Maria Hansen

Persistence of DNMT3A mutations at long‐term remission in adult patients with AML

Mutations in DNMT3A, the gene encoding DNA methyltransferase 3 alpha, have been identified as molecular drivers in acute myeloid leukaemia (AML) with possible implications for minimal residual disease monitoring and prognosis. To further explore the utility of DNMT3A mutations as biomarkers for AML, we developed assays for sensitive detection of recurrent mutations affecting residue R882. Analysis of DNA from 298 diagnostic AML samples revealed DNMT3A mutations in 45 cases (15%), which coincided with mutations in NPM1, FLT3 and IDH1. DNMT3A mutations were stable in 12 of 13 patients presenting with relapse or secondary myelodysplastic syndrome, but were also present in remission samples from 14 patients (at allele frequencies of <1–50%) up to 8 years after initial AML diagnosis, despite the loss of all other molecular AML markers. The mutant DNMT3A allele burden was not related to the clinical course of disease. Cell sorting demonstrated the presence of DNMT3A mutations in leukaemic blasts, but also at lower allele frequencies in T and B‐cells from the same patients. Our data are consistent with the recent finding of preleukaemic stem cells in AML, which are resistant to chemotherapy. The persistence of DNMT3A mutations during remission may have important implications for the management of AML.

Sortilin Facilitates Signaling of Ciliary Neurotrophic Factor and Related Helical Type 1 Cytokines Targeting the gp130/Leukemia Inhibitory Factor Receptor β Heterodimer

ABSTRACT Sortilin is a member of the Vps10p domain family of neuropeptide and neurotrophin binding neuronal receptors. The family members interact with and partly share a variety of ligands and partake in intracellular sorting and protein transport as well as in transmembrane signal transduction. Thus, sortilin mediates the transport of both neurotensin and nerve growth factor and interacts with their respective receptors to facilitate ligand-induced signaling. Here we report that ciliary neurotrophic factor (CNTF), and related ligands targeting the established CNTF receptor α, binds to sortilin with high affinity. We find that sortilin may have at least two functions: one is to provide rapid endocytosis and the removal of CNTF, something which is not provided by CNTF receptor α, and the other is to facilitate CNTF signaling through the gp130/leukemia inhibitory factor (LIF) receptor β heterodimeric complex. Interestingly, the latter function is independent of both the CNTF receptor α and ligand binding to sortilin but appears to implicate a direct interaction with LIF receptor β. Thus, sortilin facilitates the signaling of all helical type 1 cytokines, which engage the gp130/LIF receptor β complex.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

ABSTRACT Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

Sensitive ligand-based protein quantification using immuno-PCR: A critical review of single-probe and proximity ligation assays.

Quantitative PCR (qPCR) of reverse-transcribed mRNA has revolutionized gene expression analyses. qPCR analysis is based on the prevalent assumption that mRNA transcript numbers provide an adequate measure of specific biomarker expression. However, taking the complexity of protein turnover into account, there is a need to correlate qPCR-derived transcriptional patterns with protein translational patterns so as to not leave behind important pathobiological details. One emerging approach in protein analysis is PCR-coupled protein quantification, often denoted as immuno-PCR (iPCR), which targets soluble proteins. Here we review recent trends and applications in iPCR assays that may bridge the gap between classical enzyme-linked immunosorbent assays and mass spectrometry methodologies in terms of sensitivity and multiplexing.

Delineation of known and new transcript variants of the SETMAR (Metnase) gene and the expression profile in hematologic neoplasms

SET domain and mariner transposase fusion gene (SETMAR), also known as Metnase, has previously been shown to suppress the formation of chromosomal translocation in mouse fibroblasts. Despite the fact that hematologic malignancies are often characterized by chromosomal rearrangements, no studies have hitherto investigated the expression pattern of the gene in these disorders. We hypothesized that a high expression of SETMAR protected the cells from chromosomal rearrangements; thus, we examined the mRNA expression of SETMAR transcript variants in hematologic patients. We identified six transcript variants (var1, var2, var5, varA, varB, varC), of which three had not been reported previously. Expression levels were quantified by transcript-specific quantitative polymerase chain reaction in 15 healthy individuals, 70 acute myeloid leukemia (AML) patients (translocation positive, n= 30 [AML(TPos)], translocation negative, n = 40 [AML(TNeg)]), seven patients with mantle cell lymphoma (t [11,14] positive), and 13 patients with chronic myeloid leukemia (t [9,22] positive). All variants were significantly overexpressed in both subgroups of AML compared with healthy individuals (var1 and var2: p < 0.00001 for both AML subgroups, varA and varB: p = 0.0002, var5: p = 0.0008, and varC: p = 0.0001 for AML(TNeg); varA: p = 0.0048, varB and var5: p = 0.0001, varC: p = 0.0017). When comparing the expression in AML(TNeg) and AML(TPos), we found a significantly increased expression of the full length SETMAR in AML(TNeg) (var1: p = 0.047), suggesting a protective effect of high SETMAR expression on formation of chromosomal translocations. In conclusion, we have found known and novel SETMAR splice variants to be significantly increased in AML. To our knowledge, this is the first study that describes an expression profile of SETMAR in subgroups of hematologic malignancies, which can be linked to the incidence of chromosomal rearrangements.

Acidic Environment Induces Dimerization and Ligand Binding Site Collapse in the Vps10p Domain of Sortilin

Sortilin is a neuronal receptor involved in transmembrane signaling, endocytosis, and intracellular sorting of proteins. It cycles through a number of cellular compartments where it encounters various acidic conditions. The crystal structure of the sortilin ectodomain has previously been determined at neutral pH. Here, we present the 3.5-Å resolution crystal structure of sortilin at pH 5.5, which represents an environment similar to that of late endosomes, where ligands are released. The structure reveals an overall distortion of the 10-bladed β-propeller domain. This distortion and specific conformational changes, caused by protonation of a number of histidine residues, render the currently known binding sites unavailable for ligand binding. Access to the binding sites is furthermore blocked by a reversible and pH-dependent formation of tight sortilin dimers, also confirmed by electron microscopy, size-exclusion chromatography, and mutational studies. This study reveals how sortilin binding sites are disrupted and explains pH-dependent ligand affinity.

Combination of RNA- and exome sequencing: Increasing specificity for identification of somatic point mutations and indels in acute leukaemia.

Handling of large data sets from whole exome sequencing is a challenge, not the least because of the high risk of detecting false positive variants. Furthermore, there is still no consensus regarding what stage filtering of variants is performed in the bioinformatics pipeline to produce consistent result sets, the extent of filtering and how this is most optimal performed. We hypothesized that combination of coding transcriptome and exomes enables precise identification of both somatic single nucleotide and indel variants early in the bioinformatics process, superseding the need for extensive annotation and validation from external databases. Exome and RNA-sequencing were performed on diagnosis-remission paired bone marrow samples from 5 cytogenetically normal AML, i.e. sequencing of 20 samples in total. Intersection of rare exome and RNA variants, exclusively found in the diagnostic samples, yielded a median of 6 somatic point mutations and small indels, ranging from 3 to 21. Close correlation between routine diagnostic biomarkers was observed in 29/30 laboratory tests, with the exception of a large FLT3 internal tandem repeat, whereas WT1 with nonsense mutation lacked RNA transcripts. Additionally, the assay revealed mutations in DNMT3A, IDH2, TET2 and IL32 frame shift, not encompassed by routine panel. We thus describe a procedure to effectively reduce observations to a focused subset of high specificity. The approach is applicable to precise screening of both putative driver mutations and, often heterogeneous, discrete patient specific somatic combinations.

Skewed ratio between type 1 and type 2 calreticulin mutations in essential thrombocytosis patients with concomitant Janus kinase 2 V617F mutation

Detection of somatic mutations in cardinal driver genes is a strong argument for diagnosis in classical Philadelphia-negative myeloproliferative neoplasms (MPNs). Driver mutations in Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL), are generally considered mutually exclusive, but several reports have suggested that they coexist in a small subgroup of patients. In this study, we retrospectively searched for CALR mutations in 136 suspected MPN patients with low allelic burden (≤5%) JAK2 V617F. Fifteen patients with concomitant JAK2 V617F and CALR mutations were identified, of whom 10 were diagnosed with essential thrombocytosis (ET). More than 50 different indel mutations in exon 9 of CALR have been reported, with type 1 (52 bp deletion) and type 2 (5 bp insertion) accounting for more than 80% of CALR-mutated MPN cases. Type 1 is generally considered the most common mutation, but, interestingly, our double-mutated ET patients seem to have an inversed ratio between type 1 and type 2 CALR mutations. Our findings support the possibility of coexisting JAK2 V617F and CALR mutations and stress the importance of further molecular screening in MPN patients with low allele frequencies of JAK2 V617F.

Kinetics of del(7q) driven leukemogenesis in a patient with JAK2 V617F and TET2 mutated chronic myeloproliferative neoplasm.

Chronic myeloid neoplasms have susceptibility to transform into acute myeloid leukemia due to attainment of additional molecular lesions. We here describe the kinetics of a del(7q) driven leukemogenesis in a patient with multiple TET2 mutations and JAK2 V617F mutated chronic myeloproliferative neoplasm. The del(7q) emerged in the accelerated phase of disease, which was preceded by a lag phase of almost three years with normalized peripheral blood cell counts. Our results reveal that the del(7q), independently of other lesions, acts as a leukemic driver in this patient and that the stable long-lasting normalization of peripheral blood cell values concealed pending transformation.