Title: Nuclear Survivin Expression in Mantle Cell Lymphoma Is Associated with Cell Proliferation and Survival
Abstract: Survivin is a member of the inhibitor of apoptosis protein family that is expressed in G2/M phase. Survivin is overexpressed and associated with parameters of poor prognosis in different human tumors. The role of survivin in the pathogenesis of mantle cell lymphoma (MCL) was examined in a series of typical and blastoid tumors. Survivin was detected as a nuclear pattern in a variable number of tumor cells. Mitotic figures were always positive with a strong delineation of the chromosomes. Western blot analysis confirmed the presence of survivin only in nuclear fractions. Protein expression detected by immunohistochemistry correlated with mRNA levels analyzed by quantitative real-time reverse transcription-polymerase chain reaction (P < 0.0001). Survivin expression levels were higher in blastoid MCL variants (P < 0.0001) and were associated with the proliferative activity (P = 0.001), but not with the ploidy status of the tumors. The number of apoptotic cells was independent of survivin or Ki-67 expression. Overall survival was significantly shorter in patients with high survivin expression. However, in a multivariate analysis, proliferative index was a better predictor of survival than survivin score. These findings indicate that survivin is commonly expressed in MCL with a nuclear and mitotic pattern. The expression levels are strongly associated with the proliferative activity of the tumors and the survival of the patients, suggesting a potential role in cell cycle regulation and tumor progression. Survivin is a member of the inhibitor of apoptosis protein family that is expressed in G2/M phase. Survivin is overexpressed and associated with parameters of poor prognosis in different human tumors. The role of survivin in the pathogenesis of mantle cell lymphoma (MCL) was examined in a series of typical and blastoid tumors. Survivin was detected as a nuclear pattern in a variable number of tumor cells. Mitotic figures were always positive with a strong delineation of the chromosomes. Western blot analysis confirmed the presence of survivin only in nuclear fractions. Protein expression detected by immunohistochemistry correlated with mRNA levels analyzed by quantitative real-time reverse transcription-polymerase chain reaction (P < 0.0001). Survivin expression levels were higher in blastoid MCL variants (P < 0.0001) and were associated with the proliferative activity (P = 0.001), but not with the ploidy status of the tumors. The number of apoptotic cells was independent of survivin or Ki-67 expression. Overall survival was significantly shorter in patients with high survivin expression. However, in a multivariate analysis, proliferative index was a better predictor of survival than survivin score. These findings indicate that survivin is commonly expressed in MCL with a nuclear and mitotic pattern. The expression levels are strongly associated with the proliferative activity of the tumors and the survival of the patients, suggesting a potential role in cell cycle regulation and tumor progression. Mantle cell lymphoma (MCL) is a malignant lymphoproliferative disorder characterized by the proliferation of CD5+, CD23- monoclonal B cells containing the chromosomal translocation t(11;14)(q13;q32) which places the cyclin D1 gene under the transcriptional control of the immunoglobulin heavy chain enhancer elements.1Campo E Raffeld M Jaffe ES Mantle-cell lymphoma.Semin Hematol. 1999; 36: 115-127PubMed Google Scholar, 2Bosch F Jares P Campo E Lopez-Guillermo A Piris MA Villamor N Tassies D Jaffe ES Montserrat E Rozman C Cardesa A PRAD-1/cyclin D1 gene overexpression in chronic lymphoproliferative disorders: a highly specific marker of mantle cell lymphoma.Blood. 1994; 84: 2726-2732PubMed Google Scholar Additional alterations in the tumor suppressor genes p16INK4a and p53 have been described in aggressive variants of MCL, suggesting that these genes may cooperate with cyclin D1 activation in the progression of these lymphomas.3Hernandez L Fest T Cazorla M Teruya-Feldstein J Bosch F Peinado MA Piris MA Montserrat E Cardesa A Jaffe ES Campo E Raffeld M p53 gene mutations and protein overexpression are associated with aggressive variants of mantle cell lymphomas.Blood. 1996; 87: 3351-3359PubMed Google Scholar, 4Greiner TC Moynihan MJ Chan WC Lytle DM Pedersen A Anderson JR Weisenburger DD p53 mutations in mantle cell lymphoma are associated with variant cytology and predict a poor prognosis.Blood. 1996; 87: 4302-4310PubMed Google Scholar, 5Pinyol M Hernandez L Cazorla M Balbín M Jares P Fernández PL Montserrat E Cardesa A Lopez-Otin C Campo E Deletions and loss of expression of p16INK4a and p21Waf1 genes are associated with aggressive variants of mantle cell lymphomas.Blood. 1997; 89: 272-280Crossref PubMed Google Scholar, 6Dreyling MH Bullinger L Ott G Stilgenbauer S Muller-Hermelink HK Bentz M Hiddemann W Dohner H Alterations of the cyclin D1/p16-pRB pathway in mantle cell lymphoma.Cancer Res. 1997; 57: 4608-4614PubMed Google Scholar Classical cytogenetic and comparative genomic hybridization (CGH) studies have also demonstrated complex karyotypes and high number of additional recurrent chromosomal imbalances.7Bea S Ribas M Hernandez JM Bosch F Pinyol M Hernandez L Garcia JL Flores T Gonzalez M Lopez-Guillermo A Piris MA Cardesa A Montserrat E Miro R Campo E Increased number of chromosomal imbalances and high-level DNA amplifications in mantle cell lymphoma are associated with blastoid variants.Blood. 1999; 93: 4365-4374PubMed Google Scholar, 8Bentz M Plesch A Bullinger L Stilgenbauer S Ott G Muller-Hermelink HK Baudis M Barth TF Moller P Lichter P Dohner H t(11;14)-positive mantle cell lymphomas exhibit complex karyotypes and share similarities with B-cell chronic lymphocytic leukemia.Genes Chromosomes Cancer. 2000; 27: 285-294Crossref PubMed Scopus (136) Google Scholar, 9Allen JE Hough RE Goepel JR Bottomley S Wilson GA Alcock HE Baird M Lorigan PC Vandenberghe EA Hancock BW Hammond DW Identification of novel regions of amplification and deletion within mantle cell lymphoma DNA by comparative genomic hybridization.Br J Haematol. 2002; 116: 291-298Crossref PubMed Scopus (64) Google Scholar Furthermore, a high incidence of tetraploidy has been described in blastoid variants of MCL.10Ott G Kalla J Ott MM Schryen B Katzenberger T Muller JG Muller-Hermelink HK Blastoid variants of mantle cell lymphoma: frequent bcl-1 rearrangements at the major translocation cluster region and tetraploid chromosome clones.Blood. 1997; 89: 1421-1429Crossref PubMed Google Scholar This high number of secondary chromosomal aberrations are associated with inactivation of DNA damage response genes such as ATM and CHK2.11Camacho E Hernandez L Hernandez S Tort F Bellosillo B Bea S Bosch F Montserrat E Cardesa A Fernández PL Campo E ATM gene inactivation in mantle cell lymphoma mainly occurs by truncating mutations and missense mutations involving the phosphatidylinositol-3 kinase domain and is associated with increasing numbers of chromosomal imbalances.Blood. 2002; 99: 238-244Crossref PubMed Scopus (130) Google Scholar, 12Tort F Hernandez S Bea S Martinez A Esteller M Herman JG Puig X Camacho E Hernández L Sanchez M Nayach I Lopez-Guillermo A Fernández PL Colomer D Campo E CHK2-decreased protein expression and infrequent genetic alterations mainly occur in aggressive types of non-Hodgkin's lymphomas.Blood. 2002; 100: 4602-4608Crossref PubMed Scopus (61) Google Scholar MCL has an aggressive clinical course, with a median survival of less than 5 years, and a poor response to conventional treatments. The mechanisms accounting for the resistance of these tumor cells to chemotherapeutic drugs are poorly understood. It has been postulated that most chemotherapeutic agents induce cell death by apoptosis triggering,13Reed JC Apoptosis-based therapies.Nat Rev Drug Discov. 2002; 1: 111-121Crossref PubMed Scopus (596) Google Scholar but the molecular basis for chemoresistance in this pathology is not well known.Inhibitors of apoptosis proteins (IAP) are a family of negative regulators of apoptosis.14Deveraux QL Reed JC IAP family proteins-suppressors of apoptosis.Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2264) Google Scholar Survivin is a unique mammalian IAP family protein that is expressed during mitosis in a cell-cycle-dependent manner and localized to different components of the mitotic apparatus.15Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (2997) Google Scholar, 16Li F Ambrosini G Chu EY Plescia J Tognin S Marchisio PC Altieri DC Control of apoptosis and mitotic spindle checkpoint by survivin.Nature. 1998; 396: 580-584Crossref PubMed Scopus (1719) Google Scholar Survivin is virtually undetectable in most normal adult tissues, but it is highly expressed during embryogenesis and in most human cancers. This overexpression in malignant tumors has been associated with an aggressive behavior and poor prognosis of the patients.17Kawasaki H Altieri DC Lu CD Toyoda M Tenjo T Tanigawa N Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer.Cancer Res. 1998; 58: 5071-5074PubMed Google Scholar, 18Islam A Kageyama H Takada N Kawamoto T Takayasu H Isogai E Ohira M Hashizume K Kobayashi H Kaneko Y Nakagawara A High expression of Survivin, mapped to 17q25, is significantly associated with poor prognostic factors and promotes cell survival in human neuroblastoma.Oncogene. 2000; 19: 617-623Crossref PubMed Scopus (351) Google Scholar, 19Altieri DC The molecular basis and potential role of survivin in cancer diagnosis and therapy.Trends Mol Med. 2001; 7: 542-547Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 20Ikeguchi M Ueda T Sakatani T Hirooka Y Kaibara N Expression of survivin messenger RNA correlates with poor prognosis in patients with hepatocellular carcinoma.Diagn Mol Pathol. 2002; 11: 33-40Crossref PubMed Scopus (108) Google Scholar The anti-apoptotic function and topographic distribution during cell cycle progression have suggested that survivin may play a role in the mitotic checkpoint control, regulating chromosome segregation and cell division.21Kallio MJ Nieminen M Eriksson JE Human inhibitor of apoptosis protein (IAP) survivin participates in regulation of chromosome segregation and mitotic exit.EMBO J. 2001; 15: 2721-2723Google Scholar, 22Bolton MA Lan W Powers SE McCleland ML Kuang J Stukenberg PT Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin binding and phosphorylation.Mol Biol Cell. 2002; 13: 3064-3077Crossref PubMed Scopus (275) Google Scholar Its overexpression in cancer cells may facilitate tumor progression by providing a mechanism to tolerate increasing chromosomal abnormalities and promoting resistance to treatment with anticancer drugs and irradiation.19Altieri DC The molecular basis and potential role of survivin in cancer diagnosis and therapy.Trends Mol Med. 2001; 7: 542-547Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 20Ikeguchi M Ueda T Sakatani T Hirooka Y Kaibara N Expression of survivin messenger RNA correlates with poor prognosis in patients with hepatocellular carcinoma.Diagn Mol Pathol. 2002; 11: 33-40Crossref PubMed Scopus (108) Google ScholarThe aim of this study was to determine the potential role of survivin in the pathogenesis of MCL, analyzing the relationship of its expression with the proliferative activity, genomic alterations, apoptotic levels of the tumors, as well as response to therapy and survival of the patients.Materials and MethodsPatientsTumor specimens from 80 patients diagnosed with MCL between 1988 and 2000 were obtained from the Department of Pathology of the Hospital Clinic, University of Barcelona (Spain) and the Institute of Pathology, University of Würzburg (Germany). Tumors were classified as classical MCL (n = 57; 71%), and blastoid variant, including pleomorphic (n = 9; 11%) and blastic (n = 14; 18%) subtypes.1Campo E Raffeld M Jaffe ES Mantle-cell lymphoma.Semin Hematol. 1999; 36: 115-127PubMed Google Scholar, 23Jaffe ES Harris NL Stein H Vardiman JWE World Health Organization classification of tumors.in: Jaffe ES Harris NL Stein H Vardiman JWE Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press Lyon, France2001: pp 168-170Google Scholar In addition, paired samples from two patients that had progressed from classical to blastoid MCL variants (5 months to 45 months for progression) and 10 paired samples from 5 classical MCL at different times of disease (15 months to 68 months of follow-up) were also examined. The immunophenotype of the tumors was analyzed by immunohistochemistry on tissue sections and/or by flow cytometry on cell suspensions. Cyclin D1 overexpression was demonstrated in all cases by Northern blot analysis and/or immunohistochemistry.For each patient the following initial data were recorded and evaluated for analysis: 1) clinical data: age, sex, performance status (PS) according to the Eastern Cooperative Oncology Group (ECOG) scale, and presence of B-symptoms; 2) hematological and biochemical parameters: Hb, WBC count, presence of atypical lymphocytes in peripheral blood, platelet count, serum β2-microglobulin, and serum LDH level; 3) tumor extension data: number of nodal and extranodal involved sites, spleen palpable below the left costal margin, hepatomegaly, Ann Arbor stage, and bone marrow infiltration; and 4) the International Prognostic Index (IPI), as defined by the International Non-Hodgkin's Lymphoma Prognostic Factors Project.24The International Non-Hodgkin's Lymphoma Prognostic Factors Project A predictive model for aggressive non-Hodgkin's lymphoma.N Engl J Med. 1993; 329: 987-994Crossref PubMed Scopus (4946) Google Scholar Moreover, response to treatment and survival, measured from the time the sample was obtained, was also recorded and analyzed.Cell LinesThe following human cell lines were used: Raji and Daudi, derived from a Burkitt lymphoma and Granta 519, derived from a MCL. Cell lines were purchased from the American Type Culture Collection (ATCC) and were cultured in RPMI 1640 medium (Gibco BRL, Paisley, Scotland) containing 10% heat-inactivated fetal calf serum (Gibco BRL), 2 mmol/L glutamine and 50 μg/ml penicillin-streptomycin, at 37°C in a humidified atmosphere containing 5% carbon dioxide.Immunohistochemical StudiesMCL tumors were analyzed immunohistochemically using a rabbit polyclonal antibody anti-survivin directed against the full-length recombinant survivin (Novus Biologicals, Littleton, CO), a mouse monoclonal anti-Ki-67 (Immunotech, Marseille, France) and a rabbit polyclonal antibody against cleaved caspase-3, which only recognizes the large fragment of caspase-3 that results after cleavage of procaspase-3 at Asp175 position (Cell Signaling, Beverly, MA). Antigen retrieval conditions and antibody dilutions are shown in Table 1. Briefly, paraffin sections on silane-coated slides were dewaxed and then subjected to antigen retrieval using a hot start method with the adequate buffer solution in a pressure cooker for 2 to 5 minutes at highest pressure, depending on the antibody used (Table 1). Slides were stained using an automated immunostainer TechMate 500 Plus (DAKO, Carpinteria, CA) and the Envision System (DAKO), counterstained in Gill's hematoxylin and mounted in Pertex (Histolab GmbH, Göteborg, Germany).Table 1Immunohistochemical Study: Antibodies and Conditions of UsageAntigen retrieval*AntibodySourceDilutionBufferpHTimeTemperatureSurvivinNovus1:1000Cytrate65 min100°CCleaved caspase-3Cell Signaling1:10EDTA82 min100°CKi-67 (MIB-1)Immunotech1:400Cytrate65 min100°C Open table in a new tab Simultaneous demonstration of Ki-67 or cleaved caspase-3 and survivin expression was determined in 20 tumor samples using an Universal DAKO Envision Doublestain System (DAKO) following the supplier instructions. Briefly, a unique common retrieval treatment was made for double staining of Ki-67 and survivin, but for double staining of cleaved caspase-3 and survivin, samples were subjected to pressure cooker in adequate buffer before each primary antibody incubation. Survivin immunostaining was performed using the DAB-peroxidase Envision method. After washing, slides were re-incubated with either anti-Ki-67 or anti-cleaved caspase-3 and then an APAAP-Envision method was used.Immunohistochemical results were expressed as percentage of positive cells. A minimum of 1000 cells per case was counted in representative areas of the tumor. Entrapped and involved follicular germinal centers were excluded for quantification.RT-PCR AnalysisTotal RNA was isolated from each frozen tumor sample and from cell lines using guanidinium thiocyanate method (Ultraspec; Biotecx Laboratories, Houston, TX). RNA was treated with DNase (Ambion, Austin, TX) to eliminate contaminating DNA. For cDNA synthesis, 1 μg of RNA and Taqman Reverse Transcriptions reagents (including Multiscribe reverse transcriptase and random hexamers) were used, as described by the manufacturer (Applied Biosystems, Foster City, CA). The primers and probe used to amplify and quantify survivin cDNA by real-time were designed using Primer Express software (Applied Biosystems) and were as follows: survivin F, 5′-ATT TGA ATC GCG GGA CCC-3′; survivin R, 5′-GAG AAA GGG CTG CCA GGC-3′ and survivin probe, FAM-5′-CAT GGG TGC CCC GAC GTT GC-3′-TAMRA. The primers and the probe were located in a region with no homology to the EPR-1 gene. Real-time monitoring of PCR amplification of cDNAs was done with Taqman Universal master mix (Applied Biosystems) using 200 nmol/L of probe and 100 nmol/L of each survivin primer, in the ABI Prism 7700 sequence Detection System (Applied Biosystems). Relative quantification of gene expression was performed as described in the Taqman user's manual using human β-glucoronidase (GUSB)(Applied Biosystems) as an internal control.To examine separately the three splice variants of survivin mRNA, the same forward primer but different reverse primers were used, as described previously.25Mahotka C Krieg T Krieg A Wenzel M Suschek CV Heydthausen M Gabbert HE Gerharz CD Distinct in vivo expression patterns of survivin splice variants in renal cell carcinomas.Int J Cancer. 2002; 100: 30-36Crossref PubMed Scopus (122) Google Scholar Amplification was performed in a total volume of 25 μl in the presence of 3 mmol/L MgCl2, 0.5 μmol/L of each primer and 2.5 μl of each cDNA. RT-PCR amplification was performed with an initial denaturation step of 10 minutes at 95°C, followed by 40 cycles of 30 seconds at 95°C, 30 seconds annealing at 60°C, and 30 seconds elongation at 72°C.DNA PloidyDNA ploidy of the tumors was studied by flow cytometry in 27 cases in which suitable material was available. The analysis was performed on 50-μm-thick sections obtained from formalin-fixed, paraffin-embedded tissues as previously described26Hedley DW Friedlander ML Taylor IW Rugg CA Musgrove EA Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry.J Histochem Cytochem. 1983; 31: 1333-1335Crossref PubMed Scopus (1917) Google Scholar and analyzed with an Epics Profile II flow cytometer (Coulter Company, Hialeah, FL). Non-neoplastic cells in the section under study were used as the internal standard of the diploid channel.Western Blot AnalysisWestern blot analysis was performed on whole, cytoplasmic, and nuclear protein lysates. Cryostat frozen sections were lysed in ice-cold buffer containing 10 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulphoric acid (HEPES)(pH 7.9), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.1% Nonidet P-40, 0.5 mmol/L dithiothreitol, 2 μg/ml leupeptin, 5 μg/ml aprotinin, and 0.5 mmol/L phenylmethylsulfonyl fluoride for 15 minutes. After centrifugation at 3500 rpm, the supernatant was collected (cytosolic fraction) and the nuclear fraction was obtained by lysis of the precipitated nuclei in a buffer containing 80 mmol/L Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol and 0.1 mol/L DTT. Total extracts were obtained by lysis of cryostat sections in the latter buffer. Fifty μg of protein were separated by electrophoresis on 12% polyacrylamide gel and transferred to Immobilon-P (Millipore, Bedford, MA) membranes. The membranes were incubated with a polyclonal antibody against survivin. This antibody was the same used in the immunohistochemical studies. Antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase and an enhanced chemiluminescence (ECL) detection kit (Amersham, Buckinghamshire, UK). Equal protein loading was confirmed with α-tubulin antibody (Oncogene Science, Inc, Cambridge, MA).Statistical AnalysisThe Fisher's exact, χ2, t-test and analysis of variance tests were used to analyze the association between the clinico-biological parameters and the following categorized variables: immunohistochemical survivin protein expression (cutpoint 20%), survivin mRNA expression by real-time RT-PCR (cutpoint, 2), Ki-67 (cutpoint 50%), and cleaved caspase 3 (cutpoint 0.8%). Testing and estimation of possible cutoff values for survivin and Ki-67 expression was done by maximally selected log-rank statistics. Correlation between survivin, Ki-67 and cleaved caspase-3 was ascertained by means of linear regression. Survival time was measured from the time the sample was obtained. Probability of survival was calculated by the method of Kaplan and Meier, and curves were compared by means of the log-rank test. All statistical tests were two-sided and the significance level was established at 0.05. All significant prognostic variables in the univariate study, as well as survivin expression, proliferative index and Ki-67 expression, were considered for multivariate analysis performed by the stepwise proportional hazard regression method of Cox (survival). All statistical tests were performed with the SPSS v10.06 statistical package (SPSS Inc., Chicago, IL).ResultsAnalysis of Survivin Expression by Western BlotSurvivin expression was first studied in nuclear and cytoplasmic protein lysates from Granta 519, Raji, and Daudi human lymphoma cell lines. Western blot analysis using a polyclonal antibody directed against full-length recombinant survivin showed a dominant band of approximately 16.5 kd that was only observed in nuclear extracts from all of the cell lines tested (Figure 1A). In MCL tumors, survivin was also only detected in nuclear protein lysates (Figure 1B).Analysis of Survivin Expression by ImmunohistochemistryThe nuclear expression of survivin observed by Western blot was confirmed by immunohistochemistry in all of the cell lines tested and MCL tumors (Figure 1C). Some cells showed weak cytoplasmic staining that tended to disappear with progressive antibody dilutions (data not shown). In reactive lymph nodes and adult tonsils, survivin-positive cells were found mainly in the nucleus of proliferating germinal center cells, and only scattered cells in the mantle zone were positive (Figure 2A). To further confirm this nuclear localization in other lymphoid tumors, additional immunohistochemical studies were performed in a short series of diffuse large B-cell lymphomas (DLBCL) and chronic lymphocytic leukemia (CLL). In all cases, survivin staining was exclusively nuclear as in MCL cases.Figure 2Survivin, Ki-67, and cleaved caspase-3 expression in reactive tonsil and MCL. A: Adult tonsil. Reactive tonsil showing a number of hyperplastic follicles (hematoxylin and eosin, H&E). Cells expressing survivin, Ki-67, and cleaved caspase-3 were mainly localized in the germinal center of secondary follicles. B: MCL, classical variant. An entrapped reactive germinal center was colonized by tumor cells (H&E). Cyclin D1 staining confirmed the presence of tumor cells inside the reactive germinal center (not shown). Survivin-positive tumor and reactive cells were clustered in a partially involved germinal center. Only very few scattered cells were positive outside the follicle. The proliferative activity of the tumor was low. A very low number of positive cleaved caspase-3 cells were detected in the tumor. Apoptotic activity was mostly concentrated in the entrapped follicles. C: MCL, pleomorphic variant. A high mitotic activity was observed in this tumor (H&E). Survivin-positive cells were widely distributed through the tumor. A high number of Ki-67 positive cells were also detected. A few cells were positive for cleaved caspase-3. D: MCL, blastic variant. Monotonous infiltration by medium-sized lymphocytes with rounded nuclei and dispersed chromatin (H&E). Survivin was widely expressed in tumor cells. Increased proliferative activity was noticed with Ki-67 antigen. A few number of cells were positive for cleaved caspase-3. Original magnification, ×200View Large Image Figure ViewerDownload Hi-res image Download (PPT)Survivin expression was examined immunohistochemically in 80 MCL. A variable number of survivin nuclear-positive cells ranging from 5% to 95% was detected (Figure 2, B to D). This nuclear distribution of survivin was homogeneous with nucleolar enhancement in non-mitotic nuclei and more intense and granular in prophase (Figure 3A). In metaphase, anaphase, and telophase chromosomes were strongly stained, whereas mitotic spindles were also occasionally observed with a weaker labeling (Figure 3B). Survivin-positive cells were diffusely distributed throughout the tumor. However, in some cases, reactive and colonized germinal centers were easily recognized as nodular clusters of cells with strong survivin nuclear positivity (Figure 2B). Colonization of germinal centers by tumor cells was confirmed by detection of numerous cyclin D1-positive cells within the germinal centers.Figure 3Nuclear and mitotic pattern of survivin expression in primary MCL cells. A: Survivin expression showed a homogeneous pattern with nucleolar reinforcement in non-mitotic nuclei (arrows) and a strong multi-granular pattern in large-sized prophasic appearing nuclei (arrowheads). B: In late mitotic nuclei, chromosomes were strongly stained. Original magnification, ×1000View Large Image Figure ViewerDownload Hi-res image Download (PPT)The number of survivin-positive cells correlated with the histological subtype of MCL. Thus, the highest expression of survivin was observed in the blastic variant (30 ± 29% of positive cells, n = 14), compared to classical MCL (10 ± 10%, n = 57) (analysis of variance, P < 0.0001). The expression of survivin in pleomorphic MCL was similar to that observed in classical tumors (10 ± 12%, n = 9; P = 0.89) (Figure 4).Figure 4Immunohistochemical analysis of survivin expression in MCL. Blastic MCL variant showed higher survivin expression than classical and pleomorphic MCL variants. Symbols above the bars represent outlier cases.View Large Image Figure ViewerDownload Hi-res image Download (PPT)No changes in survivin levels were observed in 10 paired samples of five classical MCL at different times of disease, without evidence of tumor progression (11 ± 9% vs. 13 ± 8%). In two additional patients who had progressed from classical to blastoid MCL, an increase in the number of survivin-positive cells was observed in the blastoid variant (1.4- and three-fold, respectively).Analysis of Survivin Expression by Real-Time RT-PCRQuantification of survivin expression was assessed by real-time RT-PCR. The comparative CT (cycle threshold) method was used for relative quantification of survivin mRNA expression, after confirming that survivin cDNA and GUS cDNA were amplified with the same efficiency. Survivin expression in normal lymphocytes was used as a reference control, and the survivin expression levels of these cells adopted the arbitrary value of 1. Granta 519, a MCL-derived cell line, showed 100-fold increase in survivin levels compared to normal lymphocytes. The expression of survivin by real-time RT-PCR was analyzed in 51 MCL tumors. Survivin mRNA was detected in all MCL samples, with a mean relative value of 4 ± 4. Higher levels of survivin mRNA were detected in the blastic MCL variants (10 ± 7, n = 7), compared to those obtained in classical (4 ± 3, n = 41) and pleomorphic (3 ± 2, n = 3) tumors (Figure 5). Moreover, a significant linear correlation was found between mRNA survivin levels and the number of survivin-positive cells analyzed by immunohistochemistry (Pearson Regression Test, P < 0.0001).Figure 5Real-time RT-PCR quantitative analysis of survivin mRNA expression in MCL. Blastic MCL variant showed higher survivin mRNA expression than classical and pleomorphic MCL variants. Survivin mRNA expression levels are given in arbitrary units using normal lymphocytes as reference control. PCR arbitrary units were defined as described in Material and Methods. Symbols above the bars represent outlier cases.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of Survivin Variants by RT-PCRUsing specific primers, the different survivin variants were detected in seven MCL tumors. The specificity of amplification products was confirmed by DNA s