Departments of 1 Oncology, 2 Hematology and 3 Biostatistics, Cukurova University Faculty of Medicine, Adana, Turkey
Received 11 January 2003; revised 3 March 2003; accepted 4 March 2003
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Abstract |
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Antiapoptotic signals are important in the development, progression and prognosis of malignant tumors. The aim of this study was to determine the two distinct antiapoptotic signalssurvivin and avenin 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
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Introduction |
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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 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.
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Patients and methods |
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Survivin and aven were detected by using real-time quantitative PCR. Detail of the method is given below.
Primers
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.
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 x 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.
Statistical analysis
Chi-square and Fishers 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.
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Results |
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Discussion |
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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 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 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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Footnotes |
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References |
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