Molecular, clinical and therapeutic determinants of outcome in NPM1 mutated AML.

Although NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcome, however only FLT3-ITD mutation and adverse karyotype are currently used for risk stratification due to inconsistent results and uncertainty around how other factors should influence treatment, particularly given the strong prognostic impact of post-induction measurable residual disease (MRD). Here we analyzed a large group of patients with NPM1mut AML enrolled in prospective trials (NCRI AML17 and AML19, n=1357) to delineate the impact of baseline molecular and clinical features, post induction MRD status and treatment intensity on outcome. FLT3-ITD (HR 1.28, 95%CI 1.01-1.63), DNMT3A (HR 1.65, 95%CI 1.32-2.05), WT1 (HR 1.74, 95%CI 1272-2.38) and non-ABD NPM1 mutations (HR 1.64, 95%CI 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients achieving MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the highest risk molecular subgroups.

Therapy for isocitrate dehydrogenase 2 (IDH2)R172 -mutant acute myeloid leukaemia.

Although we earlier reported a very poor outcome for younger adult patients with isocitrate dehydrogenase 2 (IDH2)R172 -mutated acute myeloid leukaemia (AML) entered into UK trials compared to IDH2WT and IDH2R140 -mutated patients, this was not corroborated by a study from the German-Austrian AML Study Group. We have therefore investigated a later cohort of IDH2-mutated patients to identify any changes in outcome and whether this could inform the optimal treatment for IDH2R172 AML. We found an improved outcome for IDH2R172 -mutated AML in the later trials and the data suggests that this may be due to the increased use of allogeneic transplantation to consolidate first remission.

Additional impact of mutational genotype on prognostic determination in resistant and relapsed acute myeloid leukaemia.

Outcome after failure of initial therapy in younger adult patients with acute myeloid leukaemia (AML) is highly variable. Cytogenetics, length of first remission (CR1) before relapse, and allogeneic transplantation are known prognostic factors, but the contribution of leukaemic genotype is less clear, particularly in resistant disease. Of 5,651 younger adult patients entered into UK MRC/NCRI AML trials between 1988 and 2014 with available FLT3ITD and NPM1 genotype, 326 (6%) had resistant disease and 2338 (41 %) relapsed after achieving CR1. Overall survival (OS) was significantly higher in relapsed compared to resistant disease (p = 0·03). Independent favourable prognostic factors for OS in resistant disease included lower blast cell percentage after two courses of induction therapy (p = 0.0006) and NPM1 mutant (NPM1MUT) (p = 0.04). In relapsed disease, longer CR1 was a favourable independent factor for attainment of CR2 (p < 0.0001) and OS from time of relapse (p < 0.0001), but CR2 rate and OS from relapse were significantly worse in those who had received an allograft in CR1 (respectively p < 0.05, p < 0·002). NPM1MUT was marginally beneficial for OS (p = 0.04). FLT3ITD and DNMT3AMUT were adverse factors for OS (respectively p < 0.0001, p = 0.02). Mutational analysis adds additional independent prognostic information to demographic features and previous therapy in patients with resistant and relapsed disease.

Analysis of the clinical impact of NPM1 mutant allele burden in a large cohort of younger adult patients with acute myeloid leukaemia.

Although an NPM1 mutation is generally considered to be a good prognostic marker in acute myeloid leukaemia, it has recently been suggested that a higher level of NPM1 mutant (NPM1MUT ) alleles relative to wild-type alleles is associated with poor clinical outcome. We therefore sought to confirm this finding in a larger study of 876 NPM1MUT cases entered into UK national trials. In univariate analysis, the higher NPM1MUT allele burden was associated with a lower complete remission (CR) rate, higher relapse rate and reduced overall survival, but this was largely attributable to the association of the higher NPM1MUT allele burden with other known poor risk factors, particularly the presence of a concomitant FLT3 internal tandem duplication. In multivariate analysis, there was no significant impact of the NPM1MUT allele burden on CR rates, and the impact on relapse and overall survival, whilst still significant, was greatly reduced. This impact was similar in patients who did or did not receive an allogeneic transplant in first CR. We conclude that the binary presence or absence of an NPM1 mutation, combined with minimal residual disease levels following induction therapy, should continue to be used in therapeutic management rather than stratification according to the NPM1MUT level.

Genomic landscape and clonal evolution of acute myeloid leukemia with t(8;21): an international study on 331 patients.

Acute myeloid leukemia with t(8;21)(q22;q22) is characterized by considerable clinical and biological heterogeneity leading to relapse in up to 40% of patients. We sequenced coding regions or hotspot areas of 66 recurrently mutated genes in a cohort of 331 t(8;21) patients. At least 1 mutation, in addition to t(8;21), was identified in 95%, with a mean of 2.2 driver mutations per patient. Recurrent mutations occurred in genes related to RAS/RTK signaling (63.4%), epigenetic regulators (45%), cohesin complex (13.6%), MYC signaling (10.3%), and the spliceosome (7.9%). Our study identified mutations in previously unappreciated genes: GIGYF2, DHX15, and G2E3 Based on high mutant levels, pairwise precedence, and stability at relapse, epigenetic regulator mutations were likely to occur before signaling mutations. In 34% of RAS/RTKmutated patients, we identified multiple mutations in the same pathway. Deep sequencing (∼42 000×) of 126 mutations in 62 complete remission samples from 56 patients identified 16 persisting mutations in 12 patients, of whom 5 lacked RUNX1-RUNX1T1 in quantitative polymerase chain reaction analysis. KIT high mutations defined by a mutant level ≥25% were associated with inferior relapse-free survival (hazard ratio, 1.96; 95% confidence interval, 1.22-3.15; P = .005). Together with age and white blood cell counts, JAK2, FLT3-internal tandem duplicationhigh, and KIT high mutations were identified as significant prognostic factors for overall survival in multivariate analysis. Whole-exome sequencing was performed on 19 paired diagnosis, remission, and relapse trios. Exome-wide analysis showed an average of 16 mutations with signs of substantial clonal evolution. Based on the resemblance of diagnosis and relapse pairs, genetically stable (n = 13) and unstable (n = 6) subgroups could be identified.

Immunophenotypic analysis of cell cycle status in acute myeloid leukaemia: relationship to cytogenetics, genotype and clinical outcome.

Cell cycle status may play an important role in directing patient therapy. We therefore determined the cell cycle status of leukaemic cells by immunophenotypic analysis of bone marrow trephine biopsies from 181 patients with acute myeloid leukaemia (AML) and correlated the results with biological features and clinical outcome. There was considerable heterogeneity between patients. The presenting white cell count significantly correlated with the proportion of non-quiescent cells (P < 0·0001), of cycling cells beyond G1 (P < 0·0001) and the speed of cycling (P < 0·0001). Profiles in acute promyelocytic leukaemia (APL) differed from non-APL and were consistent with more differentiated cells with reduced proliferative potential, but no significant differences were observed between non-APL cytogenetic risk groups. NPM1 mutations but not FLT3 internal tandem duplication (FLT3ITD ) were significantly associated with a higher proportion of cells beyond G1 (P = 0·002) and faster speed of cycling (P = 0·003). Resistance to standard cytosine arabinoside and daunorubicin induction chemotherapy was significantly related to a slower speed of cycling (P = 0·0002), as was a higher relapse rate (P = 0·05), but not with the proportion of non-quiescent cells or actively cycling cells. These results show a link between the cycling speed of AML cells and the response to chemotherapy, and help to identify a group with a very poor prognosis.

Variable outcome and methylation status according to CEBPA mutant type in double-mutated acute myeloid leukemia patients and the possible implications for treatment.

Although CEBPA double-mutated (CEBPADM) acute myeloid leukemia is considered to be a favorable-risk disease, relapse remains a major cause of treatment failure. Most CEBPADM patients have a classic biallelic mutant combination with an N-terminal mutation leading to production of p30 protein plus a C-terminal loss-of-function in-frame indel mutation (CEBPAClassic-DM), but approximately one-third of cases have one or more non-classic mutations, with diverse combinations reported, and there is little information on the consequences of such mutants. We evaluated outcome in a cohort of 104 CEBPADM patients, 79 CEBPAClassic-DM and 25 with non-classic mutants, and found that the latter may have poorer survival (5-year overall survival 64% vs. 46%; P=0.05), particularly post relapse (41% vs. 0%; P=0.02). However, for this analysis, all non-classic cases were grouped together, irrespective of mutant combination. As CEBPADM cases have been reported to be hypermethylated, we used methylation profiling to assess whether this could segregate the different mutants. We developed a CEBPAClassic-DM methylation signature from a preliminary cohort of 10 CEBPADM (including 8 CEBPAClassic-DM) and 30 CEBPA wild-type (CEBPAWT) samples, and independently validated the signature in 17 CEBPAClassic-DM cases. Assessment of the signature in 16 CEBPADM cases with different non-classic mutant combinations showed that only 31% had a methylation profile equivalent to CEBPAClassic-DM whereas for 69% the profile was either intermediate between CEBPAClassic-DM and CEBPAWT or equivalent to CEBPAWT These results suggest that CEBPADM cases with non-classic mutants may be functionally different from those with CEBPAClassic-DM mutants, and should not automatically be included in the same prognostic group. (AML12 is registered under ISRCTN17833622 and AML15 under ISRCTN17161961).

Activation of the LMO2 oncogene through a somatically acquired neomorphic promoter in T-cell acute lymphoblastic leukemia.

Somatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5'-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy-induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome.

Detection of FLT3/TKD and IDH1 Mutations in Pakistani Acute Myeloid Leukemia Patients by Denaturing HPLC.

Acute myeloid leukemia (AML) is characterized by an increase in the number of myeloid cells in the marrow and an arrest in their maturation. Various genetic mutations are associated with AML. FMS-like tyrosine kinase 3 (FLT3), a member of the class III receptor tyrosine kinase family, plays an important role in stem cell survival, and the development of dendritic and natural killer cells. FLT3/TKD mutations are generally missense mutations or in-frame alterations of residues D835 and I836 within the activation loop of the FLT3 protein. D835 mutations have been reported to occur in ≈ 7% of AML patients. Mutations have also been reported in exon 4 of isocitrate dehydrogenase 1 (IDH1) in ≈9% of AML patients. Mutations in FLT3/TKD and IDH1 genes were studied in AML patients from Pakistan and correlated with the laboratory findings. FLT3/TKD mutations were found in 7%, while IDH1 mutations were found in 10% Pakistani AML patients. Neither of these mutations was significantly correlated with age and sex, although the incidence of these mutations was higher in female patients. These mutations were found to be positively associated with each other. IDH1 mutations were positively associated with FAB type M1 and negatively associated with FAB type M2. In conclusion, the overall incidence of all these mutations in Pakistani patients was within the globally reported ranges. J. Cell. Biochem. 118: 1174-1181, 2017. © 2016 Wiley Periodicals, Inc.

A randomized assessment of adding the kinase inhibitor lestaurtinib to first-line chemotherapy for FLT3-mutated AML.

The clinical benefit of adding FMS-like tyrosine kinase-3 (FLT3)-directed small molecule therapy to standard first-line treatment of acute myeloid leukemia (AML) has not yet been established. As part of the UK AML15 and AML17 trials, patients with previously untreated AML and confirmed FLT3-activating mutations, mostly younger than 60 years, were randomly assigned either to receive oral lestaurtinib (CEP701) or not after each of 4 cycles of induction and consolidation chemotherapy. Lestaurtinib was commenced 2 days after completing chemotherapy and administered in cycles of up to 28 days. The trials ran consecutively. Primary endpoints were overall survival in AML15 and relapse-free survival in AML17; outcome data were meta-analyzed. Five hundred patients were randomly assigned between lestaurtinib and control: 74% had FLT3-internal tandem duplication mutations, 23% FLT3-tyrosine kinase domain point mutations, and 2% both types. No significant differences were seen in either 5-year overall survival (lestaurtinib 46% vs control 45%; hazard ratio, 0.90; 95% CI 0.70-1.15; P = .3) or 5-year relapse-free survival (40% vs 36%; hazard ratio, 0.88; 95% CI 0.69-1.12; P = .3). Exploratory subgroup analysis suggested survival benefit with lestaurtinib in patients receiving concomitant azole antifungal prophylaxis and gemtuzumab ozogamicin with the first course of chemotherapy. Correlative studies included analysis of in vivo FLT3 inhibition by plasma inhibitory activity assay and indicated improved overall survival and significantly reduced rates of relapse in lestaurtinib-treated patients who achieved sustained greater than 85% FLT3 inhibition. In conclusion, combining lestaurtinib with intensive chemotherapy proved feasible in younger patients with newly diagnosed FLT3-mutated AML, but yielded no overall clinical benefit. The improved clinical outcomes seen in patients achieving sustained FLT3 inhibition encourage continued evaluation of FLT3-directed therapy alongside front-line AML treatment. The UK AML15 and AML17 trials are registered at www.isrctn.com/ISRCTN17161961 and www.isrctn.com/ISRCTN55675535 respectively.

Acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a disorder characterized by a clonal proliferation derived from primitive haematopoietic stem cells or progenitor cells. Abnormal differentiation of myeloid cells results in a high level of immature malignant cells and fewer differentiated red blood cells, platelets and white blood cells. The disease occurs at all ages, but predominantly occurs in older people (>60 years of age). AML typically presents with a rapid onset of symptoms that are attributable to bone marrow failure and may be fatal within weeks or months when left untreated. The genomic landscape of AML has been determined and genetic instability is infrequent with a relatively small number of driver mutations. Mutations in genes involved in epigenetic regulation are common and are early events in leukaemogenesis. The subclassification of AML has been dependent on the morphology and cytogenetics of blood and bone marrow cells, but specific mutational analysis is now being incorporated. Improvements in treatment in younger patients over the past 35 years has largely been due to dose escalation and better supportive care. Allogeneic haematopoietic stem cell transplantation may be used to consolidate remission in those patients who are deemed to be at high risk of relapse. A plethora of new agents - including those targeted at specific biochemical pathways and immunotherapeutic approaches - are now in trial based on improved understanding of disease pathophysiology. These advances provide good grounds for optimism, although mortality remains high especially in older patients.

Cell cycle status in AML blast cells from peripheral blood, bone marrow aspirates and trephines and implications for biological studies and treatment.

Using immunohistochemistry and flow cytometry to define phases of the cell cycle, this study shows that a high proportion of acute myeloid leukaemia (AML) blasts obtained from trephine biopsies are cycling, whereas >95% of peripheral blood-derived blasts are arrested in G1 . Results obtained from bone marrow aspirates are more similar to those from blood rather than from trephine biopsies. These differences were confirmed by gene expression profiling in a patient with high count AML. This has implications for cell cycle and other biological studies using aspirates rather than trephine biopsies and for the use of cell mobilising agents before chemotherapy.

The value of molecular stratification for CEBPA(DM) and NPM1(MUT) FLT3(WT) genotypes in older patients with acute myeloid leukaemia.

Older adult patients (≥60 years) with acute myeloid leukaemia (AML) are generally considered to be poor-risk and there is limited information available regarding risk stratification based on molecular characterization in this age group, particularly for the double-mutant CEBPA (CEBPA(DM) ) genotype. To investigate whether a molecular favourable-risk genotype can be identified, we investigated CEBPA, NPM1 and FLT3 status and prognostic impact in a cohort of 301 patients aged 60 years or more with intermediate-risk cytogenetics, all treated intensively. Overall survival (OS) at 1 year was highest in the 12 patients (4%) that were CEBPA(DM) compared to the 76 (28%) with a mutant NPM1 and wild-type FLT3 (NPM1(MUT) FLT3(WT) ) genotype or all other patients (75%, 54%, 33% respectively), with median survival 15·2, 13·6 and 6·6 months, although the benefit was short-term (OS at 3 years 17%, 29%, 12% respectively). Combination of the CEBPA(DM) and NPM1(MUT) FLT3(WT) genotype patients defined a molecular group with favourable prognosis (P < 0·0001 in multivariate analysis), with 57% of patients alive at 1 year compared to 33% for all other patients. Knowledge of genotype in older cytogenetically intermediate-risk patients might influence therapy decisions.

Assessment of Minimal Residual Disease in Standard-Risk AML.

BACKGROUND: Despite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission. METHODS: We used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction. RESULTS: Molecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status. CONCLUSIONS: The presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors. (Funded by Bloodwise and the National Institute for Health Research; Current Controlled Trials number, ISRCTN55675535.).

Simpson's Paradox and the Impact of Different DNMT3A Mutations on Outcome in Younger Adults With Acute Myeloid Leukemia.

PURPOSE: To evaluate the impact of DNMT3A mutations on outcome in younger patients with cytogenetic intermediate-risk acute myeloid leukemia. PATIENTS AND METHODS: Diagnostic samples from 914 patients (97% < 60 years old) were screened for mutations in DNMT3A exons 13 to 23. Clinical outcome was evaluated according to presence or absence of a mutation and stratified according to type of mutation (R882, non-R882 missense, or truncation). RESULTS: DNMT3A mutations (DNMT3A(MUT)) were identified in 272 patients (30%) and associated with a poorer prognosis than wild-type DNMT3A, but the difference was only seen when the results were stratified according to NPM1 genotype. This example of Simpson's paradox results from the high coincidence of DNMT3A and NPM1 mutations (80% of patients with DNMT3A(MUT) had NPM1 mutations), where the two mutations have opposing prognostic impact. In the stratified analyses, relapse in patients with DNMT3A(MUT) was higher (hazard ratio, 1.35; 95% CI, 1.07 to 1.72; P = .01), and overall survival was lower (hazard ratio, 1.37; 95% CI, 1.12 to 1.87; P = .002). The impact of DNMT3A(MUT) did not differ according to NPM1 genotype (test for heterogeneity: relapse, P = .4; overall survival, P = .9). Further analysis according to the type of DNMT3A mutation indicated that outcome was comparable in patients with R882 and non-R882 missense mutants, whereas in those with truncation mutants, it was comparable to wild-type DNMT3A. CONCLUSION: These data confirm that presence of a DNMT3A mutation should be considered as a poor-risk prognostic factor, irrespective of the NPM1 genotype, and suggest that further consideration should be given to the type of DNMT3A mutation.