The prognostic significance of IDH2 mutations in AML depends on the location of the mutation

We have investigated the prognostic significance of isocitrate dehydrogenase 2 (IDH2) mutations in 1473 younger adult acute myeloid leukemia patients treated in 2 United Kingdom Medical Research Council trials. An IDH2 mutation was present in 148 cases (10%), 80% at R140 and 20% at R172. Patient characteristics and outcome differed markedly between the 2 mutations. IDH2(R140) significantly correlated with nucleophosmin mutations (NPM1(MUT)), whereas IDH2(R172) cases generally lacked other molecular mutations. An IDH2(R140) mutation was an independent favorable prognostic factor for relapse (P = .004) and overall survival (P = .008), and there was no significant heterogeneity with regard to NPM1 or FLT3 internal tandem duplication (FLT3/ITD) genotype. Relapse in FLT3/ITD(WT)NPM1(MUT)IDH2(R140) patients was lower than in favorable-risk cytogenetics patients in the same cohort (20% and 38% at 5 years, respectively). The presence of an IDH2(R172) mutation was associated with a significantly worse outcome than IDH2(R140), and relapse in FLT3/ITD(WT)NPM1(WT)IDH2(R172) patients was comparable with adverse-risk cytogenetics patients (76% and 72%, respectively).     

Prognostic Impact of NPM1 Mutations in Serbian Adult Patients with Acute Myeloid Leukemia

Based on current findings, the presence of NPM1 mutations in acute myeloid leukemia (AML) patients is associated with an increased probability of complete remission (CR) and better overall survival (OS). We determined the incidence and prognostic relevance of NPM1 mutations, their association with FLT3 and IDH mutations, and other clinical characteristics in Serbian adult AML patients. Samples from 111 adult de novo AML patients, including 73 AML cases with a normal karyotype (NK-AML), were studied. NPM1, FLT3, and IDH mutations were detected by PCR and direct sequencing. NPM1 mutations were detected in 22.5% of patients. The presence of NPM1 mutations predicted a low CR rate and shorter OS. NPM1 mutations showed an association with both FLT3 and IDH mutations. Survival analysis based on NPM1/FLT3 mutational status revealed a lower OS for NPM1(+)/FLT3(-) compared to the NPM1(-)/FLT3(-) group in NK-AML patients. The lack of impact or unfavorable prognostic effect of NPM1 mutations found in this study can be assigned to a small cohort of analyzed AML patients, as can the presence of FLT3 and IDH mutations or other genetic lesions that cooperate with NPM1 mutations influencing prognosis.     

Gene mutation and AML pathogenesis

In their large study of 1185 patients with acute myeloid leukemia (AML), Shen and colleagues have dissected the overlapping incidences and prognostic significances of mutations of the 12 genes most frequently mutated in AML,including FLT3, NPM1, CEBPA, KIT, N-RAS, MLL, WT1, IDH1/2, TET2, DNMT3A, and ASXL1.     

The combination of NPM1, DNMT3A,and IDH1/2 mutations leads to inferior overall survival in AML

Acute myeloid leukemia (AML) is a genetically heterogeneous disease with a clinical course predicted by recurrent cytogenetic abnormalities and/or gene mutations. The NPM1 insertion mutations define the largest distinct genetic subset, ∼30% of AML, and is considered a favorable risk marker if there is no (or low allelic ratio) FLT3 internal tandem duplication (FLT3 ITD) mutation. However, ∼40% of patients with mutated NPM1 without FLT3 ITD still relapse, and the factors that drive relapse are still not fully understood. We used a next-generation sequencing panel to examine mutations at diagnosis; clearance of mutations after therapy, and gain/loss of mutations at relapse to prioritize mutations that contribute to relapse. Triple mutation of NPM1, DNMT3A and IDH1/2 showed a trend towards inferior overall survival in our discovery dataset, and was significantly associated with reduced OS in a large independent validation cohort. Analysis of relative variant allele frequencies suggests that early mutation and expansion of DNMT3A and IDH1/2 prior to acquisition of NPM1 mutation leads to increased risk of relapse. This subset of patients may benefit from allogeneic stem cell transplant or clinical trials with IDH inhibitors.     

A distinct immunophenotype identifies a subset of NPM1‐mutated AML with TET2 or IDH1/2 mutations and improved outcome

Recent work has identified distinct molecular subgroups of acute myeloid leukemia (AML) with implications for disease classification and prognosis. NPM1 is one of the most common recurrently mutated genes in AML. NPM1 mutations often co‐occur with FLT3‐ITDs and mutations in genes regulating DNA methylation, such as DNMT3A, TET2, and IDH1/2. It remains unclear whether these genetic alterations are associated with distinct immunophenotypic findings or affect prognosis. We identified 133 cases of NPM1‐mutated AML and correlated sequencing data with immunophenotypic and clinical findings. Of 84 cases (63%) that lacked monocytic differentiation (“myeloid AML”), 40 (48%) demonstrated an acute promyelocytic leukemia‐like (APL‐like) immunophenotype by flow cytometry, with absence of CD34 and HLA‐DR and strong myeloperoxidase expression, in the absence of a PML‐RARA translocation. Pathologic variants in TET2, IDH1, or IDH2 were identified in 39/40 APL‐like cases. This subset of NPM1‐mutated AML was associated with longer relapse‐free and overall survival, when compared with cases that were positive for CD34 and/or HLA‐DR. The combination of NPM1 and TET2 or IDH1/2 mutations along with an APL‐like immunophenotype identifies a distinct subtype of AML. Further studies addressing its biology and clinical significance may be especially relevant in the era of IDH inhibitors and recent work showing efficacy of ATRA therapy in NPM1 and IDH1‐mutated AML.     

Gene mutation patterns and their prognostic impact in a cohort of 1185 patients with acute myeloid leukemia

To evaluate the prognostic value of genetic mutations for acute myeloid leukemia (AML) patients, we examined the gene status for both fusion products such as AML1 (CBFα)-ETO, CBFβ-MYH11, PML-RARα, and MLL rearrangement as a result of chromosomal translocations and mutations in genes including FLT3, C-KIT, N-RAS, NPM1, CEBPA, WT1, ASXL1, DNMT3A, MLL, IDH1, IDH2, and TET2 in 1185 AML patients. Clinical analysis was mainly carried out among 605 cases without recognizable karyotype abnormalities except for 11q23. Of these 605 patients, 452 (74.7%) were found to have at least 1 mutation, and the relationship of gene mutations with clinical outcome was investigated. We revealed a correlation pattern among NPM1, DNMT3A, FLT3, IDH1, IDH2, CEBPA, and TET2 mutations. Multivariate analysis identified DNMT3A and MLL mutations as independent factors predicting inferior overall survival (OS) and event-free survival (EFS), whereas biallelic CEBPA mutations or NPM1 mutations without DNMT3A mutations conferred a better OS and EFS in both the whole group and among younger patients < 60 years of age. The use of molecular markers allowed us to subdivide the series of 605 patients into distinct prognostic groups with potential clinical relevance.     

Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype

Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3-length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1-/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM- mutations are an independent predictor for a favorable outcome in AML with normal karyotype.     

Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations

To assess the prognostic relevance of mutations in the NPM1 gene encoding a nucleocytoplasmic shuttle protein in younger adults with acute myeloid leukemia (AML) and normal cytogenetics, sequencing of NPM1 exon 12 was performed in diagnostic samples from 300 patients entered into 2 consecutive multicenter trials of the AML Study Group (AMLSG). Treatment included intensive double-induction therapy and consolidation therapy with high cumulative doses of high-dose cytarabine. NPM1 mutations were identified in 48% of the patients including 12 novel sequence variants, all leading to a frameshift in the C-terminus of the nucleophosmin 1 (NPM1) protein. Mutant NPM1 was associated with specific clinical, phenotypical, and genetic features. Statistical analysis revealed a significant interaction of NPM1 and FLT3 internal tandem duplications (ITDs). NPM1 mutations predicted for better response to induction therapy and for favorable overall survival (OS) only in the absence of FLT3 ITD. Multivariable analysis for OS revealed combined NPM1-mutated/FLT3 ITD-negative status, CEBPA mutation status, availability of a human leukocyte antigen (HLA)-compatible donor, secondary AML, and lactate dehydrogenase (LDH) as prognostic factors. In conclusion, NPM1 mutations in the absence of FLT3 ITD define a distinct molecular and prognostic subclass of young-adult AML patients with normal cytogenetics.     

急性髓细胞白血病患者NPM1与FLT3基因突变的研究

目的 探讨急性髓细胞白血病（AML）患者中NPM1基因与FLT3基因内部串联重复（ITD）突变的发生率,并了解其临床特征及预后。方法 收集86例成人AML患者初诊时骨髓单个核细胞,采用基因组DNA-PCR方法分别扩增其NPM1和FLT3基因,毛细管电泳方法分析NPM1第12外显子突变,琼脂糖电泳分析FLT3-ITD突变,随访判断其预后。结果 86例AML患者中共检出NPM1基因突变29例（33．7％）,FLT3-ITD突变15例（17,4％）。两种突变在50例染色体正常核型AML中的发生率分别为46．0％和24．0％,明显高于异常核型AML患者。7例中6例NPM1^＋／FLT3-ITD^＋双阳性AML患者表现为正常核型,外周血白细胞均〉50×10^9几。单纯NPM1^＋或FLT3-ITD^＋AML患者初诊时也表现为高白细胞（P〈0.05）,NPM1^＋AML患者CD34表达率较低（P〈0．001）、临床完全缓解（CR）率略高（66.7％、53.3％,P〉0.05）、总生存率（OS）高（P〉0.05）;而FLT3-ITD^＋AML患者CR率略低（50．0％、58．8％,P〉0.05）、OS低（P〉0.05）。NPM1^＋／FLT3-ITD^-、NPM^-／FLT3-ITD^-、NPM^＋／FLT3-ITD^＋、NPM1-／FLT3-ITD^＋四组患者的CR率分别为66．7％、62．5％、50．0％、42．9％（P〉0．05）,各组间OS差异无统计学意义（P〉0．05）。结论 NPM1和FLT3-ITD突变是AML（尤其正常核型AML）患者常见的分子学异常,且与其临床特点、疗效及预后有一定相关性。     

Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML)

Mutations of the nucleophosmin (NPM1) gene have recently been described in patients with acute myeloid leukemia (AML). To clarify the prevalence as well as the clinical impact of this mutation, we investigated 1485 patients with AML for NPM1 exon 12 mutations using fragment analysis. A 4 bp insert was detected in 408 of 1485 patients (27.5%). Sequence analysis revealed known mutations (type A, B, and D) as well as 13 novel alterations in 229 analyzed cases. NPM1 mutations were most prevalent in patients with normal karyotype (NK) (324 of 709; 45.7%) compared with 58 of 686 with karyotype abnormalities (8.5%; P < .001) and were significantly associated with several clinical parameters (high bone marrow [BM] blasts, high white blood cell [WBC] and platelet counts, female sex). NPM1 alterations were associated with FLT3-ITD mutations, even if restricted to patients with NK (NPM1-mut/FLT3-ITD: 43.8%; versus NPM1-wt/FLT3-ITD: 19.9%; P < .001). The analysis of the clinical impact in 4 groups (NPM1 and FLT3-ITD single mutants, double mutants, and wild-type [wt] for both) revealed that patients having only an NPM1 mutation had a significantly better overall and disease-free survival and a lower cumulative incidence of relapse. In conclusion, NPM1 mutations represent a common genetic abnormality in adult AML. If not associated with FLT3-ITD mutations, mutant NPM1 appears to identify patients with improved response toward treatment.     

Characteristics,clinical outcome,and prognostic significance of IDH mutations in AML

The pathophysiology of IDH mutations in tumorigenesis is increasingly described, yet the prognostic significance of IDH1 and IDH2 mutations in AML remains controversial. The primary objective of this study was to define the natural history and prognosis of patients with AML and IDH1 or IDH2 mutations and provide historical survival expectations. A total of 826 patients treated from 2010 to 2014 at a single institution were evaluated, including 167 patients (20%) with AML and IDH1 or IDH2 mutations. Median age was 62 years (range 18–92). There were 59 IDH1‐R132, 83 IDH2‐R140, and 23 IDH2‐R172 mutations. Clinicopathologic characteristics associated with IDH‐mutations included older age, less frequent therapy‐related status, and increased incidence of intermediate‐risk cytogenetics, FLT3‐ITD mutations, and NPM1 mutations. Remission rates (CR/CRi) by AML treatment status were: induction, 68%; Salvage‐1 (S1), 42%; and Salvage‐2 and beyond (S2+), 27%. No difference in response was identified by IDH mutation status. Similarly, overall survival (OS) was not dependent on IDH status within any cohort. The median OS was 15.4 months in induction, 8.7 months in S1, and 4.8 months in S2+. This analysis defines the clinical outcome associated with IDH‐mutations in both the front‐line and salvage AML treatment settings, and confirms that response rate and OS for both IDH‐mutated and IDH wild‐type AML patients is comparable. This provides contemporary data to be used for comparison with results of novel investigational (e.g., selective IDH inhibitor) strategies. Am. J. Hematol. 90:732–736, 2015. © 2015 Wiley Periodicals, Inc.     

Acute myeloid leukemia with myelodysplasia-related changes are characterized by a specific molecular pattern with high frequency of ASXL1 mutations

To determine whether the distinct and heterogeneous WHO category called "AML with myelodysplasia-related changes" (MRC-AML), presents specific molecular alterations we searched for mutations in genes known to be mutated in malignant myeloid diseases. In 48 MRC-AML patients analyzed, we found 17 mutations in ASXL1 (35%), eight in RUNX1 (17%), seven in TET2 (15%), 12 in IDH (n = 2) or IDH2 (n = 10) (25%), four in DNMT3A (8%), four in NPM1 (8%), and one in FLT3 (2%). Mutations were more frequent in the intermediate cytogenetic (IC) subgroup of 36 patients than in the unfavorable karyotype subgroup, with an average ratio mutations/patients of 1.36 [0-3] vs. 0.33 [0-2] (P < 0.001). Then, we compared these 36 patients with IC MRC-AML with a control panel of 37 no-MRC-AML patients, who had both IC and no dysplasia. IC MRC-AMLs were associated with higher incidence of ASXL1 mutations (47% vs. 0%, P < 0.001) and lower incidence of DNMT3A (6% vs. 38%, P = 0.001), NPM1 (11% vs. 62%, P < 0.001) and FLT3 (3% vs. 49%, P < 0.001) mutations. No difference was found in the incidence of IDH1/2 or TET2 mutations according to the presence of dysplasia. Complete remission rate after intensive treatment was lower in the MRC-AML group than in the no-MRC-AML group (48% vs. 78%, P = 0.023) and in wild type NPM1 patients (50% vs. 84%, P = 0.009). Our study showed that MRC-AML as defined in the WHO 2008 classification presents a specific mutation pattern characterized by a high frequency of ASXL1 mutations and a low rate of NPM1, FLT3, and DNMT3A mutations.     

Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype

Among acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.     

IDH1 and IDH2 mutation analysis in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome

The somatic mutations of isocitrate dehydrogenase genes (IDH1 and IDH2) have been identified in a proportion of hematologic malignancies. We examined IDH1 R132 and IDH2 R140/R172 mutations by high resolution melting analysis and direct sequencing in Chinese patients with different myeloid malignancies including 198 acute myeloid leukemia (AML), 82 myelodysplastic syndrome (MDS), 85 chronic myeloid leukemia, and 57 myeloproliferative neoplasms. IDH1 and IDH2 mutations were found in four (2.0%) and ten (5.0%) AML and in two (2.4%) and three (3.6%) MDS cases, but not in other patients. IDH1 and IDH2 mutations were heterozygous and mutually exclusive. IDH1/2 mutations were significantly more frequently observed in cytogenetically normal AML or MDS compared to those without mutations. There was no difference in overall survival of both AML and MDS patients with or without IDH1/2 mutations (P = 0.177 and 0.407, respectively). In conclusion, IDH1/2 mutations are recurrent but rare molecular aberrations in Chinese AML and MDS.     

Nucleophosmin mutation in Southeast Asian acute myeloid leukemia: eight novel variants, FLT3 coexistence and prognostic impact of NPM1/FLT3 mutations

NPM1 mutations were investigated in 400 Southeast Asian leukemia patients and were detectable in 105 cases (26.25%) of acute myeloid leukemia but in no cases of acute lymphoid leukemia or chronic myeloid leukemia. Eight novel and 5 known mutations were identified. All predicted novel proteins shared the last five amino acids VSLRK with the similar gain of nuclear exporting signal motif as known variants. Older age, high white blood cell and platelet counts, normal cytogenetics, and CD34-negativity were associated with NPM1 mutation. FLT3 mutation was more frequent in mutant NPM1 than wild-type cases (56.8% vs. 25.6%) whereas RAS and AML1 mutations were rarely found. Overall survival analysis based on the NPM1/FLT3 mutational status revealed a better outcome for the NPM1-positive/FLT3-negative subgroup. We conclude that: i) NPM1 mutation represents a common genetic hallmark in Southeast Asian acute myeloid leukemia with a normal karyotype; ii) NPM1 mutants coexisted mainly with FLT3 mutants, but not RAS or AML1; iii) FLT3 mutation had a negative prognostic impact on patients with mutant NPM1.     

Distinct patterns of mutations occurring in de novo AML versus AML arising in the setting of severe congenital neutropenia

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Like most other bone marrow failure syndromes, it is associated with a marked propensity to transform into a myelodysplastic syndrome (MDS) or acute leukemia, with a cumulative rate of transformation to MDS/leukemia that exceeds 20%. The genetic (and/or epigenetic) changes that contribute to malignant transformation in SCN are largely unknown. In this study, we performed mutational profiling of 14 genes previously implicated in leukemogenesis using 14 MDS/leukemia samples from patients with SCN. We used high-throughput exon-based resequencing of whole-genome-amplified genomic DNA with a semiautomated method to detect mutations. The sensitivity and specificity of the sequencing pipeline was validated by determining the frequency of mutations in these 14 genes using 188 de novo AML samples. As expected, mutations of tyrosine kinase genes (FLT3, KIT, and JAK2) were common in de novo AML, with a cumulative frequency of 30%. In contrast, no mutations in these genes were detected in the SCN samples; instead, mutations of CSF3R, encoding the G-CSF receptor, were common. These data support the hypothesis that mutations of CSF3R may provide the "activated tyrosine kinase signal" that is thought to be important for leukemogenesis.     

IDH1 and IDH2 mutations confer an adverse effect in patients with acute myeloid leukemia lacking the NPM1 mutation

We examined the incidence and prognostic effect of IDH1 and IDH2 mutations in 233 Japanese adults with acute myeloid leukemia (AML). IDH1 R132 mutations were detected in 20 (8.6%) patients with AML. IDH2 mutations were found in 19 (8.2%, 17 R140 and two R172) patients. IDH1 and IDH2 mutations were mutually exclusive and were associated with normal karyotype AML, cytogenetic intermediate‐risk group, and NPM1 mutations. Five‐year overall survival (OS) rates were significantly lower (15.6%) in patients harboring the IDH mutations than in patients lacking the IDH mutation (32.0%) in the entire cohort of AML (P = 0.005). Among patients aged 59 yr or younger with IDH mutations, 5‐yr OS in patients who underwent allogeneic stem cell transplantation (SCT) was significantly higher than that in those not receiving allogeneic SCT (50% vs. 10.6%, P = 0.020). Of 51 patients with NPM1 mutations, there was no significant difference in 5‐yr OS rates between patients with and those without the IDH mutations. In contrast, among 175 patients lacking the NPM1 mutations, 5‐yr OS rate in patients with IDH mutations was significantly lower than that in those without IDH mutations (0% vs. 34.7%, P = <0.001). These data suggest that IDH mutations have an unfavorable effect in AML, especially AML with the NPM1 wild type and younger AML patients with IDH mutations may benefit from allogeneic SCT.     

Acute myeloid leukaemia genomics

Acute myeloid leukaemia (AML) is a biologically complex, molecularly and clinically heterogeneous disease. Despite major advances in understanding the genetic landscape of AML and its impact on the pathophysiology and biology of the disease, standard treatment options have not significantly changed during the past three decades. AML is characterized by multiple somatically acquired mutations that affect genes of different functional categories. Mutations in genes encoding epigenetic modifiers, such as DNMT3A, ASXL1, TET2, IDH1, and IDH2, are commonly acquired early and are present in the founding clone. By contrast, mutations involving NPM1 or signalling molecules (e.g., FLT3, RAS gene family) are typically secondary events that occur later during leukaemogenesis. This review aims to provide an overview of advances in new prognostic markers, including targetable mutations that will probably guide the development and use of novel molecularly targeted therapies.     

NPM1 mutations are more stable than FLT3 mutations during the course of disease in patients with acute myeloid leukemia

NPM1 mutations have been reported to be the most frequent mutations in acute myeloid leukemia (AML). They are associated with a wide spectrum of morphologic subtypes of AML, normal karyotype and FLT3 mutations. The high frequency of NPM1 mutations might provide a suitable marker for monitoring residual disease of AML.