Purpose 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. Methods As 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/µL or thrombocytopenia < 100,000/μL (CRi) by clinicians, with CR/CRi subdivided by MFC-MRD assay into MRD+ and MRD-. Patients without high-risk factors, including Flt3 internal tandem duplication wt/- NPM1-wt subgroup, received a second daunorubicin/cytosine arabinoside induction; course 2 (C2) was intensified for patients with high-risk factors. Results Survival 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% MRD-; 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% MRD-, 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 MRD- (HR, 1.68 [95% CI, 0.75 to 3.85]; P = .16 for interaction). Conclusion MFC-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.
Approximately 1–2% of acute myeloid leukaemia (AML) patients have been shown to harbour a combination of FLT3 gene mutations of both the internal tandem duplication (ITD) and tyrosine kinase domain (TKD) variety (Chen et al, 2005; Mills et al, 2005). It has been unclear, however, whether these mutations have occurred within the same allele or within different clones. Mutations within the TKD are usually point mutations affecting individual codons: D835, I836 or I839 (Mills et al, 2005). Multiple TKD mutations within the same patient have not previously been reported. We have identified a patient who had three TKD mutations in tandem within the same allele and therefore within the same clone. A 69-year-old female recently presented with Hb 10·4 g/dl, total white cell count (WCC) 31·4 × 109/l and platelet count 58 × 109/l. Bone marrow examination confirmed AML French-British-American (FAB) classification type M4, with blasts accounting for 24% of total bone marrow nucleated cells, with a normal karyotype. Molecular diagnostic analyses included analysis of RNA for the presence of either an ITD or TKD mutation in the FLT3 gene. Such mutations occur in 25–35% of AML patients. ITD mutation analysis was negative. However, TKD analysis performed by reverse transcription polymerase chain reaction (RT-PCR) followed by EcoRV digestion with the products run on an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA) (Mills et al, 2005) indicated the presence of mutated undigested fragments and also the presence of two hetero-duplex products. The PCR products were cloned using TOPO TA Cloning Kit (Invitrogen, Paisley, UK) and sequenced (Fig 1). This revealed five base mutations within three sequential codons leading to three amino acid changes. In addition to the previously reported D835V mutation, the patient also had an I836F mutation (these mutations have a previously reported incidence of around 2–9% (Andersson et al, 2004; Mills et al, 2005)) and also a previously unreported M837P mutation. Sequencing indicated that all three mutations occurred in one allele. Sequence analysis of the tyrosine kinase domain region showing mutations of five bases in three codons involving Asp835Val, Ile836Phe and Met837Pro in a patient with acute myeloid leukaemia. The patient was treated initially with 6 d of oral hydroxycarbamide therapy, which lowered the WCC to <10 × 109/l, and subsequently with the orally-administered small molecule FLT3 inhibitor CEP701. CEP701 was withdrawn after 14 d when an increase in bone marrow blasts to 60% was identified. The patient died from progressive disease 2 months later. The identification of a complex, and previously undescribed, FLT3 TKD mutation gives further insight into the range and variety of mutations occurring in AML.
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.
Receptors for the natriuretic peptide family have been characterized in the adrenocorticotrophic hormone (ACTH)-secreting AtT-20 pituitary tumour cell line. Northern blot analysis detected mRNA transcripts for the guanylate cyclase-linked GC-B receptor subtype. There was no evidence for the expression of either guanylate cyclase-linked GC-A receptor or atrial natriuretic peptide (ANP)-C (clearance) receptor mRNAs. Cyclic GMP production in AtT-20 cells was stimulated up to 200-fold by C-type natriuretic peptide (CNP), which was 10- and 20 times as effective as equivalent concentrations of brain natriuretic peptide and ANP respectively. Cyclic GMP dose-response curves to CNP failed to show any signs of saturation even at concentrations up to 30 microM, indicating a relatively low affinity of CNP for the GC-B receptor. Although CNP induced large stimulations in cyclic GMP production, specific binding of [125I-Tyr0]CNP could not be demonstrated in AtT-20 cells. The absence of specific binding with this radiolabelled analogue is possibly due to a reduced affinity for the GC-B receptor, as CNP analogues with N-terminal modifications such as [Tyr0]CNP and [127I-Tyr0]CNP exhibited reduced abilities to stimulate cyclic GMP production in these cells. Despite elevating cyclic GMP levels, CNP had no effect on basal or corticotrophin-releasing factor-stimulating ACTH release from the cells. These results show that the guanylate cyclase-coupled GC-B receptor is the only natriuretic peptide receptor subtype expressed in AtT-20 cells. Although CNP can markedly stimulate cyclic GMP production in these cells, there is incomplete expression of the normal natriuretic peptide-induced inhibitory pathway of ACTH secretion at some point distal to the production of cyclic GMP.
Younger patients with acute myeloid leukemia (AML) harboring NPM1 mutations without FLT3-internal tandem duplications (ITDs; NPM1-positive/FLT3-ITD-negative genotype) are classified as better risk; however, it remains uncertain whether this favorable classification can be applied to older patients with AML with this genotype. Therefore, we examined the impact of age on the prognostic significance of NPM1-positive/FLT3-ITD-negative status in older patients with AML.
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