|Year : 2018 | Volume
| Issue : 3 | Page : 952-956
Evaluation of survivin gene expression as a prognostic biomarker in pediatric B-acute lymphoblastic leukemia
Samar M Kamal Elden1, Ayman Azzam2, Fathia Elbassal1, Mahmoud A El-Hawy3, Nagwan Y Saleh3
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Biochemistry, National Liver Institute, Menoufia, Egypt
3 Department of Pediatrics, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||14-Feb-2017|
|Date of Acceptance||03-Apr-2017|
|Date of Web Publication||31-Dec-2018|
Samar M Kamal Elden
Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebin el Kom, Menoufia
Source of Support: None, Conflict of Interest: None
The aim of this study was to evaluate the level of survivin mRNA expression in Egyptian patients with acute pediatric lymphoblastic leukemia and to outline any significant relation between the level of survivin expression and the clinical and hematological findings in those patients.
Survivin, a unique member of the inhibitor of apoptosis protein family, plays an important role in regulating both apoptosis and cell division and was found to be highly expressed in various kinds of tumors.
Patients and methods
Survivin expression was quantitatively determined by real-time (RT) PCR in 30 newly diagnosed acute lymphoblastic leukemia (B-ALL) pediatric patients before and after induction therapy and in 15 age-matched and sex-matched immune thrombocytopenic purpura individuals serving as a control group.
A highly significant elevation (P < 0.001) was found in survivin mRNA level in B-ALL children at diagnosis compared with controls. After induction therapy, a significant decrease of survivin mRNA expression level was found in B-ALL patients compared with those at diagnosis (P < 0.001). Survivin mRNA level was significantly higher in the nonsurvived group and in patients with induction failure compared with patients with favorable response (P = 0.01). In addition, a positive correlation was found between survivin mRNA expression level and initial leukocyte count (r = 0.72; P < 0.001) and bone marrow blast cells percent (r = 0.44; P = 0.015).
Survivin is overexpressed in B-ALL at presentation and may be used as a prognostic marker for ALL in pediatric patients.
Keywords: acute lymphoblastic leukemia, apoptosis, biomarkers, prognosis
|How to cite this article:|
Kamal Elden SM, Azzam A, Elbassal F, El-Hawy MA, Saleh NY. Evaluation of survivin gene expression as a prognostic biomarker in pediatric B-acute lymphoblastic leukemia. Menoufia Med J 2018;31:952-6
|How to cite this URL:|
Kamal Elden SM, Azzam A, Elbassal F, El-Hawy MA, Saleh NY. Evaluation of survivin gene expression as a prognostic biomarker in pediatric B-acute lymphoblastic leukemia. Menoufia Med J [serial online] 2018 [cited 2019 Mar 21];31:952-6. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/952/248718
| Introduction|| |
Acute lymphocytic leukemia (ALL) remains among the most common childhood cancer. It is characterized by clonal proliferation and accumulation of progenitor cells exhibiting cell markers of the earliest stage of lymphoid maturation with features of either B-cell or T-cell commitment. ALL represents around 80% of pediatric cancers, with peak incidence between 2 and 5 years; however, it can affect all ages with a slight male predominance.
Impaired apoptosis is mediated by the inhibitor of apoptosis proteins family, such as survivin. Survivin was described in some tumors, including hematologic malignancies, where it correlates with poor prognosis. Survivin expression is absent or nearly absent in most differentiated normal tissues. Higher levels of survivin are associated with cell proliferation and metastasis in many cancers,. Elevated tumor survivin levels are also linked to resistance to chemotherapy and radiation,.
Survivin is a unique member of the inhibitor of apoptosis proteins family with the smallest molecular weight; 16.5 kDa and 142 amino acid. It is the product of a single gene (BIRC5) located on chromosome 8, 17q25. It consists of three introns and four exons and exists as a functioning homodimer,.
It plays an important role in regulating both apoptosis and cell division, where it is upregulated during cell division and is related to centrosomes and mitotic spindle microtubules. It controls chromosome spindle check point assembly for normal cell division and is maximally upregulated in the G2M phase and facilitates accurate stabilization of microtubules in late stages of mitosis,.
Interactions of survivin with caspase-3 and Fas were revealed to be associated with the development of leukemia. Being almost restricted to malignant tissues makes survivin an excellent target for immunotherapeutics.
The aim of this study was to evaluate the level of survivin mRNA expression in Egyptian patients with acute pediatric lymphoblastic leukemia and to delineate any significant correlation between the level of survivin with the clinical and hematological findings in those patients.
| Patients and Methods|| |
This study was conducted on thirty B-ALL patients (group I) – 17 male patients and 13 female patients, with a mean age ± SD of 10.70 ± 4.73 years (range: 2–18 years) – who had been diagnosed at the Hematology Unit, Clinical Pathology Departments between May 2015 and June 2016. B-ALL patients were compared with 15 immune thrombocytopenic purpura (ITP) patients (group II) with morphologically normal bone marrow with no evidence of dysplasia matched with the patients in age and sex (nine male patients and six female patients), with a mean age of 12.80 ± 3.73 years (range: 6–16 years).
ALL patients were newly diagnosed; they were subjected to full history taking, clinical examination, and laboratory investigations at time of presentation. They were followed up and re-evaluated at the end of induction therapy for the outcome of induction.
B-ALL was diagnosed and classified on the basis of full history taking, thorough clinical examination, imaging studies, and laboratory investigations including complete blood count and peripheral blood smear, bone marrow aspirate examination, immunophenotyping, and cytogenetic analysis.
Informed consent was obtained from parents or legal guardians of the children at the time of enrollment, according to the guidelines of ethics committee of Faculty of Medicine, Menoufia University.
Methods and procedures
Bone marrow samples were aspirated from patients before any therapeutic measures. After 4 weeks of induction therapy in ALL patients, bone marrow expression of survivin was re-evaluated. Survivin gene expression was quantitatively determined by using the real-time (RT)-PCR technique.
Total RNA was isolated using the Gene JET RNA purification kit provided by Fermentas (Promegacorporation, Madison, Wisconsin, USA), according to the manufacturer's protocol. The purified RNA was used for reverse transcription protocol. The concentration and purity of RNA was determined by measuring its absorbance at 260 nm (A260) and 280 nm (A280) in a ultraviolet-visible spectrophotometer. cDNA synthesis was immediately carried out.
First standard cDNA synthesis was done using kit (Promegacorporation, Madison, Wisconsin, USA). Briefly, for the preparation of first-strand cDNA, l. 0 μl of random hexamer primer and 6.0 μl of nuclease-free water were added to 5.0 μl of template RNA in a sterile, nuclease-free tube placed on ice, followed by incubation at 65°C for 5 min and addition of 4.0 μl of reaction buffer, l. 0 μl of Ribolock RNAse inhibitor, l μl of deoxynucleotide triphosphates mix, and l μl of μ-MULV Revert Aid (Moloney Murineleukemia virus reverse transcriptase). The contents of the tube were mixed gently and centrifuged, and then the tubes were placed in a thermal cycler for 5 min at 25°C, followed by 60 min at 42°C. The reaction was terminated by heating at 70°C for 5 min. cDNA were stored at −20°C until RT quantitative PCR was carried out.
Real-time quantitative PCR assay
The mRNA expression levels of survivin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene were measured by quantitative RT-PCR by using SYBR green by the ABI 7500 Real-time PCR system (Applied Bio Systems Inc., Foster City, California, USA) according to the manufacturer's protocol.
The sequence of primers for the survivin gene was as follows: forward: 5'-GGACCA CCG CAT CTC TAC AT-3'; and reverse: 5'-GCA CTT TCT TCG CAG TTT CC-3'. And primers for the GAPDH gene were as follows: forward: 5'-TGCACCA CCAAACT GCT TAGC-3'; and reverse: 5'-GGC ATG GAC TGT GGT CAT GAG-3'.
The expression levels of the survivin gene were quantitated using comparative threshold, used to determine the change in expression of a nucleic acid sequence (target) in the test and control samples. The comparative threshold value for any sample was normalized to the endogenous housekeeping gene GAPDH. All reactions were performed in duplicates and a negative control without template was included in each run (experiment). Relative expression values were detected by the and were utilized in statistical analysis.
Data were collected, tabulated, and statistically analyzed using the statistical package for the social sciences (SPSS, version 13; SPSS Inc., Chicago, Illinois, USA) and P value less than 0.05 was considered statistically significant. Quantitative data were expressed as mean and SD. Median was used in case of nonparametric variables. Mann–Whitney U-test was applied to compare two quantitative nonparametric groups. The correlation of survivin expression level with other prognostic markers was analyzed using Spearmen's correlation.
| Results|| |
A total of 30 patients with B-ALL were included in the study; the clinical data showed that 13 patients had an enlarged liver, 18 patients had an enlarged spleen, and 19 patients had lymphadenopathy. The immunophenotypic classification was as follows: 18 common B-ALL patients, 11 precursor B-ALL patients, and one case mature B-ALL. t(9;22) was positive in nine cases, whereas t (8;14) was positive in one case. No cytogenetic abnormalities were present in 16 cases and no data were available for four cases. As regards treatment response at the end of induction therapy, according to National Comprehensive Cancer Network guidelines, 19 patients (63.3%) achieved complete morphological remission (CR), eight (26.7%) patients showed induction failure (IF), and three (10%) patients died during the induction therapy (early death) [Table 1].
|Table 1: Clinical and laboratory characteristics of patients with acute lymphoblastic leukemia|
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For the survivin levels, a highly significant elevation (P < 0.001) was found in survivin mRNA level in acute lymphoblastic leukemia (ALL) patients at diagnosis (day 0) when compared with controls [Table 2].
|Table 2: Comparison between survivin expression in group I (day 0) and group II (control)|
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A significant decrease of survivin mRNA expression was found in patients after induction therapy when compared with their results at presentation (day 0) (P < 0.001) [Table 3].
|Table 3: Comparison of level of survivin expression in B-acute lymphoblastic leukemia patients at presentation (day 0) and its level at the end of induction (day 28)|
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Patients with unfavorable outcome; IF and early death were found to have significantly higher survivin level both at presentation and after the end of induction therapy when compared with those achieving complete remission (P = 0.01 and P < 0.001, respectively) [Table 4] and [Figure 1].
|Table 4: Difference in survivin gene levels regarding treatment outcome in B-acute lymphoblastic leukemia patients|
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|Figure 1: Comparison between survivin gene expression according to therapy response.|
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Studying the level of survivin expression on day 0 in relation to the high-risk cytogenetics revealed a significantly higher survivin level in patients with t(9;22) when compared with those with normal karyotype (P = 0.02) [Table 5].
|Table 5: Relation between survivin expression day 0 and cytogenetic analysis|
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Pearson's correlation between survivin mRNA expression level and laboratory data showed a positive correlation between survivin mRNA expression level and established risk factors such as initial leukocyte count (r=+0.72; P < 0.001) [Figure 2], peripheral blood blasts percentage (r=+0.4; P = 0.03), and bone marrow blasts percentage (r=+0.44; P = 0.015) with a negative correlation to hemoglobin level (r=−0.40; P = 0.03).
|Figure 2: Correlation between survivin gene expression and white blood cells as a standard risk factor.|
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| Discussion|| |
ALL remains the most common malignancy in children. Improvement in chemotherapy raised the five-year event-free survival of children with ALL above 80%. However, a considerable number of children with ALL still suffer from relapse.
Survivin is an apoptosis inhibitor that is believed to have a role in oncogenesis. The prognostic value of survivin has been studied in hemopoietic neoplasms, specially lymphomas and acute myeloid leukemias.
In the present study, we evaluated survivin mRNA levels in the bone marrow aspirates of children with newly diagnosed untreated B-ALL and after initial induction treatment in a trial to assess its involvement in the development of ALL and to prospect its prognostic value.
This study showed significantly increased levels of survivin mRNA in B-ALL compared with the ITP control group. This is in accordance with Ahmed et al.who reported that survivin overexpression was significantly increased in ALL patients compared with controls. Similarly, Yahya et al. reported a highly significant elevation of survivin protein in ALL child patients at diagnosis when compared with controls.
Our results were also supported by those of Troeger et al. who found that survivin was detected in 65% of the leukemic samples in contrast to negligible expression in nonmalignant hematopoietic cells, suggesting that survivin has relevant biological activity in ALL patients.
In our study, a significant decrease in survivin expression was clearly detected in response to treatment, and survivin mRNA level was significantly higher in the nonsurvived group and in patients with IF compared with patients with favorable response (P = 0.01). This was in agreement with Ahmed et al. who found a significant reduction of survivin level in ALL patients after therapy in relation to its level before therapy. Survivin overexpression has been identified as an adverse prognostic factor in various cancer types and was implicated in resistance to apoptosis induction by anticancer agents.
Troeger et al.and Esh et al.reported that pediatric ALL patients who were suffering from relapse of the disease or death had significant higher basal level of survivin, detected by flowcytometery, compared with patients still in remission; however, they revealed that, there was no significant difference in survivin expression according to response to induction therapy.
In our study, higher survivin expression was observed in Philadelphia positive ALL than in cases of normal karyotype. It was also high in the only one mature ALL case in our study. In a study of Ahmed et al., Philadelphia positive ALL also showed higher survivin levels.
In the current study, survivin mRNA expression level was positively correlated with established risk factors such as high initial leukocyte count. It was also positively correlated to peripheral and bone marrow blast counts and negatively correlated to hemoglobin level, with no correlation to age or platelet count. Ahmed et al. reported that survivin level showed a positive significant correlation with bone marrow blast cells and blast cell count in the peripheral blood. Similarly, in accordance with our results, Esh et al. reported a significant association between survivin expression and high white blood cells count at diagnosis. However, Troeger et al. and Li et al. reported no correlation between survivin overexpression and standard risk factors for bad prognosis. The cause of this controversy between our results and their results may be attributed to the age difference between our patients and theirs, which may affect the maturity of the apoptotic and antiapoptotic protein system.
In summary, the present study highlighted the association of survivin with development of childhood ALL. It revealed that survivin was significantly higher in B-ALL children at diagnosis than its levels in both ITP control and B-ALL children after induction; it was higher in ALL patients with t (9;22) and was related to bad treatment outcome.
| Conclusion|| |
High survivin expression is a new independent unfavorable prognostic factor in childhood B-ALL and should be considered for prognostic stratification and thus used in the design and evaluation of modern therapeutic trials. Further large-scale studies and longer follow-up periods are needed to confirm our findings and may suggest survivin as a target for immune therapy of ALL.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Hanna JRA. Expression of CD95 in acute lymphocytic leukemia (ALL) in Egyptian children before and after treatment. J Blood Disord Transfus 2015; 6
Pui CH, Robinson LL, Look AT. Acute lymphpblastic leukemia. Lancet. 2008; 371
Salman T, Argon A, Kebat T, Vardar E, Erkan N, Alacacıoğlu A. The prognostic significance of survivin expression in gallbladder carcinoma. APMIS. 2016; 124
Ferrario A, Luna M, Rucker N, Wong S, Lederman A, Kim J, et al.
Targeting survivin enhances chemosensitivity in retinoblastoma cells and orthotopic tumors. PLoS One. 2016; 11
Kennedy SM, O'Driscoll L, Purcell R, Fitzsimons N, McDermott EW, Hill AD, et al.
Prognostic importance of survivin in breast cancer. Br J Cancer 2003; 88
Duffy MJ, O'Donovan N, Brennan DJ, Gallagher WM, Ryan BM. Survivin: a promising tumor biomarker. Cancer Lett 2007; 249
Altieri DC. Validating survivin as a cancer therapeutic target. Nat Rev Cancer 2003; 3
Altieri DC. Targeting survivin in cancer. Cancer Lett 2013; 332
Rödel F, Hoffmann J, Distel L, Herrmann M, Noisternig T, Papadopoulos T, et al.
Survivin as a radioresistance factor, and prognostic and therapeutic target for radiotherapy in rectal cancer. Cancer Res 2005; 65
Chantalat L, Skoufias DA, Kleman JP, Jung B, Dideberg O, Margolis RL. Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions. Mol Cell 2000; 6
Pennati M, Folini M, Zaffaroni N. Targeting survivin in cancer therapy: fulfilled promises and open questions. Carcinogenesis 2007; 28
Ruchaud S, Carmena M, Earnshaw WC. The chromosomal passenger complex: one for all and all for one. Cell 2007; 131
Yang D, Welm A, Bishop JM. Cell division and cell survival in the absence of surviving. Proc Nati Acad Sci USA 2004; 101
Cooper S, Brown P. Treatment of pediatric acute lymphoblastic leukemia. Pediatr Clin North Am 2015; 62
Paydas S, Tanriverdi K, Yavuz S, Disel U, Sahin B, Burgut R. Survivin and aven: two distinct antiapoptotic signals in acute leukemias. Ann Oncol 2003; 14
Ahmed MB, Shehata HH, Moussa M, Ibrahim TM. Prognostic significance of survivin and tumor necrosis factor-alpha in adult acute lymphoblastic leukemia. Clin Biochem 2012; 45
Yahya RS, Fouda MI, El-Baz HA, Mosa TE, El Maksoud MDA. Serum survivin and TP53 gene expression in children with acute lymphoblastic leukemia. Iran J Public Health 2012; 41
Troeger A, Siepermann M, Escherich G, Meisel R, Willers R, Gudowius S, et al.
Survivin and its prognostic significance in pediatric acute B-cell precursor lymphoblastic leukemia. Haematologica 2007; 92
Altieri DC. Survivin, cancer networks and pathway-directed drug discovery. Nat Rev Cancer 2008; 8
Esh A, Atfy M, Azizi N, El Naggar M, Khalil E, Sherie L. Prognostic significance of survivin in pediatric acute lymphoblastic leukemia. Indian J Hematol Blood Transfus. 2011; 27
Li WQ, Li XL, Wang GP, Fu B. Gene expression of livin and survivin in adult patients with acute lymphoblastic leukemia and its clinical significance. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2011; 19
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]