The clinical and biological consequences of different FLT3 mutations in patients with AML

Characterisation of pathogenic markers in acute myeloid leukaemia (AML) may benefit patients through refinement of risk stratification, application of molecularly targeted therapy and improved understanding of AML biology. Whilst the presence of an internal tandem duplication (ITD) within the fms-like tyrosine kinase-3 (FLT3) gene is known to predict adverse outcome in young adults with AML, the clinical significance of activating mutations in the tyrosine kinase domain (TKD) of FLT3 is unclear. Therefore, a highly sensitive and specific denaturing-HPLC technique was developed to screen for FLT3/TKDs in 1339 young adult patients with AML. Mutations were detected in 161 (12%) cases, with a high incidence in patients with inv(16) (24%; P=.009), a group in which FLT3/ITDs are uncommon. Unlike FLT3/ITDs, FLT3/TKDs were associated with a favourable long-term outcome with a 10-year overall survival (OS) of 36% for FLT3 WT, 51% for FLT3/ITD-TKD+ and 24% for FLT3/ITD+TKDpatients (P<.001). The relative FLT3/TKD mutant level was highly variable with the favourable prognosis residing in those patients with greater than 25% mutant alleles (10-year OS of 59%), possibly reflecting the stage at which the mutation is acquired. The mechanism of FLT3 activation also influenced sensitivity to FLT3-inhibitor induced cytotoxicity, with FLT3/ITD+ blast cells more sensitive than FLT3/TKD+ cells. Following lentiviral transduction, FLT3/ITD-transduced 32Dcl3 and Ba/F3 cells demonstrated more rapid proliferation than FLT3/TKD-transduced cells. In an NB4 cell line model of ATRA-induced myeloid differentiation, the presence of a FLT3/ITD inhibited differentiation unlike a FLT3/TKD mutation which increased differentiation. Furthermore, FLT3/ITD-transduced CD34 positive haematopoietic stem cells showed greater cytokine-free survival of colony forming cells than FLT3/TKD-transduced cells. Signalling studies also revealed that a FLT3/ITD induced stronger STAT5 activation than a FLT3/TKD mutation. This unexpected genotype-phenotype relationship is of direct relevance to current clinical decision making in AML, and may also provide insights into mechanisms of chemoresistance

Caudal–rostral progression of alpha motoneuron degeneration in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Mice with transgenic expression of human SOD1G93A are a widely used model of ALS, with a caudal–rostral progression of motor impairment. Previous studies have quantified the progression of motoneuron (MN) degeneration based on size, even though alpha (α-) and gamma (γ-) MNs overlap in size. Therefore, using molecular markers and synaptic inputs, we quantified the survival of α-MNs and γ-MNs at the lumbar and cervical spinal segments of 3- and 4-month SOD1G93A mice, to investigate whether there is a caudal–rostral progression of MN death. By 3 months, in the cervical and lumbar spinal cord, there was α-MN degeneration with complete γ-MN sparing. At 3 months, the cervical spinal cord had more α-MNs per ventral horn than the lumbar spinal cord in SOD1G93A mice. A similar spatial trend of degeneration was observed in the corticospinal tract, which remained intact in the cervical spinal cord at 3- and 4- months of age. These findings agree with the corticofugal synaptopathy model that α-MNs and CST of the lumbar spinal cord are more susceptible to degeneration in SOD1G93A mice. Hence, there is a spatial and temporal caudal–rostral progression of α-MN and CST degeneration in SOD1G93A mice

Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways

Background Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by “bulk” assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined. Results To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlate the surface immunophenotype, transcriptional profile, and differentiation potential of individual MEP cells. Highly purified, single MEP cells were analyzed using index fluorescence-activated cell sorting and parallel targeted transcriptional profiling of the same cells was performed using a specifically designed panel of genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrate that immunophenotypic MEP comprise three distinct subpopulations: “Pre-MEP,” enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity; “E-MEP,” strongly biased towards erythroid differentiation; and “MK-MEP,” a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally defined MEP are a mixed population, as a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally primed to generate exclusively single-lineage output. Conclusions Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders, and erythromegakaryocytic leukemias

Clinical and molecular characterization of HER2 amplified-pancreatic cancer

&lt;p&gt;Background: Pancreatic cancer is one of the most lethal and molecularly diverse malignancies. Repurposing of therapeutics that target specific molecular mechanisms in different disease types offers potential for rapid improvements in outcome. Although HER2 amplification occurs in pancreatic cancer, it is inadequately characterized to exploit the potential of anti-HER2 therapies.&lt;/p&gt; &lt;p&gt;Methods: HER2 amplification was detected and further analyzed using multiple genomic sequencing approaches. Standardized reference laboratory assays defined HER2 amplification in a large cohort of patients (n = 469) with pancreatic ductal adenocarcinoma (PDAC).&lt;/p&gt; &lt;p&gt;Results: An amplified inversion event (1 MB) was identified at the HER2 locus in a patient with PDAC. Using standardized laboratory assays, we established diagnostic criteria for HER2 amplification in PDAC, and observed a prevalence of 2%. Clinically, HER2- amplified PDAC was characterized by a lack of liver metastases, and a preponderance of lung and brain metastases. Excluding breast and gastric cancer, the incidence of HER2-amplified cancers in the USA is &#62;22,000 per annum.&lt;/p&gt; &lt;p&gt;Conclusions: HER2 amplification occurs in 2% of PDAC, and has distinct features with implications for clinical practice. The molecular heterogeneity of PDAC implies that even an incidence of 2% represents an attractive target for anti-HER2 therapies, as options for PDAC are limited. Recruiting patients based on HER2 amplification, rather than organ of origin, could make trials of anti-HER2 therapies feasible in less common cancer types.&lt;/p&gt

Genetic variation at MECOM, TERT, JAK2 and HBS1L-MYB predisposes to myeloproliferative neoplasms

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations in JAK2, CALR or MPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance in JAK2V617F-negative MPN: rs12339666 (JAK2; meta-analysis P=1.27 × 10−10) and rs2201862 (MECOM; meta-analysis P=1.96 × 10−9). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when including JAK2V617F-positive cases. rs9376092 has a stronger effect in JAK2V617F-negative cases with CALR and/or MPL mutations (Breslow–Day P=4.5 × 10−7), whereas in JAK2V617F-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelic χ2 P=7.3 × 10−7). Reduced MYB expression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences in MYB expression to disease phenotype

Diagonal Representation for a Generic Matrix Valued Quantum Hamiltonian

A general method to derive the diagonal representation for a generic matrix valued quantum Hamiltonian is proposed. In this approach new mathematical objects like non-commuting operators evolving with the Planck constant promoted as a running variable are introduced. This method leads to a formal compact expression for the diagonal Hamiltonian which can be expanded in a power series of the Planck constant. In particular, we provide an explicit expression for the diagonal representation of a generic Hamiltonian to the second order in the Planck constant. This last result is applied, as a physical illustration, to Dirac electrons and neutrinos in external fields.Comment: Significant revision, typos corrected and references adde

In vivo fiber optic raman spectroscopy of muscle in preclinical models of amyotrophic lateral sclerosis and Duchenne muscular dystrophy

Neuromuscular diseases result in muscle weakness, disability, and, in many instances, death. Preclinical models form the bedrock of research into these disorders, and the development of in vivo and potentially translational biomarkers for the accurate identification of disease is crucial. Spontaneous Raman spectroscopy can provide a rapid, label-free, and highly specific molecular fingerprint of tissue, making it an attractive potential biomarker. In this study, we have developed and tested an in vivo intramuscular fiber optic Raman technique in two mouse models of devastating human neuromuscular diseases, amyotrophic lateral sclerosis, and Duchenne muscular dystrophy (SOD1G93A and mdx, respectively). The method identified diseased and healthy muscle with high classification accuracies (area under the receiver operating characteristic curves (AUROC): 0.76–0.92). In addition, changes in diseased muscle over time were also identified (AUROCs 0.89–0.97). Key spectral changes related to proteins and the loss of α-helix protein structure. Importantly, in vivo recording did not cause functional motor impairment and only a limited, resolving tissue injury was seen on high-resolution magnetic resonance imaging. Lastly, we demonstrate that ex vivo muscle from human patients with these conditions produced similar spectra to those observed in mice. We conclude that spontaneous Raman spectroscopy of muscle shows promise as a translational research tool

Analysis of variance in soil research: let the analysis fit the design

Sound design for experiments on soil is based on two fundamental principles: replication and randomization. Replication enables investigators to detect and measure contrasts between treatments against the backdrop of natural variation. Random allocation of experimental treatments to units enables effects to be estimated without bias and hypotheses to be tested. For inferential tests of effects to be valid an analysis of variance (anova) of the experimental data must match exactly the experimental design. Completely randomized designs are usually inefficient. Blocking will usually increase precision, and its role must be recognized as a unique entry in an anova table. Factorial designs enable questions on two or more factors and their interactions to be answered simultaneously, and split-plot designs may enable investigators to combine factors that require disparate amounts of land for each treatment. Each such design has its unique correct anova; no other anova will do. One outcome of an anova is a test of significance. If it turns out to be positive then the investigator may examine the contrasts between treatments to discover which themselves are significant. Those contrasts should have been ones in which the investigator was interested at the outset and which the experiment was designed to test. Post-hoc testing of all possible contrasts is deprecated as unsound, although the procedures may guide an investigator to further experimentation. Examples of the designs with simulated data and programs in GenStat and R for the analyses of variance are provided as File S1

Shape Space Methods for Quantum Cosmological Triangleland

With toy modelling of conceptual aspects of quantum cosmology and the problem of time in quantum gravity in mind, I study the classical and quantum dynamics of the pure-shape (i.e. scale-free) triangle formed by 3 particles in 2-d. I do so by importing techniques to the triangle model from the corresponding 4 particles in 1-d model, using the fact that both have 2-spheres for shape spaces, though the latter has a trivial realization whilst the former has a more involved Hopf (or Dragt) type realization. I furthermore interpret the ensuing Dragt-type coordinates as shape quantities: a measure of anisoscelesness, the ellipticity of the base and apex's moments of inertia, and a quantity proportional to the area of the triangle. I promote these quantities at the quantum level to operators whose expectation and spread are then useful in understanding the quantum states of the system. Additionally, I tessellate the 2-sphere by its physical interpretation as the shape space of triangles, and then use this as a back-cloth from which to read off the interpretation of dynamical trajectories, potentials and wavefunctions. I include applications to timeless approaches to the problem of time and to the role of uniform states in quantum cosmological modelling.Comment: A shorter version, as per the first stage in the refereeing process, and containing some new reference

Flux Phase as a Dynamic Jahn-Teller Phase: Berryonic Matter in the Cuprates?

There is considerable evidence for some form of charge ordering on the hole-doped stripes in the cuprates, mainly associated with the low-temperature tetragonal phase, but with some evidence for either charge density waves or a flux phase, which is a form of dynamic charge-density wave. These three states form a pseudospin triplet, demonstrating a close connection with the E X e dynamic Jahn-Teller effect, suggesting that the cuprates constitute a form of Berryonic matter. This in turn suggests a new model for the dynamic Jahn-Teller effect as a form of flux phase. A simple model of the Cu-O bond stretching phonons allows an estimate of electron-phonon coupling for these modes, explaining why the half breathing mode softens so much more than the full oxygen breathing mode. The anomalous properties of $O^{2-}$ provide a coupling (correlated hopping) which acts to stabilize density wave phases.Comment: Major Revisions: includes comparisons with specific cuprate phonon modes, 16 eps figures, revte