Establishment of a diagnostic procedure and preliminary scanning for length mutations in cebpa/bzip domain in vietnamese acute myeloid leukemia patients

Abstract Abstract 1544 Since myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are more prevalent in the elderly, intensive chemotherapy is difficult. However, recent progress in supportive therapy, especially with anti-fungal agents, and diagnostic procedures for invasive fungal infection (IFI) such as β-D glucan (β-D), galactomannan antigen (GM) and computed tomography (CT), have resulted in dramatically enhanced safety of post-chemotherapy control of elderly patients. To evaluate the efficacy and safety of our diagnosis and treatment strategy for IFI, we examined 112 consecutive episodes in 110 patients who received remission induction therapy from December 15, 2009 to June 18, 2011, including new or recurrent patients with MDS related AML (MDS-AML) and those with AML without a history of invasive aspergillosis (IA). Diagnosis was MDS-AML in 88 episodes (relapse 18) and AML in 24 (M1 5,M2 9,M4 9,M6 1).The median age was 70 (range: 21–88). Remission induction therapy consisted of behenoyl-ara-C for 10 days and idarubicin for 4 days (For further details, please refer to 51st ASH abstract #1052; Taiichi Kyo et al). Patients were always admitted to a clean room until neutrophil recovery, and were routinely administered macrophage-colony stimulating factor (CSF) and granulocyte-CSF. Amphotericin-b syrup and itraconazole capsules were given as antifungal prophylaxis. IFI diagnostic procedures consisted of CT, GM, β-D and surveillance culture (SC). At the time of admission a control CT was taken. CT was repeated within 24 hours when pyrexia of ≥38.0°C occurred. If fever showed no improvement, CT was repeated every 3 days (X-ray was also taken). If any change suggesting infection was noted, treatment against IA was considered. GM, β-D and SC were all conducted twice a week from the time of admission until discharge. ≥0.5 GM was regarded as positive and the treatment against IA was started even if there was only one positive result. At present there is no worldwide consensus concerning β-D, thus we considered a value exceeding the cut-off value of the reagent as positive. Treatment was started when there were both a positive result and increasing fever; and treatment against IA or candidiasis depended on imaging findings. Even if β-D was negative, candida detected by SC or diarrhea combined with increasing fever was also an indication for treatment against candidiasis. IA was treated with voriconazole (VRCZ) and candidiasis with micafungin (MCFG). VRCZ and MCFC were administered at 200–300 mg/twice/day and 100–300 mg/day, respectively. When no sufficient effect was observed with VRCZ alone, MCFG was added. Complete remission (CR) and partial remission (PR) were achieved in 81/112 (72%) and 9/112 (8%) episodes, while in 19/112 (17%) no response was obtained and 3/112 (2.7%) episodes resulted in death during chemotherapy. CR rate was comparable among de novo MDS-AML (49/70, 70%), MDS-AML relapse (9/18, 50%) and AML (23/24, 96%). The cause of death associated with chemotherapy was bacteremia 1, bacteremia or IA 1, and cerebral hemorrhage 1. GM was positive in 48 (43%) episodes. The reason for this large number was probably the advanced age of the patients and the long term neutropenia [absolute neutrophil count <500 (median) 27 days]. In spite of higher IA morbidity, mortality rates seemed very low. Furthermore, although GM >2.5 indicates an unfavorable prognosis and >5.0 no hope of survival, none of our patients with GM >2.5 (10 patients) died of IA (2 died of other causes) and all patients with GM >5.0 (4 patients) survived. Candidemia was found in 2 patients (krusei 1, guilliermondii 1) and were treated succesfully. β-D was positive in 46 /112 (41%) episodes and 28/112 (25%) were also positive for GM. As for GM and β-D, GM positivity preceded that of β-D in 9/28 (32%); regarding GM and CT, GM positivity preceded the observation of CT findings in 13/30 (43%). At the beginning of this study, no control CT was obtained. But in the course of the study we found some patients who presented CT findings indicating IA, such as nodular lesions, but with no infection. Thus, we realized the need for a control CT to detect IA more accurately. Each diagnostic procedure has excellent characteristics but it is not sufficient by itself. The results of this single-center clinical study indicate that an improvement of antifungal therapy combined with a battery of diagnostic procedures may allow safe, intensive chemotherapy for many patients with MDS or AML. Disclosures: No relevant conflicts of interest to declare.

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Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease

Blood ◽

10.1182/blood.v100.1.59 ◽

2002 ◽

Vol 100 (1) ◽

pp. 59-66 ◽

Cited By ~ 667

Author(s):

Susanne Schnittger ◽

Claudia Schoch ◽

Martin Dugas ◽

Wolfgang Kern ◽

Peter Staib ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Residual Disease ◽

Remission Rate ◽

Prognostic Significance ◽

Normal Karyotype ◽

Length Mutations ◽

Acute Myeloid

Abstract FLT3 length mutation (FLT3-LM) is a molecular marker potentially useful for the characterization of acute myeloid leukemia (AML). To evaluate the distribution of FLT3-LM within biologic subgroups, we screened 1003 patients with AML at diagnosis for this mutation. FLT3-LM was found in 234 (23.5%) of all patients and thus is the most frequent mutation in AML described so far. Of all positive patients, 165 (70.5%) revealed a normal karyotype. Of the 69 patients with chromosome aberrations, 24 (34.8%) had a t(15;17). The mutation was rare in AML with t(8;21), inv(16) 11q23 rearrangements, and complex karyotypes. FLT3-LM was not distributed equally within different French-American-British (FAB) subtypes and was correlated with a high peripheral blood count in FAB M1, M2, and M4 (P < .0001). In addition, the median age of patients with the mutation was lower (54.9 vs 57.6 years;P = .043), and, at a ratio of 1.36:1 (P = .023), the mutation was more frequent in females than in males. Within the AMLCG study, FLT3-LM was of intermediate prognostic significance. The complete remission rate of 70.3% in patients with FLT3-LM was similar to that (70.4%) in patients without FLT3-LM. Overall survival was not different between patients with or without FLT3-LM. In contrast, patients with FLT3-LM had a significantly shorter event-free survival (7.4 vs 12.6 months;P = .0072) because of a higher relapse rate. Besides the importance of FLT3-LM for biologic and clinical characterization of AML, we show its value as a marker for disease monitoring based on 120 follow-up samples of 34 patients.

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Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia

Blood Advances ◽

10.1182/bloodadvances.2021004292 ◽

2021 ◽

Author(s):

Satoshi Wakita ◽

Masahiro Sakaguchi ◽

Iekuni Oh ◽

Shinichi Kako ◽

Takashi Toya ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leucine Zipper ◽

De Novo ◽

Prognostic Significance ◽

Prognostic Impact ◽

Basic Leucine Zipper ◽

Favorable Prognosis ◽

Bzip Domain ◽

Acute Myeloid

Mutations of CCAAT/enhancer binding protein alpha (CEBPAmu) are found in 10-15% of de novo acute myeloid leukemia (AML) cases. Double-mutated CEBPA (CEBPAdm) is associated with a favorable prognosis; however, single-mutated CEBPA (CEBPAsm) does not appear to improve prognosis. We investigated the CEBPAmu for prognosis in 1028 AML patients, registered in the Multi-center Collaborative Program for Gene Sequencing of Japanese AML. It was found that CEBPAmu in the basic leucine zipper domain (bZIP) was strongly associated with a favorable prognosis, but CEBPAmu out of the bZIP domain was not. The presence of CEBPAmu in bZIP was a strong indicator of a higher chance of achieving complete remission (p<0.001), better overall survival (OS; p<0.001) and a lower risk of relapse (p<0.001). The prognostic significance of CEBPAmu in bZIP was also observed in the subgroup with CEBPAsm (all patients, OS: p=0.008; the cumulative incidence of relapse (CIR): p=0.063. patients aged ≤70 years and with intermediate-risk karyotype, OS: p=0.008; CIR: p=0.026). Multivariate analysis of 744 patients aged ≤70 years showed that CEBPAmu in bZIP was the most potent predictor of OS (hazard ratio: 0.3287; p<0.001). CEBPAdm was validated as a cofounding factor, which was overlapping with CEBPAmu in bZIP. In summary, these findings indicate that CEBPAmu in bZIP is a potent marker for AML prognosis. It holds potential in the refinement of treatment stratification and the development of targeted therapeutic approaches in CEBPA mutated AML.

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CEBPα Mutations in Childhood Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v104.11.3006.3006 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3006-3006

Author(s):

Der-Cherng Liang ◽

Lee-Yung Shih ◽

Chein-Fuang Huang ◽

Iou-Jih Hung ◽

Chao-Ping Yang ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Direct Sequencing ◽

Ras Mutations ◽

Bzip Domain ◽

Childhood Aml ◽

Wbc Count ◽

Biallelic Mutations ◽

Acute Myeloid

Abstract Transcription factor CCAAT/enhancer binding protein alpha(C/EBPα) is essential for granulocyte differentiation. CEBPα mutations have been described in 7–10% of adult patients with acute myeloid leukemia (AML) at initial diagnosis and conferred a favorable prognosis. CEBPα mutations in childhood AML has not been reported yet. In this study, we sought to assess the frequency of CEBPα mutration, its clinicohematologic correlation and to analyze the cooperating mutations, including FLT3 and N-ras mutations in childhood AML. CEBPα mutations were analyzed in 117 children (age one day to 17 years, median 5 years) with de novo AML. CEBPα mutation status was examined by performing DNA polymerase chain reaction (PCR) followed by direct sequencing for each PCR product. CEBPα mutations were detected in 7 (6.0%) of 117 patients. Of the 7 patients with CEBPα mutations, 4 had FAB subtype of M2, 2 M1 and 1 M4. All had intermediate cytogenetics and comprised 11.5% (7/61) of the intermediate cytogenetic risk group. Five of the 7 mutations occurred in both N-terminal part and bZIP domain, one had N-terminal frameshift mutation and the remaining one had an inframe insertion in the bZIP domain. Clonal analysis using expand long template PCR assay with a primer pair covering entire coding sequence was performed in all 5 patients carrying two different mutations. The results demonstrated that one had homozygous combined mutation and 4 had heterozygous biallelic mutations with the cloned alleles carrying single mutations at N-terminal or bZIP domain in the different alleles or harboring combined mutations in the same alleles. None of the remission marrow samples obtained from 5 patients who had CEBPα mutations at diagnosis carried the mutations. FLT3-ITD mutations were detected in 2 of the 7 patients with CEBPα mutations compared to 15 of the 109 patients without CEBPα mutations. None of the patients carrying CEBPα mutations had FLT3-TKD mutations. Two of the 7 CEBPα(+) patients also harbored N-ras mutations compared to 10 of the 110 patients without CEBPα mutations. Taken together, 4 of 7 CEBPα(+) patients had cooperating mutations including FLT3-ITD or N-ras as compared to 27 in 109 CEBPα(−) patients (P=0.081). There were no statistical differences in age, WBC count, LDH level, 5-year overall survival and event-free survival between patients with and without CEBPα mutations; similar results were obtained if we only analyzed patients with intermediate cytogenetics. The present data showed that the frequency of CEBPα mutations in our series of childhood AML was similar to that in adults. CEBPα mutations were frequently associated with FLT3-ITD or N-ras mutations supporting the two-hit hypothesis for the pathogenesis of AML.

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The Gene Encoding Nuclear Erythroid Factor 2 (NFE2) Is Recurrently Mutated in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.1392.1392 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1392-1392

Author(s):

Mathijs A. Sanders ◽

Annelieke Zeilemakers ◽

Jasper Koenders ◽

Remco Hoogenboezem ◽

François Kavelaars ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Transcription Factor ◽

Myeloid Leukemia ◽

Sanger Sequencing ◽

Transcription Factor Gene ◽

Protein Coding ◽

Factor Gene ◽

Frame Shift ◽

Bzip Domain ◽

Acute Myeloid

Abstract Abstract 1392 Background: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of various acquired (cyto)genetic aberrations in the leukemic blasts. Novel state-of-the-art sequencing technologies enable sequencing of complete disease genomes. Methods: We have used Complete Genomics (CG) next-generation sequencing to identify novel recurrent mutations in AML. We have selected a single AML case, WHO: AML with maturation, FAB: M2, karyotype 45X, -Y and a NPM1 mutation. Mutations in FLT3, CEBPA, ASXL1, IDH1, IDH2, NRAS, KRAS and DNMT3A were absent. By whole genome sequencing of the AML and its corresponding remission sample, we identified acquired mutations in the protein coding regions of 31 genes (CG somatic score >0.1), including NPM1 and PTPN11. Results: Interestingly, a frame-shift mutation in the protein coding region of the Nuclear Erythroid Factor 2 (NFE2) transcription factor gene was identified and acquisition of this mutation was confirmed by Sanger sequencing of both AML and remission samples. The complete NFE2 gene of the index AML patient was sequenced but no additional mutation was present, nor was the remaining allele affected by deletions and/or amplifications. The index mutation introduces a premature stop codon (PTC) in the NFE2 gene, upstream of the region encoding the bZIP domain. To investigate if NFE2 would be recurrently mutated in AML, we screened a cohort of 1139 AML cases by denaturing high performance liquid chromatography (dHPLC) analyses for mutations in a 350bp region surrounding the index mutation in the NFE2 gene. We identified NFE2 mutations in 5 additional cases of AML. Subsequently, we screened the complete NFE2 gene in 254 randomly selected AML cases by Roche 454 sequencing. This analysis revealed 8 NFE2 mutant cases in total. These results indicate that approximately 3.5% (8/254) of unselected primary AML cases carry NFE2 mutations. The acquisition of the NFE2 mutations was confirmed by Sanger sequencing of all NFE2mutant AML cases and their corresponding derived T cells. Frame shift mutations upstream of the NFE2 bZIP domain introducing PTCs were present in 6 out of all identified NFE2 mutant cases (n=13). The remaining cases carried non-synonymous NFE2 mutations and a single case an in-frame insertion/deletion. In our cohort of molecularly and clinically well-characterized cohort of AML patients the NFE2 mutations were not associated with any clinical characteristic or any other (cyto)genetic aberration. Discussion: It is currently unclear if the NFE2 mutations would lead to a gain-of-function or a loss-of-function. Nfe2-deficient mice lack circulating platelets and die of hemorrhage; their megakaryocytes showed no cytoplasmic platelet formation. In addition, NFE2 transgenic mice show MPNs, including thrombocytosis, and spontaneous transformation to acute myeloid leukemia. However, the NFE2 mutations did not associate with abnormal platelet counts in the affected AML cases, nor did the AML cases have consistent megakaryocyte abnormalities. Mutant NFE2 is currently functionally studied by introduction into various cell line models and mouse primary bone marrow. Conclusion: In conclusion, we have identified recurrent mutation in the transcription factor gene NFE2 in a subset of AML cases. The exact role of mutant NFE2 is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

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A new and recurrent activating length mutation in exon 20 of the FLT3 gene in acute myeloid leukemia

Blood ◽

10.1182/blood-2002-03-0953 ◽

2002 ◽

Vol 100 (9) ◽

pp. 3423-3425 ◽

Cited By ~ 69

Author(s):

Karsten Spiekermann ◽

Ksenia Bagrintseva ◽

Claudia Schoch ◽

Torsten Haferlach ◽

Wolfgang Hiddemann ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Protein Tyrosine Kinase ◽

Catalytic Domain ◽

Genetic Alterations ◽

Activating Mutations ◽

Exon 20 ◽

Length Mutations ◽

Acute Myeloid

Abstract Activating length mutations in the juxtamembrane (JM) domain of the FLT3 gene (FLT3-LM) and mutations in the catalytic domain (FLT3D835/836) of this receptor tyrosine kinase represent the most frequent genetic alterations in acute myeloid leukemia (AML). Here, we describe a 6-bp insertion in the activation loop of FLT3 between codons 840 and 841 of FLT3 (FLT3-840GS) in 2 unrelated patients with AML. Screening for other activating mutations of FLT3, KIT, and NRASshowed no further genetic alterations in patients carrying the FLT3-840GS. In functional analyses we could show that this mutant is hyperphosphorylated on tyrosine and confers interleukin 3–independent growth to Ba/F3 cells, which can be inhibited by a specific FLT3 protein tyrosine kinase (PTK) inhibitor. Our results show for the first time that in addition to known mutations in the JM and the catalytic domain, further activating length mutations exist in theFLT3 gene.

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The Significance of BAALC Expression and CEBPa Mutations as New Prognostic Factors in Pediatric Acute Myeloid Leukemia with Normal Karyotype.

Blood ◽

10.1182/blood.v108.11.2279.2279 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2279-2279

Author(s):

Yasuhiro Mizushima ◽

Souichi Adachi ◽

Tomohiko Taki ◽

Akira Shimada ◽

Yasuhide Hayashi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Prognostic Factors ◽

Myeloid Leukemia ◽

Normal Karyotype ◽

Rt Pcr ◽

Bzip Domain ◽

Pediatric Aml ◽

Cebpa Mutations ◽

Acute Myeloid ◽

High Expression Level

Abstract Approximately half of children with acute myeloid leukemia (AML) have no chromosomal or genetic abnormalities that can predict outcome. To improve the prognostic stratification on this heterogeneous group of patients, novel markers need to be investigated. Recently, CEBPa (CCAAT/enhancer binding protein alpha) mutations have been described as a favorable prognostic factor and the high expression level of BAALC (brain and acute leukemia, cytoplasmic) was found to be a poor prognostic marker, in adult AML with normal karyotype. To explore their significance in childhood, we studied the prognostic impact of the high expression level of BAALC and CEBPa mutations in pediatric AML with normal karyotype. Samples were available from pediatric patients 3 months to 15 years of age, treated on Japanese Childhood AML Cooperative Study Group Protocol, AML99. BAALC expression was determined by comparative real-time RT-PCR assay in 29 patients. The relative BAALC expression was determined using the comparative cycle threshold method. The median value of healthy volunteers was used as cutoff. The mutational analysis of CEBPa was performed in 49 patients with RT-PCR followed by direct sequencing. For samples carrying mutations, additional subcloning analysis were carried out. 72.4% (21 of 29) of patients had high BAALC expression and 27.6% (8 of 29) had low. High BAALC expression was associated with the FAB subtypes M0, M1, and M2, whereas low BAALC expression correlated with M4 and M5. Overall survival and event-free survival was 52.3%; 49.5% (high BAALC expression group) and 75.0%; 62.5% (low BAALC expression group), respectively. CEBPa mutations were detected in four patients (8.2 %, 4 of 49; two each in M1 and M2). N-terminal frameshift mutations and inframe insertions in the basic-leucine zipper (bZIP) domain were detected. Novel mutations (212 insertion (ins) C, 214–224 deletion CCCCGCACGCG, 720 ins CGCACC, 1074 ins AGA, 1092 ins CAC) were identified. One patient had mutations occurred in both N-terminal part and bZIP domain. Moreover, above patients with CEBPa mutations had not cooperating mutations with FLT3-ITD (fms-like tyrosine kinase 3 internal tandem duplication) and maintain in complete remission for at least 21 months duration. In conclusion, BAALC expression and CEBPa mutations may be prognostic factors in pediatric AML with normal karyotype.

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Prevalence and prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia (AML): a report from the Children's Oncology Group

Blood ◽

10.1182/blood-2008-10-184747 ◽

2009 ◽

Vol 113 (26) ◽

pp. 6558-6566 ◽

Cited By ~ 105

Author(s):

Phoenix A. Ho ◽

Todd A. Alonzo ◽

Robert B. Gerbing ◽

Jessica Pollard ◽

Derek L. Stirewalt ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Prognostic Significance ◽

Normal Karyotype ◽

Risk Identification ◽

Bzip Domain ◽

Prognostic Implications ◽

Cebpa Mutations ◽

Improved Outcome ◽

Acute Myeloid

Abstract CEBPA mutations have been associated with improved outcome in adult acute myeloid leukemia (AML). We evaluated the prevalence and prognostic significance of CEBPA mutations in 847 children with AML treated on 3 consecutive pediatric trials. Two types of CEBPA mutations—N-terminal truncating mutations and in-frame bZip-domain mutations—were detected in 38 (4.5%) of 847 patients tested; 31 (82%) of 38 patients with mutations harbored both mutation types. Mutation status was correlated with laboratory and clinical characteristics and clinical outcome. CEBPA mutations were significantly more common in older patients, patients with FAB M1 or M2, and patients with normal karyotype. Mutations did not occur in patients with either favorable or unfavorable cytogenetics. Actuarial event-free survival at 5 years was 70% versus 38% (P = .015) with a cumulative incidence of relapse from complete remission of 13% versus 44% (P = .007) for those with and without CEBPA mutations. The presence of CEBPA mutations was an independent prognostic factor for improved outcome (HR = 0.24, P = .047). As CEBPA mutations are associated with lower relapse rate and improved survival, CEBPA mutation analysis needs to be incorporated into initial screening for risk identification and therapy allocation at diagnosis. The clinical trials in this study are registered at http://www.clinicaltrials.gov under NCT00002798 and NCT00070174.

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