochemical (IHC) evaluation of MMP-9 monoclonal antibodies), in relation to the clinicopathological parameters was assessed. Results: The mean of expression of MMP-9 in NHL was higher (65.4±18) than that in HL (56.7±21.4) though it did not reach the level of statistical difference P > (0.05), while the mean of expression of MMP-9 in relation to tumor grades was different as it had a value of (72±14.1) in low grade tumors, (68.3±15) in the intermediate grade tumor and (61.4±20.7) in high grade tumors; consequently, this difference did not reached the level of statistical significance P(ANOVA) >0.05 . MMP 9 had no statistical significant correlation in regard to its mean in HL subtypes. Conclusions: This study had shown that there was no significant correlation between age and mean of expression of MMP-9 in HL and NHL. There was no statistical significant difference in the mean of expression of MMP-9 between HL and NHL subtypes. Keywords: MMP-9, local invasion, Hodgkin’s lymphoma, Non Hodgkin’s Lymphoma. (J Bagh Coll Dentistry 2015; 27(1):128-132). INTRODUCTION Malignant lymphomas can be divided into two major categories: HL and NHL. Lymphomas presenting in extranodal sites of the head and neck and these sites includes oral cavity, oropharynx, nasopharynx, paranasal sinuses, and larynx which are mainly NHLs of low or high grade, it may also present as cervical lymphadenopathy which is the most common head and neck presentation for both diseases (1,2). The metastatic process involves intravasation and extravasation of tumor cells, followed by reimplantation of tumor cells, formation of a new tumor stoma, and neoangiogenesis to consolidate a secondary tumor at a distant site (3). Degradation of the extracellular matrix and components of the basement membrane by proteases facilitates the detachment of tumor cells, their crossing of tissue boundaries, and invasion into adjacent tissue compartments. (1) M.Sc Department of Oral and Maxillofacial Pathology, College of Dentistry, University of Baghdad. (2) Professor. Head Department of Oral and Maxillofacial Pathology, College of Dentistry, University of Baghdad. (3) Assistant Professor. Head Department of Pathology& forensic medicine, College of Medicine University of Baghdad. The importance of tumor-associated proteases in invasion and metastasis has been demonstrated for a variety of solid malignant tumors. MMP family has been implicated in tumor cell invasion and metastasis. As tumors vary considerably in their behavior; the degree of their differentiation and ability to invade and metastasize are not the same, however, not all cancers have equivalent ability to metastasize. In general the more likely anaplastic and larger is the primary neoplasm, the more likely is the metastatic spread, however, exceptions abound, i.e. extremely small cancers have been known to metastasize and conversely some large ugly lesions may not spread (4,5). Because of the obscure and variable biological behavior of head and neck Hodgkin lymphoma this study will concerned with tumor dynamics such as MMP- 9 as a biological marker in HL and NHL of the head and neck. The aim of this study is to evaluate the expression of the biological marker of local invasion (MMP-9) in Hodgkin’s and Non- Hodgkin’s lymphoma via immunohistochemical technique. J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 129 MATERIALS AND METHODS This study included (67) formalin-fixed, paraffin-embedded histopathologically diagnosed lymphoma blocks (head and neck lesions). The diagnosis of each case was confirmed by the histological examination of the Hematoxylin and Eosin staining (H&E), examined by two experienced pathologists. Demographic and clinical data provided by the surgeon were obtained from the case sheets presented with the tumor specimens, including information concerning patient's age, gender, clinical presentation, site of tumor. Histological classification was determined according to the International Working Formula (IWF) criteria, where the lesions classified into Hodgkin's lymphoma (24cases) and Non-Hodgkin's lymphoma (43 cases). Positive tissue controls included in this study were Breast carcinoma for MMP-9. The diagnosis of each case was confirmed by the use of CD15, CD30 for HL and CD20, bcl2 for NHL. Sections of 5µm thickness were mounted on glass slides for routine (H&E), from each block of the studied sample and the control group for histopathological re-examination. Other sections of 4µm thickness were mounted on positively charged microscopic slides (Biocare medical and Fischre brand) to obtain a greater tissue adherence. All of these collected specimens were subjected to immunohistochemical staining using specific monoclonal antibodies included in the study MMP- 9. Abcam, Expose mouse and rabbit specific HRP/DAP detection IHC kit, Antigen Retrieval Solutions: - Citrate buffer PH 6.0., Mouse monoclonal [ab58803] to MMP9 Abcam, England. Immunohistochemical staining is accomplished with antibodies that recognize the target protein. Only the number of cells showing cytoplasm and the cell membrane expression of MMP9 was quantified by counting at least 1000 cells in five representative fields at 40X objective in each case, the intensity of staining was not considered for evaluation. Tumor cells with brown staining of the cell membrane predominately or of the cytoplasm and the cell membrane occasionally were considered positive for MMP-9 The percentage of MMP-9 positively stained cells was semi-quantitatively determined as follows (-) negative ≤5%, (+) low 6-25%, (++) moderate 26-50% and (+++) high 51-100%. Statistical Analysis An expert statistical advice was sought for. Statistical analyses were done using SPSS version 21 computer software (Statistical Package for Social Sciences) in association with Microsoft Excel 2010. RESULTS The mean of expression of MMP 9 in NHL was higher (65.4±18) (Fig 1, 2 and 3) than that in HL (56.7±21.4) (Fig 4 and 5) though it did not reach the level of statistical significant difference P >0.05, this result is shown in Table 1 Regarding NHL the mean of expression of MMP 9 in low grade was 72±14.1 and in intermediate grade the mean was 68.3±15 while in high grade it had a mean of 61.4±20.7 and even when the mean of MMP -9 was different between the grades of NHL but it did not reach the level of statistical significance. These details are found in Table 2. In HL cases the mean of expression of MMP 9 in mixed cellularity (65.9±19.4) was higher than that of nodular sclerosing (53.3±22.2). Obviously there was a difference in the mean of expression but it did not reveal any statistical significance. Table 3 gives the difference in mean of expression of MMP 9 between nodular sclerosing and mixed cellularity. Table 1: Difference in mean of expression of MMP 9 between HL and NHL MMP9 NHL compared to HL P (t-test) HL NHL Range (17 - 93) (23 - 97) 0.08 [NS] Mean 56.7 65.4 SD 21.4 18 SE 4.36 2.75 N 24 43 J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 130 Table 2: Difference in mean of expression of MMP 9 between tumor grades of NHL MMP9 Tumor grade P (ANOVA) trend Low grade Intermediate grade High grade Range (49 - 92) (38 - 97) (23 - 97) 0.21 [NS] Mean 72 68.3 61.4 SD 14.1 15 20.7 SE 5.77 3.75 4.52 N 6 16 21 Table 3: Difference in mean of expression of MMP 9 between nodular sclerosing and mixed cellularity MMP9 Morphology P Nodular sclerosing Mixed cellularity Range (17 - 93) (37 - 87) 0.21 [NS] Mean 53.3 65.9 SD 22.2 19.4 SE 5.55 7.33 N 16 7 Fig 3: NHL Small cell type MMP9 cytoplasmic expression (X400) Fig 4: HL Mixed cellularity MMP9 expression in Hodgkin’s cells. (X1000) Fig 1: NHL DLBC MMP9 cytoplasmic expression (X400) Fig 2: Burkitt’s lymphoma MMP9 expression (X400) J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 131 DISCUSSION Matrix metalloproteinase (MMP)-mediated degradation of the extracellular matrix is a key point in tumor development and expansion. A former study found elevated expression of MMP9 and MMP10 in endometrial tumor related endothelium. Xue-lian du et al (6) stated that the metastatic process involves intravasation and extravasation of tumor cells, followed by reimplantation of tumor cells, formation of a new tumor stroma, and neoangiogenesis to consolidate a secondary tumor at a distant site. Degradation of the extracellular matrix and components of the basement membrane by proteases facilitates the detachment of tumor cells, their crossing of tissue boundaries, and invasion into adjacent tissue compartments. In recent years, the importance of tumor- associated proteases in invasion and metastasis has been demonstrated for a variety of solid malignant tumor. One of the first observations that suggested a role for MMP-9 in tumor invasion relates to the fact that the release of MMP-9 is associated with the metastatic phenotype of transformed rat embryo cell 92,000 gelatinase release correlates with the metastatic phenotype in transformed rat embryo cells (7,8). The increase in pro-MMP-9 in ovarian cancer and their corresponding metastases could be attributable to the infiltration of inflammatory cells that express a high amount of pro-MMP-9 and often surround malignant tumors that growth of liver metastasis from colon cancer was associated with elevated tumor tissues MMP-9, and treatment of ulinastatin significantly reduces MMP-9 expression (9,10). MMP-9 may be particularly relevant to the progression of lymphomas. MMP-9 has been shown to be important for the in vitro degradation of extracellular matrix components by non- Hodgkin's lymphoma cells (11). In vivo, MMP-9 is also overexpressed in a subset of high grade non- Hodgkin's lymphomas, and this correlates with a poor clinical outcome. The results of this study showed that MMP-9 is consistently expressed by the malignant Hodgkin and Reed-Sternberg cells of Hodgkin's disease (12). This study had showed that the expression of MMP 9 in HL56.7±21.4mean of expression and in NHL the mean was 65.4±18 with positive staining in all cases and this is in accordance with previous studies in regard to its expression in all malignant tumors moreover its expression was higher in NHL cases than HL cases but it did not reached the level of statistical significance. The infiltration of inflammatory cells that express a high amount of pro-MMP-9 and often surround malignant tumors (10). MMP-9 may be particularly relevant to the progression of lymphomas which has been shown to be important for the in vitro degradation of extracellular matrix components by non- Hodgkin's lymphoma cells; MMP-9 is also overexpressed in a subset of high grade non- Hodgkin's lymphomas, and this correlates with a poor clinical outcome. The results of previous study showed that MMP-9 is consistently expressed by the malignant Hodgkin and Reed- Sternberg cells of Hodgkin's disease12. Regarding the grades of the tumors, MMP- 9 showed negative non-significant correlation with the tumor grades and this in agreement with other studies (13,14) whom concluded from their study that there is no correlation was found between MMP-9 expression and tumor invasion or histological grade; whereas other studies had reached to contradictory results as the levels of MMP 9 and MMP 2 are highly correlated with the histological grade of malignancy (15, 16). Moreover other study reached to a conclusion that MMP-9 has been showed a tendency to increase with increasing tumor grade (15, 17). The possible explanation for this difference in relation of MMP 9with tumor grade might be attributed to Fig 5: HL Mixed cellularity MMP9 expression in Hodgkin’s cells. (X400) J Bagh College Dentistry Vol. 27(1), March 2015 Immunohistochemical Oral Diagnosis 132 microenvironment component interaction and to small sample size of this study as compared with large studies. REFERENCES 1. Rosai J. Ackerman's Surgical Pathology. 10th ed. Elsevier, Mosby; 2011. 2. Rubin R, Strayer DS. Rubin's Pathology. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2012. 3. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 57-70. 4. Kumar A, Boriek AM. Mechanical stress activates the nuclear factor-kappaB pathway in skeletal muscle fibers: a possible role in Duchenne muscular dystrophy. FASEB J 2003; 17: 386–96. 5. Arshak SS. Review. In: (eds.) 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J Neurooncol 2002; 60: 235- 45. الخالصھ ینما كل اللمفوما ھي اورام خبیثھ وھناك اللمفوما الخبیثھ ھو مصطلح عام یعطى الى االورام التي تصیب الجھاز اللمفاوي وبصورة خاصھ الخالیا اللمفاویھ والخالیا المكونھ لھل ب: خلفیھ عملیة االنتشار تتضمن الدخول . الجطیف واسع من التصرف العیادي حیث بعضھا یتبع نمطا عیادیا بطیأ واالخر یتصرف بطریقھ عدوانیھ مسببا الموت بفتره قصیره اذا ترك بدون ع تحلل المصفوفھ الخارجیھ للخالیا ومكونات الغشاء . بوعة باستزراع الخالیا السرطانیھ مكونھ حشوه سرطانیھ جدیدهفي االوعیھ الدمویھ والخروج منھا بالنسبھ للخالیا السرطانیھ مت لالورام في اھمیھ محلالت البروتین المصاحبھ . القاعدي بواسطھ محلالت البروتین یسھل أنفصال الخالیا السرطانیھ وعبورھا حدود االنسجھ وھجومھا الى االنسجھ المجاوره المستقلھ .قد تم تضمینھا على خالیا الورم اثناء الغزو واالنتشار MMP 9عائلة . عملیة الغزو واالنتشار قد تم استیضاحھا النواع من السرطانات الخبیثھ الصلبھ اربعھ وعشرون حالة ھودجكن لمفوما وثالثھ وأربعون .سبعھ وستین نسیج مثبت بالفورمالین ومطموره بشمع البرافین، مشخصھ نسیجیا لمفوما من منطقة الراس والعنق :المواد والطرق .لمعاییر العیادیھ المرضیھكمضاد احادي النسل وربط ذلك مع ا MMP 9تقنیة المناعھ الكیمیائیھ النسیجھ قد استعملت لتقییم تعبیر . الھوجكن لمفوما ورغم ذلك لم تصل الى مستوى االختالف االحصائي 21.4± 65من ما في ھودجكن لمفوما، 18± 65.4في ھودجكن والھودجكن لمفوما كان اعلى MMP 9معدل تعبیر :النتائج P>0.05 بینما معدل تعبیرMMP 9 في الورم متوسط الدرجھ و 15±68.3في الورم واطيء الدرجھ 72± 14.1وعالقتھ بدرجة الورم كان مختلف حیث كانت لھ قیمھ لم تكن لھ عالقھ أحصائیھ مھمھ بالنسبھ الى أنواع ھودجكن .P>0.05 MMP 9في الورم عالي الدرجھ وبالنتیجھ ھذا االختالف لم یصل الى المستوى االحصائي المھم 61.4±20.7 .لمفوما لم تكن ھناك أختالفات أحصائیھ مھمھ بالنسبھ الى معدل . في ھودجكن وال ھوجكن لمفوما MMP9ھ بأنھ لم تكن ھناك عالقھ مھمھ بین معدل تعبیر أظھرت ھذه الدراس :االستنتاجات .بین ھودجكن والھودجكن وأنواعھا الفرعیھ MMP 9تعبیر