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[Downloaded free from http://www.oncotargets.com on Tuesday, June 14, 2016, IP: 51.39.71.125] Original Article Access this article online Quick Response Code: Website: www.oncotargets.com DOI: 10.4103/2395-4469.177565 Livin expression by semi‑quantitative immunofluorescent staining in Hodgkin lymphoma: A promising marker or a leading role in pathogenesis? Amin Ziaei1,2, Amir Pouya Tanhaei1,2, Ahmad Reza Salehi Chaleshtori3, Safoura Mazrouei4, Mona Kharaji2, Kianoosh Keyhanian5, Mansoor Salehi1,3 Abstract: Introduction: A novel human inhibitor of apoptosis protein family member, termed livin, is enrolled in pathogenesis of different human malignancies and it was studied as a potential treatment target for malignancies. Methods: This study was designed to explore the clinical significance of livin expression in Hodgkin’s lymphoma (HL). We evaluated livin expression in 78 paraffin‑embedded blocks including 39 stage defined cases with defined stages of HL and 39 control subjects. Tissue microarray‑based semi‑quantitative immunofluorescent staining was applied for protein expression profiling in both infiltrating nonneoplastic cells and neoplastic cells of cases and control samples. Results: Our results represented that the mean ratio (M.R) of livin/glyceraldehyde 3‑phosphate dehydrogenase (GAPDH) expression was significantly increased between infiltrating background cells in HL and control cases (0.54596 vs. 0.50827, P < 0.001). In addition, significant difference was found in M.R of livin/GAPDH expression in neoplastic cells regarding the background cells in tumor microenvironment (0.59024 vs. 0.54596, P < 0.001). In addition, this study confirmed a significant increase of livin expression through more advanced stages of HL (0.52888 vs. 0.580146, P < 0.01). Conclusion: These findings recommended a possibly critical role of livin in pathogenesis of HL. It also could be a novel prognostic marker and a potential therapeutic target in this disease. Key words: Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, 2Medical Students Research Center, School of Medicine, Isfahan University of Medical Sciences, 3Medical Genetic Center of Genome, Isfahan, Iran, 4Faculty of Medicine, Friedrich Schiller University of Jena, Jena, Germany, 5 Ottawa Hospital Research Institute, Ontario, Canada 1 Address for correspondence: Dr. Mansoor Salehi, Isfahan University of Medical Sciences, Hezar Jirib Street, Isfahan, Iran. E‑mail: m_salehi@med. mui.ac.ir Submission: 15‑08‑2015 Accepted: 05‑12‑2015 Apoptosis, Hodgkin lymphoma, immunofluorescent staining, livin protein Introduction H odgkin lymphoma (HL) is the most striking example of tightly tumor‑host correlation,[1] and its classic form is responsible for 95% of the cases.[2] Classic HL is sub‑divided into four entities such as nodular sclerosis classical HL, mixed cellularity classical HL, lymphocyte‑rich classical HL, and lymphocyte depleted classical HL. [3] The remaining 5% represent nodular lymphocyte predominant HL (NLPHL).[2,3] Unlike most cancers, malignant cells in HL are outnumbered by background cells in the microenvironment of tumor.[2,4] This accounts for only around 1% of all cells in the tumor tissue.[1,2,4] In addition, as for HL, major reactive cells in lymph nodes are lymphocytes (mostly T‑cell),[5] macrophages/histiocyte cells, eosinophils, mast cells, plasma cells, fibroblast, and neutrophils.[5,6] Despite increasing attention to HL, its molecular biology is still poorly understood [2,7] and supplementary studies are required to discover unresolved molecular pathobiological issues of HL (details described elsewhere).[2,8] Some advances in molecular profiling of Hodgkin and Reed‑Sternberg (HRS) cells revealed the potential © 2016 Oncobiology and Targets | Published by Wolters Kluwer - Medknow role of these cells in pathogenesis of HL. This capability is manifested via secretion of cytokines and chemokines[2,4,9] and eventually escape from apoptosis.[9,10] Apoptosis is essentially important in n e u r o d e g e n e r a t i o n , [8‑10] c a n c e r , a n d hematolymphoid neoplasm. Progression of hematological malignancies is related to impaired regulation of apoptosis. [10‑14] Bcl‑2 and inhibitor of apoptosis proteins (IAPs) are recognized as hallmarks of apoptosis regulation [13] and key players in mentioned malignancies. Upregulation of IAPs correlates with poor prognosis in a variety of cancers such This is an open access article distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as the author is credited and the new creations are licensed under the identical terms. For reprints contact: [email protected] How to cite this article: Ziaei A, Tanhaei AP, C h a l e s h t o r i A R , M a z r o u e i S , Kh a r a j i M , Keyhanian K, et al. Livin expression by semiquantitative immunofluorescent staining in Hodgkin lymphoma: A promising marker or a leading role in pathogenesis?. Oncobiol Targets 2016;3:1. 1 [Downloaded free from http://www.oncotargets.com on Tuesday, June 14, 2016, IP: 51.39.71.125] Ziaei, et al.: Livin expression in Hodgkin lymphoma as hematological malignancies.[14] The neuronal apoptosis inhibitory protein (NAIP)/BIRC1, cIAP‑1/BIRC2, cIAP‑2/ BIRC3, X‑linked inhibitor of apoptosis/BIRC4, SURVIVIN/ BIRC5, BRUCE/BIRC6, ILP‑2/BIRC8, and livin/ML‑IAP/ BIRC7 are eight identified IAP members in human.[11,15] Livin plays a prominent role in apoptosis inhibition.[16] It consists of a single baculoviral IAP repeat (BIR) domain and RING finger motif which forms a globular conformation (conserved by four α‑helices) and three‑stranded anti‑parallel β‑sheets, respectively.[17,18] It was reported that livin directly interacts with executioner caspase‑3 and caspase‑7 in vitro and initiator caspase‑9 in vivo through its BIR domain.[18,19] BIR domain has a critical role in IAP function and mutations in this domain decline inhibition of caspase‑9 and general anti‑apoptotic capability of cell.[20] In addition, truncated form of livin (tLivin) not only loses its anti‑apoptotic effect but also acquires a pro‑apoptotic effect.[17,21] There are some evidence for elevated levels of livin expression in colon cancer,[22] bladder cancer,[23] lung cancer,[24] neuroblastoma,[25] pancreatic cancers,[26] osteosarcoma, renal cell carcinoma,[17] and leukemia including colon cancer[27] and certainly non‑HL.[10,28] There is no report about the quantity of livin expression in HL and discussed the prognostic significance of livin as an informative biomarker in HL. In addition, there are some controversial reports around prognostic importance of livin expression in different malignancies. Hence, the present study was designed to examine the livin expression in HL and evaluate the potential role of this molecule as a promising marker in different stages of HL. Materials and Methods Patients and tissue blocks Livin expression was examined in 78 paraffin‑embedded blocks including 39 HL Formalin‑Fixed Paraffin‑Embedded (FFPE) tissue blocks and 30 and 9 FFPE with diagnosed normal and reactive hyperplasia lymph nodes, respectively. The lymph nodes were excised in the department of surgery, Seyed Al Shohada and Alzahra Hospitals, Isfahan University of Medical Sciences, 2008–2011. Based on histological examination, primary diagnosis of CHL was performed through H and E staining and morphologically confirmed RS cells. Based on immunohistochemical assessment, RS cells (to consider CHL cases) were verified. Subsequently, suitable lymph node blocks were confirmed morphologically with supplementary immunohistochemical staining (data were not shown). Clinical features of all patients including sex, age, B‑symptom (fever, night sweats, and weight loss), lymph node involvement sites (cervical, axillary, mediastinum, para‑aortic, perihilar, and inguinal), and extra‑lymphatic organ involvement (lung, liver, spleen, and bone marrow) were documented. The clinical staging was performed based on Ann Arbor staging procedure criteria. In addition, we determined another staging style which assumed stage I and II as early‑stages and stage III and IV as advanced‑stages.[29] Some cases did not have the required clinical information for staging, 2 so they were considered in undefined stage group. Informed consent was obtained from all donor materials. Immunostaining FFPE samples were subjected to histological studies using standard method. Histological sections (3 μm) were cut using a Jung rotary microtome (Wetzlar, Germany), floated out on a 50–50 volume mixture of absolute ethanol‑distilled water in 48°C, and mounted on glass microscopic slides which were previously coated with (3‑Aminopropyl) triethoxysilane (Sigma‑Aldrich, USA) prior to overnight storage at 37°C. The semi‑quantitative immunofluorescent staining method used in this study was described elsewhere.[30,31] Expression of livin and glyceraldehyde 3‑phosphate dehydrogenase (GAPDH) proteins was measured by semi‑quantitative immunofluorescent assay. We used livin primary antibody of BIR detecting amino acids 264–280 of the short form and 281–298 of the long form of human livin. This sequence is identical between α and β isoforms of the livin protein. Therefore, the antibody detects both α and β isoforms of intact livin protein but not truncated livin proteins. GAPDH primary antibody was GAPDH mouse anti‑Human. Fluorochrome‑conjugated secondary antibody against the species of the anti‑livin and anti‑GAPDH antibodies were fluorescein isothiocyanate (FITC) affini pure goat anti‑rabbit, immunoglobulin G (IgG) (H + L), and Texas Red Affini Pure Goat anti mouse, IgG (H + L), respectively. These reagents were provided by Jackson Immuno Research Laboratories (West Grove, PA, USA). The staining of two types of antibodies were used against livin and GAPDH, made on the same slide. Primary antibodies (livin and GAPDH) were diluted to its optimal dilution (1/100) in diluents. Then, 50 μl of the primary antibodies was added to the slides and incubated for 45 min at 37°C before rinsing slides with PBS. The following steps must be performed in darkness. The secondary conjugated antibodies were diluted to its optimal dilution (1/50) in diluents. Thereafter, 50 μl of secondary antibody was added to each slide; these slides were then incubated for 1 h at 37°C before washing in PBS (2 × 10 min). In this study, livin and GAPDH were stained with FITC and Texas Red conjugation, respectively. Finally, all slides were covered with a cover slip, sealed with clear nail polish, and kept in a cold and dark place. Image analysis Prepared slides were examined using LEICA fluorescence microscope (BZ00) (LEICA, Germany) with filters set appropriately for FITC and Texas Red dyes (Chroma, USA). Two images were captured from each microscope field, one with blue filter (350–450 nm) and the other with green filter (550–650 nm). Furthermore, 20–30 images were captured for each sample from different area of the tissue section randomly. Images were captured using a cooled charge‑coupled device camera (LEICA: DC 350F) (Wetzlar, Germany) interfaced with a PC. We analyzed these captured images using image processing algorithms in MATLAB 7 software (http://www. mathworks.com). Oncobiology and Targets - Vol 3 | 2016 [Downloaded free from http://www.oncotargets.com on Tuesday, June 14, 2016, IP: 51.39.71.125] Ziaei, et al.: Livin expression in Hodgkin lymphoma The whole boundary of desired cells in both green and red planes was selected from our available images and then the ratio of intensity between the mean of pixels in the green and red plane of selected cells was computed by following ratio formula: Mean of pixels intensity for a cell in the Ratio = green plane ( indicated livin expression ) Mean of pixels intensity for the same cell in the red plane ( indicated GAPDH expression ) Unlike some previous studies,[25,31] samples were analyzed one by one to acquire highly accurate and trustworthy results. Among all captured images for every sample, 30 cell‑collections were selected and analyzed by MATLAB 7 (http://www.mathworks.com). We excluded fluorescence artifacts from the evaluation by selecting appropriate cells. Typical lymphocytes were our desired cells for analysis. We selected and confirmed 20 morphologically definite typical neoplastic HRS cells in HL samples. Statistical analysis One‑way analysis of variance and Student’s t‑test were run to analyze the mean level of livin expression ratio between case and control groups and their subdivisions. In addition, χ2 and Fisher’s exact tests were applied to analyze data in clinical features of this study [Tables 1 and 2]. Results Among 39 HL cases, patient number 10, 9, 4, and 8 were diagnosed to be at stages I, II, III, and IV, respectively. The total cell count was around 2060 cells in all prepared HL and control sections. To determine livin upregulation in neoplastic tissues, 1030 infiltrating cells were analyzed in the tumor environment and approximately the same numbers of background cells in normal and reactive environment of lymph node were considered. Mean ratio (M.R) of livin/GAPDH expression in all HL cases was 0.5459 ± 0.0612 and, in the control group, it was equivalent to 0.508 ± 0.0267. Accordingly, livin expression was significantly higher in HL than normal and reactive samples (P < 0.001, t‑test) [Figure 1]. Surprisingly, no statistical difference between the livin expression in normal (M.R of livin/GAPDH expression = 0.5085 ± 0.0284) and reactive hyperplastic lymph nodes (M.R of livin/GAPDH expression = 0.5068 ± 0.0213) (P > 0.05) was not observed [Figure 2]. In addition, all diagnosed malignant cells (HRS) in HL [Figure 3] were analyzed for livin expression (M.R of livin/GAPDH expression = 0.5902 ± 0.0135) and compared with all infiltrating cells (M.R of livin/GAPDH expression = 0.5459 ± 0.06126) in HL, (P < 0.001, paired t‑test). The expression of livin in malignant cells was significantly higher than reactive inflammatory cells either (P < 0.001, paired t‑test) [Figure 4]. Cases number 19 and 13 were recognized as early‑stage (I and II) and late‑stage tumors (III and IV), respectively. In the first Oncobiology and Targets - Vol 3 | 2016 group, M.R of livin/GAPDH expression was 0.5288 ± 0.0418 and, for the second one, it was 0.5801 ± 0.06941. The differences between stages in HL were statistically significant (P < 0.01, t‑test) [Figures 2 and 5]. Based on varying proportion of neoplastic and nonneoplastic cells between 39 HL samples, 18, 14, 4, 2, and 1 cases were diagnosed with NHCHL, MCCHL, LRCHL, LDCHL, and NLPHL histological patterns, respectively. There was no significant relationship between livin expressions in different entities of HL [Figures 3 and 6]. Although the pattern of livin expression is the same for all histological types of Hodgkin lymphoma, it may be acceptable for NHCHL and MCCHL. However, there are a few number of other types such as LRCHL, LDCHL, and NLPHL [Figure 6]. For difficult cases which area under the cureve (AUC) of 0.761, the receiver operating characteristic (ROC) curve determine Table 1: Clinical features of patients with Hodgkin lymphoma No. of cases Sex Male Female Age (years) B symptom Positive Fever Night sweat Weight loss Negative Lymph node involvement Cervical Axillary Mediastinal Para-aortic Perihilar Inguinal Extra-lymphatic organ involvement Lung Liver Spleen Bone Marrow Others 21 17 33.6±15.1 14 9 out of 14 7 out of 14 8 out of 14 17 26 5 9 3 3 4 3 2 3 0 4 Table 2: The cut‑off points to discriminate Hodgkin’s lymphoma from non neoplastic lymph node according to mean ratio of livin/glyceraldehyde‑3‑phosphate dehydrogenase expression Optimal Cut-off point Cut-off point with higher sensitivity Cut-off point with higher specificity Points Sensitivity Specificity AUC % % 0.5231 74.9 74.1 0.761 (95% CI 0.648 to 0.874) 0.5032 82.1 35.9 0.761 (95% CI 0.648 to 0.874) 0.5334 61.5 89.7 0.761 (95% CI 0.648 to 0.874) AUC: Areas Under the Curve, 95% CI: 95% confidence interval 3 [Downloaded free from http://www.oncotargets.com on Tuesday, June 14, 2016, IP: 51.39.71.125] Ziaei, et al.: Livin expression in Hodgkin lymphoma Discussion the optimal cut‑off point for mean ratio of livin/GAPDH expression at 0.5231. This maximizes true positives and true negatives (sensitivity 74.9%, specificity 74.4%) which enabled us to distinguish HL from nonneoplastic lymph node samples as control group [Figure 7] in difficult cases with AUC of 0.761 [Figure 4 and Table 2]. This study revealed prognostic significance of livin protein in HL. Livin expression in some human malignancies described before[13,32] as well as malignant melanoma that is illustrated as melanoma IAP.[21] Since upregulation and/or abnormal livin expression is proved in tumor genesis and progression of cancer,[17] over expression of livin is illustrated as a potential prognostic and diagnostic biomarker. [21] The prognostic importance of livin has explained in neuroblastoma, bladder cancer, and Non‑Hodgkin’s Lymphoma (NHL).[21,28,33,34] The ROC curve revealed that the mean ratio of livin/GAPDH expression could be an accurate indicator to distinguish HL from control group (AUC = 0.761, P < 0.0001). Clinical features of case groups are shown in Table 1. Figure 1: Livin/glyceraldehyde 3‑phosphate dehydrogenase expression in 39 Hodgkin’s lymphoma patients and 39 controls (normal and reactive) lymph nodes. The expression of livin protein in Hodgkin’s lymphoma and control groups was significant statistically (P < 0.001) a Figure 2: Livin/glyceraldehyde 3‑phosphate dehydrogenase expression in 30 normal and nine ones with reactive hyperplasia lymph nodes. The livin protein expression between these groups was not significant statistically b c d Figure 3: Glyceraldehyde 3‑phosphate dehydrogenase expression detected by fluorescein and Texas Red conjugated antibodies, respectively (a and b) are related to infiltrating reactive cells in Hodgkin’s lymphoma. Parts (c and d) show Hodgkin and Reed‑Sternberg cells in Hodgkin’s lymphoma 4 Oncobiology and Targets - Vol 3 | 2016 [Downloaded free from http://www.oncotargets.com on Tuesday, June 14, 2016, IP: 51.39.71.125] Ziaei, et al.: Livin expression in Hodgkin lymphoma Figure 4: Livin/glyceraldehyde 3‑phosphate dehydrogenase expression in malignant (Hodgkin and Reed‑Sternberg) and nonmalignant cells in Hodgkin’s lymphoma. Quantification of livin expression between these different populations was significant (P < 0.001) Figure 6: The mean ratio of livin/glyceraldehyde 3‑phosphate dehydrogenase expression among five histological entities of Hodgkin’s lymphoma. No significant relevance is noticed between livin expressions Gazzaniga et al.[34] explained that livin can be utilized as a marker of early recurrence. They proved that median relapse‑free time of livin‑positive patients is shorter than livin‑negative patients. Subsequently, Kim et al.[21,33] had shown positive correlation of livin with prognosis and outcome of the disease in neuroblastoma cells. Gao et al.[28] found that expression of livin mRNA and protein was positively correlated with clinical stage of NHL, B symptom, and some other clinical features. Figure 5: Comparison of livin/glyceraldehyde 3‑phosphate dehydrogenase expression between Early‑stage and advanced‑stage groups of Hodgkin’s lymphoma patients. Alteration of livin expression was statistically significant in different clinical stages of Hodgkin’s lymphoma (P < 0.001) Figure 7: Diagnostic statistics = Receiver operating characteristic curve for livin/glyceraldehyde 3‑phosphate dehydrogenase expression in discriminating classical Hodgkin’s lymphoma from nonneoplastic lymph node samples as control group (area under the cureve = 0.761) livin in both neoplastic and nonneoplastic cell population are statistically significant regarding the cell population presenting in the control lymph nodes. Surprisingly, we found that livin expression in malignant cells is elevated regarding the nonneoplastic background cells. As a result, increased livin expression leads to decreased apoptosis and tumorigenesis. In contrast, there are some studies on Lung and colon cancers, [35] colon cancer, [27] and metastatic melanoma, persisting on correlation between livin overexpression and clinical outcome despite the up‑regulation of livin in these malignancies. Considering the role of nuclear factor‑kappa B (NFκB) on up‑regulation of IAPs,[36] it is interpreted that TF (NFκB) is activated in the reprogrammed HRS cells.[2] This activation can be effective in upregulation of IAPs (livin is one of them) in the malignant cells compared with inflammatory background cells. More comprehensive molecular profiling studies are required to underpin this issue. The present study is the first description of evaluation and clinical significance of livin expression in HL. We evaluated livin expression not only in the bulk of infiltrating background cells but also in malignant ones and then compared livin expression in involved lymph nodes to nonneoplastic lymphoid tissues. We found that differences in expression of Our findings revealed that difference in livin expression between the early stage cases and advanced‑stage HL samples may be significant. In addition, our results unveiled no correlation between the level of the livin expression and other factors such as age, gender, B symptom, and lymphoid and extra‑lymphoid tissues involvements. Oncobiology and Targets - Vol 3 | 2016 5 [Downloaded free from http://www.oncotargets.com on Tuesday, June 14, 2016, IP: 51.39.71.125] Ziaei, et al.: Livin expression in Hodgkin lymphoma The present study proposed that livin might be considered a prognostic factor for determination of stages in HL as well as to be targeted in treatment of HL patients. In the present study, LDCHL samples (the most invasive form of HL[4]) had the lowest M.R of livin/GAPDH expression while LRCHL cases (with a rich background of normal lymphocytes) had the highest M.R. of livin/GAPDH expression [Figure 6]. This might be attributed to the restricted numbers of samples which were studied here. Because the expression of GAPDH is constant in normal and neoplastic lymphoid tissues, any differences in livin/GAPDH ratio indicate changes in livin protein expression.[37] Hence, the authors recommended more sophisticated studies with more expanded samples of HL. It is concluded that livin over expression is significant in pathogenesis and progression of HL. It may become an attractive candidate for targeted therapies through different strategies as listed elsewhere.[21] Nevertheless, it is unidentified whether pan‑IAP inhibitors or specific livin inhibitors are the most useful therapeutically for treatment of malignancies. Evaluation of all members of IAP family is, therefore, recommended to find a more vivid picture of their role in different stages of HL. We have extended our study to evaluate NAIP expression profile in HL.[37] Acknowledgments This research work was supported by Grant No. 187070 from the Deputy for Research, Isfahan University of Medical Sciences, Isfahan, Iran. We would like to appreciate Dr. Ardeshir Talebi (Department of Pathology, Isfahan University of Medical Sciences) for pathology diagnosis of cases, Dr. Mohammad Reza Mohajeri and Ms. Sahar Mahmoodi for sample preparation and immune staining of samples. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. References 1. 2. 3. 4. 5. 6. 7. 6 Yurchenko M, Sidorenko SP. Hodgkin’s lymphoma: The role of cell surface receptors in regulation of tumor cell fate. Exp Oncol 2010;32:214‑23. Kuppers R. Molecular biology of Hodgkin lymphoma. Hematology Am Soc Hematol Educ Program 2009;1:491‑6. Mani H, Jaffe ES. Hodgkin lymphoma: An update on its biology with new insights into classification. Clin Lymphoma Myeloma 2009;9:206‑16. 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