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Journal of Clinical Oncology, Vol 22, No 9 (May 1), 2004: pp. 1682-1688 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.10.172 Survivin Expression Predicts Poorer Prognosis in Anaplastic Large-Cell LymphomaFrom the Departments of Hematopathology and Lymphoma-Myeloma, The University of Texas M.D. Anderson Cancer Center, Houston, TX Address reprint requests to L. Jeffrey Medeiros, MD, Department of Hematopathology, Box 72, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; jmedeiro{at}mail.mdanderson.org
PURPOSE: Survivin, a member of the inhibitor of apoptosis (IAP) family, is not detected in normal adult tissues but is overexpressed in various cancers, including some types of lymphoma. The frequency and prognostic significance of survivin expression in anaplastic large-cell lymphoma (ALCL) is unknown. MATERIALS AND METHODS: We assessed for survivin expression in 62 ALCL tumors (30 anaplastic lymphoma kinase [ALK]-positive and 32 ALK-negative) obtained before doxorubicin-based chemotherapy. Given that survivin is a target of the STAT3 signaling pathway and STAT3 is activated in ALCL, survivin expression was also correlated with STAT3 activation. RESULTS: Survivin was expressed in 34 tumors (55%) and did not correlate with ALK. A significant association between survivin expression and STAT3 activation was observed (P = .007, Fisher's exact test). For the ALK-positive group, the 5-year failure-free survival (FFS) was 34% for patients with survivin-positive ALCL compared with 100% for patients with survivin-negative ALCL (P = .009, log-rank test). For the ALK-negative group, the 5-year FFS was 46% for patients with survivin-positive tumors compared with 89% for patients with survivin-negative tumors (P = .03, log-rank test). Overall survival was similarly worse for patients with survivin-positive tumors in both the ALK-positive and ALK-negative groups. Furthermore, multivariate analysis confirmed the independent prognostic value of survivin expression, along with age older than 60 years and Ann Arbor stage III or IV. CONCLUSION: Survivin is expressed in approximately half of ALCL tumors and independently predicts unfavorable clinical outcome. Modulation of survivin expression or function may provide a novel target for experimental therapy in patients with ALCL.
Survivin, a member of the inhibitor of apoptosis (IAP) family, is expressed during embryonic and fetal development but is not expressed in terminally differentiated adult tissues.1 Evidence from in vitro studies suggests that survivin inhibits cell death initiated by both the extrinsic and intrinsic (mitochondrial) apoptotic pathways.2 Survivin is also involved in regulation of mitotic spindle assembly and cytokinesis.2 Overexpression of survivin has been demonstrated in a variety of human cancers.1 Among hematologic malignancies, survivin is expressed in adult T-cell lymphoma and leukemia,3 Hodgkin's disease,4 and high-grade non-Hodgkin's lymphomas.1,5 Moreover, in one study survivin expression predicted clinical outcome in patients with diffuse large B-cell lymphoma.6 Anaplastic large-cell lymphoma (ALCL) accounts for approximately 3% of adult and 10% to 30% of childhood non-Hodgkin's lymphomas.7 A subset of ALCLs carry the t(2;5)(p23;q35) or variant translocations involving the 2p23 locus, resulting in ALK overexpression.8,9 Several pathways are proposed to be involved in ALCL oncogenesis, including the ras, PLC-gamma and PI-3-kinase pathways, resulting in deregulation of cell proliferation and apoptosis.10–12 In addition, BCL-2 family proteins are differentially expressed in ALK-positive and ALK-negative ALCL, indicating that pro- or antiapoptotic signals from the intrinsic pathway may be involved in ALCL pathogenesis.13,14 Of note, apoptosis regulating proteins correlate with outcome in these tumors,15 although the biologic or clinical significance of survivin in ALCL is undefined. In this study, we investigated survivin expression in 62 ALCL tumors. Our findings suggest that survivin expression independently predicts unfavorable clinical outcome in patients with ALCL.
Cell Lines Four ALK-positive ALCL cell lines that carry the t(2;5) were assessed including Karpas 299 (gift from M. Kadin, Boston, MA), SR-786, SU-DHL-1 (both from DSMZ, Braunschweig, Germany), and JB-6 (gift from D. Jones, Houston, TX). Cell lines were maintained in RPMI 1640 medium supplemented with 1% nonessential amino acids, 10% fetal calf serum (Invitrogen Corp, Grand Island, NY), and 1% streptomycin-penicillin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Subcellular Fractionation and Western Blot Analysis
ALCL Tumors The diagnosis of ALCL was based on criteria specified by the WHO classification.7 All ALCLs uniformly expressed CD30. Fifty-two ALCLs (84%) were of T-cell lineage, and 10 (16%) were of null-cell lineage. ALK expression, assessed using the ALK-1 antibody (DAKO), was positive in 30 tumors (48%). The pattern of ALK immunostaining was nuclear and cytoplasmic in 23 tumors (77%) and cytoplasmic only in seven (23%). A nuclear and cytoplasmic pattern has been shown to strongly correlate with the t(2;5) and a cytoplasmic pattern occurs with variant abnormalities affecting the alk gene.7
Tissue Microarray and Immunohistochemical Methods Based on the results from subcellular fractionation studies, only cytoplasmic survivin expression was considered positive, irrespective of intensity. Because the distribution of data showed that most tumor cells were positive in cases of survivin-positive ALCL, no cutoff for survivin expression was used in the statistical analysis. As expected, many mitotic figures also stained positively for survivin.18 The survivin immunostains were reviewed without knowledge of clinical data by multiple observers.
Statistical Analysis
Expression and Subcellular Localization of Survivin in ALCL Western blot analysis after subcellular fractionation demonstrated predominantly cytoplasmic localization of survivin in Karpas 299 and SUDHL-1 cells (Fig 1A). Low level nuclear expression, most likely attributable to the presence of mitoses, was also observed in accord with published data.18 Using immunohistochemistry, cytoplasmic survivin was detected in 34 of 62 ALCL tumors (55%; Fig 1B), 19 ALK-positive tumors (63%), and 15 ALK-negative tumors (47%; P = .2, Fisher's exact test). In the ALK-positive group, ALCLs with a nuclear and cytoplasmic staining pattern for ALK expressed survivin in 16 of 23 tumors (70%) compared with three of seven (42%) ALCLs with cytoplasmic staining pattern for ALK (P = .37, Fisher's exact test).
Survivin expression correlated with elevated serum ß2-microglobulin levels but not with other clinical or laboratory parameters (Table 1). In addition, an inverse correlation between survivin and BCL-2 expression was observed (P = .048, Fisher's exact test; Table 2).
Survivin Expression Is Associated With STAT3 Activation in ALCL Tumors Because survivin is a target of the STAT3 signaling pathway22 and STAT3 is activated (Tyr-705 phosphorylated) in ALCL,23 we correlated survivin expression with STAT3 activation assessed immunohistochemically (pSTAT3) in 53 tumors.16 When pSTAT3 expression was considered as a categoric variable using a 20% cutoff, survivin expression correlated significantly with pSTAT3 (P = .007, Fisher's exact test; Table 2). Moreover, when pSTAT3 expression levels were analyzed as a continuous variable, the mean percentage of pSTAT3-positive tumor cells was 59% for survivin-positive ALCL, compared with 34% for survivin-negative ALCL (P = .02, Mann-Whitney test).
Univariate Survival Analysis
ALK-positive and ALK-negative tumors were also analyzed separately. For the ALK-positive group, the 5-year FFS was 34% for patients with survivin-positive tumors versus 100% for patients with survivin-negative tumors (P = .009, log-rank test; Fig 3A). For the ALK-negative group, the 5-year FFS was 46% for patients with survivin-positive tumors versus 89% for patients with survivin-negative tumors (P = .03, log-rank test; Fig 3B).
Survivin also correlated with OS. For the ALK-positive group, the five-year OS was 56% for patients with survivin-positive tumors versus 100% for patients with survivin-negative tumors (P = .08, log-rank test; Fig 4A). For the ALK-negative group, the 5-year OS was 60% for patients with survivin-positive tumors versus 92% for patients with survivin-negative tumors (P = .04, log-rank test; Fig 4B).
Multivariate Survival Analysis For the entire study group, the independent prognostic value of survivin was evaluated by multivariate analysis using the Cox’s proportional hazards model. As shown in Table 3, survivin expression was independently associated with inferior FFS along with two clinical parameters included in the IPI: age at least 60 years and Ann Arbor stage III or IV. The other components of the IPI, performance status, number of extranodal sites and serum lactate dehydrogenase level were not significantly associated with FFS when these factors were entered in the model (Table 3). When IPI score (0 to 1 v = > 2) and survivin entered the model, survivin expression remained an independent adverse prognostic factor (P = .02) for FFS.
In this study, we report that survivin, a member of the IAP family, is expressed in approximately half of ALCL tumors, regardless of ALK status. Apart from its cytoplasmic localization, survivin has been shown to localize to various components of the mitotic apparatus and can be expressed in mitoses in a cell-cycle dependent manner.2 To address the question whether survivin is predominantly expressed in the nucleus or cytoplasm of ALCL cells, we performed Western blot analysis after subcellular fractionation. Our findings demonstrated that survivin is predominantly localized in the cytoplasm of ALCL cells. Using univariate survival analysis, we have shown that survivin expression was significantly associated with a poorer clinical outcome for the entire group of ALCL patients (Fig 2). This was true for both FFS and OS. Furthermore, multivariate analysis for FFS demonstrated the independent prognostic significance of survivin in this series, along with age older than 60 years and Ann Arbor stage III or IV. Similar analysis for OS also showed the independent prognostic significance of survivin. These results are in agreement with a previous study in which survivin expression predicted clinical outcome in patients with diffuse large B-cell lymphoma.6 Similar associations of survivin with adverse clinical outcome have been reported in other malignancies.3,24–27 Given that ALCL as defined in the WHO classification7 is an heterogeneous entity, and it has been reported that ALK-positive ALCL is associated with a younger patient age and better prognosis than ALK-negative tumors,28–30 we also analyzed the ALK-positive and ALK-negative ALCL cases separately. Survivin expression also correlated significantly with inferior FFS and OS within ALK-positive and ALK-negative groups (Figs 3 and 4). Previous studies have shown that survivin is a target of the STAT3 signaling pathway,22 and Zamo et al23 reported that STAT3 is activated by the NPM-ALK fusion transcript in ALCL. Furthermore, we have recently demonstrated that STAT3 also can be activated in a subset of ALK-negative ALCL,16 and this is also true for many other tumor types.31 We therefore correlated survivin expression with STAT3 activation as assessed by pSTAT3tyr705 immunostaining.16 A significant correlation between survivin and active STAT3 levels was observed (P = .007). These associations between survivin and STAT3 are further supported by Aoki et al,32 who showed that STAT3 inhibition repressed survivin transcription in primary effusion lymphoma cell lines, suggesting that constitutive activation of STAT3 stimulates survivin expression. In addition, infection of ALCL cells with an adenoviral vector that carries dominant negative STAT3 (mutation at residue 705tyr->phe) results in downregulation of survivin.33 Survivin, by inhibiting chemotherapy-induced apoptosis, may provide a biologic explanation for the worse prognosis of patients with survivin-positive ALCL. Moreover, because an association between STAT3 activation and poor survival was reported recently in ALK-positive and ALK-negative ALCL,16 and expression of survivin and pSTAT3 are strongly correlated, the adverse prognostic impact of survivin expression in ALCL also may be related to STAT3 activation. Regulation of survivin expression and function is complex. The survivin gene promoter is specifically induced in G2/M phase of the cell cycle34 and is located within a CpG island,2 but its methylation status has not been studied in lymphomas. Expression of survivin may be regulated at the posttranscriptional level via degradation by the ubiquitin-proteasome pathway.35 In addition, the antiapoptotic function of survivin is regulated by its Thr34-phosphorylation by the cyclin-dependent kinase p34cdc2-cyclin B1 complex.36 In conclusion, survivin expression in ALCL predicts unfavorable clinical outcome in both ALK-positive and ALK-negative tumors. Survivin expression,37 or its Thr34-phosphorylation that enhances tumor cell apoptosis,38 may be targets appropriate for novel therapies in ALCL.
The authors indicated no potential conflicts of interest.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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14. Rassidakis GZ, Lai R, McDonnell TJ, et al: Overexpression of mcl-1 in anaplastic large cell lymphoma cell lines and tumors. Am J Pathol 160:2309–2310, 2002
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18. Fortugno P, Wall NR, Giodini A, et al: Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function. J Cell Sci 115:575–585, 2002 19. Kaplan EL, Meier P: Non-parametric estimation from incomplete observations. J Am Stat Assoc 53:257–481, 1958 20. Mantel N: Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother Rep 50:163–170, 1966[Medline] 21. Cox DR: Regression models and life tables (with Discussion). J R Stat Soc B 34:187–220, 1972 22. Mahboubi K, Li F, Plescia J, et al: Interleukin-11 up-regulates survivin expression in endothelial cells through a signal transducer and activator of transcription-3 pathway. Lab Invest 81:327–334, 2001[Medline] 23. Zamo A, Chiarle R, Piva R, et al: Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death. Oncogene 21:1038–1047, 2002[CrossRef][Medline] 24. Adida C, Berrebi D, Peuchmaur M, et al: Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet 351:882–883, 1998[CrossRef][Medline]
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28. Shiota M, Nakamura S, Ichinohasama R, et al: Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: A distinct clinicopathologic entity. Blood 86:1954–1960, 1995
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30. Gascoyne RD, Aoun P, Wu D, et al: Prognostic significance of anaplastic lymphoma kinase (ALK) protein expression in adults with anaplastic large cell lymphoma. Blood 93:3913–3921, 1999 31. Bromberg J: Stat proteins and oncogenesis. J Clin Invest 109:1139–1142, 2002[CrossRef][Medline]
32. Aoki Y, Feldman GM, Tosato G: Inhibition of STAT3 signaling induces apoptosis and decreases survivin expression in primary effusion lymphoma. Blood 101:1535–1542, 2003 33. Amin HM, McDonnell TJ, Ma Y, et al: Selective inhibition of STAT3 induces apoptosis and G1 cell cycle arrest in ALK-positive anaplastic large cell lymphoma. Oncogene, 2004 [in press] 34. Li F, Ambrosini G, Chu EY, et al: Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396:580–584, 1998[CrossRef][Medline] 35. Zhao J, Tenev T, Martins LM, et al: The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner. J Cell Sci 113:4363–4371, 2000[Abstract]
36. O'Connor DS, Grossman D, Plescia J, et al: Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc Natl Acad Sci U S A 97:13103–13107, 2000 37. Shen C, Buck A, Polat B, et al: Triplex-forming oligodeoxynucleotides targeting survivin inhibit proliferation and induce apoptosis of human lung carcinoma cells. Cancer Gene Ther 10:403–410, 2003[CrossRef][Medline]
38. Wall NR, O'Connor DS, Plescia J, et al: Suppression of survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis. Cancer Res 63:230–235, 2003 Submitted October 24, 2003; accepted February 20, 2004.
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 and Fisher's exact tests were used to compare survivin expression with various clinicopathologic parameters. The Mann-Whitney U test was chosen for nonparametric correlation of survivin expression with continuous variables. Failure-free survival (FFS) was defined as time from initiation of therapy to last follow-up, primary treatment failure, or relapse. Overall survival (OS) was measured from the onset of treatment to the time of last follow-up or death from any cause. Actuarial survival analysis was based on the method of Kaplan and Meier.
2) International Prognostic Index (IPI) score were analyzed separately. 
