|Year : 2018 | Volume
| Issue : 3 | Page : 131-137
Gravin and survivin gene expression levels as possible therapeutic targets for acute myeloid leukemia in adult Egyptian patients
Hadeer A Abbassy1, Dalia A Elneely1, Ahmed A Shehata2
1 Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Alexandria University, Alexandria, Egypt
|Date of Submission||24-Feb-2017|
|Date of Acceptance||14-Mar-2017|
|Date of Web Publication||3-Dec-2018|
Hadeer A Abbassy
34, Ahmed Yehia Street, Zezenia, Alexandria
Source of Support: None, Conflict of Interest: None
Background Despite the recent progress in diagnosis and management, acute myeloid leukemia (AML) still remains a fatal hematologic malignancy, which invites the need for accurate predictors of clinical outcome.
Objective The aim of the present study was to explore the possible prognostic importance of gravin and survivin gene expression in adult patients with de-novo AML by correlating their expression levels with response to induction therapy, disease-free survival (DFS), and overall survival (OS).
Patients and methods This study was conducted on 105 patients with de-novo AML, and 45 age-matched and sex-matched patients were selected as a control group. RNA isolation from bone marrow aspirates or peripheral blood and cDNA preparation followed by quantitative real-time reverse transcription-PCR were done to assess expression of gravin and survivin.
Results Gravin expression was markedly downregulated whereas survivin gene showed an overexpression in AML cases. There was a significant association between gravin and survivin expression levels with poor clinical outcome. OS and DFS were significantly lower in patients with low gravin and high survivin expression levels.
Conclusion Survivin overexpression and gravin downregulation were significantly associated with adverse clinical outcome and tendency to chemoresistance in AML. The degree of their expression derangement has been found to be correlated with a lower complete remission rate and shorter OS and disease-DFS which renders them as future candidates for target through adjuvant immunotherapy.
Keywords: acute myeloid leukemia, expression, gravin, survivin
|How to cite this article:|
Abbassy HA, Elneely DA, Shehata AA. Gravin and survivin gene expression levels as possible therapeutic targets for acute myeloid leukemia in adult Egyptian patients. Egypt J Haematol 2018;43:131-7
|How to cite this URL:|
Abbassy HA, Elneely DA, Shehata AA. Gravin and survivin gene expression levels as possible therapeutic targets for acute myeloid leukemia in adult Egyptian patients. Egypt J Haematol [serial online] 2018 [cited 2019 Apr 18];43:131-7. Available from: http://www.ehj.eg.net/text.asp?2018/43/3/131/246773
| Introduction|| |
Acute myeloid leukemia (AML) remains a fatal hematologic malignancy characterized by the clonal expansion of myeloid blasts in the peripheral blood, bone marrow (BM), and/or other tissues . AML is the commonest acute leukemia in adult patients. With intensive induction therapy, complete remission (CR) rates between 65 and 75% are achieved in younger patients (<60 years). Unfortunately, more than 50% of these patients experience relapse, thus reducing the overall survival (OS) rate to only 30–40% after 5 years. Results are more devastating in patients older than 60 years . Consequently, accurate predictors of clinical outcome are necessary for the design of appropriate treatment for individual patients .
The tumor suppressor gene (TSG) gravin/AKAP12 encodes a scaffolding protein and is a member of the cyclic AMP-dependent kinase-anchoring proteins (AKAPs) . Evidence that AKAP12 might be a tumor and/or metastasis suppressor comes from its downregulation by specific oncogenes in cancer cell lines and human cancer tissues compared with normal controls. Moreover, its upregulation by treatments that suppress oncogenic growth by direct demonstrations shows that its re-expression suppresses in-vitro and in-vivo oncogenic growth, especially metastasis formation, or that its loss produces a tumor-prone or metastasis-prone condition . Epigenetic silencing of AKAP12 transcription might occur in myeloid malignancies and showed that AKAP12 is a target of aberrant DNA methylation in AML cell lines and in primary hematopoietic cells of children with myelodysplastic syndrome or AML .
One of the most frequently upregulated transcripts in cancer and almost undetectable in many normal adult tissues is survivin, which is a member of the inhibitors of apoptosis protein family . Because of being crucial in regulation of cell proliferation and cell death and its differential expression in many cancers, survivin is considered as a promising therapeutic target. Currently several clinical trials employing different approaches including antisense oligonucleotides, small molecule inhibitors, and immunotherapy are in progress . Overexpression of survivin has been identified in several hematologic malignancies . Significantly higher levels of survivin were discovered in CD34+38− AML stem/progenitor cells than in bulk blasts and total CD34+ AML cells, and the higher levels of survivin were associated with a shorter OS and event-free survival. Furthermore, the expression of survivin correlated with the expression of multiple proteins involved in proliferation and cell survival .
The aim of the present study was to determine gravin and survivin expression levels in adult patients with de-novo AML and to explore their possible prognostic importance by correlating their expression levels with response to induction therapy, disease-free survival (DFS), and OS.
| Patients and methods|| |
This study was conducted on 105 patients with de novo AML. Patients who met the diagnostic criteria for AML were selected from the Hematology Unit of Alexandria Main University Hospital between December 2013 and December 2015. There were 81 males and 24 females with age range from 18 to 65 years (median: 45 years). Moreover, 45 age-matched and sex-matched normal BM transplantation donors were selected as a control group. There were 27 male and 18 female, with age range from 20 to 60 years (median: 38 years). The selection of these patients was based on the following criteria: full history taking; thorough clinical examination; and standard diagnostic methods, including cytomorphological, cytochemical, cytogenetic, and immunophenotypic evaluation, which was established using FACS Calibur flow cytometer equipped with Cell Quest Software (Becton Dickinson, San Diego, California, USA). Positivity by flow cytometry was defined as an expression in at least 20% of cells in the gated population of interest, compared with internal negative control cells. Newly diagnosed AMLs with different FAB subtypes and normal karyotyping by conventional cytogenetics on bone marrow aspirate (BMA) at the time of diagnosis were selected for the study. To establish cytogenetically normal AML, 20 or more metaphase cells from the samples had to be examined to assure normal karyotypes. Patients with therapy-related AML were excluded from the study. RNA isolation from BMAs or peripheral blood and cDNA preparation followed by quantitative real-time PCR were done to determine the expression of gravin and survivin levels in both cases and controls.
Then patients received the ‘3+7’ standard induction chemotherapy protocol: doxorubicin (45 mg/m2/day) for 3 days and cytarabine (100 mg/m2/day as a continuous 24 h intravenous infusion) for 7 days. BMA was done between 21 and 28 days after the initiation of chemotherapy to demonstrate the morphological remission. Consolidation comprised three to four courses of high-dose cytosine arabinoside (3 g/m2 every 12 h on days 1, 3, and 5; total, 18 g/m2). Patients were followed up once every 3 months with clinical examination and complete blood counts. BMA was done if there was any doubt of a relapse on clinical examination or peripheral smear. CR was a normocellular BM containing less than 5% blasts and showing evidence of normal maturation of other BM elements, with neutrophil count of up to 1×109/l and a platelet (PLT) count of up to 100×109/l. OS was the time from diagnosis to the date of death. For patients achieving CR, DFS was the time from the date of first CR to an event (death in first CR or relapse).
The study was approved by the medical ethics committee, and informed consents were obtained from all participants involved in the study. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008.
Gravin and survivin expression
Purification of total cellular RNA from human whole blood was done using the PureLink RNA Mini Kit (Ambion by life technologies, Waltham, Massachusetts, USA). The concentration and purity of RNA were estimated by measuring the absorbance at 260, 280, and 230 nm using Nanodrop2000 spectrophotometer (Thermo Scientific, USA). A260 : A230 ratio greater than 1.9 and A260 : A280 ratio greater than 2.1 indicate highly pure RNA. Reverse transcription (cDNA synthesis) was done using high-capacity cDNA reverse transcription kit (Applied Biosystems, USA) by Biometra Thermal Cycler (Gottingen, Germany). Quantitative reverse transcription-PCR for gravin and survivin expression was performed using TaqMan Universal Master Mix II (Applied Biosystems). Thermocycling was carried out for 5 min at 95°C, followed by 45 cycles of denaturation for 30 s at 95°C and combined annealing/extension for 30 s at 60°C. For quantification of gene expression, the comparative Ct method was used. Expression data were normalized to the geometric mean of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase to control the variability in expression levels by reverse transcription-PCR, using real-time cycler Rotor-Gene Q (Qiagen, USA). Data analysis was done using the 2-ΔΔCT method as relative gene expression values.
Data were fed to the computer and analyzed using IBM statistical package for the social sciences software package version 20.0 (SPSS; SPSS Inc., Chicago, Illinois, USA). Comparisons between groups for categorical variables were done using χ2-test. Student’s t-test was used to compare two groups for normally distributed quantitative variables. Mann–Whitney test and Kruskal–Wallis test were used to compare two or more groups for abnormally distributed quantitative variables. Paired t-test and Wilcoxon signed ranks test were performed for comparison between different periods. Spearman’s coefficient was used to correlate between quantitative variables. Kaplan–Meier test was used for survival analysis, and the statistical significance of differences among curves was determined by log-rank test. Confidence interval was 95%. Significance of the obtained results was judged at the 5% level.
| Results|| |
Demographic findings, initial peripheral blood counts, and tumor burden parameters in cases compared with the control group are shown in [Table 1]. Gravin expression was markedly downregulated whereas survivin gene showed an overexpression in leukemic blasts of AML cases. The difference in gravin as well as survivin expression level between patients and controls was statistically significant (P<0.001). There was a significant association between both gravin as well as survivin expression levels and demographic data (Page<0.001, Psex=0.001) ([Table 2]). A significant negative correlation was found between gravin expression level and age (rs=−0.608, P<0.001) as well as BM blasts percentage (rs=−0.250, P=0.010) ([Table 3]). Conversely, a significant positive correlation was detected between gravin expression level and PLT count (rs=0.277, P=0.004). Moreover, there was a significant positive correlation between survivin expression level and age (rs=0.615, P<0.001) as well as BM blasts percentage (rs=0.242, P=0.013). On the contrary, a significant negative correlation was found between survivin expression level and PLT count (rs=−0.281, P=0.004). There was a significant association between gravin expression level and the clinical outcome of patients with AML, where it was highest in patients who achieved a CR and lowest in relapsed patients and those who died (P<0.001) ([Table 4]). Another significant relation was detected between survivin expression level and induction outcome, where it was markedly lower in patients with continuous CR than in relapsed patients and highest in those with induction death (P<0.001). OS and DFS times according to gravin and survivin expression levels are shown in [Table 5] and [Figure 1]. OS and DFS were lower in patients with low gravin expression level (cumulative survival: 70.6, 0.0%) than in patients with high gravin expression level (cumulative survival: 100, 83.3%) with a statistical significance (P<0.001). OS and DFS times were higher in patients with low survivin expression level (cumulative survival: 100, 88.2%) than in patients with high survivin expression level (cumulative survival: 72.2, 0.0%) with a statistical significance (P<0.001).
|Table 1 Comparison between the two studied groups according to different parameters|
Click here to view
|Table 2 Relation between demographic data with gravin and survivin relative quantitative expression levels in cases|
Click here to view
|Table 3 Correlation between gravin and survivin relative quantitative expression levels in cases with different parameters in cases|
Click here to view
|Table 4 Relation between different clinical outcomes with gravin and survivin relative quantitative expression levels in cases|
Click here to view
|Table 5 Overall and disease-free survival with gravin and survivin relative quantitative expression in cases|
Click here to view
|Figure 1 Kaplan–Meier survival curves for overall and disease-free survival with gravin and survivin expression|
Click here to view
| Discussion|| |
In the present study, gravin expression level was significantly lower in AML cases in comparison with control samples (P<0.001). In harmony with our results, Mostafa et al.  found that the relative quantitative expression level of gravin gene was 16 folds lower in patients than in controls. Boultwood et al.  reported that gravin expression was downregulated in all patients with de-novo AML and at all phases of the disease, thus concluding that gravin was consistently downregulated in myeloid malignancies, and its expression was absent in several cases.
On the contrary, survivin expression was significantly higher in AML cases in comparison with control samples (P<0.001). This agrees with Sun et al.  who revealed that survivin expression in patients with AML was higher than that in controls (P<0.001).
Moreover, Wang et al.  found that survivin mRNA was detected in all the cell lines and most of the patients with acute leukemia. However, in normal peripheral blood and anemia samples, expression of survivin gene was not detected . A quantitative analysis of the expression of the antiapoptotic gene survivin in malignant hematopoietic cells revealed that survivin mRNA levels were demonstrable in all malignant cell lines, but no survivin mRNA expression was detected in normal leukocyte fractions . Furthermore, in a study on hematopoietic cells transformed by FLT3-ITD (FMS-like tyrosine kinase 3-internal tandem duplication) frequently expressed in AML, Abe et al.  demonstrated, that the FLT3-ITD mutations increased survivin expression which was associated with enhanced or accelerated cell proliferation.
The discrepancy in the expression levels of gravin and survivin between patients with AML and controls also agrees with Yildirim et al.  who observed that the expression levels of gravin were decreased in 77.5% of AML cases as compared with the control group. Moreover, Azzazi et al.  found that survivin gene was constitutively expressed in AML cases but was barely detectable in the control group (P<0.001). Zaimy et al.  found that survivin was one of the genes that were overexpressed in AML and have antiapoptotic properties.
Considering the demographic data, the present study identified a significantly lower expression of gravin and a significantly higher expression of survivin in male than in female patients (P<0.001). Meanwhile, a significant negative correlation was found between gravin expression and age, and a significant positive correlation was found between survivin expression and age (P<0.001). These results are in agreement with another study, which reported a significant positive correlation between survivin expression and age but not with the patients’ sex . These data point out that the older the patient, the lower the expression level of gravin and the higher the expression level of survivin and that was significantly predominating in the studied male patients. Wilhelm et al.  demonstrated that epigenetic silencing of AKAP12 has been linked to the process of tumorigenesis. They found that AKAP12α promoter showed DNA hypermethylation in juvenile myelomonocytic leukemia samples, which was associated with decreased AKAP12α expression and correlated with older age at diagnosis, elevated levels of fetal hemoglobin, and poor prognosis .
In the current study, it was found that there was a significant positive correlation between gravin and PLT count (P=0.004) and conversely a significant negative correlation between survivin and PLT count (P=0.004). On the contrary, gravin and survivin expression levels showed no statistically significant correlations with hemoglobin concentration (P=0.080, 0.084) or white blood cells (WBC) count (P=0.177 and 0.219, respectively). These results are in accordance with data reported by Azzazi et al.  who noticed that there was no significant correlation between survivin expression and WBC count, hemoglobin, PLT count, or BM blasts. Furthermore, Yildirim et al.  showed no correlation between gravin expression values and WBC count, hemoglobin, or PLT count in patients with acute leukemia. Regarding BM blasts, there was a statistically significant negative correlation between gravin expression and BM blast percentage (P=0.010). On the contrary, a significant positive correlation was found between survivin expression and BM blasts (P=0.013). Likewise, Sadek et al.  reported a statistically positive correlation between survivin expression and BM blasts.
Regarding remission after induction chemotherapy, Kern et al.  considered that failure of achieving blast clearance from BMAs after 1 or 2 weeks of induction chemotherapy a poor prognosis. Liso et al.  evaluated the response to induction chemotherapy by estimating the degree of residual leukemic infiltration in the BM after 14 days of chemotherapy.
In the present study, owing to BM aplasia present on day 14 and poor general condition of the patients, the evaluation of response to induction chemotherapy was carried out on day 28, where only 45 (42.9%) patients achieved a CR whereas 60 (57.1%) patients failed to respond to induction therapy. There was a statistically significant association between gravin expression level and the clinical outcome of patients with AML where it was highest in patients who achieved a CR and lowest in relapsed patients and those who died (P<0.001). In agreement with these results, Mostafa et al.  found that gravin showed a significantly lower expression in patients who failed to respond to therapy than those who achieved a CR (P<0.001). Similarly, Raslan and Heikel  detected a significant difference in gravin expression in patients with CR in comparison with those with induction failure (P=0.032). As for the risk of relapse, Matsushita et al.  reported that hypermethylation of TSGs might be involved in the relapse of childhood acute lymphoblastic leukemia. Moreover, Agrawal et al.  reported that increased level of methylation of TSGs in the BM of patients with acute leukemia in clinical remission provides a powerful indicator for a high risk of leukemia relapse in these patients.
On the contrary, a significant relation was detected between survivin expression level and induction outcome where it was markedly lower in patients with continuous CR than in relapsed patients and highest in those who died (P<0.001). Concordant with our results, another study by Azzazi  et al. had reported that the difference in survivin expression level between patients who achieved a CR and those who failed to respond to induction therapy was statistically significant (P=0.005). Zhu et al.  also showed that CR in survivin-positive patients with AML was significantly lower than that reported in survivin-negative patients (P=0.018). Carter et al.  recently profiled survivin expression in samples from 511 newly diagnosed patients with AML and reported that survivin levels predict poor clinical outcomes in AML. Similarly, Smith et al.  revealed that high expression of survivin was associated with poor clinical outcome in pediatric patients with AML. This indicates that lower gravin expression and overexpression of survivin were associated with an unfavorable response to induction chemotherapy which coincides with the detected correlation in this study with lower PLT count and higher BM blasts.
The present study demonstrated that OS and DFS were lower in patients with low gravin expression than in patients with high gravin expression with a statistical significance (P<0.001). Boultwood et al.  reported that the expression levels of gravin were decreased in both the accelerated phase and plastic crisis when compared with the chronic phase and thus they suggested that a progressive reduction in the expression levels of gravin may be associated with rapid progression and decreased survival. In contrast, Yildirim et al.  hypothesized that OS was longer in cases with lower gravin expression. Nevertheless, Mostafa et al.  found that gravin expression was not correlated with OS or DFS, and this was confirmed by multivariable analysis. The current study also revealed that OS and DFS times were higher in patients with low survivin expression than in patients with high survivin expression with a statistical significance (P<0.001). This is in agreement with Huang et al.  who demonstrated that higher levels of survivin correlated with a worse OS in patients with AML.
| Conclusion|| |
Survivin overexpression and gravin downregulation were significantly associated with adverse clinical outcome and tendency to chemoresistance in AML. The degree of their expression derangement has been found to be correlated with a lower CR rate and shorter OS and DFS, which renders them as future candidate for targets through adjuvant immunotherapy.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
O’Donnell MR, Abboud CN, Altman J, Appelbaum FR, Arber DA, Attar E et al.
Acute myeloid leukemia. J Natl Compr Canc Netw
Zhao J, He A, Zhang W, Meng X, Gu L. Quantitative assessment of MLAA-34 expression in diagnosis and prognosis of acute monocytic leukemia. Cancer Immunol Immunother
Döhner H, Estey EH, Amadori S, Appelbaum FR, Büchner T, Burnett AK et al.
Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood
Nauert JB, Klauck TM, Langeberg LK, Scott JD. Gravin, an autoantigen recognized by serum from myasthenia gravis patients, is a kinase scaffold protein. Curr Biol
Gelman IH. Emerging roles for SSeCKS/Gravin/AKAP12 in the control of cell proliferation, cancer malignancy, and barriergenesis. Genes Cancer
Flotho C, Kratz C, Niemeyer CM, Targeting RAS. Signaling pathways in juvenile myelomonocytic leukemia. Curr Drug Targets
Velculescu VE, Madden SL, Zhang L, Lash AE, Yu J, Rago C et al.
Analysis of human transcriptomes. Nat Genet
Carter BZ, Andreeff M. Targeting survivin in leukemia. Oncol Rev
Fulda S. Inhibitor of apoptosis proteins in hematological malignancies. Leukemia
Carter BZ, Qiu Y, Huang X, Diao L, Zhang N, Coombes KR et al.
Survivin is highly expressed in CD 34+
leukemic stem/progenitor cells and predicts poor clinical outcomes in AML. Blood
Mostafa MR, Yahia RS, Abd El Messih HM, El-Sisy E, El Ghannam DM. Gravin gene expression in acute myeloid leukemia. Med Oncol
Boultwood J, Pellagatti A, Watkins F, Campbell LJ, Esoof N, Cross NC et al.
Low expression of the putative tumour suppressor gene gravin in chronic myeloid leukaemia, myelodysplastic syndromes and acute myeloid leukaemia. Br J Haematol
Sun WX, Zhang PH, Fang LH, Tian Z, Tang KJ, Rao Q et al.
Expression of survivin in patients with acute myeloid leukemia. Zhongguo Shi Yan Xue Ye Xue Za Zhi
Wang XJ, Dai GY, Cao LM, Wang GH, Zhu HF, Zhang Y et al.
Prognostic significance of expression of survivin in acute leukemia. Chin J Cancer Res
Moriai R, Asanuma K, Kobayashi D, Yajima T, Yagihashi A, Yamada M et al.
Quantitative analysis of the anti-apoptotic gene survivin expression in malignant haematopoietic cells. Anticancer Res
Abe M, Pelus LM, Singh P, Hirade T, Onishi C, Purevsuren J et al.
Internal tandem duplication in FLT3 attenuates proliferation and regulates resistance to the FLT3 inhibitor AC220 by modulating p21 Cdkn1a and Pbx1 in hematopoietic cells. PLoS One
Yildirim M, Paydas S, Tanriverdi K, Seydaoglu G, Disel U, Yavuz S Gravin gene expression in acute leukaemias: clinical importance and review of the literature. Leuk Lymphoma
Azzazi M, El-Arab SE, Hegab HM, Elsalakawy W, Ibrahim R, Shazly M. Prognostic significance of intracellular survivin in myeloid blast cells as an inhibitor of apoptosis in Egyptian adult acute myeloid leukemia patients. Egypt J Haematol
Zaimy MA, Jebali A, Bazrafshan B, Mehrtashfar S, Shabani S, Tavakoli A et al.
Coinhibition of overexpressed genes in acute myeloid leukemia subtype M2 by gold nanoparticles functionalized with five antisense oligonucleotides and one anti-CD33(+)/CD34(+) aptamer. Cancer Gene Ther
Wilhelm T, Lipka DB, Witte T, Wierzbinska JA, Fluhr S, Helf M et al.
Epigenetic silencing of AKAP12 in juvenile myelomonocytic leukemia. Epigenetics.
Sadek H, Ragab S, Rasmy H, Guindy NM, Ezzat W, Hamed M. Expression of the antiapoptotic gene survivin in acute leukemias. J Am Sci
Kern W, Haferlach T, Schoch C, Löffler H, Gassmann W, Heinecke A et al.
Early blast clearance by remission induction therapy is a major independent prognostic factor for both achievement of complete remission and long-term outcome in acute myeloid leukemia: data from the German AML Cooperative Group (AMLCG) 1992 Trial. Blood
Liso V, Albano F, Pastore D, Carluccio P, Mele G, Lamacchia M et al.
Bone marrow aspirate on the 14th day of induction treatment as a prognostic tool in de novo adult acute myeloid leukemia. Haematologica
Raslan H, Heikel A. Expression of the putative tumor suppressor gene gravin and ß actin in acute leukaemias: clinical importance and prognostic value by real-time quantitative PCR. Med J Cairo Univ
Matsushita C, Yang Y, Takeuchi S. Aberrant methylation in promoter-associated CpG islands of multiple genes in relapsed childhood acute lymphoblastic leukemia. Oncol Rep
Agrawal S, Unterberg M, Koschmieder S, zur Stadt U, Brunnberg U, Verbeek W et al.
DNA methylation of tumor suppressor genes in clinical remission predicts the relapse risk in acute myeloid leukemia. Cancer Res
Zhu FB, Wang SY, Zhang YW. Expression of antigen CD40 and survivin gene and their clinical implications in acute myeloid leukemia. Zhonghua Nei Ke Za Zhi
Smith AM, Little EB, Zivanovic A, Hong P, Liu AK, Burow R et al.
Targeting survivin with YM155 (Sepantronium Bromide): a novel therapeutic strategy for paediatric acute myeloid leukaemia. Leuk Res
Huang J, Lyu H, Wang J, Liu B. Influence of survivin-targeted therapy on chemosensitivity in the treatment of acute myeloid leukemia. Cancer Lett
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]