BACKGROUNDDespite 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. METHODSWe 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. RESULTSMolecular 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. CONCLUSIONSThe presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors.

The relationships between normal and leukemic stem/progenitor cells are unclear. We show that in ∼80% of primary human CD34+ acute myeloid leukemia (AML), two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. Both populations have leukemic stem cell (LSC) activity and are hierarchically ordered; one LSC population gives rise to the other. Global gene expression profiling shows the LSC populations are molecularly distinct and resemble normal progenitors but not stem cells. The more mature LSC population most closely mirrors normal granulocyte-macrophage progenitors (GMP) and the immature LSC population a previously uncharacterized progenitor functionally similar to lymphoid-primed multipotential progenitors (LMPPs). This suggests that in most cases primary CD34+ AML is a progenitor disease where LSCs acquire abnormal self-renewal potential.

Gene expression profiling is a robust technology for the diagnosis of hematologic malignancies with high accuracy. It may complement current diagnostic algorithms and could offer a reliable platform for patients who lack access to today's state-of-the-art diagnostic work-up. Our comprehensive gene expression data set will be submitted to the public domain to foster research focusing on the molecular understanding of leukemias.

We investigated the effect on outcome of measurable or minimal residual disease (MRD) status after each induction course to evaluate the extent of its predictive value for acute myeloid leukemia (AML) risk groups, including NPM1 wild-type (wt) standard risk, when incorporated with other induction response criteria. MethodsAs part of the NCRI AML17 trial, 2,450 younger adult patients with AML or high-risk myelodysplastic syndrome had prospective multiparameter flow cytometric MRD (MFC-MRD) assessment. After course 1 (C1), responses were categorized as resistant disease (RD), partial remission (PR), and complete remission (CR) or complete remission with absolute neutrophil count , 1,000/mL or thrombocytopenia , 100,000/mL (CRi) by clinicians, with CR/CRi subdivided by MFC-MRD assay into MRD+ and MRD2. Patients without high-risk factors, including Flt3 internal tandem duplication wt/2NPM1-wt subgroup, received a second daunorubicin/cytosine arabinoside induction; course 2 (C2) was intensified for patients with high-risk factors. ResultsSurvival outcomes from PR and MRD+ responses after C1 were similar, particularly for good-to standard-risk subgroups (5-year overall survival [OS], 27% RD v 46% PR v 51% MRD+ v 70% MRD2; P , .001). Adjusted analyses confirmed significant OS differences between C1 RD versus PR/MRD+ but not PR versus MRD+. CRi after C1 reduced OS in MRD+ (19% CRi v 45% CR; P = .001) patients, with a smaller effect after C2. The prognostic effect of C2 MFC-MRD status (relapse: hazard ratio [HR], 1.88 [95% CI, 1.50 to 2.36], P , .001; survival: HR, 1.77 [95% CI, 1.41 to 2.22], P , .001) remained significant when adjusting for C1 response. MRD positivity appeared less discriminatory in poor-risk patients by stratified analyses. For the NPM1-wt standard-risk subgroup, C2 MRD+ was significantly associated with poorer outcomes (OS, 33% v 63% MRD2, P = .003; relapse incidence, 89% when MRD+ $ 0.1%); transplant benefit was more apparent in patients with MRD+ (HR, 0.72; 95% CI, 0.31 to 1.69) than those with MRD2 (HR, 1.68 [95% CI, 0.75 to 3.85]; P = .16 for interaction). ConclusionMFC-MRD can improve outcome stratification by extending the definition of partial response after first induction and may help predict NPM1-wt standard-risk patients with poor outcome who benefit from transplant in the first CR. J Clin Oncol 36:1486-1497. © 2018 by American Society of Clinical Oncology INTRODUCTIONIn acute myeloid leukemia (AML), failure to achieve morphologic complete remission (CR) after a first cycle of induction in previously untreated patients is an established independent prognostic factor from earlier studies. 1-3 Thus, morphologic response at this time point is often incorporated with genetic and pretreatment clinical parameters to guide further therapy, 4 including second induction courses, choice of consolidation, and whether intensification from allogeneic stemcell transplantation (SCT) may be appropriate in otherwise intermediate-risk patients. Despite morphologic response criteria being standard, a differe...

The prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established and is of particular interest given the opportunities for targeted therapies using FLT3 inhibitors. We studied 203 patients with PML-RARA-positive APL; 43% of the patients had an FLT3 mutation (65 internal tandem duplications [ITDs], 19 D835/I836, 4 ITD؉D835/I836). Both mutations were associated with higher white blood cell (WBC) count at presentation; 75% of the patients with WBC counts of 10 ؋ 10 9 /L or greater had mutant FLT3. FLT3/ITDs were correlated with M3v subtype (P < .001), bcr3 PML breakpoint (P < .001), and expression of reciprocal RARA-PML transcripts (P ‫؍‬ .01). Microarray analysis revealed differences in expression profiles among patients with FLT3/ITD, D835/I836, and wild-type FLT3. Patients with mutant FLT3 had a higher rate of induction death (19% vs 9%; P ‫؍‬ .04, but no significant difference in relapse risk (28% vs 23%; P ‫؍‬ .5) or overall survival (59% vs 67%; P ‫؍‬ .2) at 5 years. In in vitro differentiation assays using primary APL blasts (n ‫؍‬ 6), the FLT3 inhibitor CEP-701 had a greater effect on cell survival/proliferation in FLT3/ITD ؉ cells, but this inhibition was reduced in the presence of ATRA. IntroductionMost cases of acute promyelocytic leukemia (APL) are characterized by t(15;17)(q22;q21) leading to formation of the promyelocytic leukemia-retinoic acid receptor ␣ (PML-RARA) fusion protein. 1 PML-RARA plays a critical role in determining disease phenotype, mediating the characteristic differentiation block through the repression of genes implicated in myelopoiesis, which is overcome by pharmacologic levels of retinoic acid. 1 However, evidence derived largely from transgenic mouse models has suggested that PML-RARA is insufficient for leukemogenesis, 2,3 although the precise nature of the cooperating events implicated in generating the full disease phenotype remains uncertain. A number of potential candidates have been proposed to play a role in this process. These include the reciprocal fusion gene product RARA-PML, which is expressed in approximately 75% of patients [4][5][6] and has been postulated to contribute to leukemogenesis by promoting genomic instability, thereby predisposing to the acquisition of additional oncogenic lesions. 7 There has also been considerable interest in the potential role of activating mutations of genes encoding receptor tyrosine kinases (RTKs), which commonly accompany acute myelocytic leukemia (AML)-associated translocations including t(15;17), giving rise to the proposition that they could provide a common class of cooperating mutation in the development of the disease. 8 Fms-like tyrosine kinase 3 (FLT3) is an RTK expressed on hematopoietic progenitors. Mutation of the FLT3 gene is common in AML. [9][10][11][12] Numerous mutations have been identified. The majority, present in approximately 25% of patients, are internal tandem duplications (ITDs) that lead to in-frame insertions within the juxtamembrane region of the receptor. Le...

SummaryGene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r 2 correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.

Relapse remains the most common cause of treatment failure for patients with acute myeloid leukemia (AML) who undergo allogeneic stem cell transplantation (alloSCT), and carries a grave prognosis. Multiple studies have identified the presence of measurable residual disease (MRD) assessed by flow cytometry before alloSCT as a strong predictor of relapse, but it is not clear how these findings apply to patients who test positive in molecular MRD assays, which have far greater sensitivity. We analyzed pretransplant blood and bone marrow samples by reverse-transcription polymerase chain reaction in 107 patients with NPM1-mutant AML enrolled in the UK National Cancer Research Institute AML17 study. After a median follow-up of 4.9 years, patients with negative, low (&lt;200 copies per 105ABL in the peripheral blood and &lt;1000 copies in the bone marrow aspirate), and high levels of MRD had an estimated 2-year overall survival (2y-OS) of 83%, 63%, and 13%, respectively (P &lt; .0001). Focusing on patients with low-level MRD before alloSCT, those with FLT3 internal tandem duplications(ITDs) had significantly poorer outcome (hazard ratio [HR], 6.14; P = .01). Combining these variables was highly prognostic, dividing patients into 2 groups with 2y-OS of 17% and 82% (HR, 13.2; P &lt; .0001). T-depletion was associated with significantly reduced survival both in the entire cohort (2y-OS, 56% vs 96%; HR, 3.24; P = .0005) and in MRD-positive patients (2y-OS, 34% vs 100%; HR, 3.78; P = .003), but there was no significant effect of either conditioning regimen or donor source on outcome. Registered at ISRCTN (http://www.isrctn.com/ISRCTN55675535).

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 -internal tandem duplication mutations, 23%-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; = .3) or 5-year relapse-free survival (40% vs 36%; hazard ratio, 0.88; 95% CI 0.69-1.12; = .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 -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.