Incidence and Clinical Significance of Nucleophosmin Mutations in Childhood AML: A Childrens Oncology Group Study.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 221-221 ◽  
Author(s):  
Patrick Brown ◽  
Emily McIntyre ◽  
Rachel Rau ◽  
Todd A. Alonzo ◽  
Robert Gerbing ◽  
...  
Keyword(s):  
Clinical Significance ◽  
Genomic Dna ◽  
Direct Sequencing ◽  
Prognostic Significance ◽  
Pcr Amplification ◽  
Normal Karyotype ◽  
Phase Iii ◽  
Remission Induction ◽  
Therapeutic Trial ◽  
Childhood Aml

Abstract Nucleophosmin mutations (NPM-mu) result in aberrant cytoplasmic localization of the NPM protein and occur in 25–35% of adult AML. NPM-mu are most commonly found in cases with normal karyotype, and are frequently associated with FLT3/ITD mutations. NPM-mu have been associated with high remission induction rates and improved survival, especially in patients with normal karyotype that lack FLT3/ITD mutations. The incidence and clinical significance of NPM-mu in childhood AML are less well-characterized. The AIEOP in Italy reported NPM-mu in 7 of 107 (6.5%) children treated on its AML02 protocol, and a Taiwanese group reported NPM-mu in 1 of 47 (2.1%) of children. The prognostic significance of NPM-mu in childhood AML is not known. The purpose of this study was to determine the incidence and clinical significance of NPM-mu in two large cohorts of children with newly-diagnosed AML treated on U.S. cooperative group phase III clinical trials (CCG-2961 and POG-9421). Criteria for selection of study patients included enrollment on the therapeutic trial and availability of banked genomic DNA (for CCG-2961) or RNA (for POG-9421). 919 patients met these criteria (566 from CCG-2961, 353 from POG-9421). For the genomic DNA samples, exon 12 of the nucleophosmin gene was directly amplified by PCR. The RNA samples were reverse transcribed to cDNA prior to PCR amplification. Mutations were detected using SSCP gel electrophoresis and confirmed by direct sequencing. The incidence of NPM-mu was 8.8% (9.5% for CCG, 7.7% for POG). As in prior reports, all of the mutations consisted of 4-bp insertions that resulted in changes in the 2 trytophan residues at AA positions 288 and 290 (important for nuclear localization). Only 48% of the mutations were of the “A” type (compared to 70–80% in adult AML), and 36% were novel mutations. FLT3/ITD mutations were more common in NPM-mu than NPM-wild type (wt) patients (17% vs. 9%, p=0.0381). NPM-mu patients were older than NPM-wt (median age 12 vs. 9 years, p=0.018). NPM-mu was particularly uncommon in children less than 3 years (1 mutation in 178 patients). Females accounted for 62% of the NPM-mut patients vs. 46% of the NPM-wt patients (p=0.0154). 73% of NPM-mut patients had normal cytogenetics, vs. 25% of NPM-wt patients (p<0.0001). There were no significant differences between NPM-mut and NPM-wt patients in median WBC, platelet or hemoglobin counts, FAB classification, hepatosplenomegaly or CNS disease. As shown in the table, there were no significant differences in EFS or OS for either cohort, although there was a trend towards improved survival for NPM-mu patients on POG 9421, particularly within the normal karyotype subset. In conclusion, NPM mutations are less common in children than adults and appear to have less prognostic relevance, although prospective studies will be needed to determine whether NPM-mu may contribute to risk stratification in children with normal karyotype. CCG-2961 N EFS OS NPM-mu 45 36% 49% NPM-wt 407 41% 53% logrank p=0.762 p=0.909 POG-9421 N EFS OS NPM-mu 23 48% 52% NPM-wt 270 33% 49% logrank p=0.284 p=0.660 POG-9421 (nl karyotype) N EFS OS NPM-mu 16 56% 56% NPM-wt 69 28% 39% logrank p=0.065 p=0.150

scholarly journals The incidence and clinical significance of nucleophosmin mutations in childhood AML

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 979-985 ◽  
Author(s):  
Patrick Brown ◽  
Emily McIntyre ◽  
Rachel Rau ◽  
Soheil Meshinchi ◽  
Norman Lacayo ◽  
...  
Keyword(s):  
Clinical Significance ◽  
Myeloid Leukemia ◽  
Normal Karyotype ◽  
Remission Induction ◽  
Cooperative Group ◽  
Cytoplasmic Localization ◽  
Newly Diagnosed ◽  
Childhood Aml ◽  
Normal Cytogenetics ◽  
Acute Myeloid

Abstract Frameshift mutations in exon 12 of the nucleophosmin gene (NPM1) result in aberrant cytoplasmic localization of the NPM protein (NPMc+) and occur in 25% to 35% of adult acute myeloid leukemia (AML). In adults with AML, NPMc+ has been associated with normal karyotype, FLT3/ITD mutations, high remission induction rates, and improved survival (particularly in patients lacking FLT3/ITD). NPMc+ has not been well characterized in childhood AML. This study examines the incidence and clinical significance of NPMc+ in 295 children with newly diagnosed AML treated on a large cooperative group clinical trial (POG-9421). We find that NPMc+ is relatively uncommon in childhood AML (23 of 295 patients, 8%); and is significantly associated with FLT3/ITD mutations (P = .046), female sex (P = .029), older age (P = .047), and normal cytogenetics (P < .001). There is a favorable impact of NPMc+ on survival in children lacking FLT3/ITD (5-year EFS, 69% vs 35%; hazard ratio, 0.39; P = .051), which is similar in magnitude to the favorable impact of t(8;21) and inv(16). We conclude that NPMc+ is relatively rare in childhood AML, particularly in younger children. NPMc+ does not abrogate the negative prognostic influence of FLT3/ITD mutations, but may contribute to risk stratification in children who lack FLT3/ITD mutations by identifying a group with superior prognosis.

Prevalence and Prognostic Implications of WT1 Mutations in Pediatric AML Â: Report from Children’s Oncology Group

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 143-143 ◽  
Author(s):  
Jessica A Pollard ◽  
Rong Zeng ◽  
Phoenix Ho ◽  
Todd Alonzo ◽  
Robert Gerbing ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Direct Sequencing ◽  
Prognostic Significance ◽  
Normal Karyotype ◽  
Wt1 Gene ◽  
Molecular Alterations ◽  
Microcapillary Electrophoresis ◽  
Pediatric Aml ◽  
Wbc Count ◽  
Wt1 Mutation

Abstract Molecular alterations of the WT1 gene have been associated with clinical outcome in adult AML. We evaluated the prevalence and prognostic significance of WT1 mutations in a cohort of 842 pediatric patients treated on pediatric AML trials CCG-2941, CCG-2961, or COG-AAML03P1. Exons 6–10 of the WT1 gene were evaluated by microcapillary electrophoresis and direct sequencing. Of the 842 samples diagnostic specimens analyzed, 68 (8%) contained mutations in exon 7 (n=62), exon 8 (n= 5), or exon 9 (n=1). Correlation analyses were done to determine whether the presence of WT1 mutations is associated with laboratory and disease characteristics and clinical outcome. There were no differences in sex, race, median diagnostic blast %, or median diagnostic WBC count between samples from patients carrying WT1 mutations and those from patients who did not; however, such mutations were less common in the younger patients (age, 0–2 years; p&lt;0.001) and in those with FAB M5 AML (p=0.009). Our evaluation of clinical outcome showed that the rate of complete remission after the first round of induction chemotherapy for those with and without WT1 mutations 74% and 80%, respectively (P=0.3) Actuarial overall survival (OS) from the time of study entry for patients with WT1 mutations was 35% vs. 52% for those without WT1 mutations; p=0.014. Corresponding event-free survival (EFS) was also significantly worse for those with WT1 mutations (27% vs. 41%; p=0.013). We also evaluated associations between WT1 mutations and cytogenetic and molecular alterations. In the patients with WT1 mutations, 31% had inv(16) or t(8;21). There was also substantial overlap between WT1 mutation and FLT3/ITD, i.e., 29% of those carrying a WT1 mutation were FLT3/ITD- positive, whereas only 7% of patients without WT1 were FLT3/ITD-positive (p&lt;0.001). In addition, 11q23 alterations were rare in patients with WT1 mutations (4% vs. 24%; p=0.002). Prognostic significance of WT1 mutations in FLT3/ITD-negative patients was determined. In a comparison of samples from FLT3/ITD-negative patients with WT1 mutations and those from patients who did not carry the 2 mutations, the OS (51% vs. 54%, respectively; p=0.5) and the corresponding EFS (34% and 43%, respectively; p=0.22) were not significantly different. The prognostic significance of the WT1 mutation was also determined in patients with a normal karyotype who were FLT3/ITD-negative. No significant differences were found in the OS (40% and 55%, respectively; p=0.23) or in the corresponding EFS values (31% and 45%, respectively; p=0.335). We conclude that about 8% of children with AML carry WT1 mutations, including novel mutations identified in exon 8. These mutations are associated with other cytogenetic and molecular alterations, and despite their overall association with poor outcome, the prognostic significance of WT1 mutations is less pronounced once the data are corrected for FLT3/ITD and cytogenetic abnormalities.

Comprehensive Assessment of Genetic and Molecular Features Predicting Outcome in Patients With Chronic Lymphocytic Leukemia: Results From the US Intergroup Phase III Trial E2997

2007 ◽  
Vol 25 (7) ◽  
pp. 799-804 ◽  
Author(s):  
Michael R. Grever ◽  
David M. Lucas ◽  
Gordon W. Dewald ◽  
Donna S. Neuberg ◽  
John C. Reed ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Prognostic Significance ◽  
Variable Region ◽  
Hazard Ratio ◽  
Lymphocytic Leukemia ◽  
Phase Iii ◽  
Therapeutic Trial ◽  
Cd38 Expression ◽  
Molecular Features ◽  
Mutational Status

Purpose Genomic features including unmutated immunoglobulin variable region heavy chain (IgVH) genes, del(11q22.3), del(17p13.1), and p53 mutations have been reported to predict the clinical course and overall survival of patients with chronic lymphocytic leukemia (CLL). In addition, ZAP-70 and Bcl-2 family proteins have been explored as predictors of outcome. Patients and Methods We prospectively evaluated the prognostic significance of a comprehensive panel of laboratory factors on both response and progression-free survival (PFS) using samples and data from 235 patients enrolled onto a therapeutic trial. Patients received either fludarabine (FL; n = 113) or fludarabine plus cyclophosphamide (FC; n = 122) as part of a US Intergroup randomized trial for previously untreated CLL patients. Results Complete response (CR) rates were 24.6% for patients receiving FC and 5.3% for patients receiving FL (P = .00004). PFS was statistically significantly longer in patients receiving FC (median, 33.5 months for patients receiving FC and 19.9 months for patients receiving FL; P < .0001). The occurrence of del(17p13.1) (hazard ratio, 3.428; P = .0002) or del(11q22.3) (hazard ratio, 1.904; P = .006) was associated with reduced PFS. CR and overall response rates were not significantly different based on cytogenetics, IgVH mutational status, CD38 expression, or p53 mutational status. Expression of ZAP-70, Bcl-2, Bax, Mcl-1, XIAP, Caspase-3, and Traf-1 was not associated with either clinical response or PFS. Conclusion These results support the use of interphase cytogenetic analysis, but not IgVH, CD38 expression, or ZAP-70 status, to predict outcome of FL-based chemotherapy. Patients with high-risk cytogenetic features should be considered for alternative therapies.

scholarly journals FLT3, NPM1 and CEBPA Mutations in 195 Children with Acute Myeloid Leukemia in Argentina

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5318-5318
Author(s):  
Cristina N. Alonso ◽  
Patricia L. Rubio ◽  
Adriana Medina ◽  
Silvia Eandi Eberle ◽  
Andrea Bernasconi ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Normal Karyotype ◽  
Prognostic Impact ◽  
Sequencing Analysis ◽  
Rt Pcr ◽  
Genetic Abnormalities ◽  
Childhood Aml ◽  
The Mean ◽  
Cebpa Mutations

Abstract Background: Mutations of FLT3, NPM1 and CEBPA are found in 25 to 35% of adult-AML. These mutations correlate with outcome, especially in AML with normal karyotype. There are few reports concerning the incidence and prognostic significance of these mutations in childhood-AML and there is no data from Argentina. Objectives: To describe the prevalence of FLT3, NPM1 and CEBPA mutations and to analyze the prognostic impact in the outcome in our setting. Methods: Samples from 195 children treated with AML protocols were retrospectively analyzed. The mean age at diagnosis was 6.8 [0.0-17.9] years, including 65 patients younger than 2 years of age. FAB subtypes were M2: 18%, M3: 15%, M4: 12%, M5: 34%, M6: 3%, M7: 10%, while 16 cases (8%) disclosed an ambiguous lineage immunophenotype. Genetic abnormalities of AML cases were characterized by cytogenetic analysis (97%) and/or RT-PCR for AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF4, MLL-AF9, MLL-ENL and MLL-AF10 fusion transcripts (95%). The distribution of the genetic abnormalities was: AML1-ETO: 11%, PML-RARA: 15%, CBFB-MYH11: 6%, MLL/11q23: 23%, other abnormalities: 25% and normal karyotype: 16%. Detection of NPM1 and CEBPA mutations was performed by Gene-scanning; FLT3-ITD and FLT3-TKD were studied by RT-PCR and RFLP respectively. Positive cases were further characterized by sequencing analysis. Results: The prevalences of the studied mutations were: FLT3-ITD: 10.3%, FLT3-TKD: 8.2%, NPM1mut: 4.6% and CEBPAmut: 2.1%. Within the group of AML with normal karyotype the incidences were: FLT3-ITD: 12.5%, FLT3-TKD: 6.3%, NPM1mut: 25.0% and CEBPAmut: 12.5%. The mean age for each subgroup was: FLT3-ITD: 14 years, FLT3-TKD: 9 years, NPM1mut: 12 years and CEBPAmut: 12 years. Simultaneous presence of FLT3-ITD and NPM1 mutations was detected in 2 cases while 1 patient disclosed both FLT3-TKD and CEBPAmut. FLT3-ITD and FLT3-TKD showed significant association with the presence of PML-RARA (p<0.00001 and p=0.055 respectively). Eight out of nine patients with NPM1mut and 4/4 patients with CEBPAmut were AML with normal karyotype. The FAB subtypes more frequently observed for each subgroup were: FLT3-ITDmut: M3 (n:10/20; p<0.00001), FLT3-TKDmut: M5 (n:8/16; p=n.s.), NPM1mut: M2 (n:4/9; p=0.062) and CEBPAmut: M2 (n:3/4; p=0.019). The mean ages of patients with FLT3-ITDmut, NPM1mut and CEBPAmut were significantly higher (p<0.00001, p=0.006 and p=0.033, respectively). FLT3-TKD was the only mutation detected in 5/45 (11%) of patients younger than 1 year of age. The five-years leukemia-free survival probabilities (pLFS) and standard error (SE) were: Total AML: 49 (4)%, FLT3-ITDmut:68 (12)%, FLT3-TKDmut:46 (17)%, NPM1mut: 75 (15)%, CEBPAmut: 100 (0)% and NPM1mut/CEBPAmut/FLT3-ITDneg: 83 (15)% (p<0.00001). The pLFS (SE) of patients with normal karyotype and FLT3-ITDneg and NPM1mut or CEBPAmut was 88 (12)% (p=0.066). Conclusions: This is the first report of the frequencies of FLT3, NPM1 and CEBPA mutations in childhood AML in our country. The incidences of NPM1mut and CEBPAmut were significantly higher in AML with normal karyotype. Our data confirm the favorable prognosis of AML with NPM1mut/FLT3-ITDneg and CEBPAmut/FLT3-ITDneg genotypes, especially in cases with normal karyotype. The present results support the notion that this group should be considered as a new AML subset with better outcome. This group of AML patients with better outcome could be included in the standard risk group, thus avoiding intensive treatments and related toxicity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lack of TEK Gene Mutation in Patients with Cutaneomucosal Venous Malformations from the North-Western Region of Algeria

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Nabila Brahami ◽  
Mourad Aribi ◽  
Badr-Eddine Sari ◽  
Philippe Khau Van Kien ◽  
Isabelle Touitou ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Direct Sequencing ◽  
Venous Malformation ◽  
Pcr Amplification ◽  
Venous Malformations ◽  
Dominant Form ◽  
The North ◽  
Vascular Morphogenesis ◽  
Western Algeria ◽  
North Western

Background. Venous malformations (VM) result from an error in vascular morphogenesis. The first gene suspected in their development is the TEK gene (tyrosine kinase, endothelial). Mutations of this gene have been identified in several Belgian families with a dominant form of the disease. Therefore, we investigated whether mutations in this TEK gene could explain the MV development in patients of families from Tlemcen region (north-western Algeria). Methods. Genomic DNA was extracted from leucocytes of ten patients. The search for mutations in all the 23 exons and in the 5′ and 3′ intronic sequences flanking the TEK gene was performed using PCR amplification and direct sequencing of amplified genomic DNA. Additionally, a search for somatic mutations of the gene TEK was performed on a biopsy of the venous malformation from one of the ten eligible patients. Results. The sequencing of the 23 exons of the TEK gene revealed neither germinal mutation in our ten patients nor somatic mutation in the tissue of the biopsy. Conclusion. The absence of mutation in the TEK gene in the population studied suggests that the TEK gene is not necessarily involved in the onset of VM; its association with these malformations may differ from one population to another.

DNMT3A Mutation inChinese Childhood Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5227-5227
Author(s):  
Weijing Li ◽  
Chao Gao ◽  
Lei Cui ◽  
Shuguang Liu ◽  
Xiaoxi Zhao ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Clinical Characteristics ◽  
Conflicts Of Interest ◽  
Direct Sequencing ◽  
Prognostic Significance ◽  
Fusion Genes ◽  
Childhood Acute Myeloid Leukemia ◽  
Childhood Aml ◽  
Acute Myeloid

Abstract Purpose DNMT3A mutation has been found in approximately 20% of adult acute myeloid leukemia (AML) patients and in 0~1.4% of children with AML, and the hotspots of mutations are mainly located in the catalytic methyltransferase domain, thereinto, mutation R882 accounts for 60%. Although the prognosis of adult patients with DNMT3A mutations is dismal, the prognosis of DNMT3A mutation in childhood AML is still unclear. Here, we tried to determine the incidence and prognostic significance of DNMT3A mutations in Chinese childhood AML. Methods We detected the mutations in DNMT3A exon 23 by PCR and direct sequencing in 342 children with AML (0~16 years old) from January in 2005 to June in 2013, treated on BCH-2003 AML protocol. The correlation of DNMT3A mutations with clinical characteristics, fusion genes, other molecular anomalies (FLT3-ITD, NPM1, C-KIT and WT1 mutations), and treatment outcome were analyzed. Results DNMT3A mutations were detected in 4 out of 342 (1.2%) patients. The mutations in coding sequences included S892S, V912A, R885G, and Q886R. Furthermore, there were two intronic mutations (c.2598-15C>T and c.2739+55A>C) found in one patient. No association of DNMT3A mutations with common clinical features was found. Two patients with DNMT3A mutations died of relapse or complications during treatment, one patient gave up treatment due to introduction failure in day33. Only one patient achieved continuous complete remission. Conclusions DNMT3A mutations were rare in Chinese children with AML. The mutation positions were different from the hotspots reported in adult AML. DNMT3A mutations may have adverse impact on prognosis of children with AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sequencing of Genomic DNA by Combined Amplification and Cycle Sequencing Reaction

2005 ◽  
Vol 51 (1) ◽  
pp. 35-39 ◽  
Author(s):  
Kathleen M Murphy ◽  
Karin D Berg ◽  
James R Eshleman
Keyword(s):  
Genomic Dna ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Target Sequence ◽  
Factor V ◽  
Cycle Sequencing ◽  
Bacterial Speciation ◽  
Set Up ◽  
The Cost ◽  
Single Reaction

Abstract Background: Despite considerable advances, DNA sequencing has remained somewhat time-consuming and expensive, requiring three separate steps to generate sequencing products from a template: amplification of the target sequence; purification of the amplified product; and a sequencing reaction. Our aim was to develop a method to routinely combine PCR amplification and cycle sequencing into one single reaction, enabling direct sequencing of genomic DNA. Methods: Combined amplification and sequencing reactions were set up with Big Dye™ sequencing reagents (Applied Biosystems) supplemented with variable amounts of forward and reverse primers, deoxynucleotide triphosphates (dNTPs), and input DNA. Reactions were thermal-cycled for 35 or 45 cycles. Products were analyzed by capillary electrophoresis to detect sequencing products. Results: Reactions using two oligonucleotide primers at a ratio of 5:1 (500 nM primer 1 and 100 nM primer 2), 125 μM supplemental dNTPs, and 35–45 thermal cycles optimally supported combined amplification and cycle sequencing reactions. Our results suggest that these reactions are dominated by PCR during early cycles and convert to cycle sequencing in later cycles. We used this technique for a variety of sequencing applications, including the identification of germline mutations/polymorphisms in the Factor V and BRCA2 genes, sequencing of tumor DNA to identify somatic mutations in the DPC4/SMADH4 and FLT3 genes, and sequencing of 16S ribosomal DNA for bacterial speciation. Conclusions: PCR amplification and cycle sequencing can be combined into a single reaction using the conditions described. This technique allows direct sequencing of genomic DNA, decreasing the cost and labor involved in gene sequencing.

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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