Activating Mutations of the FMS-Like Tyrosine Kinase-3 (FLT3) At Complete Response and Relapse in Patients with Acute Myeloid Leukemia,

Abstract Abstract 3557 Background – Activating mutations of the transmembrane receptor tyrosine kinase, FLT3, occur in approximately 30% of patient with acute myeloid leukemia (AML) and predict for a shorter relapse-free and overall survival. There is limited data on loss or persistence of the mutated clones at the time of complete response (CR) and their recurrence at the time of relapse. Objectives and Methods – To evaluate patterns of loss and recurrence of FLT3 mutated clones in relation to response and relapse in patients with FLT3 mutated AML treated with idarubicin and cytarabine (IA) with or without sorafenib (S), vorinostat (V), or tipifarnib (T). Bone marrow samples at diagnosis, CR and relapse were examined for the presence of FLT3 mutated clones using reverse transcription polymerase chain reaction. Results – Among 361 patients with AML treated from October 2004 to March 2010 on one of the 4 induction regimens of IA, IAS, IAV, and IAT, 321 had presentation bone marrow samples tested and 72 had FLT3 mutations (including 50 with ITD and 16 D835 with 6 having both). The median age for the entire group was 53 years (range, 17–73) and for the FLT3 mutated patients 52 years (range, 17 to 73). Cytogenetics at diagnosis in FLT3 mutated patients included normal karyotype in 48 (67%) patients, chromosome 5 and 7 abnormalities in 4(6%), trisomy 8 in 4(6%), 11q abnormalities in 2 (3%), insufficient metaphases in 3(4%), and miscellaneous in 11(16%). 271 (75%) patients overall, and 64 (89%) patients with mutated FLT3 achieved CR. Among the 56 patients with presentation FLT3-ITD, 51 achieved CR. Among 13 patients with available samples at CR, none had FLT3-ITD; 8 of these patients relapsed and 5 had FLT3-ITD positive clones at relapse (2 negative and 1 not done); Among the 38 patients with no samples at CR, 17 relapsed, 8 with a FLT3-ITD clone (1 negative and 8 not done). Among the 201 patients without FLT3-ITD at diagnosis, who achieved CR, 8 patients acquired a clone with FLT3-ITD at relapse. Conclusions – FLT3-ITD mutant clones are unstable at follow-up. Relapse may occur in their absence and they may occur for the first time at relapse. Therefore, FLT3-ITD cannot be used reliably for minimal residual leukemia monitoring. Disclosures: Ravandi: Bayer Onyx: Honoraria, Research Funding.

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BCOR Mutations in Acute Myeloid Leukemia: Clonal Evolution and Prognosis

Blood ◽

10.1182/blood-2020-137127 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 4-4

Author(s):

Ashley Zhang ◽

Yuntao Liu ◽

Shuning Wei ◽

Benfa Gong ◽

Chunlin Zhou ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Risk Stratification ◽

Myeloid Leukemia ◽

Statistical Difference ◽

Tp53 Mutation ◽

Clonal Evolution ◽

Normal Karyotype ◽

Abnormal Karyotype ◽

Acute Myeloid

Background BCOR gene is a transcription repressor that may influence normal hematopoiesis and is associated with poor prognosis in acute myeloid leukemia (AML) with normal karyotype. However, due to the rare mutation frequency in AML (3.8%-5%), clinical characteristics and prognosis of AML patients with BCOR mutation including abnormal karyotype are still unknown. In addition, the clonal evolution of AML patients with BCOR mutation has not been fully investigated. Methods By means of next generation of sequencing, we performed sequencing of 114 genes related to hematological diseases including BCOR on 509 newly diagnosed AML patients (except for acute promyelocytic leukemia) from March 2017 to April 2019. The 2017 European Leukemia Net (ELN) genetic risk stratification was used to evaluate prognosis. Overall survival (OS) was defined as the time from diagnosis to death or last follow-up. Relapse-free survival (RFS) was measured from remission to relapse or death. Clonal evolution was investigated through analyzing bone marrow samples at diagnosis, complete remission (CR) and relapse from the same patient. Result Among 509 AML patients, we found BCOR mutations in 23 patients (4.5%). BCOR mutations were enriched in patients with mutations of RUNX1 (p = 0.008) and BCORL1 (p = 0.0003). Patients with BCOR mutation were more at adverse ELN risk category compared to patients without BCOR mutation (p = 0.007). Besides, there was a larger proportion of patients with normal karyotype in BCOR mutation group but it had not reached statistical difference (62.5% vs 45.5%, p = 0.064). The abnormal karyotype in patients with BCOR mutations included trisomy 8, t(9;11), inv(3), -7 and complex karyotype.There were no significant differences in age, sex, white blood cell count, hemoglobin or platelet count between the two groups. More patients died during induction (13.0% vs 3.5%, p = 0.56) and fewer patients achieved CR after 2 cycles of chemotherapy when patients had BCOR mutations (69.6% vs 82.5%, p = 0.115) but the difference had not reached statistical difference . Patients with BCOR mutations had inferior 2-year OS (52.1% vs 70.7%, p = 0.0094) and 2-year RFS (29.8% vs 61.1%, p = 0.0090). After adjustment for ELN risk stratification, BCOR mutation was still remain a poor prognostic factor. However, the adverse prognostic impact of BCOR mutation is overcome by hematopoietic stem cell transplantation (HSCT), in which there was no difference between BCOR mutation group and wild type group (p = 0.474) (Figure 1). Through analysis of paired bone marrow sample at diagnosis, remission and relapse, we revealed the clonal evolution that BCOR mutation was only detected at diagnosis sample as a subclone and diminished at CR and relapse while TP53 mutation was only detected at relapse with a variant allele frequency (VAF) of 25.5%. We also found BCOR mutation at another patient's diagnosis and relapse sample while TP53 mutation was detected at relapse with VAF of 11.8%. Conclusion BCOR is associated with RUNX1 mutation and higher ELN risk. AML patients with BCOR mutation including normal and abnormal karyotype conferred a worse impact on OS that can be overcome by HSCT. BCOR mutation is a subclone at diagnosis or relapse in some patients, in which TP53 mutation clone occurred at relapse. Disclosures No relevant conflicts of interest to declare.

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Monocytic Maturation Induced by FLT3 Inhibitor Therapy of Acute Myeloid Leukemia: Morphologic and Immunophenotypic Characteristics

Laboratory Medicine ◽

10.1093/labmed/lmz094 ◽

2019 ◽

Vol 51 (5) ◽

pp. 478-483

Author(s):

Cade D Arries ◽

Sophia L Yohe

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Tyrosine Kinase ◽

Cell Population ◽

Myeloid Leukemia ◽

Inhibitor Therapy ◽

Molecular Abnormalities ◽

Flt3 Inhibitor ◽

Flt3 Inhibitors ◽

Acute Myeloid

Abstract Background FMS-like tyrosine kinase-3 (FLT3-ITD) mutations are some of the most common mutations in acute myeloid leukemia (AML), and patient outcomes have improved since the advent of tyrosine kinase inhibitors. First, granulocytic differentiation was described in FLT3-positive AML treated with FLT3 inhibitors, and more recently, monocytic differentiation was reported. Methods Two patients with myelomonocytic cells in their bone marrow were identified during routine follow-up after AML treatment that included FLT3 inhibitors. The bone marrow study was done as standard of care. Results Both patients had FLT3-ITD+ AML and showed an atypical maturing monocytic cell population and a decrease in the leukemic blast cell population after FLT3 inhibitor therapy. Concurrent genetic testing revealed persistent genetic abnormalities. Conclusions These cases illustrate monocytic maturation in FLT3+ AML after FLT3 inhibitor treatment. It is critical for pathologists and clinicians to be aware of the differentiation phenomenon, as these patients have persistent molecular abnormalities despite response to treatment and normalization of blast counts.

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Rapid Detection of Nucleophosmin (NPM1) Mutations to Determine Prognostic Implications in Acute Myeloid Leukemia Patients with a Normal Karyotype.

Blood ◽

10.1182/blood.v114.22.4676.4676 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4676-4676

Author(s):

Seo-Jin Park ◽

Hyun-Sook Chi ◽

Kyung Ran Jun ◽

Sook Kyoung Min ◽

Seongsoo Jang ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Direct Sequencing ◽

Screening Method ◽

Normal Karyotype ◽

Npm1 Mutation ◽

Prognostic Groups ◽

Acute Myeloid

Abstract Abstract 4676 INTRODUCTION Mutations of the nucleophosmin gene (NPM1) occur in up to 40-50% of adult acute myeloid leukemia (AML) with a normal karyotype and are associated with a higher frequency of fms-like tyrosine kinase-3 internal tandem duplications (FLT3-ITD) and responsiveness to induction chemotherapy. The incidence of NPM1 mutations in Caucasians have been previously reported in several studies whereas there have been few reports from Asian countries including Japan, China, and Taiwan. The objectives of our study was to determine the prevalence of NPM1 mutations and distribution of AML subtypes in the normal karyotype AML Korean population in addition to establishing an easily applicable yet reliable method to indentify these mutations. We also examined treatment outcomes and survival (relapse-free survival (RFS) and overall survival (OS)) by stratifying them into groups according to NPM1 and FLT3-ITD mutation status. METHODS We retrospectively analyzed the prevalence of NPM1 mutations in 185 patients with normal karyotype AML diagnosed between 2002 and 2009. Genomic DNA extracted from bone marrow aspirate specimens obtained at diagnosis was amplified by PCR, followed by analysis on an ABI 3130 Genetic Analyzer (Applied Biosystems) by capillary electrophoresis. Cases found to have mutation peaks at 174bp by Gene Mapper ID v3.2 software (Applied Biosystems) were further analyzed by direct sequencing of exon 12 of NPM1 gene. Follow-up data was reviewed by retrospective chart review for treatment outcome and survival analyses. Among the 185 AML patients, 18 with less than a 1-month follow-up period were excluded since they could not be sufficiently evaluated. RESULTS Mutations in exon 12 of NPM1 were found in 37 of 185 (20.0%) normal karyotype AML patients and were composed of TCTG duplications (Type A, 32/37, 86.5%), 3 previously reported variants, and 2 new variants previously not reported. Mutations were most frequently seen in AML M1 patients (12/37, 32.4%) and other subtypes such as M2, and M4 were often observed. NPM1 mutations were particularly associated with CD34-negativity (<0.0001) and higher bone marrow blast (%) at diagnosis (p=0.0067). There was a mild trend towards frequent FLT3-ITD mutations in NPM1+ patients in comparison to the NPM1- group (35.1% and 19.6%, p=0.0787). After exclusion of the 18 patients lost during follow-up, no significant differences in RFS (8.5 and 10.8 months, p=0.7922) and OS (11.5 and 13.6 months, p=0.6147) were observed between the NPM1+ and NPM1- groups. Stratification into good (NPM1+/FLT3-ITD-), intermediate (NPM1-/FLT3-ITD- & NPM1+/FLT3-ITD+), and poor (NPM1-/FLT3-ITD+) prognostic groups did not reveal significant differences in median values of RFS and OS (in months; RFS, 16.0 and 13.8 and 7.3, p=0.1872; OS, 16.0 and 10.8 and 7.3, p=0.3661). However, the Kaplan-Meier survival analysis of these stratified prognostic groups showed a trend toward a difference in RFS (p=0.084) and a significantly longer OS in the NPM1+/FLT3-ITD- (good prognostic) group (p=0.031). CONCLUSIONS The prevalence of NPM1 mutations in normal karyotype AML patients in Koreans was lower than those reported in Western studies. In areas with low prevalence, a screening method to detect mutations enables rapid reporting with only selective cases requiring the labor-intensive direct sequencing step. In accordance with previous studies, a significantly longer OS in the NPM1+/FLT3-ITD- group suggests that NPM1+ may be associated with a favorable outcome. However, discordant parameters such as prevalence and RFS may signify that elucidation of the prognostic significance of NPM1 mutations in different ethnic groups may be necessary. Thus, NPM1 mutation studies should be considered in the diagnostic work-up of all AML patients with a normal karyotype given its role as a prognostic marker. Disclosures: No relevant conflicts of interest to declare.

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The Prognostic Impact and Stability of IDH2 Mutation and the Insufficiency of the Mutation Alone for Leukemogenesis In Adult Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.2697.2697 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2697-2697

Author(s):

Weng-Chi Lei ◽

Wen-Chien Chou ◽

Bor-Shen Ko ◽

Hsin-An Hou ◽

Hwei-Fang Tien

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Normal Karyotype ◽

Idh1 Mutation ◽

Group 12 ◽

Worse Prognosis ◽

Idh2 Mutation ◽

Acute Myeloid

Abstract Abstract 2697 Purpose: Although the clinical and biological features of Isocitrate dehydrogenase (IDH) mutations in acute myeloid leukemia (AML) have been characterized, its stability and in vivo sufficiency of the mutation alone for leukemogenesis remain uninvestigated. Patients and Methods: Mutations of IDH and other clinically relevant genes were analyzed in the bone marrow from 446 adult patients with de novo non-M3 AML. IDH2 mutations were examined serially in 140 patients at diagnosis and after chemotherapy. Results: Among the 446 adults with de novo non-M3 AML, IDH2 R172, R140, and IDH1 R132 mutations occurred at a frequency of 2.9%, 9.2%, and 6.1%, respectively. IDH2 mutation was associated with higher platelet counts (p=0.046), intermediate-risk (p=0.002) or normal karyotype (p=0.023), and isolated +8 (p=0.014), but was inversely correlated with expression of HLA-DR (p=0.002), CD34 (p=0.039), CD15 (p=0.003), CD7 (p=0.010), and CD56 (p=0.048), and was mutually exclusive with WT1 mutation (p=0.037) and core-binding factor translocations (p=0.001). All these correlations became stronger when IDH1 and IDH2 mutations were considered together, suggesting similarity of biological roles between these 2 mutations. However, IDH2 but not IDH1 mutation conferred a better prognosis (Fig 1), especially in those with normal karyotype or intermediate cytogenetics (median overall survival: not reached vs. 58 months, p=0.044 and not reached vs. 19 months, p=0.027 for normal and intermediate karyotype group, respectively). Importantly, IDH2 but not IDH1 mutation was an independent favorable prognostic factor (HR: 0.332, 95% CI: 0.159–0.694; p=0.003). Patients with IDH2−/FLT3-ITD+ genotype had especially worse prognosis (median OS of IDH2−/FLT3-ITD+ vs. IDH2+/FLT3-ITD− group: 12 months vs. not reached; p=0.003; median OS of IDH2−/FLT3-ITD+ vs. IDH2+/FLT3-ITD+ or IDH2−/FLT3-ITD− group : 12 months vs. 35 months; p<.0001) (Fig 2A). The worse prognosis was also seen in patients with IDH−/FLT3-ITD+ genotype (Fig 2B). Serial analyses of IDH2 mutations during the clinical course of 140 patients confirmed the stability of this mutation; all the patients with IDH2 mutations at diagnosis harbored the same mutation at relapse with the exception of one patient who had extramedullary but not bone marrow relapse, while none of the IDH2-wild patients acquired this mutation at relapse. Importantly, sequential samples from two patients in long-term remission retained the original R140Q mutation while other accompanied mutations, FLT3-ITD in the first patient and NPM1 in the second, respectively, disappeared. In the first patient, the skin tissue was absent of the mutation and in the second, the mutation was restricted in myeloid cells but spared in lymphocytes indicating the mutation was acquired in these two patients. Conclusion: IDH2 mutation is a stable marker during disease evolution and confers favorable prognosis. FLT3-ITD combined with wild type IDH2 exerted synergistic negative impact on survival. IDH2 mutation alone is insufficient for leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Development of Somatic NRAS Mutation Associated with Rapid Transition from Germline GATA2 Mutation Associated Myelodysplastic Syndrome to Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.3616.3616 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3616-3616 ◽

Cited By ~ 1

Author(s):

Yanqin Yang ◽

Yubo Zhang ◽

Jun Zhu ◽

Catherine E. Lai ◽

Jingrong Tang ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Myelodysplastic Syndrome ◽

Myeloid Leukemia ◽

Research Funding ◽

Bone Marrow Aspirate ◽

Nras Mutation ◽

Trisomy 8 ◽

Acute Myeloid

Abstract There is increasing recognition of the role of inherited germline predisposition for myeloid disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The additional somatic genetic events required for development of a malignant phenotype are however poorly understood. A 25 year old woman was referred to the NHLBI hematology branch in March 2014 for a seven year history of pancytopenia. Her medical history included recurrent pneumonias, oral ulcers, severe varicella infection and arthralgias. Prior bone marrow examinations at ages 21 and 23 at outside institutions reported normocellular marrow, tri-lineage hematopoiesis and mild dyspoiesis. Cytogenetics were remarkable for trisomy 8 in 80% (aged 21) or 90% (aged 23) of metaphases. Previously unrecognized lymphedema was noted on examination. Peripheral blood counts showed WBC 2.28 K/ul [normal range: 3.98-10.04], HGB 9.9 g/dL [11.2-15.7], PLT: 67 K/ul [173-369], ALC: 0.36 K/ul [1.18-3.74] and AMC: 0.06 [0.24-0.86]. Peripheral blood flow cytometry demonstrated decreased CD3+ CD4+ (T) cells, CD19+ (B) cells and NK cells. HLA-DR15 negative. Bone marrow examination showed trilineage hematopoiesis, 50-60% cellularity, mild erythroid predominance and mildly increased, mildly atypical megakaryocytes. Blasts less than 5%. Bone marrow flow cytometry revealed severely decreased B-cells and monocytes, absent B-cell precursors, absent dendritic cells, inverted CD4:CD8 ratio, and atypical myeloid maturation pattern. Cytogenetics demonstrated stable trisomy 8 in 90% of metaphases. On the basis of this assessment the diagnosis of MDS was confirmed. Sanger sequencing revealed a GATA2 L375S mutation in the second zinc finger of known pathogenic significance. Four months later she developed increased fatigue and easy bruising with worsening thrombocytopenia (PLT: 10K/ul). Bone marrow was dramatically changed; now markedly hypercellular (90-100%) with diffuse sheets of immature cells consistent with blasts having fine chromatin, distinct or prominent nucleoli, and visible cytoplasm. Blasts were positive for CD33, CD56, CD64, CD123, and CD163; and were negative for CD34, CD14, and myeloperoxidase. Cytogenetics showed a new trisomy 20 in 65% of metaphases, in addition to previously seen trisomy 8 in 100%. A diagnosis of acute monoblastic leukemia (M5a subtype) was made. At both clinic visits bone marrow aspirate was collected on an IRB approved research sample acquisition protocol. Whole exome sequencing of 1ug DNA was performed using Agilent SureSelect v5 Exome enrichment Kits on an Illumina HiSeq 2000 with 100-bp paired-end reads (Macrogen, Rockville, MD). Data was mapped to hg19 (BWA) and processed using an in-house pipeline (Samtools/Picard/GATK/VarScan/Annovar). Mean read depth of target regions was 157 and 149. There was high correlation between both samples with the exception of a NRAS:NM_002524:exon3:c.C181A:p.Q61K mutation (57 of 180 reads) seen only in the later sample. Confirmatory ultra-deep sequencing for NRAS was performed using Illumina TruSight Myeloid Sequencing Panel on an Illumina MiSeq. No evidence of the NRAS Q61K mutation was found in the earlier March MDS bone marrow sample even when sequenced to a depth greater than 1750 reads (see figure). The mutation was confirmed in the August AML sample at a variant allele frequency of 35%. If heterozygous this would reflect a clone size of 70%, consistent with data from both cytogenetics (new trisomy 20 in 65% of metaphases) and the 76% blasts documented by bone marrow aspirate smear differential. We report here the rapid progression to AML in a patient with germline GATA2 MDS associated with development of a new trisomy 20 karyotype and a NRAS Q61K mutation. The NRAS mutation was not detectable after the patient achieved a complete remission following induction chemotherapy further supporting this association. This NRAS mutation has been implicated in the pathogenesis of multiple cancers by constitutive activation of proliferative signaling. GATA2 associated MDS is a high-risk pre-leukemic condition with the potential for rapid evolution to AML. This is the first report of acquired somatic mutations in the RAS/RTK signaling pathway in the context of germline GATA2 insufficiency associated with acute leukemic transformation. Figure 1. Figure 1. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding.

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Effect of Complete Remission Achievement on Survival In Acute Myeloid Leukemia of the Elderly.

Blood ◽

10.1182/blood.v116.21.1048.1048 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1048-1048

Author(s):

Felicetto Ferrara ◽

Cira Riccardi ◽

Salvatore Palmieri ◽

Tiziana Izzo ◽

Antonella Carbone

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Complete Remission ◽

Myeloid Leukemia ◽

Normal Karyotype ◽

Loading Dose ◽

Refractory Disease ◽

Bone Marrow Blast ◽

Blast Count ◽

Acute Myeloid

Abstract Abstract 1048 The achievement of complete remission (CR) is considered an essential prerequisite for cure in acute myeloid leukemia (AML). Notwithstanding, in older AML patients recent data suggest that, at least for patients receiving new compounds such as hypomethilating agents Azacytidine and Decitabine, the benefit on survival can be independent from CR achievement, namely in patients with low bone marrow blast count (< 30%) at diagnosis. In this study we evaluated the impact of CR achievement on overall survival from a series of 140 patients aged over 60 years; all patients received a therapeutic program including continuous infusion of fludarabine (F) and cytarabine (ARA-C) as induction and consolidation, followed whenever possible by autologous stem cell transplantation (Ferrara et al, Haematologica, 2005). Briefly, F was administered at a loading dose of 10 mg/m2 over 15 min at day 0 followed 6 hours and half later by continuous infusion (c.i.) of 20 mg/m2/24 hours for 72 hours (days 0–2); ARA-C was given at a loading dose of 390 mg/m2 three hours and half after F and then as c.i. over 96 hours at 1440 mg/m2/24 hours (days 0–3). G-CSF was added at day +15 at a dose of 5 μg/kg. A second identical course was planned for patients obtaining partial response, defined as less than 5% blasts in peripheral blood and less than 30% of blasts in the bone marrow. Patients achieving CR, established as less than 5% blasts in the bone marrow, normal blood count and differential and absence of extramedullary leukemia, were programmed to receive an additional identical course as consolidation, reduced of one day (i.e. two days c.i. of F and three days c.i, of ARA-C). The effect of CR was separately analyzed according to karyotype, bone marrow blast count and, in patients with normal karyotype, NPM1 and FLT3 positivity. Of note, patients dead in induction were excluded from survival benefit evaluation. The median age was 69 years (range 61–82). Cytogenetic analysis was successfully in 134/140 patients (96%). Among these 89 (66%) were found as having normal karyotype (NK) and 45 (34%) with different chromosomal abnormalities, mostly complex or involving chromosomes 5 and/or 7, classified as unfavorable (UK). Overall 94 patients (67%) achieved CR; the CR rate was 77 % in NK and 47% in unfavorable karyotype (p:<0.001). Of note, rates of either death in induction (22% vs 14%) or primary refractory disease (33 % vs 8%) were significantly higher in patients with adverse cytogenetics. The median survival for the whole patient population was 10 months; survival was significantly influenced by cytogenetics at diagnosis (12 months for NK vs 7 months for UK), p:<0.001). The median duration of CR was 11 months (16 months for patients with NK as opposed to 7 months for those with UK). The overall impact of CR achievement on survival was remarkable and remained statistically significant after exclusion of patients dead in induction (18 months vs. 6 months, p:< 0.001). The advantage of achieving CR was found in patients with NK, independently from molecular assessment at diagnosis, i.e. NPM1+/FLT3-, NPM1-FLT3-, NPM-FLT3+, NPM+/FLT3+). Of interest, no difference was found as bone marrow blast count at diagnosis, i.e. more or less than 30 %, was concerned in the rate of CR achievement, CR duration and impact of CR on survival either in univariate or multivariate analysis. By separately analyzing patients with UK, the advantage of CR achievement was found only when patients dead in induction were excluded and was limited to 4 months (11 months for remitters vs. 7 months for refractory patients, p:0.04). We conclude that older AML patients with unfavorable karyotype have lower CR rates following conventional chemotherapy, because of higher mortality in induction and more frequent refractory disease; in addition, CR is shorter when compared to patients with normal karyotype and has limited impact on survival. Accordingly, even when clinically eligible for aggressive chemotherapy, such patients should be included into therapeutic programs based on experimental programs including agents with alternative mechanisms of action. Disclosures: No relevant conflicts of interest to declare.

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TET2, ANPM1 and FLT3-ITD Gene Mutations in Normal Karyotype Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.1413.1413 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1413-1413

Author(s):

Aining Sun ◽

Xiaopeng Tian ◽

Jia Yin ◽

Weiyang Li ◽

Suning Chen ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Gene Mutations ◽

Normal Karyotype ◽

Genetic Mutation ◽

Wild Type ◽

Cell Percentage ◽

High Bone ◽

Acute Myeloid

Abstract Abstract 1413 Objective: Analyze the molecular genetics characteristics of acute myeloid leukemia with normal karyotype and explore the relationship between different genetic mutation patterns and prognosis. Methods: A total of 373 acute myeloid leukemia (AML) with normal karyotype diagnosed and treated in the First Affiliated Hospital of Soochow University from 2005 to 2010 were recruited in this research to assess the genetic mutation patterns. The target genes which was extracted from bone marrow cell were amplified by PCR and analyzed by massively DNA sequencing. All of the TET2, DNMT3A, IDH1, IDH2, EZH2, CBL, ASXL1, MLL-PTD, NPM1, WT1, RUNX1, c-KIT, FLT3-ITD, FLT3-TKD, N-RAS and JAK2V617F gene mutations were detected in our study. Results: (1). A total of 16.1% of patients had TET2 mutations, 31.6% had FLT3 internal tandem duplications (ITDs), 6.2% had FLT3 tyrosine kinase domain mutations, 2.4% had c-KIT mutations, 37.8% had NPM1 mutations, 11.3% had WT1 mutations, 5.9% had RUNX1 mutations, 11.5% had ASXL1 mutations, 3.8% had MLL partial tandem duplications (PTDs), 7.8% had IDH1 mutations, 7.8% had NRAS mutations, 12.3% had IDH2 mutations, 1.6% had EZH2 mutations, 14.7% had DNMT3A mutations and no mutations were found of CBL and JAK2V617F. In conclusion, there are 77% (287/373) gene mutations hide in normal karyotype AML patients.(2). We found that the TET2 gene mutations were associated with DNMT3A (P = 0.041) and RUNX1 (P <0.001) mutations, but mutually exclusive with IDH2 (P = 0.021), or IDH1/2 (P = 0.006) gene mutations. NPM1 gene mutations were highly correlated with DNMT3A mutations (P <0.0001), IDH1 mutations (P <0.0001) and IDH2 mutations (P = 0.001), but mutually exclusive with RUNX1 mutations (P=0.003). IDH2 mutations and WT1 mutations were mutually exclusive (P = 0.01); DNMT3A mutations were associated with NRAS mutations (P = 0.01). In addition, study have shown that the number of gene mutations was closely associated with older age, high white blood cell and high bone marrow blast cell percentage, but wasn't correlated with gender, hemoglobin and platelet levels.(3). In the NPM1m+ patients, TET2 mutations were associated with shorter median OS in contrast to TET2 wild type (9.9 vs. 27.0 months, P= 0.023). Surprisingly, in NPM1m+/FLT3-ITDm- group, TET2 mutations was also an unfavorable prognostic factor, which was closely associated with shorter median OS compared to TET2 wild type (9.5 vs. 32.2 months, P=0.013). Conclusion: Gene mutations incidence was high in normal karyotype AML patients. TET2 mutations was an unfavorable prognostic factor which was closely associated with shorter median OS in contrast to TET2 wild type in NPM1m+/FLT3-ITDm-group. In addition, The number of gene mutation was closely associated with older age, high white blood cell levels and high bone marrow blast cell percentage. Disclosures: No relevant conflicts of interest to declare.

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Activating FLT3 Mutations Display a Wide Range Of Transforming Potential and a Characteristic Pathway Profile Associated With Prognosis In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1312.1312 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1312-1312

Author(s):

Hanna Janke ◽

Friederike Schneider ◽

Daniela Schumacher ◽

Tobias Herold ◽

Hopfner Karl-Peter ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Tyrosine Kinase ◽

Gene Expression Profile ◽

Myeloid Leukemia ◽

Normal Karyotype ◽

Wide Range ◽

Flt3 Mutations ◽

Acute Myeloid

Abstract Background Internal tandem duplication (ITD) and pointmutations in the tyrosine kinase domain (TKD) of the receptor tyrosine kinase FLT3 occur in about 30% of patients with acute myeloid leukemia (AML). In contrast to the negative prognostic impact of FLT3-ITD in normal karyotype AML, FLT3 pointmutations occurring in the TKD and juxtamembrane (JM) region are less frequent and of unclear clinical impact. Although TKD mutations can induce resistance to tyrosine kinase inhibitors the individual transforming potential of FLT3 pointmutations has not been analysed in detail. In this study we have performed a comprehensive analysis of various FLT3 mutants in a comparative setting in vitro and analyzed gene expression profiles, and clinical outcome with respect to FLT3mutation status. Material and Methods We analyzed relapse and survival in 672 cytogenetically normal AML patients and the FLT3 status at diagnosis and relapse in 156 patients. In the murine Ba/F3 cell model we analyzed the transforming potential, subcellular localization, phosphorylation status and signaling properties of eight different FLT3 mutants. The investigated FLT3 mutations include three ITD of different length and insertion site, V592A in the JM region, common FLT3-TKD mutations D835V and D835Y as well as D839G and I867S in the second TKD. FLT3-D839G and -I867S were recently found in AML patients by our group during routine diagnostics but have not been functionally characterized before. The corresponding remission samples did not express these mutations. Further a gene expression profile analysis with respect to FLT3-ITD and -TKD mutation status and evaluation of differences in activation of predefined STAT5 target gene set was performed. Results In 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with an inferior relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%). In vitro, FLT3-ITD induced a fully transformed phenotype in Ba/F3 cells, whereas FLT3 pointmutations showed a weaker but clearly transformed phenotype with gradual increase in proliferation and protection from apoptosis. The transforming capacity of the investigated mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD transformed cells, further gene expression profile analyses displayed differences in predefined STAT5 target genes between FLT3-ITD and FLT3-TKD mutations. In contrast, FLT3-non-ITD mutants had an enhanced signal of AKT and MAPK activation. Further differences were found on mRNA level presenting deregulation of SOCS2, ENPP2, PRUNE2 and ART3 expression between FLT3-ITD, FLT3-TKD and FLT3-WT. Conclusion Although apparently divergent in response to treatment all functionally characterized mutants showed a clear gain-of-function phenotype with a wide range of transforming activity associated with clinical prognosis and signaling. Disclosures: No relevant conflicts of interest to declare.

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Distinct clinical and biological features of de novo acute myeloid leukemia with additional sex comb-like 1 (ASXL1) mutations

Blood ◽

10.1182/blood-2010-05-283291 ◽

2010 ◽

Vol 116 (20) ◽

pp. 4086-4094 ◽

Cited By ~ 131

Author(s):

Wen-Chien Chou ◽

Huai-Hsuan Huang ◽

Hsin-An Hou ◽

Chien-Yuan Chen ◽

Jih-Luh Tang ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Human Leukocyte ◽

Normal Karyotype ◽

Trisomy 8 ◽

Disease Evolution ◽

Biological Features ◽

Sex Comb ◽

Acute Myeloid

Abstract Mutations in the additional sex comb-like 1 (ASXL1) gene were recently shown in various myeloid malignancies, but they have not been comprehensively investigated in acute myeloid leukemia (AML). In this study, we analyzed ASXL1 mutations in exon 12 in 501 adults with de novo AML. ASXL1 mutations were detected in 54 patients (10.8%), 8.9% among those with normal karyotype and 12.9% among those with abnormal cytogenetics. The mutation was closely associated with older age, male sex, isolated trisomy 8, RUNX1 mutation, and expression of human leukocyte antigen–DR and CD34, but inversely associated with t(15;17), complex cytogenetics, FLT3–internal tandem duplication, NPM1 mutations, WT1 mutations, and expression of CD33 and CD15. Patients with ASXL1 mutations had a shorter overall survival than patients without, but the mutation was not an independent adverse prognostic factor in multivariate analysis. Sequential analyses showed that the original ASXL1 mutations were lost at relapse and/or refractory status in 2 of the 6 relapsed ASXL1-mutated patients studied, whereas 2 of the 109 ASXL1-wild patients acquired a novel ASXL1 mutation at relapse. In conclusion, AML bearing ASXL1 mutations showed distinct clinical and biological features. The ASXL1 mutation status can change during disease evolution in a few patients.

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Role of CYP3A4 in bone marrow microenvironment–mediated protection of FLT3/ITD AML from tyrosine kinase inhibitors

Blood Advances ◽

10.1182/bloodadvances.2018022921 ◽

2019 ◽

Vol 3 (6) ◽

pp. 908-916 ◽

Cited By ~ 15

Author(s):

Yu-Ting Chang ◽

Daniela Hernandez ◽

Salvador Alonso ◽

Minling Gao ◽

Meng Su ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Bone Marrow Microenvironment ◽

Clinical Activity ◽

Protective Effects ◽

Tki Resistance ◽

Acute Myeloid

Abstract An intriguing aspect of the clinical activity of FMS-like tyrosine kinase 3 inhibitors (FLT3 TKIs) is their apparent higher activity against peripheral blasts from FLT3/internal tandem duplication (ITD) acute myeloid leukemia than marrow disease in the same patients. Accordingly, studies showed that the bone marrow microenvironment plays a role in FLT3 TKI resistance, although the underlying mechanisms are unclear. We recently identified a previously undescribed mechanism by which the bone marrow microenvironment can contribute to drug resistance: expression of cytochrome P450 enzymes (CYPs). In fact, bone marrow stromal cells (BMSCs) expressed most CYPs, including CYP3A4. Because hepatic CYP3A4 plays a role in the inactivation of several FLT3 TKIs, we explored the potential role of CYP3A4 in bone marrow microenvironment–mediated FLT3 TKI resistance. We found that CYP3A4 plays a major role in BMSC-mediated inhibition in the activity of 3 different FLT3 TKIs (sorafenib, quizartinib, and gilteritinib) against FLT3/ITD acute myeloid leukemia (AML). Furthermore, clarithromycin, a clinically active CYP3A4 inhibitor, significantly reversed the protective effects of BMSCs. We show, for the first time, that bone marrow stromal CYP3A4 contributes to FLT3 TKI resistance in the bone marrow. These results suggest that combining FLT3 TKIs with CYP3A4 inhibitors could be a promising strategy toward improving the activity of FLT3 TKIs.

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