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Background Still 30–40% of pediatric acute myeloid leukemia (pedAML) patients relapse. Delineation of the transcriptomic profile of leukemic subpopulations could aid in a better understanding of molecular biology and provide novel biomarkers. Methods Using microarray profiling and quantitative PCR validation, transcript expression was measured in leukemic stem cells (LSC, n = 24) and leukemic blasts (L-blast, n = 25) from pedAML patients in comparison to hematopoietic stem cells (HSCs, n = 19) and control myeloblasts (C-blast, n = 20) sorted from healthy subjects. Gene set enrichment analysis was performed to identify relevant gene set enrichment signatures, and functional protein associations were identified by STRING analysis. Results Highly significantly overexpressed genes in LSC and L-blast were identified with a vast majority not studied in AML. CDKN1A, CFP, and CFD (LSC) and HOMER3, CTSA, and GADD45B (L-blast) represent potentially interesting biomarkers and therapeutic targets. Eleven LSC downregulated targets were identified that potentially qualify as tumor suppressor genes, with MYCT1, PBX1, and PTPRD of highest interest. Inflammatory and immune dysregulation appeared to be perturbed biological networks in LSC, whereas dysregulated metabolic profiles were observed in L-blast. Conclusion Our study illustrates the power of taking into account cell population heterogeneity and reveals novel targets eligible for functional evaluation and therapy in pedAML. Impact Novel transcriptional targets were discovered showing a significant differential expression in LSCs and blasts from pedAML patients compared to their normal counterparts from healthy controls. Deregulated pathways, including immune and metabolic dysregulation, were addressed for the first time in children, offering a deeper understanding of the molecular pathogenesis. These novel targets have the potential of acting as biomarkers for risk stratification, follow-up, and targeted therapy. Multiple LSC-downregulated targets endow tumor suppressor roles in other cancer entities, and further investigation whether hypomethylating therapy could result into LSC eradication in pedAML is warranted.

Juvenile myelomonocytic leukemia (JMML) treatment primarily relies on hematopoietic stem cell transplantation and results in long-term overall survival of 50–60%, demonstrating a need to develop novel treatments. Dysregulation of the non-coding RNA transcriptome has been demonstrated before in this rare and unique disorder of early childhood. In this study, we investigated the therapeutic potential of targeting overexpressed long non-coding RNAs (lncRNAs) in JMML. Total RNA sequencing of bone marrow and peripheral blood mononuclear cell preparations from 19 untreated JMML patients and three healthy children revealed 185 differentially expressed lncRNA genes (131 up- and 54 downregulated). LNA GapmeRs were designed for 10 overexpressed and validated lncRNAs. Molecular knockdown (≥ 70% compared to mock control) after 24 h of incubation was observed with two or more independent GapmeRs in 6 of them. For three lncRNAs (lnc-THADA-4, lnc-ACOT9-1 and NRIR) knockdown resulted in a significant decrease of cell viability after 72 h of incubation in primary cultures of JMML mononuclear cells, respectively. Importantly, the extent of cellular damage correlated with the expression level of the lncRNA of interest. In conclusion, we demonstrated in primary JMML cell cultures that knockdown of overexpressed lncRNAs such as lnc-THADA-4, lnc-ACOT9-1 and NRIR may be a feasible therapeutic strategy.

Juvenile myelomonocytic leukemia (JMML), a rare myelodysplastic/myeloproliferative neoplasm of early childhood, is characterized by clonal growth of RAS signaling addicted stem cells. JMML subtypes are defined by specific RAS pathway mutations and display distinct gene, microRNA (miRNA) and long non-coding RNA expression profiles. Here we zoom in on circular RNAs (circRNAs), molecules that, when abnormally expressed, may participate in malignant deviation of cellular processes. CirComPara software was used to annotate and quantify circRNAs in RNA-seq data of a “discovery cohort” comprising 19 JMML patients and 3 healthy donors (HD). In an independent set of 12 JMML patients and 6 HD, expression of 27 circRNAs was analyzed by qRT-PCR. CircRNA-miRNA-gene networks were reconstructed using circRNA function prediction and gene expression data. We identified 119 circRNAs dysregulated in JMML and 59 genes showing an imbalance of the circular and linear products. Our data indicated also circRNA expression differences among molecular subgroups of JMML. Validation of a set of deregulated circRNAs in an independent cohort of JMML patients confirmed the down-regulation of circOXNAD1 and circATM, and a marked up-regulation of circLYN, circAFF2, and circMCTP1. A new finding in JMML links up-regulated circMCTP1 with known tumor suppressor miRNAs. This and other predicted interactions with miRNAs connect dysregulated circRNAs to regulatory networks. In conclusion, this study provides insight into the circRNAome of JMML and paves the path to elucidate new molecular disease mechanisms putting forward circMCTP1 up-regulation as a robust example.

© 2020 Linskens, Diks, Neirinck, Perez-Andres, De Maertelaere, Berkowska, Kerre, Hofmans, Orfao, van Dongen, Haerynck, Philippé and Bonroy., Background: Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results. Methods: FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt™ software (Cytognos). For comparison, percentage differences in absolute cell counts/µL were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples. Results: The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (, The coordination and innovation processes of this study were supported by the EuroFlow Consortium. The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA). JN is granted by the Fund for Scientific Research, TBM Funding, Belgium (T000119N). This work was also supported by a grant of the Grand Challenges Program of VIB.

Background: Light microscopy and antigen-based rapid diagnostic tests are the primary diagnostic tools for detecting malaria, although being labour-intensive and frequently challenged by lack of personnel's experience and low levels of parasite density. The latter being especially important in non-endemic settings. Novel molecular techniques aim to overcome this drawback. The objective of this study was to assess the diagnostic performance of the illumigene malaria assay (R) (Meridian Bioscience) compared to microscopy, RDT and real-time PCR. This loop-mediated isothermal amplification (LAMP) assay is a qualitative in vitro diagnostic test for the direct detection of Plasmodium spp. DNA in human venous whole blood samples. Methods: The illumigene assay was assessed on a retrospective panel of stored blood samples (n = 103) from returned travellers and external quality control samples (n = 12). Additionally the assay was prospectively assessed on 30 fresh routine samples with a request for malaria diagnosis. The illumigene assay was compared to microscopy, RDT and Plasmodium species specific real-time PCR. Results: In the retrospective evaluation, the illumigene assay showed 100% agreement with the real-time PCR, RDT and microscopy yielding a sensitivity and specificity of 100% (95% CI 95.1-100% and 89.7-100%, respectively). Seven samples from patients recently treated for Plasmodium falciparum infection that were RDT positive and microscopy negative yielded positive test results. The performance of the illumigene assay equals that of microscopy combined with RDT in the prospective panel with three false negative RDT results and one false negative microscopy result. Excellent concordance with PCR was observed. The limit of detection of the assay approached 0.5 parasites/mu L for both P. falciparum and Plasmodium vivax. Conclusion: In non-endemic regions where the diagnostic process for malaria infections is questioned by lack of experience and low levels of parasite densities, the illumigene assay can be of value. Due to its high sensitivity, the LAMP assay may be considered as primary diagnostic test. The results of this study indicate that negative screen results do not need further confirmation. However, before implementation, this approach needs to be confirmed in larger, prospective studies. A shortcoming of this assay is that no species identification nor determination of parasite density are possible.

The natural history of primary eosinophilia remains highly variable and is characterized by underlying disease heterogeneity. Chronic eosinophilic leukemia, not otherwise specified (CEL-NOS) is a rare and aggressive disease characterized by non-specific cytogenetic abnormalities or elevated blasts, with high risk of transformation to acute leukemia. We describe a case of CEL-NOS with two hierarchically related non-specific cytogenetic rearrangements, associated with an NPM1 mutation and followed by evolution to secondary AML. NPM1 mutations are not previously described in CEL-NOS. Keywords: CEL-NOS, Hypereosinophilia, Secondary AML, NPM1 mutation, Decitabine

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive myelodysplastic and myeloproliferative disorder of early childhood. It is characterized by proliferation of granulocytic and monocytic cells.[1][1] Currently, hematopoietic stem cell transplantation (HSCT) is the standard of care and

BACKGROUND Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive myelodysplastic/myeloproliferative disorder of early childhood. Hematopoietic stem cell transplantation results in long-term overall survival of only 50-60% and is fraught with frequent relapse and toxicity. Consequently, there is need to develop novel treatments. Evidence is growing that non-coding RNAs can serve as alternative therapeutic targets. Long non-coding RNAs (lncRNAs) are a recently discovered class of RNAs, with a minimum length of 200 nucleotides, of which perturbation can impact the cancer phenotype in vivo. Recently, we documented for the first time the lncRNA landscape in 44 JMML patients using microarrays and have associated lncRNA expression with clinical and molecular characteristics. Also, we demonstrated that JMML patients exhibit a distinct lncRNA expression profile compared to healthy controls and we identified 15 lncRNAs overexpressed in JMML patients. AIM The aim of this study is to identify overexpressed lncRNAs in JMML and to provide proof-of-concept for lncRNA perturbation as novel therapeutic strategy in this disease. METHODS To further identify overexpressed lncRNAs in JMML, total RNA from isolated mononuclear cell preparations from 19 previously untreated JMML patients (median age: 2.02 years) and 3 normal pediatric bone marrow (NPBM) samples from healthy controls (siblings screened for transplantation) was sequenced (HiSeq, Illumina). Differential gene expression (adjusted P-value < 0.05 and FDR < 0.10) was performed with DESEQ2 and EdgeR (R Bioconductor). Functional analysis of differentially expressed lncRNAs was performed through a lncRNA-mRNA interaction network (lncPath, R Bioconductor). A subset of overexpressed lncRNAs, based on fold change ≥2 and low to absent expression in NPBM, were subsequently validated by quantitative reverse-transcriptase PCR (qPCR). Antisense LNA™ GapmeRs (Qiagen), potent antisense oligonucleotides used for highly efficient inhibition of mRNA and lncRNA function, were designed for a subset of significantly overexpressed lncRNAs. As no JMML cell lines are available, cell lines from other pediatric and adult haematopoietic malignancies were selected based on publically available RNA-seq data and lncRNA expression was verified on qPCR. The effect of GapmeR treatment on lncRNA expression and cell viability was tested with qPCR and flow cytometry viability assays (7-AAD and annexin V staining). RESULTS Total RNA sequencing additionally revealed presence of 122 upregulated and 47 downregulated lncRNAs, most of which not present on the microarray. JMML patients clustered together and divergent from healthy controls on principal component analysis using only lncRNAs. Different pathways associated with RAS-MAPK signaling, cancer development, DNA metabolism and cell cycle were identified as being synergistically regulated by these differentially expressed lncRNAs through a lncRNA-mRNA interaction network. Forty-two lncRNAs (55 different transcripts) showing overexpression in JMML were validated with qPCR and in 13 transcripts from 11 lncRNAs a significantly higher expression could be observed in JMML patients compared to NPBM (Figure 1A), thus providing potential therapeutic targets. A minimum of four different antisense LNA GapmeRs were subsequently designed for each of these validated lncRNAs, overexpressed in JMML. For each lncRNA, one or more hematopoietic cell lines, showing expression of that lncRNA, could be identified and validated with qPCR. In vitro incubation of these cell lines with GapmeRs against lncRNA with expression in these cell lines, revealed concentration-dependent molecular knockdown in ≥ 2 GapmeRs for 4/5 lncRNAs (Figure 1B). For two targets, lnc-THADA4-1 and lnc-ACOT9, additional cell viability assays were performed and an effect on cell viability could be observed in GapmeR treated wells (Figure 1C). CONCLUSION Using microarray and total RNA sequencing, we documented the lncRNA landscape in JMML, and identified deregulated lncRNAs associated with key processes in JMML pathogenesis. We further provided proof-of-concept that knockdown of these lncRNAs using LNA GapmeRs can be a feasible therapeutic strategy in vitro in hematopoietic cell lines. Subsequently, safety and efficacy of this novel therapeutic strategy needs to be validated in JMML xenograft models in vivo. Figure 1 Disclosures Niemeyer: Celgene: Consultancy.

In September 2013, an 87-year-old man presented to his general practitioner with progressive dyspnoea, mild chest discomfort and marked muscle weakness since three weeks. He was referred to our eme...

Staphylococcus saprophyticus is a well-known cause of uncomplicated urinary tract infections, especially in young and sexually active women. Presence in blood cultures is rare and often attributed to contamination. When bacteremia is significant, it occurs mostly in patients with hematologic malignancies and is predominantly catheter-related. However, we describe a case of significant bacteremia with S. saprophyticus associated with urinary tract infection after extracorporeal shock wave lithotripsy of an ureterolithiasis in an otherwise healthy patient.