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

Hagag AA, Badraia IM, Hassan SM, Abd El-Lateef AE - Mediterr J Hematol Infect Dis (2016)

Bottom Line: Positive WT-1 gene expression was found in 22 cases (55%) and negative expression in 18 cases (45%).There were a significantly higher relapse and death rate and a lower rate of CR, DFS, and OAS in negative WT-1 gene expression group.MRD at end of induction therapy was found in 14 cases out of 40 patients.

View Article: PubMed Central - PubMed

Affiliation: Pediatrics Departments, Faculty of Medicine, Tanta University, Egypt.

ABSTRACT

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer representing 23% of pediatric cancers. Wilms' tumor -1 gene is a novel prognostic factor, minimal residual disease marker and therapeutic target in acute leukemia.

Aim of the work: The aim of this work was to study the impact of WT-1 gene expression in the prognosis of ALL.

Patients and methods: This study was conducted on 40 Egyptian children with newly diagnosed ALL who were subjected to full history taking, thorough clinical examination and laboratory investigations including; complete blood count, LDH, BM aspiration, cytochemistry, immunophenotyping, FISH technique for detection of t(12;21) and t(9;22) and assessment of WT-1 Gene by real-time PCR in BM samples at time of diagnosis.

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 presentation of 5.261 ± 0.811 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 with significantly older age 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. There were no significant differences in platelets and WBCs counts, hemoglobin and LDH levels and the 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. There were no statistically significant differences between positive and negative WT-1 gene expression groups regarding immunophenotyping and chromosomal translocations including t(12;21) and t(9;22). There were a significantly higher relapse and death rate and a lower rate of CR, DFS, and OAS in negative WT-1 gene expression group. MRD at end of induction therapy was found in 14 cases out of 40 patients. There were significantly higher number of patients with MRD+ in negative WT-1 gene expression group (After the 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).

Conclusions and recommendation: WT-1 gene expression is an important prognostic factor in patients with ALL, being able to prognosticate a negative MRD. Therefore, we can recommend its incorporation into novel risk-adapted therapeutic strategies in patients with ALL.

No MeSH data available.


Related in: MedlinePlus

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).
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f1-mjhid-8-1-e2016008: 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).

Mentions: 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


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

Hagag AA, Badraia IM, Hassan SM, Abd El-Lateef AE - Mediterr J Hematol Infect Dis (2016)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696468&req=5

f1-mjhid-8-1-e2016008: 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).
Mentions: 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

Bottom Line: Positive WT-1 gene expression was found in 22 cases (55%) and negative expression in 18 cases (45%).There were a significantly higher relapse and death rate and a lower rate of CR, DFS, and OAS in negative WT-1 gene expression group.MRD at end of induction therapy was found in 14 cases out of 40 patients.

View Article: PubMed Central - PubMed

Affiliation: Pediatrics Departments, Faculty of Medicine, Tanta University, Egypt.

ABSTRACT

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer representing 23% of pediatric cancers. Wilms' tumor -1 gene is a novel prognostic factor, minimal residual disease marker and therapeutic target in acute leukemia.

Aim of the work: The aim of this work was to study the impact of WT-1 gene expression in the prognosis of ALL.

Patients and methods: This study was conducted on 40 Egyptian children with newly diagnosed ALL who were subjected to full history taking, thorough clinical examination and laboratory investigations including; complete blood count, LDH, BM aspiration, cytochemistry, immunophenotyping, FISH technique for detection of t(12;21) and t(9;22) and assessment of WT-1 Gene by real-time PCR in BM samples at time of diagnosis.

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 presentation of 5.261 ± 0.811 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 with significantly older age 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. There were no significant differences in platelets and WBCs counts, hemoglobin and LDH levels and the 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. There were no statistically significant differences between positive and negative WT-1 gene expression groups regarding immunophenotyping and chromosomal translocations including t(12;21) and t(9;22). There were a significantly higher relapse and death rate and a lower rate of CR, DFS, and OAS in negative WT-1 gene expression group. MRD at end of induction therapy was found in 14 cases out of 40 patients. There were significantly higher number of patients with MRD+ in negative WT-1 gene expression group (After the 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).

Conclusions and recommendation: WT-1 gene expression is an important prognostic factor in patients with ALL, being able to prognosticate a negative MRD. Therefore, we can recommend its incorporation into novel risk-adapted therapeutic strategies in patients with ALL.

No MeSH data available.


Related in: MedlinePlus