Prognostic Impact of WT-1 Gene Expression in Egyptian Children with Acute Lymphoblastic Leukemia 

Adel A Hagag¹, Ibrahim M Badraia¹, Samir M Hassan¹ and Amal E Abd El-Lateef²

¹ Pediatrics Departments, Faculty of Medicine, Tanta University, Egypt
² Clinical Pathology Departments, Faculty of Medicine, Tanta University, Egypt

Published: January 1, 2016
Received: August 7, 2015
Accepted: November 11, 2015
Mediterr J Hematol Infect Dis 2016, 8(1): e2016008, DOI 10.4084/MJHID.2015.008
This article is available on PDF format at:

Introduction

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer representing 23% of cancer diagnoses among children younger than 15 years. ALL occurs at an annual rate of approximately 30-40 per million. A sharp peak in ALL incidence is observed among children aged 2-3 years (>80 per million per year), with rates decreasing to 20 per million for ages 8-10 years.[1]
With improvements in diagnosis and treatment, overall cure rates for children with ALL have reached 90%. The use of risk-adapted treatment protocols has improved cure rates while limiting the toxicity of therapy.[2] Among children with ALL, more than 95% attain remission and 75%-85% survive free of leukemia recurrence at least 5 years from diagnosis with current treatments that incorporate systemic chemotherapy and specific central nervous system preventive therapy.[3]
Relapse is the leading cause of treatment failure for patients with ALL.[4] Relapse originates from leukemic cells that are resistant to chemotherapy but become undetectable after initial treatment in most cases. Nevertheless, methods more sensitive than microscopic examination can demonstrate leukemic cells in a proportion of samples with no morphologic evidence of leukemia, a finding termed “minimal residual disease (MRD).[5] MRD is currently the most powerful prognostic indicator in childhood ALL[6] and has been introduced into many treatment protocols for risk assignment and selection of therapeutic regimens.[4]
WT1 gene has been identified in the developing kidney and in various malignancies.[7] Real-time quantitative PCR (RQ-PCR) is an accurate method of quantifying WT1 expression. WT1 gene over-expression was demonstrated in diagnostic samples of many types of leukemia[8] and this gene is considered to be an excellent tool for monitoring minimal residual disease in 70% of acute myeloid leukemia patients.[9]
The scope of the present work is to study the impact of WT-1 gene expression in children with acute lymphoblastic leukemia.

Subjects and Methods

After approval from ethical committee of Tanta University research center and written consent from the parents of all children included in this study, the present study was conducted on 40 patients with newly diagnosed ALL who were admitted in Hematology Unit, Pediatric department, Tanta University Hospital in the period from January 2011 to January 2015 including 26 males and 14 females with their ages ranging from 4-15 years with mean age value of 7.24 ± 3.35 years. Patients included in this study were followed up clinically and by blast cells count in BM on day 21 of induction and for MRD in BM after induction and thereafter for 2 years to assess prognosis and were treated according to ALL-protocol of therapy.[10-14]
All patients were subjected to the following:
•    Full history taking.
•    Thorough clinical examination with particular attention to fever, pallor, purpura, hepatomegaly, splenomegaly, and lymphadenopathy.
•    Laboratory investigations
Specimen collection and handling: Three ml of venous blood were collected under complete aseptic technique. They were delivered into 2 tubes: 1 ml blood into a tube containing EDTA for complete blood count and 2 ml blood into a plain tube for assessment of Lactate dehydrogenase levels. Two ml of bone marrow aspirate were drawn into a sterile tube containing EDTA for mononuclear cell separation for polymerase chain reaction (PCR).
Laboratory investigations include the following:
- Complete blood count.
- Lactate dehydrogenase (LDH)
- Bone marrow aspiration with cytochemical examination with Sudan black and Myeloperoxidase and immunophenotyping which was performed on gated blast cells from bone marrow samples by flow cytometry using an extensive panel of Fluorescin Isothiocyanate [FITC] and Phycoerythrin [PE] conjugated monoclonal antibodies [MoAbs] for diagnosis and subtyping of ALL including T-cell lymphoid markers (CD3, CD5, CD7), B-cell markers (CD10, CD19, CD22 and cy-immunoglobulin) and Myeloid cell markers (CD13, CD33).[15]
- Fluorescent in situ hybridization (FISH) for detection of t(9;22) and t(12;21): BM specimens were cultured for 72 hours at 37º^C in RPMI medium supplemented with 10% fetal bovine serum without the addition of any mitogen (un-stimulated). Colcemid (0.02ug/ml) was added to the cultures 30 minutes before harvest. After 30 minutes of hypotonic treatment with 0.075M KCL, the cells were fixed with methanol and acetic acid (3:1) and cells were made into slide preparations. Hybridization mixture (10ul) was then applied to each slide, which was covering slipped and sealed. Hybridization solution contained (hybridization buffer, purified water, and a specific probe). Specific BCR-ABL dual-color DNA probe hybridizes to chromosome 9q34 and chromosome 22q11.2 to detect t(9;22)(q34,q11.2) and TEL-AML dual-color DNA probe hybridizes to chromosome 12p13and chromosome 21q22 to detect t(12;21)(p13,q22) were used. FISH assay was performed according to (Vysis, Abbott # 32-190022) manufacturer's instructions. The analysis was done under a fluorescence microscope equipped with Quips spectra vision hard and software.[16]
Assessment of WT-1 Gene by real-time PCR in bone marrow samples at the time of diagnosis: Mononuclear cells were separated from the aspirated bone marrow samples by centrifugation on Density gradient medium (Lymph Prep). RNA was isolated from the samples using an RNA easy Mini Kit according to the Qiagen Protocol (QIAGEN GmbH, Hilden, Germany) for isolation of total RNA and the amount of extracted total RNA was estimated quantitatively by spectrophotometric measurement and qualitatively by electrophoresis.  Patients RNA was reverse transcripted using transcription first strand cDNA synthesis kit (ROCH diagnostic GmbH, Mannheim Germany) and then stored at -80ºC until PCR amplification. The cDNA is used as a template to amplify WT-1 gene and the cDNA normalized using glyceraldehyde -3 phosphate dehydrogenase as a housekeeping gene. The expression is calculated relative to Housekeeping gene, and so the value as a ratio to this gene is either expressed (positive WT-1 gene expression) or not expressed (negative WT-1 gene expression). WT1 expression in peripheral blood was examined from 10 healthy children as control, and the cut-off value is determined relative to controls. DNA amplification of the WT-1gene was done by real-time PCR using Gene Amp 5700 Sequence Detection System (Applied Biosystems). Using the primers and probe for WT1 (Applied Biosystems, Foster City, California, USA) and Standard, primers and probes of GAPDH (Applied Biosystems, Foster City, California, USA), the Light Cycler TaqMan Master protocol was followed according to manufacturer's instructions (Figure 1).[17]
Real-time PCR, Primers, and Probes:
WT1 was amplified using: 
Forward WT-1 Gene primer: 5’-GATAACCACACAACGCCCATC-3’,
Reverse WT-1 Gene primer: 5’-CACACGTCGCACATCCTGAAT-3’; with annealing temperature 56 °C.
WT-1 Gene probe: 5’-FAM-ACACCGTGCGTGTGTATTCTGTATTGG-TAMRA-3’ was designed using Primer-Express software (PE Biosystems, Foster city, CA, USA). Real-time PCR amplification and data analysis were performed using the ABI Prism 5700 Sequence Detector System (PM Biosystems). Reactions were performed using real-time PCR 7000 sequence detection system (Applied Biosystems, Foster city, CA, USA)[11].
Detection of MRD by RT- PCR of antigen receptor gene rearrangements: This method aims to generate clone-specific primers with a sensitivity of at least 10-4. The first step involves identification and sequencing of clone-specific antigen receptor gene rearrangements. This step is followed by a primer design in which the clone-specific sequence is examined to identify potential allele-specific oligonucleotides. The specificity and sensitivity of these primers are then tested. Post-induction DNA is then subjected allele-specific priming and the results compared with those produced from dilution of leukemic DNA into BC DNA. Controlled for variation in amplifiability of individual DNA samples is achieved using a control PCR, in this case, β-globin.
Primers were purchased from a commercial supplier (Invitrogen, Carlsbad, CA). Direct sequencing of PCR product was done. Clonal PCR products were excised and purified using QIA quick gel extraction kits (QIAGEN, Valencia, CA). Purified PCR fragments were sequenced directly by the Dana-Farber/Harvard Cancer Center Core Sequencing Facility (Boston, MA). Sequence reactions were analyzed on an Applied Biosystems 3700 capillary sequencer using Big Dye Terminator Chemistry version 2 (Applied Biosystems, Foster City, CA). The relevant consensus forward and reverse primers were used as sequence primers to obtain the sequence of both strands Nucleotide sequences were aligned using DNA star software (DNASTAR, Madison, WI). TCR gene segments were identified using the Immunogenetics Database (http://imgt.cines.fr, IMGT; European Bioinformatics Institute, Montpellier, France).[18]
Protocol of treatment utilized in the studied ALL patients at diagnosis:[10-14]
Induction (6 weeks): IV Vincristine 1.5mg/kg/m2/week (days 0, 7, 14, 21, 28, 35), Doxorubicin 25mg/m2/ week IV infusion (days 0, 7, 14, 21, 28, 35), asparaginase 6000 u/m2 SC on alternate days (10 doses) and oral prednisone 40mg/m2/day for 6 weeks. On day 21, BM aspiration was done; If BM blast cells is more than 5%, we add etoposide 100 mg/m2/dose IV (days 22, 25, 29), cyclophosphamide 750 mg/m2/dose IV infusion (days 22, 25, 29), aracytin 100/m2/dose IV (days 22, 25, 29), and methotrexate (MTX) 5g/m2 over 4 hours on day 28.[10]
Consolidation (9 weeks): IV methotrexate 1gm/m2/dose over 24 hour infusion on days 0, 21, 42 and 63, oral mercaptopurine 60 mg/m2 daily on days 0-13 and 28-41, IV vincristine 1.5 mg/m2 on days 14, 21, 42 and 49, PEG asparaginase 2,500 units/m2 IM on days 14 and 22, cyclophosphamide 750 mg/m2/dose IV infusion on days 0 and 28, aracytin 100/m2/dose IV on days 1-4, 8-11, 29-32 and 36-39 and age-adjusted intrathecal methotrexate on days 1,8,15 and 22.[11,12]
Interim maintenance (6 weeks): IV Vincristine 1.5 mg/m2 on days 0, 10, 20, 30, 40, IV methotrexate starting dose of 100 mg/m2/dose over 10-15 minutes on day 0 thereafter escalate by 50 mg/m2/dose on days 10, 20, 30 and 40, PEG asparaginase 2,500 units/m2 IM on days 1 and 21 and age-adjusted intrathecal methotrexate on days 0 and 30.[12]
Delayed–intensification (6 weeks): Oral dexamethasone (10 mg/m2/day on days 1-7 and 14-21, IV vincristine 1.5 mg/m2 on days 0, 7 and 14, IM or IV pegylated L-asparaginase 2500 u/m2 on day 4, doxorubicin 25 mg/m2 IV on days 0, 7 and 14, IV cyclophosphamide 1gm/m2 over 30 minutes on day 28, oral 6-thioguanine 60 mg/m2 on days 28-41, aracytin 75mg/m2 on days 29-32 and 36–39 and age-adjusted intrathecal MTX on day 28.[13]
Maintenance (30 months): Weekly IV methotrexate 20 mg/m2, prednisone 120 mg/m2/day for 5 days every 3 weeks, vincristine 2mg/m2 IV every 3 weeks, oral 6-mercaptopurine 50 mg/m2/day for 14 days every 3 weeks and age-adjusted intrathecal MTX every 18 weeks.[14]
Definition of disease response and relapse: Complete remission (CR) is defined as a cellularity of more than 20% with fewer than 5% blasts in BM after induction chemotherapy. Relapse is defined by appearance of more than 50% lymphoblasts in a single BM aspirate or more than 25% lymphoblasts of two or more BM aspirate and 2% or more circulating lymphoblasts or progressive repopulation of lymphoblasts in excess of 5% culminating in more than 25% of two or more BM samples separated by 1 week or more or leukemic cell infiltration in extramedullary organs as gonads or lymphoblasts in CSF with cell count greater than 5 WBCs/mm3.[13]
Statistical analysis: The collected data were organized, tabulated and statistically analyzed using SPSS version 13. All Data expressed in terms of mean values ± SD. The difference between two means was statistically analyzed using the student (t) test. Chi-square test (X2) and Fischer exact test were used as a test of significance. For comparison between means of two different groups, parametric analysis (t-test) and non-parametric analysis (Mann- Whitney U test) were used. Significance was adapted at P<0.05. Log-rank test were used to assess survival.[19]

Figure 1. A case with positive expression of WT-1 gene by quantitative real-time PCR. Amplification Plot showing the log of the change in the fluorescence plotted versus cycle number (ΔRn vs. Cycle).

Results

Positive WT-1 gene expression was found in 22 cases (55%) and negative expression in 18 cases (45%).
Positive WT-1 gene expression group (n=22) includes 14 males and 8 females with mean age at diagnosis of 5.261±0.811 in whom pallor was found in 22 cases, purpura in 22 cases, splenomegaly in 6 cases, hepatomegaly in 8 cases, lymphadenopathy in 8 cases and bone-ache in 2 cases while negative WT-1 gene expression group (n=18) includes 12 males and 6 females with mean age at diagnosis of 9.669±3.731 in whom pallor was found in 10 cases, purpura in 18 cases, splenomegaly in 18 cases, hepatomegaly in 18 cases, lymphadenopathy in 12 cases and bone-ache in 8 cases with significantly older age at diagnosis in negative WT-1 gene expression group but no significant differences between positive and negative WT-1 gene expression groups regarding sex and clinical presentations (Table 1).

Table 1. Comparison between positive and negative WT-1 gene expression groups regarding clinical and laboratory data at time of diagnosis.

There were no significant differences in platelets and WBCs counts, Hb and LDH levels and number of peripheral blood and BM blast cells at diagnosis between positive and negative WT-1 gene expression groups but after induction therapy there were significantly lower BM blast cells in positive WT-1 gene expression group (Mean platelets count in positive WT-1 gene expression group was 25.11±9.07 versus 48.09±37.65 in negative group with p-value of 0.09, mean WBCs count in positive WT-1 gene expression group was 26.25±8.23 versus 23.47±3.37 in negative group with p-value of 0.71, mean Hb level in positive WT-1 gene expression group was 8.39±1.02 versus 9.39±1.38 in negative group with p-value of 0.08, mean LDH level in positive WT-1 gene expression group was 1122.23±277.16 versus 1089.83±271.66 in negative group with p-value of 0.099, mean peripheral blood blast cells at diagnosis in positive WT-1 gene expression group was 36.22±10.11 versus 33.52±16.43 in negative group with p-value of 0.075, mean BM blast cells at diagnosis in positive WT-1 gene expression group was 88.72±8.73 versus 81.55±7.55 in negative group with p-value of 0.068 and mean BM blast cells after induction in positive WT-1 gene group was 4.272±2.323 versus 7.577±2.988 in negative group with p-value of 0.0122 (Table 1).
There were no significant differences between positive and negative WT-1 gene expression groups regarding immunophenotyping and chromosomal translocations) (Positive WT-1 gene expression group (n=22) includes 7 cases with early pre-B, 13 cases with pre-B, 2 cases with T-cell ALL and 5 cases having t(12;21) and 2 cases having t(9;22) while negative WT-1 gene expression (n=18) includes 5 cases with early pre-B, 9 cases with pre-B and 4 cases with T-cell ALL and 4 cases having t(12;21) and 1 case with t(9;22) (Table 1).
There were significantly higher rates of relapse and death and lower rate of CR, DFS and OAS in negative WT-1 gene expression group compared with positive group (in 22 cases with positive WT-1 gene expression; 19 achieved complete remission, 2 cases relapsed and 1 case died while in 18 cases with negative WT-1 gene expression; 6 achieved complete remission, 8 cases relapsed and 4 cases died with median DFS in positive WT-1 gene expression group of 23.52 months compared with 13.71 months in negative WT-1 gene expression group and median OAS in positive WT-1 gene expression group of 23.95 months compared with 16.8 months in negative WT-1 gene expression group). (Table 2 and 5).
Minimal residual disease at the end of induction therapy was found in 14 out of 40 patients. There was a significantly higher number of cases with MRD+ in negative WT-1 gene expression group (After the induction therapy 20 out of 22 (89%) patients with positive WT-1 gene expression attained a negative MRD, while only 6 out of 18 (33%) with negative WT-1 attained a negative MRD) (p-value = 0.006) (Table 4). MRD at the end of induction was associated with lower remission and higher relapse rates compared with MRD negativity (p-value=0.026) (In total number of 26 cases with negative MRD, 21 cases achieved and maintained CR, 3 cases died and 2 case relapsed while in 14 cases with positive MRD, only 4 cases achieved and maintained CR, 8 cases relapsed and 2 cases died). (Table 3).

Table 2. Outcome of studied patients in relation to WT-1 gene expression.

Table 3. Outcome of studied patients in relation to Minimal residual disease.

Table 4. Relation between WT-1 gene expression and MRD.

Table 5. Log Rank test of overall and disease-free survival.

Discussion

Conclusion and Recommendation

The variation between this study and other previously published studies may be explained by different age groups, times and places of research. However, it is important to note, accordingly to Boublikova et al. 2006,[24] that WT1 expression in childhood ALL is very variable and much lower than in AML or adult ALL. Therefore, WT1, will not be a useful marker for MRD detection in childhood ALL, however, it does represent a potential independent risk factor in childhood ALL. But its level could have a different significance. Interestingly, Boublikova et al.[24] report a proportion of childhood ALL patients expressing WT1 at reduced levels at higher risk of relapse. In our hands, the high level of WT1 at the onset of disease was a predictor of a good response to therapy paralleling with a negative MDR after therapy. The small number of patients in our study and the heterogeneity in age, presentation, immunophenotype of studied patients, and the intrinsic variable expression of WT1 in ALL can justify the contradictory results. More studies need. However, the assessment of WT1 at the onset of the disease, associated with MDR testing after therapy, could improve the risk evaluation and then the novel risk-adapted therapeutic strategies in patients with ALL.

References

1. Shah A and Coleman MP. Increasing incidence of childhood leukemia: A controversy re-examined. Br J Cancer 2007; 97(7): 1009-1012. PMid:17712312 PMCid:PMC2360402      
2. Hunger SP, Lu X, Devidas M, Camitta BM, Gaynon PS, Winick NJ, Reaman GH, Carroll WL. Improved survival for children and adolescents with acute lymphoblastic leukemia between 1990 and 2005: A Report from the Children's Oncology Group. J Clin Oncol 2012 May 10; 30(14):1663-9. Epub 2012 Mar 12. http://dx.doi.org/10.1200/JCO.2011.37.8018      
3. Pui CH, Campana D, Pei D, Bowman WP, Sandlund JT, Kaste SC, Ribeiro RC, Rubnitz JE, Raimondi SC, Onciu M, Coustan-Smith E, Kun LE, Jeha S, Cheng C, Howard SC, Simmons V, Bayles A, Metzger ML, Boyett JM, Leung W, Handgretinger R, Downing JR, Evans WE, Relling MV. Treating childhood acute lymphoblastic leukemia without cranial irradiation. N Engl J Med 2009; Jun 25; 360(26):2730-41. http://dx.doi.org/10.1056/NEJMoa0900386      
4. Möricke A, Zimmermann M, Reiter A, Henze G, Schrauder A, Gadner H, Ludwig WD, Ritter J, Harbott J, Mann G, Klingebiel T, Zintl F, Niemeyer C, Kremens B, Niggli F, Niethammer D, Welte K, Stanulla M, Odenwald E, Riehm H, Schrappe M. Long-term results of five consecutive trials in childhood acute lymphoblastic leukemia performed by the ALL-BFM study group from 1981 to 2000. Leukemia 2010 Feb; 24(2):265-84. Epub 2009 Dec 10 http://dx.doi.org/10.1038/leu.2009.257      
5. Campana D: Role of minimal residual disease monitoring in adult and pediatric acute lymphoblastic leukemia. Hematol Oncol Clin North Am 2009; 23:1083–1098. http://dx.doi.org/10.1016/j.hoc.2009.07.010 PMid:19825454 PMCid:PMC2762949      
6. Stow P, Key L, Chen X, Pan Q, Neale GA, Coustan-Smith E, Mullighan CG, Zhou Y, Pui CH, Campana D. Clinical significance of low levels of minimal residual disease at the end of remission induction therapy in childhood acute lymphoblastic leukemia. Blood 2010 Jun 10; 115(23):4657-63. Epub 2010 Mar 19. http://dx.doi.org/10.1182/blood-2009-11-253435      
7. Cilloni D, Gottardi E, Fava M, Messa F, Carturan S, Busca A, Guerrasio A, Saglio G. Usefulness of quantitative assessment of the WT1 gene transcript as a marker for minimal residual disease detection. Blood 2003 Jul 15; 102(2):773–774. http://dx.doi.org/10.1182/blood-2003-03-0980 PMid:12835231      
8. Oji Y, Miyoshi S, Maeda H, Hayashi S, Tamaki H, Nakatsuka S, Yao M, Takahashi E, Nakano Y, Hirabayashi H, Shintani Y, Oka Y, Tsuboi A, Hosen N, Asada M, Fujioka T, Murakami M, Kanato K, Motomura M, Kim EH, Kawakami M, Ikegame K, Ogawa H, Aozasa K, Kawase I, Sugiyama H. Overexpression of Wilms' tumor gene in de novo lung cancers. Int J Cancer. 2002 Jul 20; 100(3):297-303. http://dx.doi.org/10.1002/ijc.10476 PMid:12115544      
9. Ostergaard M, Olesen LH, Hasle H, Kjeldsen E, Hokland P. WT1 gene expression: An excellent tool for monitoring minimal residual disease in 70% of acute myeloid leukemia patients. Results from a single –centre study. Br J Haematol 2004 Jun; 125(5):590–600. http://dx.doi.org/10.1111/j.1365-2141.2004.04952.x PMid:15147374      
10. Ching-Hon Pui. Acute lymphoplastic leukemia: Overview. In: Lichtman MA, Beulter E, Seligsonn U, Kipps TO, Kaushansky K, Prchal J (editors). William Textbook of Hematology. 17th edition, Chapter 91. New York: McGraw-Hill Companies, Inc.; Part XI: Neoplastic Lymphoid Diseases, 2007; 91: 1141-53.      
11. Chauvenet AR, Martin PL, Devidas M, Linda SB, Bell BA, Kurtzberg J, Pullen J, Pettenati MJ, Carroll AJ, Shuster JJ, Camitta B. Antimetabolite therapy for lesser-risk B-lineage acute lymphoblastic leukemia of childhood: a report from the Children's Oncology Group Study P9201. Blood 2007 Aug 15; 110(4):1105-11. Epub 2007 Apr 18. http://dx.doi.org/10.1182/blood-2006-12-061689 PMid:17442849 PMCid:PMC1939894      
12. Seibel NL, Steinherz PG, Sather HN, Nachman JB, Delaat C, Ettinger LJ, Freyer DR, Mattano LA Jr, Hastings CA, Rubin CM, Bertolone K, Franklin JL, Heerema NA, Mitchell TL, Pyesmany AF, La MK, Edens C, Gaynon PS. Early post induction intensification therapy improves survival for children and adolescents with high risk acute lymphoblastic leukemia: a report from the Children's Oncology Group. Blood 2008 Mar 1; 111(5):2548-55. Epub 2007 Nov 26. http://dx.doi.org/10.1182/blood-2007-02-070342 PMid:18039957 PMCid:PMC2254538      
13. Lanzkowsky PH. Leukemias. In: Lanzkowsky Philip (editor), Manual of Pediatric Hematology and Oncology. 5th ed. Churchill livingstane. Nek, London, Madrid, 2011; 17: 518- 566.      
14. Goldberg JM, Silverman LB, Levy DE, Dalton VK, Gelber RD, Lehmann L, Cohen HJ, Sallan SE, Asselin BL. Childhood T cell acute lymphoblastic leukemia. The Dana-Farber Cancer Institute. Acute Lymphoblastic Leukemia Consortium experience. J Clin Oncol 2003 Oct 1; 21(19):3616-22. http://dx.doi.org/10.1200/JCO.2003.10.116 PMid:14512392      
15. Catovsky D and Hoffbrand AV. Acute myeloid leukaemia. In: A.V. Hoffbrand and S.M. Lewis (eds) Postgraduate Haematology, 5th edition, Oxford, UK: Reed Educational and Professional Publishing Ltd. 2005; 509-524.      
16. O'Connor C. Fluorescence in situ hybridization (FISH). Nature Education 2008; 1(1):171.      
17. Boyam A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1g. Scand J Clin Lab Invest Suppl. 1968; 77:97-99.      
18. JJ Taylor, D Rowe, IK Williamson, SE Christmas, SJ Proctor, and PG Middleton. Detection of T-cell receptor gamma chain V gene rearrangements using the polymerase chain reaction: application to the study of clonal disease cells in acute lymphoblastic leukemia. Blood 1991 may 1; (9): 77.      
19. Petrie A and Sabin C. Medical statistics at a glance. Blackwell Publishing, Oxford, 2nd edition, 2005; 978(1):4051-8051.      
20. Ribera JM, Ribera J, Genescà E. Treatment of adolescent and young adults with acute lymphoblastic leukemia. Mediterr J Hematol Infect Dis. 2014 Jul2;6(1):e2014052. doi: 10.4084/MJHID.2014.052. eCollection 2014. Review. PubMed http://dx.doi.org/10.4084/mjhid.2014.052        
21. Liu HS, Zhu MS, Zhang HL, Wei SY, Wang XN, Xi XP, Yu FF, Xi JY, Wang MC, Zhang M. WT1 gene expression difference in leukemia and non- leukemia and its clinical significance. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2014 Oct; 22(5):1217-2: http://dx.doi.org/10.7534/j.issn.1009-2137.2014.05.005       
22. Shandilya J, Roberts SG. A role of WT1 in cell division and genomic stability. Cell Cycle. 2015;14(9):1358-64. doi: 10.1080/15384101.2015.1021525. http://dx.doi.org/10.1080/15384101.2015.1021525      
23. Barragán E, Cervera J, Bolufer P, Ballester S, Martín G, Fernández P, Collado R, Sayas MJ, Sanz MA. Prognostic implications of Wilms' tumor gene (WT1) expression in patients with de novo acute myeloid leukemia. Haematologica 2004 Aug; 89(8): 926–933. PMid:15339675      
24. Boublikova L, Kalinova M, Ryan J, Quinn F, O'Marcaigh A, Smith O, Browne P, Stary J, McCann SR, Trka J, Lawler M. Wilms' tumor gene 1 (WT1) expression in childhood acute lymphoblastic leukemia: A wide range of WT1 expression levels, its impact on prognosis and minimal residual disease monitoring, Leukemia 2006 Feb; 20 (2):254–263. http://dx.doi.org/10.1038/sj.leu.2404047 PMid:16341043      
25. Kerst G, Kreyenberg H, Roth C, Well C, Dietz K, Coustan-Smith E, Campana D, Koscielniak E, Niemeyer C, Schlegel PG, Müller I, Niethammer D, Bader P. Concurrent detection of minimal residual disease in childhood ALL by flow cytometry and real-time PCR. Br J Haematol 2005 Mar; 128 (6): 774–782. http://dx.doi.org/10.1111/j.1365-2141.2005.05401.x PMid:15755280      
26. Sadek HA, El-Metnawey WH, Shaheen IA, Korshied MM, Mohamed AS. Quantitative assessment of WT1 Gene transcripts in Egyptian acute lymphoblastic leukemia Patients: Journal of Investigative Medicine 2011 Dec; 59(8): 1258-1262. http://dx.doi.org/10.231/JIM.0b013e31822a24f7.      
27. Ibrahim A, Badrawy H, Sayed H. Prognostic Implications of Expression of the Wilms Tumor 1 Gene in Acute Leukemia (Experience from South Egypt). British Journal of Medicine & Medical Research 2015; 7(1):61-71. http://dx.doi.org/10.9734/BJMMR/2015/15834        
28. Zhang R, Yang JY, Sun HQ, Jia H, Liao J, Shi YJ, Li G. Comparison of minimal residual disease (MRD) monitoring by WT1 quantification between childhood acute myeloid leukemia and acute lymphoblastic leukemia. Eur Rev Med Pharmacol Sci. 2015 Jul; 19(14):2679-88. PMid:26221900      

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