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Survivin and aven: two distinct antiapoptotic signals in acute leukemias
Annals of Oncology 14: 1045–1050, 2003 DOI: 10.1093/annonc/mdg277
Original article
Survivin and aven: two distinct antiapoptotic signals in acute leukemias S. Paydas1*, K. Tanriverdi2, S. Yavuz1, U. Disel1, B. Sahin1 & R. Burgut3 Departments of 1Oncology, 2Hematology and 3Biostatistics, Cukurova University Faculty of Medicine, Adana, Turkey Received 11 January 2003; revised 3 March 2003; accepted 4 March 2003
tumors. The aim of this study was to determine the two distinct antiapoptotic signals—survivin and aven—in acute leukemias and compare them with clinical and hematological findings and response to therapy. Real-time quantitative PCR was used and survivin and aven were detected at the messenger (m)RNA level. Patients and methods: Sixty-five patients with acute leukemia [37 with acute myeloblastic leukemia (AML) and 28 with acute lymphoblastic leukemia (ALL)] were used as the study group and 10 healthy subjects were used as the control group. Results: Survivin was between 0.0 and 0.829 copy number/cell (median 0.0721, mean 0.5424301909 ± 0.139799488589) and aven was between 0.0 and 0.853 copy number/cell (median 0.0124, mean 0.070335542 ± 0.1524685709). We found an important association between survivin and aven (P = 0.000). Both survivin and aven were higher in the study group than in the controls (P = 0.001 and 0.035, respectively). When we compared survivin and aven with other clinical and hematological parameters, there was an important association between survivin and extramedullary involvement (P = 0.033), survivin and alkaline phosphatase (P = 0.06), white blood cell (WBC) count and lactate dehydrogenase (LDH) (P = 0.000), WBC count and uric acid (P = 0.074), hemoglobin level and LDH (P = 0.072), LDH and uric acid (P = 0.057), CD7 expression and survivin (P = 0.097), and CD34 expression and aven (P = 0.058). Response to therapy was evaluated according to the survivin and aven levels. Survivin level was lower in refractory patients as compared with complete responders (P = 0.085). Aven level was higher in patients with relapse as compared with non-relapse patients (P = 0.04). There was no important association between survivin or aven and performance status, lymphadenopathy or organomegaly. Conclusions: Both survivin and aven are important antiapoptotic signals in acute leukemias, and the association between extramedullary involvement, CD7 expression and CD34 expression, which are important poor prognostic indicators in acute leukemias, suggests that survivin and/or aven may be novel prognostic indicators in acute leukemias. Further studies with a higher number of patients will be more informative. Key words: acute leukemia, antiapoptotic signals, aven, CD7 expression, CD34 expression, extramedullary involvement, response to therapy, survivin
Introduction The balance between cell death and cell viability is important in tissue homeostasis. Abnormalities in the control of programmed cell death (apoptosis) play an important role in tumorigenesis [1, 2]. Evolutionarily preserved multistep cascade is regulated by proteins that promote or inhibit apoptotic cell death [3]. Bcl-2 was the first protein shown to cause prolonged cell survival by inhibiting apoptosis [4]. Inhibitors of apoptosis proteins (IAPs) are originally identified in baculoviruses and highly conserved across species similar to their viral counterparts. Expression of human IAP genes can inhibit apoptosis induced by a variety of stimuli. Six human IAPs have been described so far: NAIP, CIAP1,
*Correspondence to: Professor S. Paydas, Department of Oncology, Cukurova University Faculty of Medicine, Adana, Turkey. Tel: +90-322-338-6060; Fax +90-322-338-6153; E-mail: [email protected] © 2003 European Society for Medical Oncology
CIAP2, XIAP, survivin and apollon [4–9]. There are several apoptosis inhibitors that cause inhibition of apoptosis. Survivin, a member of this family of proteins, is present during fetal development, but is undetectable in terminally differentiated adult tissues [10]. It suppresses apoptosis induced by fas, bax, caspases and anticancer drugs [11]. It not only controls apoptosis but also participates in cell-cycle progression, and thereby integrates apoptosis and cell division [12]. Survivin prevents apoptosis by blocking caspase activity [11]. It is expressed at the G2/M phase of the cell cycle [12]. Caspase activity occurs during each cell cycle and survivin functions to block this activity [13]. Aven, a novel antiapoptotic member, binds to both Bcl-xL and the caspase regulator, Apaf-1. Aven is broadly expressed and is conserved in mammalian species. It suppresses apoptosis induced by Apaf-1 and caspase-9 [14]. The expression of survivin may be a general feature of cancer and survivin alone or with other antiapoptosis genes such as Bcl-2
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Background: Antiapoptotic signals are important in the development, progression and prognosis of malignant
1046 may extend the viability of transformed cells and regulate their susceptibility/resistance to apoptosis-based therapy. For this reason survivin may provide an ideal therapeutic target for its selective expression in neoplasia [4]. Aven is less widely studied in cancer cells but its antiapoptotic feature is known. The aim of this study is to demonstrate two distinct antiapoptotic signals, survivin and aven, showing activity at different stages of apoptosis in acute leukemias, and to compare them with known clinical factors in leukemia.
Patients and methods
Survivin and aven were detected by using real-time quantitative PCR. Detail of the method is given below.
No. of patients
65 (37 AML and 28 ALL)
Age (years)
15–75 (median 32, mean 36.91 ± 17.61)
Male/female
40/25
Physical exam
LAP 22 patients, hepato/splenomegaly 35 patients, EMT 12 patients
Hb (g/dl)
3–13.5 (median 7.6, mean 8.01 ± 2.21)
Hct (%)
10–39 (median 22.71, mean 23.7 ± 6.6)
WBC (× 109)
0.815–250 (median 24.25, mean 42.3 ± 54.12)
Platelets (× 109)
2–434 (median 27.5, mean 43.335 ± 63.822)
Response
CR 39 patients, PR 8 patients, refractory 13 patients, early death 5 patients
Survivin (copy number/cell)
0.0–0.829 (median 0.0721, mean 0.5424301909 ± 0.139799488589)
Aven (copy number/cell)
0.0–0.853 (median 0.0124, mean 0.0703355 ± 0.152468579)
ALL, acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; CR, complete response; EMT, extramedullary involvement; LAP, lymphadenopathy; Hb, hemoglobin; Hct, hematocrit; PR, partial response; WBC, white blood cell count.
Primers
Statistical analysis
Primers were designed using the Primer Premier Software in METIS Biotechnology (Ankara, Turkey). Aven primers were AF 5′-GATTTCAGTGTCCTCCTTAG-3′ and AR 5′-CCTTGCCATCATCAGTTCTC-3′. The aven mRNA Genbank accession number is AF283508. Survivin primers were SF 5′-ACCAGGTGAGAAGTGAGGGA-3′ and SR 5′-AACAGTAGAGGAGCCAGGGA-3′. The survivin mRNA Genbank accession number is NM001168.
Chi-square and Fisher’s exact tests were used for relapse and response evaluation. Wilcoxon U-test was used for nonparametric evaluations. Survival times according to the survivin and aven levels were compared by log-rank test.
Messenger (m)RNA isolation and complementary (c)DNA synthesis Peripheral blood samples were taken into tubes with K3EDTA from the patients (study group) and from healthy subjects (control group). White blood cell (WBC) counting was performed in Beckman Coulter GenS System 2. The equivalent to 3 × 106 WBC volume was taken. Red cells were removed by lysis with red blood cell lysis buffer (Roche Applied Science, Germany). The WBCs were suspended with lysis buffer of MagNA pure LC mRNA Isolation Kit (Roche Applied Science) and lysed cells were stored at –20°C until mRNA isolation. mRNA isolations were performed using MagNA pure LC automated DNA, RNA isolating and PCR setup instrument (Roche Applied Science). Isolated mRNAs were reverse transcribed with 1st Strand cDNA Synthesis Kit (Roche Applied Science). cDNAs were stored at –20°C until real-time quantitative PCR was performed.
Real-time quantitative PCR of survivin Real-time quantitative PCR was performed using a LightCycler rapid thermal cycler instrument (Roche Applied Science). Reactions were performed in a 10 µl volume with 5 pmol primers and MgCl2 4 mM using a LightCycler FastStart DNA Master SYBR Green I kit. The amplification protocol was 10 min at 95°C for activating hot start Taq polymerase, then 0 s at 95°C, 5 s at 60°C, 10 s at 72°C and 0 s at 80°C for a total of 45 cycles. Fluorescence readings were performed at 84°C every cycle to prevent fluorescence from primer dimers. The quantification data were analyzed with the LightCycler software. Results were evaluated as copy number/cell.
Results Our study group consisted of 65 patients with acute leukemia (37 with AML and 28 with ALL) and 10 healthy subjects as the control group. Forty of our patients were male and 25 were female; age range was between 15 and 75 years (median 31, mean 36.49 ± 17.42). Baseline hematologic test results and survivin and aven levels are shown in Tables 1 and 2. Lymphadenopathy was seen in 22 patients, hepato- and/or splenomegaly (organomegaly) was found in 35 patients and extramedullary involvement was detected in 12 patients. Complete response was achieved in 39 patients, partial response in eight patients and 13 patients were refractory to remission induction therapy. Five patients died during induction therapy. Both survivin and aven were higher in study group patients than in the controls (P = 0.001 and 0.035, respectively). Survivin and aven levels were compared with baseline clinical and hematological findings, response to therapy and overall survival. Among these comparisons the most important correlation was between aven and survivin (P = 0.009 and 0.000 with correlation and Spearman’s nonparametric correlation, respectively). There were important correlations between survivin and extramedullary involvement (P = 0.033) (Figure 1), survivin and alkaline phosphatase (P = 0.06), WBC count and lactate dehydrogenase (LDH) (P = 0.000), WBC count and uric acid (P = 0.074), hemoglobin and LDH (P = 0.072), and LDH and uric acid (P = 0.057). When surface antigen expressions were evaluated we found that there was an association between CD7 expression and survivin
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Sixty-five patients with acute leukemia were analysed for survivin and aven expression. All patients were diagnosed as de novo acute leukemia, 37 with acute myeloblastic leukemia (AML) and 28 with acute lymphoblastic leukemia (ALL). Routine diagnostic procedures, including morphology, histochemistry and surface immunophenotyping, were performed in all patients. Median percentage of leukemic blast cells in peripheral blood was >90%. Ara-C + idarubicin (7 + 3 regimen) was used for AML and an intensive regimen consisting of cyclophosphamide, vincristine, L-asparaginase, prednisolone, methotrexate and Ara-C was used for ALL.
Table 1. Baseline demographic and hematological findings
1047 Table 2. Survivin and aven levels according to the baseline characteristics and statistical significance Survivin
P
Aven
P
Sex Male
0–0.829 (0.006395)
Female
0–0.704 (0.00808)
0–0853 (0.0124) 0.973
0–0.762 (0.0201)
0.521
PS Good
0–0.829 (0.00727)
Poor
0–0.704 (0.00736)
0–0.229 (0.0124) 0.399
0–0.853 (0.0130)
0.661
Disease 0–0.324 (0.00653)
ALL
0–0.829 (0.00772)
0–0.853 (0.0186) 0.233
0–0.299 (0.0060)
0.177
EMT +
0.000871–0.829 (0.0097)
–
0–0.704 (0.00653)
0–0.853 (0.022355) 0.033
0–0.762 (0.0127)
0.153
LAP +
0–0.829 (0.00594)
–
0–0.324 (0.00808)
0–0.299 (0.003695) 0.358
0–0.853 (0.0201)
0.399
Organomegaly +
0–0.829 (0.00653)
–
0–0.853 (0.00543) 0.185
0.920
ALL, acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; EMT, extramedullary involvement; LAP, lymphadenopathy; PS, performance status.
Figure 1. Extramedullary (EMT) involvement and survivin.
Figure 2. Association between aven and relapse.
(P = 0.097), CD33 expression and aven (P = 0.001), CD34 expression and aven (P = 0.058), CD8 expression and extramedullary involvement (P = 0.043), and CD10 expression and lympadenopathy (P = 0.057). Response to therapy was evaluated according to the survivin and aven levels. Aven level was found to be higher in patients with relapse as compared with non-relapse patients (P = 0.04) (Figure 2). Survivin level was lower in refractory patients as compared with complete responders (P = 0.085). There was no
important association between survivin or aven and performance status, lympadenopathy or organomegaly. We performed further analyses on survivin and aven levels using four subgroups: 16 patients (24.6%) had higher levels of both survivin and aven (group 1), both signals were lower than controls in 30 patients (46.2%) (group 2), higher survivin and lower aven were detected in 17 patients (26.2%) (group 3) and higher aven and lower survivin were detected in two patients (group 4). We
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AML
1048
Figure 4. High and low survivin and aven expressors and CD33.
compared the clinical parameters mentioned above in these four groups and only WBC count and CD33 expression were found to be higher in group 1 than group 2 (P = 0.041 and 0.028, respectively) (Figures 3 and 4).
to high in acute ATL patients and lowest in chronic ATL patients. In these studies overexpression of survivin has been suggested as responsible for malignant behavior in ATL [30, 33]. Also, it was found that survivin controls the B-cell CLL proliferative pool, so interfering with apoptosis, and that its expression may be mediated by microenvironmental stimuli [31]. Generally, survivin was expressed in all AML cell lines and most of the leukemia cells but not in normal peripheral blood mononuclear cells and/or bone marrow cells [10, 29, 30, 33, 35, 36]. Survivin and aven expression were found to be higher in our study group (in 35 patients, 54%) than in controls. In some studies survivin-expressing leukemias have been found to have a worse prognosis compared with nonexpressors [29, 30, 33, 34]. In a study covering 125 AML patients, survivin expression has been detected in 75 samples (60%) and it has been found that survivin correlated with lower WBC count and favorable/intermediate cytogenetics. There was no difference in complete response rate and overall survival between survivin-positive and -negative patients but survivin was found to be an unfavorable prognostic factor. Survivin has been studied using immunohistochemistry in 222 patients with diffuse large B-cell lymphoma. Survivin expression was found in 60% of the patients and 5-year survival was found to be inferior in patients expressing survivin (40% versus 54%) [34]. In our study we found that survivin level was higher in patients with extramedullary tumor and higher CD7 expression, parameters that are poor prognostic indicators in acute leukemias although there are some controversies [40, 41]. This is an important association in our study. However, higher levels of aven in relapsing patients than non-relapsing patients suggest that aven is a novel poor prognostic indicator in acute leukemia. Increased survivin expression in response to cytokines raises the intriguing possibility that Bcl-2 and survivin may represent complementary survival pathways that are differentially regulated by the cell-cycle status of leukemic progenitors. Quiescent progenitors are protected from apoptosis and are restrained from entering the cell cycle by the expression of Bcl-2 and possibly of
Discussion Programmed cell death is a feature of living cells, and damaged cells are eliminated in this way. Inhibitors of programmed cell death aberrantly prolong cell viability, so contributing to the occurrence and growth of tumors [3]. Survivin is a bifunctional protein that suppresses apoptosis and regulates cell division [15]. Antiapoptotic properties by such regulatory gene expressions would clinically provide a significant growth advantage and disclose malignant behavior in tumors. Survivin is a unique apoptosis inhibitor and is believed to play a role in oncogenesis [4]. Survivin is switched off during fetal development and is not found in nonneoplastic adult tissues. However, it is prominently re-expressed by all the most common human cancers including cancers of the lung, genito-urinary, malignant melanoma, neuroblastoma, hepatocellular, pancreas, stomach, colon, breast cancers and soft tissue sarcomas [16–28]. The prognostic value of survivin in hemopoietic neoplasias has not been as widely studied as in solid tumors. Although the data are limited, the prognostic value of survivin has been studied in some hemopoietic neoplasias such as high-grade lymphomas and leukemias [10, 29–38]. Various studies in solid tumors have revealed a correlation between survivin expression and a clinically unfavorable course of disease, suggesting that survivin expression is a poor prognostic factor [22, 39]. The results are not clear enough in hemopoietic neoplasias, but in some studies it has been found that survivin expression is a bad prognostic indicator [34]. Chronic B- and T-cell leukemias, such as chronic lymphocytic leukemia (CLL) and adult T-cell leukemia (ATL), seem to represent the best example of a defective apoptosis resulting in accumulation of leukemic cells. In ATL, survivin was found to be prominently and consistently expressed; expression level was low
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Figure 3. High and low survivin and aven expressors and white blood cell (WBC) count.
1049
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16. Swana HS, Grossman D, Anthony JN et al. Tumor content of the antiapoptosis molecule survivin and recurrence of bladder cancer. Lancet 1999; 341: 452–453. 17. Saitoh Y, Yaginuma Y, Ishikawa M. Analysis of Bcl-2, Bax, and survivin genes in uterine cancer. Int J Oncol 1999; 15: 137–141. 18. Grossman D, McNiff JM, Li F, Altieri DC. Expression and targeting of the apoptosis inhibitor, survivin, in human melanoma. J Invest Dermatol 1999; 113: 1076–1081. 19. Tanaka K, Iwamoto S, Gon G et al. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clin Cancer Res 2000; 6: 127–134. 20. Islam A, Kagayama H, Takada N et al. High expression of survivin mapped to 17q25, is significantly associated with poor prognostic factors and promotes cell survival in human neuroblastoma. Oncogene 2000; 19: 617–623. 21. Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res 1998; 58: 1808–1812. 22. Kawasaki H, Altieri DC, Lu CD et al. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res 1998; 58: 5071–5074. 23. Ito T, Shiraki K, Sugimoto K et al. Survivin promotes cell proliferation in human hepatocellular carcinoma. Hepatology 2000; 31: 1080–1085. 24. Sarela AI, Macadam RC, Farmery SM et al. Expression of the antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma. Gut 2000; 46: 645–650. 25. Satoh K, Kaneko K, Hirota M et al. Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors. Cancer 2001; 92: 271–278. 26. Würi P, Kappler M, Meye A et al. Co-expression of survivin and TERT and risk of tumor-related death in patients with soft-tissue sarcoma. Lancet 2002; 359: 943–945. 27. Yu J, Leung WK, Ebert MPA et al. Increased expression of survivin in gastric cancer patients and in first degree relatives. Br J Cancer 2002; 87: 91–97. 28. Yu J, Leung WK, Ebert MPA et al. Increased expression of survivin in gastric cancer patients and in first degree relatives. Br J Cancer 2002; 87: 91–97. 29. Mori A, Wada H, Nishimura Y et al. Expression of the antiapoptosis gene survivin in human leukemia. Int J Hematol 2002; 75: 161–165. 30. Kamihira S, Yamada Y, Hirakata Y et al. Aberrant expression of caspase regulatory genes in adult T-cell leukemia: survivin is an important determinant for prognosis. Br J Haematol 2001; 114: 63–69. 31. Granziero L, Ghia P, Circosta P et al. Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood 2001; 97: 2777–2783. 32. Notarbartolo M, Cervello M, Dusoncher L et al. Resistance to diverse apoptotic triggers in multidrug resistant HL60 cells and its possible relationship to the expression of P-glycoprotein, Fas and of the novel antiapoptosis factors IAP (inhibitory apoptosis proteins). Cancer Lett 2002; 180: 91–101. 33. Mori N, Yamada Y, Hata T et al. Expression of survivin in HTLV-I cell lines and primary ATL cells. Biochem Biophys Res Commun 2001; 282: 1110–1113. 34. Adida C, Haioun C, Gaulard P et al. Prognostic significance of survivin in diffuse large B-cell lymphomas. Blood 2000; 96: 1921–1925. 35. Morai R, Asanuma K, Kobayashi D et al. Quantitative analysis of the anti-apoptotic gene survivin expression in malignant hematopoietic cells. Anticancer Res 2001; 21: 595–600. 36. Shinozawa I, Inokuchi K, Wakabayashi I, Dan K. Disturbed expression of the anti-apoptosis gene, survivin, and EPR-1 in hematological malignancies. Leuk Res 2000; 24: 965–970.
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Bcl-xL. However, once recruited into the cell cycle, proliferating cells could switch to a survivin-mediated survival pathway that enables them to successfully complete mitosis and avoid a default induction of apoptosis at cell division. In our study group, survivin and aven levels were found to be parallel and association between survivin and aven was significantly important. Although they act at different levels, it has been shown that two survival pathways, Bcl-2 and survivin, may function in concert to prevent cell death [10]. This finding was supported in our study. In conclusion, it has been shown that survivin and aven are two important antiapoptotic signals in acute leukemias. The association between higher survivin expression and extramedullary involvement, CD7 expression, higher WBC count and alkaline phosphatase, which are strong negative prognostic factors in acute leukemias, suggests that survivin is a candidate parameter to determine poor prognosis in acute leukemias. Aven is a novel antiapoptotic signal, and we found higher aven levels in relapsing than non-relapsing patients. This finding must be evaluated in larger study groups.
1050 37. Andersen MH, Pederson LO, Becker JC, Straten PT. Identification of a cytotoxic T lymphocyte response to the apoptosis inhibitor protein survivin in cancer patients. Cancer Res 2001; 61: 869–872. 38. Adida C, Recher C, Raffoux E et al. Expression and prognostic significance of survivin in de novo acute myeloid leukemia. Br J Haematol 2000; 111: 196–203. 39. Adida-Adida C, Berrebi D, Paichmaur M et al. Anti-apoptosis gene survivin, and prognosis of neuroblastoma. Lancet 1998; 351: 882–883.
40. Del Poeta G, Stasi R, Venditti A et al. CD 7 expression in acute leukemia. Leuk Lymphoma 1995; 17: 111–119. 41. Byrd JC, Weiss RB, Arthur DC et al. Extramedullary leukemia adversely affects hematologic complete remission rate and overall survival in patients with t(q22;q22). Results from Cancer and Leukemia Group B8461. J Clin Oncol 1997; 15: 466–475.
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Original article
Survivin and aven: two distinct antiapoptotic signals in acute leukemias S. Paydas1*, K. Tanriverdi2, S. Yavuz1, U. Disel1, B. Sahin1 & R. Burgut3 Departments of 1Oncology, 2Hematology and 3Biostatistics, Cukurova University Faculty of Medicine, Adana, Turkey Received 11 January 2003; revised 3 March 2003; accepted 4 March 2003
tumors. The aim of this study was to determine the two distinct antiapoptotic signals—survivin and aven—in acute leukemias and compare them with clinical and hematological findings and response to therapy. Real-time quantitative PCR was used and survivin and aven were detected at the messenger (m)RNA level. Patients and methods: Sixty-five patients with acute leukemia [37 with acute myeloblastic leukemia (AML) and 28 with acute lymphoblastic leukemia (ALL)] were used as the study group and 10 healthy subjects were used as the control group. Results: Survivin was between 0.0 and 0.829 copy number/cell (median 0.0721, mean 0.5424301909 ± 0.139799488589) and aven was between 0.0 and 0.853 copy number/cell (median 0.0124, mean 0.070335542 ± 0.1524685709). We found an important association between survivin and aven (P = 0.000). Both survivin and aven were higher in the study group than in the controls (P = 0.001 and 0.035, respectively). When we compared survivin and aven with other clinical and hematological parameters, there was an important association between survivin and extramedullary involvement (P = 0.033), survivin and alkaline phosphatase (P = 0.06), white blood cell (WBC) count and lactate dehydrogenase (LDH) (P = 0.000), WBC count and uric acid (P = 0.074), hemoglobin level and LDH (P = 0.072), LDH and uric acid (P = 0.057), CD7 expression and survivin (P = 0.097), and CD34 expression and aven (P = 0.058). Response to therapy was evaluated according to the survivin and aven levels. Survivin level was lower in refractory patients as compared with complete responders (P = 0.085). Aven level was higher in patients with relapse as compared with non-relapse patients (P = 0.04). There was no important association between survivin or aven and performance status, lymphadenopathy or organomegaly. Conclusions: Both survivin and aven are important antiapoptotic signals in acute leukemias, and the association between extramedullary involvement, CD7 expression and CD34 expression, which are important poor prognostic indicators in acute leukemias, suggests that survivin and/or aven may be novel prognostic indicators in acute leukemias. Further studies with a higher number of patients will be more informative. Key words: acute leukemia, antiapoptotic signals, aven, CD7 expression, CD34 expression, extramedullary involvement, response to therapy, survivin
Introduction The balance between cell death and cell viability is important in tissue homeostasis. Abnormalities in the control of programmed cell death (apoptosis) play an important role in tumorigenesis [1, 2]. Evolutionarily preserved multistep cascade is regulated by proteins that promote or inhibit apoptotic cell death [3]. Bcl-2 was the first protein shown to cause prolonged cell survival by inhibiting apoptosis [4]. Inhibitors of apoptosis proteins (IAPs) are originally identified in baculoviruses and highly conserved across species similar to their viral counterparts. Expression of human IAP genes can inhibit apoptosis induced by a variety of stimuli. Six human IAPs have been described so far: NAIP, CIAP1,
*Correspondence to: Professor S. Paydas, Department of Oncology, Cukurova University Faculty of Medicine, Adana, Turkey. Tel: +90-322-338-6060; Fax +90-322-338-6153; E-mail: [email protected] © 2003 European Society for Medical Oncology
CIAP2, XIAP, survivin and apollon [4–9]. There are several apoptosis inhibitors that cause inhibition of apoptosis. Survivin, a member of this family of proteins, is present during fetal development, but is undetectable in terminally differentiated adult tissues [10]. It suppresses apoptosis induced by fas, bax, caspases and anticancer drugs [11]. It not only controls apoptosis but also participates in cell-cycle progression, and thereby integrates apoptosis and cell division [12]. Survivin prevents apoptosis by blocking caspase activity [11]. It is expressed at the G2/M phase of the cell cycle [12]. Caspase activity occurs during each cell cycle and survivin functions to block this activity [13]. Aven, a novel antiapoptotic member, binds to both Bcl-xL and the caspase regulator, Apaf-1. Aven is broadly expressed and is conserved in mammalian species. It suppresses apoptosis induced by Apaf-1 and caspase-9 [14]. The expression of survivin may be a general feature of cancer and survivin alone or with other antiapoptosis genes such as Bcl-2
Downloaded from http://annonc.oxfordjournals.org/ at University of Miami - Otto G. Richter Library on June 27, 2015
Background: Antiapoptotic signals are important in the development, progression and prognosis of malignant
1046 may extend the viability of transformed cells and regulate their susceptibility/resistance to apoptosis-based therapy. For this reason survivin may provide an ideal therapeutic target for its selective expression in neoplasia [4]. Aven is less widely studied in cancer cells but its antiapoptotic feature is known. The aim of this study is to demonstrate two distinct antiapoptotic signals, survivin and aven, showing activity at different stages of apoptosis in acute leukemias, and to compare them with known clinical factors in leukemia.
Patients and methods
Survivin and aven were detected by using real-time quantitative PCR. Detail of the method is given below.
No. of patients
65 (37 AML and 28 ALL)
Age (years)
15–75 (median 32, mean 36.91 ± 17.61)
Male/female
40/25
Physical exam
LAP 22 patients, hepato/splenomegaly 35 patients, EMT 12 patients
Hb (g/dl)
3–13.5 (median 7.6, mean 8.01 ± 2.21)
Hct (%)
10–39 (median 22.71, mean 23.7 ± 6.6)
WBC (× 109)
0.815–250 (median 24.25, mean 42.3 ± 54.12)
Platelets (× 109)
2–434 (median 27.5, mean 43.335 ± 63.822)
Response
CR 39 patients, PR 8 patients, refractory 13 patients, early death 5 patients
Survivin (copy number/cell)
0.0–0.829 (median 0.0721, mean 0.5424301909 ± 0.139799488589)
Aven (copy number/cell)
0.0–0.853 (median 0.0124, mean 0.0703355 ± 0.152468579)
ALL, acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; CR, complete response; EMT, extramedullary involvement; LAP, lymphadenopathy; Hb, hemoglobin; Hct, hematocrit; PR, partial response; WBC, white blood cell count.
Primers
Statistical analysis
Primers were designed using the Primer Premier Software in METIS Biotechnology (Ankara, Turkey). Aven primers were AF 5′-GATTTCAGTGTCCTCCTTAG-3′ and AR 5′-CCTTGCCATCATCAGTTCTC-3′. The aven mRNA Genbank accession number is AF283508. Survivin primers were SF 5′-ACCAGGTGAGAAGTGAGGGA-3′ and SR 5′-AACAGTAGAGGAGCCAGGGA-3′. The survivin mRNA Genbank accession number is NM001168.
Chi-square and Fisher’s exact tests were used for relapse and response evaluation. Wilcoxon U-test was used for nonparametric evaluations. Survival times according to the survivin and aven levels were compared by log-rank test.
Messenger (m)RNA isolation and complementary (c)DNA synthesis Peripheral blood samples were taken into tubes with K3EDTA from the patients (study group) and from healthy subjects (control group). White blood cell (WBC) counting was performed in Beckman Coulter GenS System 2. The equivalent to 3 × 106 WBC volume was taken. Red cells were removed by lysis with red blood cell lysis buffer (Roche Applied Science, Germany). The WBCs were suspended with lysis buffer of MagNA pure LC mRNA Isolation Kit (Roche Applied Science) and lysed cells were stored at –20°C until mRNA isolation. mRNA isolations were performed using MagNA pure LC automated DNA, RNA isolating and PCR setup instrument (Roche Applied Science). Isolated mRNAs were reverse transcribed with 1st Strand cDNA Synthesis Kit (Roche Applied Science). cDNAs were stored at –20°C until real-time quantitative PCR was performed.
Real-time quantitative PCR of survivin Real-time quantitative PCR was performed using a LightCycler rapid thermal cycler instrument (Roche Applied Science). Reactions were performed in a 10 µl volume with 5 pmol primers and MgCl2 4 mM using a LightCycler FastStart DNA Master SYBR Green I kit. The amplification protocol was 10 min at 95°C for activating hot start Taq polymerase, then 0 s at 95°C, 5 s at 60°C, 10 s at 72°C and 0 s at 80°C for a total of 45 cycles. Fluorescence readings were performed at 84°C every cycle to prevent fluorescence from primer dimers. The quantification data were analyzed with the LightCycler software. Results were evaluated as copy number/cell.
Results Our study group consisted of 65 patients with acute leukemia (37 with AML and 28 with ALL) and 10 healthy subjects as the control group. Forty of our patients were male and 25 were female; age range was between 15 and 75 years (median 31, mean 36.49 ± 17.42). Baseline hematologic test results and survivin and aven levels are shown in Tables 1 and 2. Lymphadenopathy was seen in 22 patients, hepato- and/or splenomegaly (organomegaly) was found in 35 patients and extramedullary involvement was detected in 12 patients. Complete response was achieved in 39 patients, partial response in eight patients and 13 patients were refractory to remission induction therapy. Five patients died during induction therapy. Both survivin and aven were higher in study group patients than in the controls (P = 0.001 and 0.035, respectively). Survivin and aven levels were compared with baseline clinical and hematological findings, response to therapy and overall survival. Among these comparisons the most important correlation was between aven and survivin (P = 0.009 and 0.000 with correlation and Spearman’s nonparametric correlation, respectively). There were important correlations between survivin and extramedullary involvement (P = 0.033) (Figure 1), survivin and alkaline phosphatase (P = 0.06), WBC count and lactate dehydrogenase (LDH) (P = 0.000), WBC count and uric acid (P = 0.074), hemoglobin and LDH (P = 0.072), and LDH and uric acid (P = 0.057). When surface antigen expressions were evaluated we found that there was an association between CD7 expression and survivin
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Sixty-five patients with acute leukemia were analysed for survivin and aven expression. All patients were diagnosed as de novo acute leukemia, 37 with acute myeloblastic leukemia (AML) and 28 with acute lymphoblastic leukemia (ALL). Routine diagnostic procedures, including morphology, histochemistry and surface immunophenotyping, were performed in all patients. Median percentage of leukemic blast cells in peripheral blood was >90%. Ara-C + idarubicin (7 + 3 regimen) was used for AML and an intensive regimen consisting of cyclophosphamide, vincristine, L-asparaginase, prednisolone, methotrexate and Ara-C was used for ALL.
Table 1. Baseline demographic and hematological findings
1047 Table 2. Survivin and aven levels according to the baseline characteristics and statistical significance Survivin
P
Aven
P
Sex Male
0–0.829 (0.006395)
Female
0–0.704 (0.00808)
0–0853 (0.0124) 0.973
0–0.762 (0.0201)
0.521
PS Good
0–0.829 (0.00727)
Poor
0–0.704 (0.00736)
0–0.229 (0.0124) 0.399
0–0.853 (0.0130)
0.661
Disease 0–0.324 (0.00653)
ALL
0–0.829 (0.00772)
0–0.853 (0.0186) 0.233
0–0.299 (0.0060)
0.177
EMT +
0.000871–0.829 (0.0097)
–
0–0.704 (0.00653)
0–0.853 (0.022355) 0.033
0–0.762 (0.0127)
0.153
LAP +
0–0.829 (0.00594)
–
0–0.324 (0.00808)
0–0.299 (0.003695) 0.358
0–0.853 (0.0201)
0.399
Organomegaly +
0–0.829 (0.00653)
–
0–0.853 (0.00543) 0.185
0.920
ALL, acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; EMT, extramedullary involvement; LAP, lymphadenopathy; PS, performance status.
Figure 1. Extramedullary (EMT) involvement and survivin.
Figure 2. Association between aven and relapse.
(P = 0.097), CD33 expression and aven (P = 0.001), CD34 expression and aven (P = 0.058), CD8 expression and extramedullary involvement (P = 0.043), and CD10 expression and lympadenopathy (P = 0.057). Response to therapy was evaluated according to the survivin and aven levels. Aven level was found to be higher in patients with relapse as compared with non-relapse patients (P = 0.04) (Figure 2). Survivin level was lower in refractory patients as compared with complete responders (P = 0.085). There was no
important association between survivin or aven and performance status, lympadenopathy or organomegaly. We performed further analyses on survivin and aven levels using four subgroups: 16 patients (24.6%) had higher levels of both survivin and aven (group 1), both signals were lower than controls in 30 patients (46.2%) (group 2), higher survivin and lower aven were detected in 17 patients (26.2%) (group 3) and higher aven and lower survivin were detected in two patients (group 4). We
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AML
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Figure 4. High and low survivin and aven expressors and CD33.
compared the clinical parameters mentioned above in these four groups and only WBC count and CD33 expression were found to be higher in group 1 than group 2 (P = 0.041 and 0.028, respectively) (Figures 3 and 4).
to high in acute ATL patients and lowest in chronic ATL patients. In these studies overexpression of survivin has been suggested as responsible for malignant behavior in ATL [30, 33]. Also, it was found that survivin controls the B-cell CLL proliferative pool, so interfering with apoptosis, and that its expression may be mediated by microenvironmental stimuli [31]. Generally, survivin was expressed in all AML cell lines and most of the leukemia cells but not in normal peripheral blood mononuclear cells and/or bone marrow cells [10, 29, 30, 33, 35, 36]. Survivin and aven expression were found to be higher in our study group (in 35 patients, 54%) than in controls. In some studies survivin-expressing leukemias have been found to have a worse prognosis compared with nonexpressors [29, 30, 33, 34]. In a study covering 125 AML patients, survivin expression has been detected in 75 samples (60%) and it has been found that survivin correlated with lower WBC count and favorable/intermediate cytogenetics. There was no difference in complete response rate and overall survival between survivin-positive and -negative patients but survivin was found to be an unfavorable prognostic factor. Survivin has been studied using immunohistochemistry in 222 patients with diffuse large B-cell lymphoma. Survivin expression was found in 60% of the patients and 5-year survival was found to be inferior in patients expressing survivin (40% versus 54%) [34]. In our study we found that survivin level was higher in patients with extramedullary tumor and higher CD7 expression, parameters that are poor prognostic indicators in acute leukemias although there are some controversies [40, 41]. This is an important association in our study. However, higher levels of aven in relapsing patients than non-relapsing patients suggest that aven is a novel poor prognostic indicator in acute leukemia. Increased survivin expression in response to cytokines raises the intriguing possibility that Bcl-2 and survivin may represent complementary survival pathways that are differentially regulated by the cell-cycle status of leukemic progenitors. Quiescent progenitors are protected from apoptosis and are restrained from entering the cell cycle by the expression of Bcl-2 and possibly of
Discussion Programmed cell death is a feature of living cells, and damaged cells are eliminated in this way. Inhibitors of programmed cell death aberrantly prolong cell viability, so contributing to the occurrence and growth of tumors [3]. Survivin is a bifunctional protein that suppresses apoptosis and regulates cell division [15]. Antiapoptotic properties by such regulatory gene expressions would clinically provide a significant growth advantage and disclose malignant behavior in tumors. Survivin is a unique apoptosis inhibitor and is believed to play a role in oncogenesis [4]. Survivin is switched off during fetal development and is not found in nonneoplastic adult tissues. However, it is prominently re-expressed by all the most common human cancers including cancers of the lung, genito-urinary, malignant melanoma, neuroblastoma, hepatocellular, pancreas, stomach, colon, breast cancers and soft tissue sarcomas [16–28]. The prognostic value of survivin in hemopoietic neoplasias has not been as widely studied as in solid tumors. Although the data are limited, the prognostic value of survivin has been studied in some hemopoietic neoplasias such as high-grade lymphomas and leukemias [10, 29–38]. Various studies in solid tumors have revealed a correlation between survivin expression and a clinically unfavorable course of disease, suggesting that survivin expression is a poor prognostic factor [22, 39]. The results are not clear enough in hemopoietic neoplasias, but in some studies it has been found that survivin expression is a bad prognostic indicator [34]. Chronic B- and T-cell leukemias, such as chronic lymphocytic leukemia (CLL) and adult T-cell leukemia (ATL), seem to represent the best example of a defective apoptosis resulting in accumulation of leukemic cells. In ATL, survivin was found to be prominently and consistently expressed; expression level was low
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Figure 3. High and low survivin and aven expressors and white blood cell (WBC) count.
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Bcl-xL. However, once recruited into the cell cycle, proliferating cells could switch to a survivin-mediated survival pathway that enables them to successfully complete mitosis and avoid a default induction of apoptosis at cell division. In our study group, survivin and aven levels were found to be parallel and association between survivin and aven was significantly important. Although they act at different levels, it has been shown that two survival pathways, Bcl-2 and survivin, may function in concert to prevent cell death [10]. This finding was supported in our study. In conclusion, it has been shown that survivin and aven are two important antiapoptotic signals in acute leukemias. The association between higher survivin expression and extramedullary involvement, CD7 expression, higher WBC count and alkaline phosphatase, which are strong negative prognostic factors in acute leukemias, suggests that survivin is a candidate parameter to determine poor prognosis in acute leukemias. Aven is a novel antiapoptotic signal, and we found higher aven levels in relapsing than non-relapsing patients. This finding must be evaluated in larger study groups.
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