Clonal evolution of acute myeloid leukemia with FLT3-ITD mutation under treatment with midostaurin.

In the international randomized phase 3 RATIFY (Randomized AML Trial In FLT3 in patients less than 60 Years old) trial, the multikinase inhibitor midostaurin significantly improved overall and event-free survival in patients 18 to 59 years of age with FLT3-mutated acute myeloid leukemia (AML). However, only 59% of patients in the midostaurin arm achieved protocol-specified complete remission (CR), and almost half of patients achieving CR relapsed. To explore underlying mechanisms of resistance, we studied patterns of clonal evolution in patients with FLT3-internal tandem duplications (ITD)-positive AML who were entered in the RATIFY or German-Austrian Acute Myeloid Leukemia Study Group 16-10 trial and received treatment with midostaurin. To this end, paired samples from 54 patients obtained at time of diagnosis and at time of either relapsed or refractory disease were analyzed using conventional Genescan-based testing for FLT3-ITD and whole exome sequencing. At the time of disease resistance or progression, almost half of the patients (46%) became FLT3-ITD negative but acquired mutations in signaling pathways (eg, MAPK), thereby providing a new proliferative advantage. In cases with FLT3-ITD persistence, the selection of resistant ITD clones was found in 11% as potential drivers of disease. In 32% of cases, no FLT3-ITD mutational change was observed, suggesting either resistance mechanisms bypassing FLT3 inhibition or loss of midostaurin inhibitory activity because of inadequate drug levels. In summary, our study provides novel insights into the clonal evolution and resistance mechanisms of FLT3-ITD-mutated AML under treatment with midostaurin in combination with intensive chemotherapy.

Impact of gemtuzumab ozogamicin on MRD and relapse risk in patients with NPM1-mutated AML: results from the AMLSG 09-09 trial.

Monitoring of measurable residual disease (MRD) provides prognostic information in patients with Nucleophosmin1-mutated (NPM1mut) acute myeloid leukemia (AML) and represents a powerful tool to evaluate treatment effects within clinical trials. We determined NPM1mut transcript levels (TLs) by quantitative reverse-transcription polymerase chain reaction and evaluated the prognostic impact of NPM1mut MRD and the effect of gemtuzumab ozogamicin (GO) on NPM1mut TLs and the cumulative incidence of relapse (CIR) in patients with NPM1mut AML enrolled in the randomized phase 3 AMLSG 09-09 trial. A total of 3733 bone marrow (BM) samples and 3793 peripheral blood (PB) samples from 469 patients were analyzed. NPM1mut TL log10 reduction ≥ 3 and achievement of MRD negativity in BM and PB were significantly associated with a lower CIR rate, after 2 treatment cycles and at end of treatment (EOT). In multivariate analyses, MRD positivity was consistently revealed to be a poor prognostic factor in BM and PB. With regard to treatment effect, the median NPM1mut TLs were significantly lower in the GO-Arm across all treatment cycles, resulting in a significantly greater proportion of patients achieving MRD negativity at EOT (56% vs 41%; P = .01). The better reduction in NPM1mut TLs after 2 treatment cycles in MRD positive patients by the addition of GO led to a significantly lower CIR rate (4-year CIR, 29.3% vs 45.7%, P = .009). In conclusion, the addition of GO to intensive chemotherapy in NPM1mut AML resulted in a significantly better reduction in NPM1mut TLs across all treatment cycles, leading to a significantly lower relapse rate.

Impact of NPM1/FLT3-ITD genotypes defined by the 2017 European LeukemiaNet in patients with acute myeloid leukemia.

Patients with acute myeloid leukemia (AML) harboring FLT3 internal tandem duplications (ITDs) have poor outcomes, in particular AML with a high (≥0.5) mutant/wild-type allelic ratio (AR). The 2017 European LeukemiaNet (ELN) recommendations defined 4 distinct FLT3-ITD genotypes based on the ITD AR and the NPM1 mutational status. In this retrospective exploratory study, we investigated the prognostic and predictive impact of the NPM1/FLT3-ITD genotypes categorized according to the 2017 ELN risk groups in patients randomized within the RATIFY trial, which evaluated the addition of midostaurin to standard chemotherapy. The 4 NPM1/FLT3-ITD genotypes differed significantly with regard to clinical and concurrent genetic features. Complete ELN risk categorization could be done in 318 of 549 trial patients with FLT3-ITD AML. Significant factors for response after 1 or 2 induction cycles were ELN risk group and white blood cell (WBC) counts; treatment with midostaurin had no influence. Overall survival (OS) differed significantly among ELN risk groups, with estimated 5-year OS probabilities of 0.63, 0.43, and 0.33 for favorable-, intermediate-, and adverse-risk groups, respectively (P < .001). A multivariate Cox model for OS using allogeneic hematopoietic cell transplantation (HCT) in first complete remission as a time-dependent variable revealed treatment with midostaurin, allogeneic HCT, ELN favorable-risk group, and lower WBC counts as significant favorable factors. In this model, there was a consistent beneficial effect of midostaurin across ELN risk groups.

Genetic alterations in Thai adult patients with acute myeloid leukemia and myelodysplastic syndrome-excess blasts detected by next-generation sequencing technique.

Several molecular aberrations affect the prognosis of patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with excess blasts (EB). This study aimed to determine the incidence and clinical impact of molecular genetic aberrations in Thai patients with AML and MDS-EB, detected by the next-generation sequencing (NGS) technique. This prospective, observational study was conducted between 2018 and 2020 on newly diagnosed Thai AML or MDS-EB patients aged above 15 years. NGS was performed using a custom amplicon-based targeted enrichment assay for 42 genes recurrently mutated in myeloid neoplasms. The molecular results were correlated with baseline patient and disease characteristics as well as outcomes. Forty-nine patients were enrolled in this study. The median age was 56 years (interquartile range [IQR], 44-64), with nearly equal proportions of males and females. The median number of mutations was 3 (IQR, 2-4). The most frequent alterations were FLT3 internal tandem duplications (ITD) (28.6%), DNMT3A (24.5%), and WT1 (22.4%) mutations. FLT3-ITD was more frequent in the de novo AML group than in the MDS/secondary AML group, whereas in the MDS/secondary AML group, ASXL1, ETV6, and SRSF2 mutations were more frequent. Patients aged greater than 65 years and patients with mutated TP53 were more likely to have inferior overall survival from multivariate analysis. FLT3-ITD was the most common mutation among newly diagnosed Thai AML patients. TP53 mutation and advanced age were independent adverse factors for survival outcome. The genetic landscapes of AML patients vary between national populations. Thai Clinical Trials Registry identifier: TCTR20190227003.

Engineering polyshell spherical microcapsules for optimal localized light absorption.

Optical radiation absorption in a polylayer spherical microparticle simulating an inorganic/organic polyshell absorbing microcapsule is considered. With the aim of the finite-difference time-domain technique, the spatial distribution of the absorbed light power in microcapsules of various sizes and internal structure is numerically calculated. For the purpose of light absorption enhancement, we have engineered the optimal structure of a capsule consisting of a strong-refracting transparent outer coating and an absorbing layer which covers a liquid core. The proposed microcapsule prototype provides for a manifold increase in the absorbed light power density in comparison with the usual single-layer absorbing capsule. We show that for light-wavelengths-scaled microcapsules it is optimal to use a material with the refractive index larger than two as an outer shell, for example, titanium dioxide (TiO2). The highest values of the absorbed power density can be obtained in microcapsules with absorbing shell thickness of approximately a tenth of a laser wavelength.

The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts.

The Bordetella type III secretion system (T3SS) effector protein BteA is necessary and sufficient for rapid cytotoxicity in a wide range of mammalian cells. We show that BteA is highly conserved and functionally interchangeable between Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis. The identification of BteA sequences required for cytotoxicity allowed the construction of non-cytotoxic mutants for localization studies. BteA derivatives were targeted to lipid rafts and showed clear colocalization with cortical actin, ezrin and the lipid raft marker GM1. We hypothesized that BteA associates with the cytoplasmic face of lipid rafts to locally modulate host cell responses to Bordetella attachment. B. bronchiseptica adhered to host cells almost exclusively to GM1-enriched lipid raft microdomains and BteA colocalized to these same sites following T3SS-mediated translocation. Disruption of lipid rafts with methyl-beta-cyclodextrin protected cells from T3SS-induced cytotoxicity. Localization to lipid rafts was mediated by a 130-amino-acid lipid raft targeting domain at the N-terminus of BteA, and homologous domains were identified in virulence factors from other bacterial species. Lipid raft targeting sequences from a T3SS effector (Plu4750) and an RTX-type toxin (Plu3217) from Photorhabdus luminescens directed fusion proteins to lipid rafts in a manner identical to the N-terminus of BteA.

Genomic heterogeneity in core-binding factor acute myeloid leukemia and its clinical implication.

Core-binding factor (CBF) acute myeloid leukemia (AML) encompasses AML with inv(16)(p13.1q22) and AML with t(8;21)(q22;q22.1). Despite sharing a common pathogenic mechanism involving rearrangements of the CBF transcriptional complex, there is growing evidence for considerable genotypic heterogeneity. We comprehensively characterized the mutational landscape of 350 adult CBF-AML [inv(16): n = 160, t(8;21): n = 190] performing targeted sequencing of 230 myeloid cancer-associated genes. Apart from common mutations in signaling genes, mainly NRAS, KIT, and FLT3, both CBF-AML entities demonstrated a remarkably diverse pattern with respect to the underlying cooperating molecular events, in particular in genes encoding for epigenetic modifiers and the cohesin complex. In addition, recurrent mutations in novel collaborating candidate genes such as SRCAP (5% overall) and DNM2 (6% of t(8;21) AML) were identified. Moreover, aberrations altering transcription and differentiation occurred at earlier leukemic stages and preceded mutations impairing proliferation. Lasso-penalized models revealed an inferior prognosis for t(8;21) AML, trisomy 8, as well as FLT3 and KIT exon 17 mutations, whereas NRAS and WT1 mutations conferred superior prognosis. Interestingly, clonal heterogeneity was associated with a favorable prognosis. When entering mutations by functional groups in the model, mutations in genes of the methylation group (ie, DNMT3A, TET2) had a strong negative prognostic impact.

Whispering-gallery mode excitation in a microdroplet illuminated by a train of chirped ultrashort laser pulses.

The peculiarities of resonant optical field excitation inside a water microdroplet under illumination by a spatially bounded Gaussian beam with a temporal regime of a single chirped ultrashort laser pulse and a chirped pulse train are considered. It is established that the coupling efficiency of incident radiation to a selected high-Q whispering-gallery mode significantly depends on the interpulse interval in the train and chirping parameter of pumped laser radiation. The influence of the geometry of particle illumination by a laser beam and of the number of pulses in the train on the whispering-gallery mode buildup and its peak intensity is investigated.

PFIT and PFRIT: bioinformatic algorithms for detecting glycosidase function from structure and sequence.

The identification of the enzymes involved in the metabolism of simple and complex carbohydrates presents one bioinformatic challenge in the post-genomic era. Here, we present the PFIT and PFRIT algorithms for identifying those proteins adopting the alpha/beta barrel fold that function as glycosidases. These algorithms are based on the observation that proteins adopting the alpha/beta barrel fold share positions in their tertiary structures having equivalent sets of atomic interactions. These are conserved tertiary interaction positions, which have been implicated in both structure and function. Glycosidases adopting the alpha/beta barrel fold share more conserved tertiary interactions than alpha/beta barrel proteins having other functions. The enrichment pattern of conserved tertiary interactions in the glycosidases is the information that PFIT and PFRIT use to predict whether any given alpha/beta barrel will function as a glycosidase or not. Using as a test set a database of 19 glycosidase and 45 nonglycosidase alpha/beta barrel proteins with low sequence similarity, PFIT and PFRIT can correctly predict glycosidase function for 84% of the proteins known to function as glycosidases. PFIT and PFRIT incorrectly predict glycosidase function for 25% of the nonglycosidases. The program PSI-BLAST can also correctly identify 84% of the 19 glycosidases, however, it incorrectly predicts glycosidase function for 50% of the nonglycosidases (twofold greater than PFIT and PFRIT). Overall, we demonstrate that the structure-based PFIT and PFRIT algorithms are both more selective and sensitive for predicting glycosidase function than the sequence-based PSI-BLAST algorithm.

Regulation of biosynthesis and transport of aromatic amino acids in low-GC Gram-positive bacteria.

Computational comparative techniques were applied to analysis of the aromatic amino acid regulon in Gram-positive bacteria. A new candidate transcription regulation signal of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and shikimate kinase genes was identified in Streptococcus and Lactococcus species. New T-boxes were found upstream of aromatic amino acid biosynthesis and transport genes in the Bacillus/Clostridium group. The substrate specificity of proteins from the PabA/TrpG family was assigned based on metabolic reconstruction and analysis of regulatory signals and phylogenetic patterns. New candidate tryptophan transporters were identified; their specificity was predicted by analysis of T-box regulatory sites. Comparison of all available genomes shows that regulation of genes of the aromatic amino acid biosynthesis pathway is quite labile and involves at least four regulatory systems, two at the DNA level and two more involving competition of alternative RNA secondary structures for transcription and/or translation regulation at the RNA level.

A genome-wide screen identifies a Bordetella type III secretion effector and candidate effectors in other species.

Bordetella bronchiseptica utilizes a type III secretion system (TTSS) for induction of non-apoptotic cytotoxicity in host cells and modulation of host immunity. The identity of Bordetella TTSS effectors, however, has remained elusive. Here we report a genome-wide screen for TTSS effectors based on shared biophysical and functional characteristics of class I chaperones and their frequent colocalization with TTSS effectors. When applied to B. bronchiseptica, the screen identified the first TTSS chaperone-effector locus, btcA-bteA, and we experimentally confirmed its function. Expression of bteA is co-ordinated with expression of TTSS apparatus genes, BteA is secreted through the TTSS of B. bronchiseptica, it is required for cytotoxicity towards mammalian cells, and it is highly conserved in the human-adapted subspecies B. pertussis and B. parapertussis. Transfection of bteA into epithlieal cells results in rapid cell death, indicating that BteA alone is sufficient to induce potent cytotoxicity. Finally, an in vitro interaction between BteA and BtcA was demonstrated. The search for TTSS chaperones and effectors was then expanded to other bacterial genomes, including mammalian and insect pathogens, where we identified a large number of novel candidate chaperones and effectors. Although the majority of putative effectors are proteins of unknown function, several have similarities to eukaryotic protein domains or previously identified effectors from other species.

Comparative genomics of bacterial zinc regulons: enhanced ion transport, pathogenesis, and rearrangement of ribosomal proteins.

Zinc is an important component of many proteins, but in large concentrations it is poisonous to the cell. Thus its transport is regulated by zinc repressors ZUR of proteobacteria and Gram-positive bacteria from the Bacillus group and AdcR of bacteria from the Streptococcus group. Comparative computational analysis allowed us to identify binding signals of ZUR repressors GAAATGTTATANTATAACATTTC for gamma-proteobacteria, GTAATGTAATAACATTAC for the Agrobacterium group, GATATGTTATAACATATC for the Rhododoccus group, TAAATCGTAATNATTACGATTTA for Gram-positive bacteria, and TTAACYRGTTAA of the streptococcal AdcR repressor. In addition to known transporters and their paralogs, zinc regulons were predicted to contain a candidate component of the ATP binding cassette, zinT (b1995 in Escherichia coli and yrpE in Bacillus subtilis). Candidate AdcR-binding sites were identified upstream of genes encoding pneumococcal histidine triad (PHT) proteins from a number of pathogenic streptococci. Protein functional analysis of this family suggests that PHT proteins are involved in the invasion process. Finally, repression by zinc was predicted for genes encoding a variety of paralogs of ribosomal proteins. The original copies of all these proteins contain zinc-ribbon motifs and thus likely bind zinc, whereas these motifs are destroyed in zinc-regulated paralogs. We suggest that the induction of these paralogs in conditions of zinc starvation leads to their incorporation in a fraction of ribosomes instead of the original ribosomal proteins; the latter are then degraded with subsequent release of some zinc for the utilization by other proteins. Thus we predict a mechanism for maintaining zinc availability for essential enzymes.