Mutations of the NPM1 gene (NPM1mut) are among the most common genetic alterations in acute myeloid leukemia and are suitable for minimal residual disease detection. We retrospectively investigated ...the prognostic impact of NPM1mut-based minimal residual disease detection from bone marrow for development of relapse by using a newly developed real-time polymerase chain reaction based on locked nucleic acid–containing primers in 174 patients, 155 of whom were treated within prospective protocols. The prognostic value of 5 cutoff values after completion of treatment or after allogeneic transplantation was studied by using cause-specific hazard models. Subsequent validation using cross-validated partial likelihood analysis revealed that an increase of more than 1% NPM1mut/ABL1 was most prognostic for relapse after chemotherapy, whereas an increase of more than 10% NPM1mut/ABL1 was most prognostic for relapse after allogeneic transplantation. Univariate and multivariate analysis of disease-free survival and overall survival revealed a significantly worse outcome in patients with >1% NPM1mut/ABL1 and >10% NPM1mut/ABL1, respectively, which remained significant after adjustment for FLT3–internal tandem duplication status. Our results in a large data set define and optimize cutoff values for early diagnosis of molecular relapse. These results may be especially important for defining triggers for early therapeutic intervention.
• NPM1 RT-PCR levels >1% are associated with poor overall and disease-free survival in AML patients treated with chemotherapy.• NPM1 MRD levels >10% are associated with poor overall and disease-free survival in AML patients after allogeneic transplantation.
Understanding the genetic, molecular and evolutionary basis of cysteine-stabilized antifungal proteins (AFPs) from fungi is important for understanding whether their function is mainly defensive or ...associated with fungal growth and development. In the current study, a transcriptome meta-analysis of the Aspergillus niger γ-core protein AnAFP was performed to explore co-expressed genes and pathways, based on independent expression profiling microarrays covering 155 distinct cultivation conditions. This analysis uncovered that anafp displays a highly coordinated temporal and spatial transcriptional profile which is concomitant with key nutritional and developmental processes. Its expression profile coincides with early starvation response and parallels with genes involved in nutrient mobilization and autophagy. Using fluorescence- and luciferase reporter strains we demonstrated that the anafp promoter is active in highly vacuolated compartments and foraging hyphal cells during carbon starvation with CreA and FlbA, but not BrlA, as most likely regulators of anafp. A co-expression network analysis supported by luciferase-based reporter assays uncovered that anafp expression is embedded in several cellular processes including allorecognition, osmotic and oxidative stress survival, development, secondary metabolism and autophagy, and predicted StuA and VelC as additional regulators. The transcriptomic resources available for A. niger provide unparalleled resources to investigate the function of proteins. Our work illustrates how transcriptomic meta-analyses can lead to hypotheses regarding protein function and predict a role for AnAFP during slow growth, allorecognition, asexual development and nutrient recycling of A. niger and propose that it interacts with the autophagic machinery to enable these processes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Autotaxin (ATX) has been reported to act as a motility and growth factor in a variety of cancer cells. The ATX protein acts as a secreted lysophospholipase D by converting lysophosphatidylcholine ...(LPC) to lysophosphatidic acid (LPA), which signals via G-protein–coupled receptors and has important functions in cell migration and proliferation. This study demonstrates that ATX expression is specifically upregulated and functionally active in acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) mutation of the FLT3 receptor gene. Moreover, ATX expression was also found in normal human CD34+ progenitor cells and selected myeloid and lymphoid subpopulations. Enforced expression of mutant FLT3-ITD by retroviral vector transduction increased ATX mRNA in selected cell lines, whereas inhibition of FLT3-ITD signaling by sublethal doses of PKC412 or SU5614 led to a significant downregulation of ATX mRNA and protein levels. In the presence of LPC, ATX expression significantly increased proliferation. LPA induced proliferation, regardless of ATX expression, and induced chemotaxis in all tested human leukemic cell lines and human CD34+ progenitors. LPC increased chemotaxis only in cells with high expression of endogenous ATX by at least 80%, demonstrating the autocrine action of ATX. Inhibition of ATX using a small molecule inhibitor selectively induced killing of ATX-expressing cell lines and reduced motility in these cells. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration during hematopoiesis and that deregulation of ATX contributes to the pathogenesis of AML.
Abstract 886
The diagnosis acute myeloid leukemia (AML) describes a heterogeneous group of myeloid stem cell disorders. Based on current concepts of disease development, the combination of at least ...two mutations is necessary for transformation, typically affecting transcription factors (e.g. RUNX1) blocking normal differentiation and growth promoting genes, e.g. receptor tyrosine kinases like FLT3. This model has been challenged by more recent results of genome-wide mutational analysis, which revealed a typical load of 8–12 mutations affecting several additional pathways, e.g. epigenetic regulation (DNMT3A, TET2 or IDH1). However, the sequence of acquisition and the individual impact of these mutations are largely unknown because these aspects are difficult to study. Here we describe a unique case of a donor cell leukemia giving unexpected insights into the development of AML in man.
In May 2004, a 51-year old male (P1) with an 8-year history of B-CLL received G-CSF mobilized peripheral blood stem cells after dose reduced conditioning from his HLA-identical sister, because he had relapsed after several lines of conventional therapy. He rapidly engrafted and showed complete donor chimerism (DC). In February 2012, he was admitted to the hospital with elevated WBC counts and circulating blasts. Bone marrow (BM) aspiration and morphology revealed an infiltration of the BM with 94% myeloid blasts (FAB M1). Cytogenetic and standard molecular assessment showed a normal female karyotype and NPM1 and FLT3-ITD mutations. STR-based analysis also revealed a persistent, 100% DC, thus the diagnosis of a donor cell AML was made, which developed almost 8 years after SCT. Interestingly, his sister, the donor (P2) had been also diagnosed with AML (FAB M2, cytogenetics: 47, XX,+8; NPM1 and FLT3-ITD neg.) only 3 months before her brother in Nov. 2011. Currently, P1 is in CR after re-SCT from an unrelated donor, whereas P2 relapsed and is scheduled for SCT after reinduction. Since DNA material of both individuals was available and due to this unique constellation, we performed next generation sequencing of whole exome enriched material using an Illumina HiSEQ 2000 platform after obtaining informed consent to compare both AMLs. Identified mutations were then confirmed using conventional Sanger sequencing and traced back by 454-based amplicon deep sequencing in a pre-SCT sample of the donor/P2 as well as several post SCT samples collected from P1 for the documentation of chimerism.
Comparison of the two AML-samples with a pre-SCT donor sample and a sample taken after 1.SCT as well as the HG19 and dbSNP135 releases revealed more than 100 unknown SNPs. Confirmation focused on cancer related changes or genes in critical pathways. In P1, in addition to the known NPM1 and FLT3-ITD mutations, we found somatic changes in CLCA1, PKHD1 and TET2, whereas in P2, we identified and confirmed somatic mutations in CDCA2, CBL, IDH1, NEK9 and PHF6. In addition, a typical DNMT3A R882C mutation was found in both leukemias. Interestingly, this mutation was also detectable by conventional Sanger sequencing in the pre-SCT sample of P2, but not in P2-germline DNA derived from buccal swaps. As shown in the figure, the 454-amplicon sequencing revealed a gradual increase of the TET2 (R1167G) mutational load over time in P1, and showed also that this mutation was present at low levels (4%) already in the pre-SCT sample of P2, but not in her final AML. FLT3-ITD and NPM1 mutations were detectable only in the AML-sample of P1, but not at any prior time points or in P2.
These data indicate that mutations like the DNMT3A R882C can be present in normal appearing hematopoiesis at high levels years before the development of AML. The presence of the mutation in the absence of overt leukemia or MDS indicates that these mutations might not have a direct effect on the development of the disease, but favor the development of aberrant clones which then acquire additional changes in a latency phase. Other mutations (e.g. TET2) might give a small clonal advantage, but only the final acquisition of abnormalities like NPM1 and FLT3-ITD might transform this latency phase into a rapidly proliferating status, consistent with the “driver” status of these aberrations. The divergent mutational pattern found in the two AMLs emerging from the same DNMT3A-starting clone points to the high clonal diversity which might develop even within a single individual. Display omitted
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