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ANALYSIS OF NF-κB, CASPASE-3, MMP-2, MMP-9 EXPRESSIONS, AND THEIR RELATIONSHIP WITH EBV-LMP-1 IN CLASSICAL HODGKIN LYMPHOMA “NF-κB, CASPASE-3, MMP-2, MMP-9 AND EBV-LMP-1 IN CHL” 1. İbrahim Metin Çiriş, M. D., Associate Professor, Süleyman Demirel University, Faculty of Medicine, Department of Pathology, Isparta, Turkey 2. Sema Bircan, M. D., Professor, Süleyman Demirel University, Faculty of Medicine, Department of Pathology, Isparta, Turkey 3. Şirin Başpınar, M. D., Assistant Professor, Süleyman Demirel University, Faculty of Medicine, Department of Pathology, Isparta, Turkey 4. Gülsün İnan Mamak, M. D., Consultant, Burdur State Hospital, Department of Pathology, Burdur, Turkey 5. Kemal Kürşat Bozkurt, M. D., Assistant Professor, Süleyman Demirel University, Faculty of Medicine, Department of Pathology, Isparta, Turkey Corresponding Author’s Address: Kemal Kürşat Bozkurt, Süleyman Demirel Üniversitesi Tıp Fakültesi Araştırma ve Uygulama Hastanesi Tıbbi Patoloji Anabilim Dalı 32260 Isparta/Türkiye, +90 246 2119421 (Phone), +90 246 2112830 (Fax) This study was presented as a poster presentation in the 21st National Congress of Pathology in İzmir/Turkey in 2011. 1 ف ي EBV-LMP-1وع الق تها ال ت ع ب يرات 3، MMP-2، MMP-9 ،ك ا س باس ك ي لوب اي ت NF ،ت ح ل يل ال ل يم فاوي ة دال غد سرطان هودج ك ين ال ك ال س ي ك ية ابراهِيم ِمتين ت ِشريش ,د.ابرهِيم ِمتين تشِريش/مساعد برفيسور /جامعة سليمان ديميرال /كلية الطب/قسم علم األمراض/اسبارطة –تركيا 2. ِسما بِيرجان ,دِ .سما ِبيرجان /برفيسورة /جامعة سليمان ديميرال /كلية الطب/قسم علم األمراض/اسبارطة – تركيا 3. ِشرين باشبِِّينار ,دِ .شرين باشبِِّينار /مساعدة برفيسور /جامعة سليمان ديميرال /كلية الطب/قسم علم األمراض/اسبارطة –تركيا 4. سن إينَان َما َماك /اختصاصية /مستشفى مدينة بوردور /قسم علم األمراض /بوردور – سن إينَان َما َماك ,دُ .چل ُ ُچل ُ تركيا شات ُب ْوزكورت ,د.كَمال ُكر ً كَمال ُكر ً 5. شات ب ُْوزكورت /مساعد برفيسور /جامعة سليمان ديميرال /كلية الطب/قسم علم األمراض/اسبارطة –تركيا 1. عنوان المؤلف المناظر :كَمال ُكر ً شات ب ُْوزكورت /مساعد برفيسور /جامعة سليمان ديميرال /كلية الطب/مستشفى االيحاث والتجارب /قسم علم األمراض /32260اسبارطة –تركيا هاتف +90 246 2119421 فاكس +90 246 2112830 2 ABSTRACT Objectives: Though the histological and clinical features of Classical Hodgkin Lymphoma (CHL) are well known, the mechanisms of tumorigenesis has not been fully elucidated. The aim of this study is to examine the association of nuclear factor kappa B (NF-κB), caspase-3, matrix metalloproteinase (MMP)-2, MMP-9 and latent membrane protein-1 (LMP-1) expressions in CHL patients. Methods: This study was carried out in 2011 at the Süleyman Demirel University, Faculty of Medicine, Department of Pathology, Isparta, Turkey. The study included 40 CHL, including 29 mixed cellularity CHL (MCCHL) and 11 nodular sclerosis CHL (NSCHL). The immunohistochemical NF-κB, MMP-2, MMP-9, caspase-3 and LMP-1 stainings were performed in tissue microarray sections. Results: The study included 27 (67.5%) male and 13 (32.5%) female patients, and the mean age was 36.8 ±20.9 (3-89). LMP-1 expression was found in 23 (57.5%) of all CHL cases. Twenty two (75.9%) cases of MCCHL showed LMP-1 expression, while 1 (9.1%) case of NSCHL was positive, which was statistically significant (p<0.0001). The expressions of NF-κB, caspase-3, MMP-2 and MMP-9 were found in 38 (95%), 39 (97.5%), 8 (20%), and 40 (100%) of all cases, respectively. There was no significant relationship between NF-κB, MMP-2, MMP-9, caspase-3, LMP-1 expressions and clinicopathological features of the patients (p>0.05). However, a significant association was found between the immunostaining levels of NF-κB and caspase-3 (p=0.001), NF-κB and MMP-9 (p=0.002), caspase-3 and MMP-9 (p<0.0001). Conclusion: NF-κB, caspase-3, MMP-9 are consistently expressed in Hodgkin and ReedSternberg (HRS) cells. Although these expressions do not seem to be related with LMP-1 expression, our results suggest that there may be possible relationship between NF-κB, caspase-3 and MMP-9 expressions in CHL patients. Keywords: Hodgkin lymphoma; NF-κB; MMP-2; MMP-9; Caspase-3; LMP-1 3 INTRODUCTION Classical Hodgkin lymphoma (CHL) is a monoclonal lymphoid neoplasm composed of Hodgkin and Reed-Sternberg (HRS) cells in a background of variable mixture of nonneoplastic inflammatory cells, fibroblasts and collagen fibres [1,2]. It is now widely accepted as a malignancy of B cell origin [2,3]. Despite its histological and clinical features are well known, the mechanisms of tumorigenesis have not been fully elucidated [1,4]. Epstein-Barr virus (EBV) has been postulated to play a role in the pathogenesis of CHL, and is consistently associated with a proportion of the cases [2,5,6]. This assocation is believed to be causal, although exact mechanisms are still unclear [5-7]. EBV has been detected in approximately 40% of cases of CHL, but this proportion varies (20%-85%) depending on geographic region and clinicopathological features of cases [1,3,7-9]. In EBV positive CHL, tumor cells are characterized by expression of latent membrane protein (LMP)-1 and 2, EBV nuclear antigen (EBNA)-1, and EBV-encoded RNAs (EBERs) [7]. LMP-1 is particularly interesting because of its oncogenic, strong transforming and antiapoptotic potential [2,4,7]. The growth transforming effect of LMP-1 derives from its ability to activate various signaling pathways, and has been shown to bring about cellular changes via the activation of NF-κB pathway [3,10]. NF-κB is a transcription factor that regulates many genes involved in cell proliferation, inflammatory response, and apoptosis [11,12]. When activated, it translocates to the nucleus from the cytoplasm of the cell, and induces the expression of different genes involved immune system, inflammation, cell proliferation, inhibition of apoptosis, angiogenesis, invasion and metastasis [3,11,13]. The studies have reported that aberrant NF-κB activation is a hallmark of CHL [3,11,13,14]. Strong constituve activation of NF-κB is a characteristic of HRS cells which induces survival factors and stimulates proliferation, and confers resistance to apoptosis, results in a growth advantage of HRS cells [11,13,15]. According to widely accepted hypothesis, apoptosis in HRS cells is thought to play a role in the pathogenesis of CHL [16]. Apoptosis is a form of cellular suicide, and execution of apoptosis depends on proper functioning of effector caspases, and among the effector caspases, caspase-3 is important for the execution of apoptotic cell death [17-20]. The significance of apoptosis in CHL has remained controversial. Although, physiologically, HRS cells should undergo apoptosis, they succeed in surviving and even proliferate. It is suggested this surviving to be linked with NF-κB and LMP-1 of EBV [16]. Some authors 4 believe that apoptosis is not found in HRS cells; while others describe variable degree of HRS cell apoptosis [16,19]. Although the studies have reported high expression of caspase-3 in HRS cells, it is exactly unkown that HRS cells are not killed by caspase-3 [18-22]. Matrix metalloproteinase (MMP)-2 and MMP-9 are proteinases that have physiological roles in degrading the extracellular matrix (ECM). They are expressed in a wide variety of tissues, and play integral role in physiologic tissue degradation and remodeling. Their expressions are usually linked with invasive behaviour in malignant tumors [23-25]. It has been reported that MMPs play important role in ECM degradation in non-Hodgkin lymphomas (NHL) [26]. Some studies have reported that MMP-9 expression is related with a poor clinical outcome in NHLs, and plays a crucial role in the aggressiveness of these neoplasms [24,25,27]. However, the exact function of MMPs in CHL is largely unknown. Their biologic function in lymphoma may be much more complicated and linked to the other mechanism of inflammation, immunologic processes, and apoptosis [24]. Although NF-κB and caspase-3 expressions are investigated in CHL tissues, MMPs are relatively at limited number. Moreover, there is no data investigating all these markers together with EBV-LMP-1 in CHL tissues immunohistochemically. Thus, we aimed to investigate the relationship between cell proliferation, apoptosis and tumor spread by using NF-κB, caspase-3, MMP-2 and MMP-9 expressions, and to examine their association with LMP-1 in CHL tissues using immunohistochemistry. METHODS Patients and samples This study was carried out in 2011 at the Süleyman Demirel University, Faculty of Medicine, Department of Pathology, Isparta, Turkey. The cases diagnosed as HL were retrieved retrospectively from the archival material of the Department of Pathology. Their hematoxylin eosin slides and routine immunoperoxidase staining sections performed at the time of diagnosis (CD30, CD45, CD20, CD3) were reevaluated according to WHO 2008 classification [2]. The study included 40 cases of CHL diagnosed as mixed cellularity CHL (MCCHL) (n=29) and nodular sclerosis CHL (NSCHL) (n=11). The tissue microarray (TMA) blocks were prepared from archival formalin fixed paraffin embedded tissue blocks of cases. Briefly, the areas containing HRS cells were marked on hematoxylin eosin slides by light microscopy. These areas, three cores of 2 mm diameter, were punched from an each case 5 and inserted into a recipient paraffin block using TMA Builder (Labvision Tissue Microarray Builder, LOT 80428, ThermoFisher Scientific, Fremont CA USA). Immunohistochemistry Immunohistochemistry was performed on TMA slides using the streptavidin-biotinperoxidase technique. A 4µm histologic section was deparaffinized in xylene and dehydrated in descending dilutions of ethanol. Antigen retrieval was achieved by heat treatment in PT Module (citratebuffer [pH¼ 6.0]) for 20min. The immunostaining was performed using LabVision Autostainer360 (Fremont, CA, USA). Endogenous peroxidase activity was blocked by 20 min of incubation with 0.3% hydrogen peroxidase. Slides were tested with NF-κB (p65 (RelA) Ab-1, Rabbit PAb, Thermoscientific, 1/100 dilution), MMP-2 (SC-13595, Santa Cruz Biotechnology, 1/100 dilution), MMP-9 (92kDa Collagenase IV Ab-9, Rabbit PAb, Neomarkers, 1/100 dilution), Caspase-3 (CPP32 Ab-4, Rabbit PAb, Thermoscientific, 1/200 dilution), LMP-1 antibodies (MS-1458-51, Mouse Monoclonal Coctail, Neomarkers, 1/25 dilution). Sections were tested with streptavidin-biotin-peroxidase kit (UltraVision Large Volume Detection System Anti-Polyvalent, HRP, LabVision, USA), and the reaction product was detected using diaminobenzidine (DAB). Finally, the sections were counterstained with Mayer’s haematoxylin, and mounted with Aqueous mounting medium. Background lymphocytes were used as internal controls for the staining. The immunoexpression was evaluated only in neoplastic HRS cells. LMP-1 expression in HRS cells was evaluated as positive and negative immunostaining. The expressions of other biomarkers were scored semiquantitatively according to the percentage of staining cells as follows: 0, negative (no staining); +1, low (<10%); +2, moderate (10-50%) and +3, high (>50%) staining of HRS cells. Statistical Analysis The relationship between clinicopathologic parameters and immunohistochemical data, and also the association of biologic markers with each other were assessed by Pearson Chi-Square, nonparametric Mann Whitney U and Spearman’s rank correlation coefficient tests (SPSS, version 15.0, Chicago,Ill, USA). A value of p <0.05 was considered as significant. RESULTS The clinicopathologic features of all CHL cases were shown in Table 1. The study included 40 CHL patients, 29 (72.5%) MCCHL and 11 (27.5%) NSCHL. The ages of patients changed from 3 to 89 (mean: 36.8 ±20.9), which were categorized into four age groups as 6 children, young adult, older adult and older: <15 (n=5), 15-35 (n=13), 36-49 (n=13) and >50 (n=9) patients. There were 27 (67.5%) male and 13 (32.5%) female patients. There was a significant association between histological type and age groups. MCCHL type was significantly higher in children (80%), older adults (92.3%) and older age groups (100%), while NSCHL was higher in young adult group (69.2%) (p=0.001). In addition, MCCHL subtype was also significantly higher in male (92.6%) than female gender, whereas NSCHL type was higher in female patients (69.2%) (p<0.0001) (Table 1). LMP-1 expresssion was detected in 23 (57.5%) of all cases as cytoplasmic, membranous or golgi zone staining in HRS cells (Fig 1A). According to the histological subtype, 22 (75.9%) MCCHL were positive for LMP-1, while 1 (9.1%) case of NSCHL showed LMP-1 expression, which was statistically different (p<0.0001) (Table 1). LMP-1 expression was tended to be related with gender. Eighteen (66.7%) of 27 male patients were positive for LMP-1, while 5 (38.5%) of 13 female patients showed LMP-1 expression (p=0.089). No significant association was found between LMP-1 expression and age groups. Cytoplasmic immunostaining was detected for NF-κB, MMP-2, MMP-9 and caspase-3 in HRS cells (Fig 1B-E). The NF-κB expression was found in 38 (95%) cases of CHL, and according to immunostaining level, 2 (5%), 9 (22.5%), 27 (67.5%) cases showed 1+, 2+ and 3+ staining, respectively. MMP-2 expression was detected in 8 (20%) of all CHL, 4 (50%) cases had 2+ and 4 (50%) cases had 3+ MMP-2 expression levels. All cases (100%) showed positive immunoreactivity for MMP-9. One (2.5%), 7 (17.5%), 32 (80%) cases had 1+, 2+ and 3+ expression levels, respectively. Caspase-3 expression was found in 39 (97.5%) of cases, and 1 (2.5%), 2 (5%) and 36 (90%) cases showed 1+, 2+ and 3+ staining levels, respectively. Considering histological subtypes, as seen in Table 1, 28 (96.6%) cases of MCCHL and 10 (90.9%) of NSCHL showed NF-κB expression. All MCCHL and NSCHL cases showed MMP-9 expression, and for MMP-2, 4 (13.8%) cases of MCCHL and 4 (36.4%) cases of NSCHL were positive. For caspase-3, all MSCHL and 10 (90.9%) of NSCHL cases were positive. There was not any significant relationship between NF-κB, MMP-2, MMP-9, caspase-3 expressions and LMP-1 or histologic subtypes, age and gender of the patients (p>0.05). No significant association was also found between these markers in relation to positive/negative immunostaining results (p>0.05). Considering the immunostaining level, there was a significant association between the levels of NF-κB and caspase-3; NF-κB and MMP-9; and 7 also caspase-3 and MMP-9 expressions (p=0.001, p=0.002, p<0.0001, respectively). The cases having +2 or +3 immunostaining level for NF-κB or MMP-9 showed high level of staining for caspase-3 and MMP-9. DISCUSSION Classical Hodgkin lymphoma shows bimodal age distribution, with two peaks in young adult (15-35 years) and older age group in industrialized countries, and in childhood and older age groups in developing countries [7]. MCCHL is relatively more frequent in children and older adults and more common in developing countries; whereas NSCHL is the predominant type in industrialized countries and accounts for the young adult age [7,8]. In our study, most of the cases were young and older adult patients. Thus, MCCHL type was significantly higher in childhood and older ages and also in males in contrast to NSCHL which was higher in young adults and also in female patients. The EBV is thought to play a critical role in a part of cases in CHL (20%-85%), and this proportion rates vary with different geographic areas, age, sex, and histologic subtype [1,58]. It ranges from 22% to 52% in Western countries and 61% to 100% in less developed countries [7,9]. In particular, 50%-85% of MCCHL and 10%-40% of NSCHL show LMP-1 and/or EBER expression, and its incidence is the highest in MCCHL type [1,2,7]. It has been reported that EBV prevalence is higher in male patients than in females [7]. The association with Epstein-Barr virus varies with age. Childhood Hodgkin lymphoma and most elderly patients are almost invariably associated, while the lowest rates are reported in young adults [4]. The studies have showed a significant correlation between EBV positivity and young children and older patients [3,7]. In this study, in accordance with the above data, LMP-1 expression was detected in 23 (57.5%) of all CHL cases. Furthermore, LMP-1 positivity was significantly higher in MCCHL subtype than NSCHL (75.9% vs 9.1%), and it was also higher in males although not significant. Hovewer, a significant association was not found between LMP-1 expression and age. This might result from limited number of cases or from immunohistochemistry method instead of insitu hybridization for EBV. In EBV associated CHL, EBV gene products appear to be contributed to HRS cell survival, proliferation and reprogramming, and most probably leads to NF-κB activation [5]. The transcription factor NF-κB is constitutively activated in HRS cells, and involved in the regulation of multiple signaling pathways including apoptotic response of cells and survival [3,11,13,14,28]. Its expression is induced by LMP-1 or other signals, and results in the 8 upregulation of different genes including apoptotic regulators, cytokines, intracellular signaling molecules, growt factors and transcription factors [3,11,13,14,29]. Moreover, it has been reported that NF-κB inhibitors could suppress NF-κB activation, and induce cell death by enhancing the pro-apoptotic pathway [11,21]. The studies have reported intense NF-κB staining in HRS cells of all CHL cases by immunohistochemistry [21,30]. Similar with these studies, in our study, NF-κB expression was found in most of cases (95%), with moderate and high staining levels. However, any significant association was not found between NF-κB expression and LMP-1 or clinicopathological features of cases. According to our knowledge, these are the first data investigating such a relationship in CHL immunohistochemically. Further studies should be performed to reveal the association of NF-κB expression with LMP1 and clinicopathologic features in CHL patients. Caspase-3 is important for the execution of apoptotic cell death [17-20]. Many studies have reported that caspase-3 is commonly expressed in HRS cells and/or CHL cell lines [1822]. In the current study, we detected the high expression of caspase-3 almost in all cases (97.5%), with high level of immunostaining. However, we did not demonstate any significant association between caspase-3 expression and clinicopathological features. Dürkop et al. [20] noted that although HRS cells highly expressed caspase-3 in CHL, why caspase-3 did not kill HRS cells and apoptosis resistance of HRS cells seemed to be paradoxical. They suggested that HRS cells expressed not only caspase-3 expression, but also its inhibitor, and showed that cIAP2 was important for the survival of HRS cells by blocking caspase-3, and contributed to the apoptosis resistance of HRS cells by inhibiting effector caspases [20]. Benharroch et al. [16] also suggested that two factors considered responsible for apoptosis inhibition in HRS cell, NF-κB and LMP-1. Furthermore, Izban et al. [21] reported that the inhibition of NF-κB expression resulted in caspase-independent apoptosis which demonstates a critical role for NF-κB in HRS cell surviving. On the other hand Dukers et al. [19] reported that high numbers of active caspase-3 positive HRS cells in pretreatment biopsy predict a highly favorable clinical outcome in CHL patients. In our study, we found a significant correlation between the immunostaining levels of caspase-3 and NF-κB expressions, as this finding may consider a possible relationship between caspase-3 and NF-κB in CHL patients. However, additional studies are needed to determine caspase-3 functions and also association with NF-κB expression. 9 The exact role of MMP-2 and MMP-9 expressions in lymphoid tumors is not clearly known. The studies have shown that MMP-9 expression is overexpressed in a subset of high grade aggressive NHLs, and correlated with a poor clinical outcome. Moreover, a correlation between tumor grade and MMP-9 expression has been reported in NHLs [24-26]. However, other studies have found neither MMP-2 nor MMP-9 expression to be correlated with clinicopathologic features of NHLs [31-33]. Considering CHL, the studies related to MMPs are relatively limited. MMP-2 and MMP-9 expressions in HRS cells have been reported not to be correlated with age, sex, disease stage, bulky disease, extranodal infiltrates [23,33-35]. However, Kuittinen et al. [23] found that strong MMP-2 expression correlated with a favorable prognosis, while MMP-9 was an adverse prognostic factor in CHL patients. Flavell et al. [35] showed that MMP-9 expression was not related with histological type, EBV-LMP-1 status or survival. However, it has been shown that MMP-9 expression is increased in EBV latency type III lymphoma cell line and also LMP-1 trasfection upregulated MMP-9 in the C33A cell line [36]. MMP-9 expression were consistently expressed in all CHL cases examined [33-35], while MMP-2 was positive in 53% of cases [34]. However, Thorns et al. [37] reported that MMP-9 and MMP-2 were negative in 97% and 82% of CHL, respectively. In our study, MMP-9 expression was present in all CHL cases at moderate and high staining levels, while 80% of cases were negative for MMP-2. They were not related with clinicopathological features or LMP-1 expression. However, we detected that the high level of MMP-9 expression correlated with high levels of NF-κB and caspase-3 immunostaining. These findings may suggest that NF-κB or caspase 3 expressions may be associated with MMP-9 expression in HRS cells biologically, and also consider that high expression of MMP-9 may contribute to HRS cells surviving. However, additional studies are needed to clarify their biologic function in CHL patients. In conclusion, we examined NF-κB, caspase-3, MMP-2 and MMP-9 expressions, and also their association with EBV-LMP-1 in CHL with immunohistochemistry. This is the first study investigating such a relationship in the CHL tissues. According to our results, NF-κB, caspase-3, and MMP-9 are consistently expressed by HRS cells. Furthermore, our findings suggest that there may be possible relationship between NF-κB, caspase-3 and MMP-9 expressions in CHL patients. These expressions do not appear to be related with EBV-LMP-1 in our study which is based on immunohistochemistry. The method we used can be the limitation of our study as EBER by insitu hybridization is the best method for investigating its 10 role in CHL patients. Therefore, further studies including more cases and EBER are needed to clarify the relationship of these biomarkers and effects on clinicopathological features in CHL patients. ACKNOWLEDGMENTS We would like to thank Dr. Ahmad Jubran for his contributions. REFERENCES 1- Piccaluga PP, Agostinelli C, Gazzola A, Tripodo C, Bacci F, Sabattini E et al. Pathobiology of Hodgkin Lymphoma. Adv Hematol 2011;doi: 10.1155/2011/920898. Epub 2010 Dec 22. 2- Swerdlow SH, Campo E. Hodgkin Lymphoma. In: Harris NL, Pileri SA, Stein H, Thiele J et al., editors. 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The expression of matrix metalloproteinase 9 is enhanced by Epstein-Barr virus latent membrane protein 1. Proc Natl Acad Sci U S A. 1998;95(7):3621-6. 37- Thorns C, Bernd HW, Hatton D, Merz H, Feller AC, Lange K. Matrix-metalloproteinases in Hodgkin lymphoma. Anticancer Res 2003;23:1555–1558. 14 TABLES Table 1: The distribution of LMP-1, NF-κB, MMP-2, MMP-9 and caspase-3 expressions in CHL patients. CHL MCCHL NSCHL n=40 (%) n=29 (%) n=11 (%) Mean Age 36.8 (3-89) 41.9 (3-89) 23.3 (13-42) <15 5 (12.5) (80) (20) 15-35 13 (32.5) (30.8) (69.2) 36-49 13 (32.5) (92.3) (7.7) 9 (22.5) (100) - Male 27 (67.5) 25 (92.6) 2 (30.8) Female 13 (32.5) 4 (7.4) 9 (69.2) Positive 23 (57.5) 22 (75.9) 1 (9.1) Negative 17 (42.5) 7 (24.1) 10 (90.9) Positive 38 (95) 28 (96.6) 10 (90.9) Negative 2 (5) 1 (3.4) 1 (9.1) Positive 8 (20) 4 (13.8) 4 (36.4) Negative 32 (80) 25 (86.2) 7 (63.6) Positive 40 (100) 29 (100) 11 (100) Negative - - - Positive 39 (97.5) 29 (100) 10 (90.9) Negative 1 (2.5) - 1 (9.1) p* Age >50 p=0.001 Gender p<0.0001 LMP-1 p<0.0001 NF-κB p>0.05 MMP-2 p>0.05 MMP-9 p>0.05 Caspase-3 p>0.05 p* = Pearson Chi-Square, MCCHL vs NSCHL 15 FIGURE LEGENDS Figure 1: Positive immunostaining in HRS cells were seen with LMP-1 (A, x400), NF-κB (B, x400), MMP-2 (C, x400), MMP-9 (D, x400), caspase-3 (E, x400) in CHL tissues. 16