Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia

2011 ◽  
Vol 91 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Oscar Fuster ◽  
Eva Barragán ◽  
Pascual Bolufer ◽  
Esperanza Such ◽  
Ana Valencia ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Fragment Length ◽  
Myeloid Leukemia ◽  
Intermediate Risk ◽  
Cebpa Mutations ◽  
Length Analysis ◽  
Acute Myeloid

Patients with FLT3 Negative Acute Myeloid Leukemia and Normal Cytogenetic who Have the Combination Mutations (NPM1-/CEBPA-) Have Better Survival Than Patients Who Have the Combination Mutations (NPM1+/CEBPA+)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4874-4874
Author(s):  
Shamail Butt ◽  
Pascal Akl ◽  
Himanshu Bhardwaj ◽  
Samer A Srour ◽  
Terry Dunn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Acute Myeloid Leukemia ◽  
Mutation Analysis ◽  
Myeloid Leukemia ◽  
Risk Group ◽  
Intermediate Risk ◽  
Cebpa Mutations ◽  
Normal Cytogenetics ◽  
Acute Myeloid

Abstract Abstract 4874 Introduction: Acute Myeloid Leukemia (AML) is the most common type of acute leukemia in adults. About 50% of patients with AML have normal karyotype, and are categorized as intermediate risk group. However, the clinical behavior and response to treatment in this group is heterogeneous. As a result, there is strong interest in characterizing molecular genetic features in the intermediate-risk AML patients that might rectify their stratification risk. In this group, FLT3-ITD (Internal Tandem Duplication) and FLT3-TKD (Tyrosine Kinase Domain) mutations are known to confer unfavorable risk whereas NPM1 and CEBPA mutations are known to be favorable risk markers. The purpose of this study is to analyze the combination of NPM1 and CEBPA mutations in presence or absence of FLT3 mutations on prognosis of AML patients referred to the State's largest tertiary care center over a period of 10 years for the treatment of leukemia. Patients and Method: We performed a retrospective chart review of all patients with AML evaluated at University of Oklahoma Health Sciences Center between January 2000 and December 2010. Patient's age, gender, race, laboratory and clinical data as well as bone marrow biopsy and aspirate findings were reported. PCR and Fragment Analysis were conducted on all available DNA preserved bone marrow materials to test the FLT3, NPM1 and CEBPA mutations. For statistical analysis, Kaplan-Meyer curve was used. Results: A total of 239 patients were evaluated. Male to female ratio was 2/1. Median age at diagnosis was 46y. 21 out of the 239 patients were less than 18 year old. DNA samples were present on 132 patients and mutation analysis for FLT3, CEBPA and NPM1 was performed. Correlation between mutations and AML prognosis was determined. 67/132 (50.8 %) patients were categorized into intermediate risk group (majority of patients had normal cytogenetics). 14/67 (20.9%) pts were FLT3+ (FLT3-ITD or FLT3-TKD mutation). 17/67 (23.9%) were NPM1+. 7/67 (10.4%) were CEBPA +. Kaplan-Meier curve was used to identify cumulative proportion surviving over time. FLT3 presence or absence itself was not identified to be statistically significant (p 0.416) in terms of overall survival. Interestingly, presence or absence of combined NPM1/CEBPA mutation in FLT3 negative patients, among intermediate risk group, was found to be statistically significant (p<0.05) in determining overall survival. In this subgroup, presence of NPM1/CEBPA combination (NPM1+/CEBPA+) was associated with poor prognosis (figure 2, lower curve), while absence of NPM1/CEBPA combination (NPM1-/CEBPA-) carries a better prognosis (figure 2, upper curve). Conclusion: Results of our study highlight the importance of performing combinations of mutation analysis in evaluation of overall prognosis in AML patients. FLT3-/NPM1+ profile in patients with normal cytogenetics is thought to confer a favorable prognosis. We demonstrated in this study that using combination mutation analysis in patients with FLT3- can change the risk stratification in patients with intermediate risk group and might affect therapeutic interventions in this patient population. Larger prospective studies are needed in the future for further validation of our findings. Disclosures: No relevant conflicts of interest to declare.

Characterization of CEBPA Mutations in Acute Myeloid Leukemia in Taiwan - Most Patients with CEBPA Mutations Have Biallelic Mutations and Show a Distinct Immunophenotype of the Leukemic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4321-4321
Author(s):  
Liang In Lin ◽  
Chien Yuan Chen ◽  
Dong Tsamn Lin ◽  
You Chia Yeh ◽  
Hwei Fang Tien
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Topoisomerase Ii ◽  
Normal Karyotype ◽  
Leukemic Cells ◽  
Intermediate Risk ◽  
Hla Dr ◽  
Cebpa Mutations ◽  
Biallelic Mutations ◽  
Acute Myeloid

Abstract The transcription factor CCAAT/enhancer binding protein alpha (C/EBPa) encoded by the CEBPA gene, is crucial for the differentiation of immature granulocytes. Diminished or abnormal C/EBPa activity resulting from CEBPA gene mutations is widely known to contribute to the transformation of myeloid progenitors via reduction of their differentiation potential. The CEBPA mutations have been detected in approximately 7% of total acute myeloid leukemia (AML) and in 15% of those with intermediate-risk cytogenetics or those with normal karyotype. However, the age distribution of the patients with the CEBPA mutations and the immunophenotype of their leukemic cells are not known. Sequential studies of the CEBPA gene in AML patients are also limited. In this study, 104 patients with de novo acute myeloid leukemia (AML) were evaluated for the CEBPA mutation by direct sequencing. Excluding the silent mutations, 16 (15%) of the total 104 AML patients, 15 (25%) of the 61 patients with intermediate-risk cytogenetics and 11 (35%) of the 31 patients with normal karyotype showed CEBPA mutations, frequencies higher than those reported in the West. Further cloning and subsequent nucleotide sequence analysis revealed that 14 patients had heterozygous biallelic mutations: 11 had mutations involving both the N-terminal transactivation domain (TAD) and the C-terminal basic leucine zipper domain (bZIP) and three, in either the TAD region or the bZIP region. The remaining two patients had only one allele mutation in the TAD1 region. Most mutations in TAD region were repeat-number changes of simple sequence repeats and those in bZIP region were internal tandem duplications. Sequence analysis revealed that in the region spanning the bZIP mutations, there was hot spot for concensus topoisomerase II sites, which has also been shown in other AML-related mutations FLT3-ITD and MLL duplication. All but one patient with CEBPA mutations had M1 or M2 subtype of AML. The patients with CEBPA mutations had significantly higher incidences of CD7 (73%), CD15 (100%), CD34 (93%) and HLA-DR (93%) expression than others and the majority of them showed a distinct immunophenotype of the leukemic cells: HLA-DR+ CD7+ CD13+ CD14− CD15+ CD33+ CD34+. The incidence of the CEBPA mutation in children with AML was similar to that in adults. The CEBPA mutation was serially analyzed in 27 patients; the mutations disappeared at CR, but reappeared at relapse. No one developed novel mutation during the follow-up period. In conclusion, the CEBPA mutation may play an important role in the development, but not progression, of AML. The patients with the CEBPA mutations showed a distinct immunophenotype of the leukemic cells. Potential topoisomerase II cleavage sites locating in the bZIP region were first reported and we propose that this is relevant to the process of illegitimate recombination generating the internal tandem duplication pattern of bZIP mutations.

Prognostic Impact and Concurrent Genetic Alterations of CEBPA Double Mutations in Adults with Cytogenetically Intermediate-Risk Acute Myeloid Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2538-2538
Author(s):  
Shunichiro Yamaguchi ◽  
Kenji Tokunaga ◽  
Eisaku Iwanaga ◽  
Tomoko Nanri ◽  
Taizo Shimomura ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Clinical Outcome ◽  
Myeloid Leukemia ◽  
Gene Mutations ◽  
Prognostic Impact ◽  
Intermediate Risk ◽  
Disease Entity ◽  
Cebpa Mutations ◽  
Acute Myeloid

Abstract Abstract 2538 Aims: Among acute myeloid leukemia (AML) patients with intermediate-risk cytogenetics, C/EBPa mutations represent a distinct disease entity with a favorable clinical outcome and is adopted in the current WHO classification of AML as a provisional disease entity in the category AML with recurrent genetic abnormalities. CEBPA encodes a transcription factor that is essential for neutrophil development. AML patients with CEBPA mutations can be separated into two subgroups with a single mutation in the CEBPA (CEBPA sm) and double mutations (CEBPA dm). Biallelic mutations consisted of an N-terminal frameshift mutation and a C-terminal inframe bZIP mutation were detected in the majority of CEBPA dm, whereas CEBPA sm occurs in either N-terminal or C-terminal regions. More recent data indicate that favorable outcome is mainly observed in AML patients with CEBPA dm but not with CEBPA sm. In addition, concurrent gene mutations may occur more frequently in AML with CEBPA sm than in CEBPA dm. In contrast, transcription factor GATA2 mutations are frequently identified in AML with CEBPA dm. In this study, we examined incidence, concurrent gene mutations and clinical significance of CEBPA dm and CEBPA sm in Japanese adults with cytogenetically intermediate-risk AML. Methods: To identify the prevalence and prognostic impact of CEBPA dm and CEBPA sm, we examined 111 patients with intermediate-risk AML who were mainly treated with the JALSG protocols. Age ranged from 16 to 86 years, with a median of 58.5 years. DNA was extracted from bone marrow or peripheral blood mononuclear cells at diagnosis and subjected to PCR amplification and direct sequencing of the CEBPA, FLT3, NPM1, IDH1, IDH2, DNMT3A and GATA2 genes. This study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the revised Declaration of Helsinki. Results: Of 111 cytogenetically intermediate-risk AML, we found 12 (10.8%) CEBPA dm and 7 (6.3%) CEBPA sm. In 7 CEBPA sm, one NPM1 mutation and one FLT3-ITD were detected. Two FLT 3-ITD and no concurrent mutation of NPM1 were found in CEBPA dm. No mutation in the IDH1, IDH2, DNMT3A exon 23 was identified in both patients with CEBPA sm and CEBPA dm. On the other hand, mutations in the GATA2 zinc finger domains were detected in 3 of 12 (25%) patients with CEBPA dm. No GATA2 mutations were found in 7 CEBPA sm. One of 21 patients with wild-type CEBPA (CEBPA wt) had a GATA2 mutation. Patients with CEBPA double or single mutations showed a better 5-year overall survival (OS) compared to CEBPA wt (51.3% vs 16.0%, P=0.0048). CEBPA dm AML was associated with a significant superior clinical outcome compared with CEBPA wt (5-year OS, 55.6% vs 16.0%, P=0.0025). However, no significant difference was identified between CEBPA dm and CEBPA sm AML (5-years OS, 55.6% vs 42.9%, P=0.1375) or between CEBPA sm and CEBPA wt AML (5-year OS, 42.9% vs 16.0%, P=0.4827). In addition, the presence of additional GATA2 mutations did not significantly influence the clinical outcome of AML patients with CEBPA dm. Conclusions: A total of 19 (17.1%) patients with cytogenetically intermediate-risk AML harbored CEBPA mutations. Our study indicates that the presence of the CEBPA dm but not CEBPA sm is associated with favorable outcome in Japanese patients with cytogenetically intermediate-risk AML. Disclosures: No relevant conflicts of interest to declare.

Clinical Impact of GATA2 Mutations in Acute Myeloid Leukemia Patients Harboring CEBPA Mutations: A Study of the AML Study Group (AMLSG)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1332-1332
Author(s):  
Frauke Theis ◽  
Andrea Corbacioglu ◽  
Verena I. Gaidzik ◽  
Peter Paschka ◽  
Daniela Späth ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Clinical Impact ◽  
Large Cohort ◽  
Favorable Outcome ◽  
Intermediate Risk ◽  
Exon 4 ◽  
Cebpa Mutations ◽  
Acute Myeloid

Abstract Background Based on their association with certain biological and clinical features as well as their prognostic significance, mutations in the CCAAT/enhancer-binding protein-alpha (CEBPA) gene have been included as a provisional entity into the 2008 World Health Organization (WHO) classification of myeloid neoplasms. CEBPA mutations (CEBPAmut) are mainly found in acute myeloid leukemia (AML) with normal cytogenetics, and approximately 60% of the mutated patients (pts) carry biallelic mutations. Several studies showed that in particular pts with double mutant CEBPA (CEBPAdm) have a favorable outcome compared to all others. Recently, mutations in the transcription factor GATA2 were identified as genetic lesions potentially cooperating with CEBPAdm. Both, CEBPA and GATA2 are involved in the control of proliferation and differentiation of myeloid progenitors, and mutations in both genes are discussed as pre-disposing events in myeloid leukemia. Based on functional studies there is an important interplay between the two genes, e.g. through the formation of direct protein complexes. Finally, preliminary data suggest that the genotype CEBPAdm/GATA2 mutated (GATA2mut) is associated with a favorable outcome in AML pts. Aims To evaluate the frequency and the clinical impact of GATA2mut within a large cohort of CEBPAmut AML pts and to further analyze the CEBPAmut/GATA2mutgenotype within the context of other genetic alterations. Methods In total 202 AML pts (age 18 to 78 years) with CEBPA single mutations (n=89) or CEBPAdm (n=113) were analyzed for the presence of GATA2mut. All pts were enrolled on one of 6 AMLSG treatment trials applying intensive therapy [AMLHD93 n=15; AMLHD98A (NCT00146120) n=53; AMLHD98B n=13; AMLSG 07-04 (NCT00151242) n=74; AMLSG 06-04 (NCT00151255) n=25 and AMLSG 12-09 (NCT01180322) n=22]. GATA2 mutation screening was performed using a DNA-based PCR-assay covering exons 2 to 6 followed by Sanger sequencing. Results GATA2 mut were restricted to the cytogenetic intermediate-risk group; in total we detected 42 GATA2mut in 40 of the 202 pts (20.7%); 36 pts had CEBPAdm (36/113, 31.8%), 4 were CEBPA single mutated (4/89, 4.4%). All mutations were heterozygous, with 2 pts having two mutations (in exon 4 and 5, respectively). 31 (73.8%) of the 42 mutations were located in zinc-finger 1 (ZF1, exon 4) and 11 (26.1%) in ZF2 (exon 5). GATA2 sequence alterations included 39 missense and 3 frameshift mutations. The median follow-up of the 202 pts was 64.2 months (95%-CI: 60.1 – 75.1). First, we evaluated the clinical impact of GATA2mut in the whole cohort. Here, we found no differences in overall (OS), event-free (EFS), and relapse-free (RFS) survival as well as for the cumulative incidence of relapse (CIR) between GATA2mut and GATA2 wildtype pts. Next, the effects of GATA2mut in CEBPAdm pts (n=113) were analyzed without seeing any differences for the clinical endpoints OS, EFS, RFS and CIR. The same was also true when we investigated the impact of GATA2mut with respect to their location in the ZF domains; there were no differences between pts with ZF1 (n=29) and ZF2 (n=9) mutations, respectively. Finally, we evaluated the possible relevance of GATA2mut in the subgroup of CEBPAdm pts <60 years with intermediate-risk cytogenetics (n=94); but again GATA2mut did not impact the endpoints OS, EFS, RFS and CIR. In contrast to recently published data, we also detected GATA2mut in a small number of pts with CEBPA single mutations (n=4); however the low pt number did not allow a meaningful analysis. In addition, in our study GATA2mut occurred in rare cases with NPM1mut, FLT3-ITD or FLT3-TKD mutations. Conclusions In our study on a large cohort of CEBPA mutated AML pts we could confirm the high coincidence of GATA2 mutations, in particular in the subgroup of pts with CEBPA double mutations. However, GATA2 mutations had no impact on clinical outcome neither in the whole cohort nor in distinct pt subgroups. Disclosures: Schlegelberger: Celgene: Consultancy. Germing:Celgene: Honoraria, Research Funding. Kindler:Novartis: Membership on an entity’s Board of Directors or advisory committees. Schlenk:Novartis: Research Funding; Amgen: Research Funding; Chugai: Research Funding; Pfizer: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Ambit: Honoraria.

scholarly journals Nanopore Targeted Sequencing for Rapid Gene Mutations Detection in Acute Myeloid Leukemia

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1026 ◽  
Author(s):  
Cumbo ◽  
Minervini ◽  
Orsini ◽  
Anelli ◽  
Zagaria ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Low Cost ◽  
Gene Mutations ◽  
Predictive Biomarkers ◽  
Molecular Testing ◽  
Sequencing Analysis ◽  
Cebpa Mutations ◽  
Targeted Gene Sequencing ◽  
Acute Myeloid

Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (NPM1, FLT3, CEBPA, TP53, IDH1 and IDH2) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis; all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer FLT3 internal tandem duplications and phasing double CEBPA mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.

Impact of race on outcomes in intermediate-risk acute myeloid leukemia

2021 ◽  
Author(s):  
Ivy Elizabeth Abraham ◽  
Anand Ashwin Patel ◽  
Heidy Wang ◽  
John Patrick Galvin ◽  
Olga Frankfurt ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Intermediate Risk ◽  
Acute Myeloid
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