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Background Systemic mastocytosis (SM) is a rare myeloproliferative disease that results from a clonal proliferation of abnormal mast cells in one or more extra-cutaneous organs. Systemic mastocytosis with an associated hematological neoplasm (SM-AHN) is the second most common subgroup and is diagnosed when WHO criteria for both SM and a hematological neoplasm of non-mast cell lineage are met. The SM-AHN category as currently proposed is highly heterogeneous in terms of pathogenesis, clinical presentation, and prognosis. Case presentation We present the first reported case of SM-AHN associated with two hematological malignancies of different lineages, a monocytic myeloid sarcoma and a B-cell chronic lymphatic leukemia. Cytogenetic and molecular analyses revealed a distinct clonal origin of the two associated malignancies. The SM-myeloid sarcoma clone demonstrated an abnormal karyotype, trisomy 8 and del(13)(q12.3q14.3), as well as mutations in KITD816V, DNMT3A and RUNX1. The DNMT3A mutation could be detected years before disease onset, supporting its potential role as early driver of leukemogenesis. No genetic aberrations could be identified in the CLL clone, which is assumed to present coincidentally. Conclusions This report highlights the importance of full diagnostic work-up in SM patients in whom an associated hematological malignancy is suspected. Moreover, the importance of genetic analysis is highlighted, as it provides additional insights in the underlying clonal pathogenesis of different phenotypes, can aid in risk stratification, and may help identify potential therapy targets.

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive clonal neoplasm of early childhood, classified as an overlap myeloproliferative/myelodysplastic neoplasm by the World Health Organization. In 90% of the patients with JMML, typical initiating mutations in the canonical Ras pathway genes NF1, PTPN11, NRAS, KRAS, and CBL can be identified. Hematopoietic stem cell transplantation (HSCT) currently is the established standard of care in most patients, although long-term survival is still only 50-60%. Given the limited therapeutic options and the important morbidity and mortality associated with HSCT, new therapeutic approaches are urgently needed. Hyperactivation of the Ras pathway as disease mechanism in JMML lends itself to the use of targeted therapy. Targeted therapy could play an important role in the future treatment of patients with JMML. This review presents a comprehensive overview of targeted therapies already developed and evaluated in vitro and in vivo in patients with JMML.

We previously showed that germline SAMD9 and SAMD9L (SAMD9/9L) disorders are the most common predisposition to childhood myelodysplastic syndromes with bone marrow failure (BMF). Heterozygous mutations in both genes are growth inhibitory and undergo negative selection by acquiring compensatory rescue events in blood. Here, we describe the first report of SAMD9L genetic rescue occurring during embryonal development and resulting in germline triple-allelic mosaicism (1 wildtype (WT) and 2 mutant alleles), where only one mutant allele was transmitted to each of the two diseased children. In a family of 2 affected siblings with hypocellular BMF, the Index case carried germline heterozygous SAMD9L V1512M mutation and her brother was heterozygous for V1512L mutation. The clinical presentation for Index with V1512M was more severe compared to her brother with V1512L mutation. Genetic assessment of the parents showed the asymptomatic mother to be a triple-allelic mosaic, carrying WT SAMD9L allele at ~50% frequency in all tissues, while both V1512M and V1512L mutant alleles “competed” in their allelic distribution depending on the tissue origin. Single-cell DNA sequencing on her peripheral blood (PB) revealed 3 independent diploid clones: V1512M in 14%, V1512L in 68%, and WT (due to revertant UPD7q) in 18% of cells. Because her parents were SAMD9L WT, one of the mutations likely arose de novo and underwent failed embryonic rescue attempt leading to a second mutation. Towards studying the effect on hematopoiesis in vitro, we knocked in V1512M and V1512L mutations in inducible pluripotent stem cells (iPSC). For in vivo functional validation, we created constitutive mouse models with ortholog mutations (V1507M and V1507L). Mutant hematopoietic progenitor cells from iPSC had severely compromised proliferative capacity and yielded fewer erythroid and myeloid cells. This effect was more severe in V1512M vs. V1512L mutants. The divergent mutational phenotypes were also replicated in our mouse models: two-thirds (13/19) of the heterozygous V1507M (V1507Mhet) pups died before 4 weeks of age. In contrast, V1507Lhet mice had overall survival equal to WT mice, while one-third of homozygous V1507L (V1507Lhom) mice showed decreased survival post 19 weeks of age. At baseline, V1507Mhet mice showed severe growth retardation with multisystemic issues, which were absent in the V1507L mutant models. PB cytopenia (anemia, B-cell lymphopenia) though observed in both V1507Mhet and V1507Lhom mice, the degree of severity was high in the V1507Mhet than in the V1507Lhom model. Meanwhile, V1507Lhet mice had blood counts similar to WT. In summary, V1512M compared to V1512L mutation resulted in a more severe phenotype in all analyzed model systems. This observation showed how different mutational permutations of the same amino acid can exert divergent phenotypic effects in SAMD9L BMF disorder. Therefore, together with our genetic analysis, we could postulate that the V1512L mutation arose as a failed rescue attempt of V1512M during embryogenesis. Citation Format: Sushree S Sahoo, Charnise Goodings, Shondra M. Pruett-Miller, Maria A Lillo, Lei Han, Baranda Hansen, Ti-Cheng Chang, Tim Lammens, Mattias Hofmans, Marta Derecka, Barbara De Moerloose, Marcin W Wlodarski. Divergent effects of MDS predisposing SAMD9L point mutations, in humans, mice, and cellular models [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A51.

Pediatric acute myeloid leukemia (pedAML) is a heterogeneous blood cancer that affects children. Although survival rates have significantly improved over the past few decades, 20-30% of children will succumb due to treatment-related toxicity or relapse. The molecular characterization of the leukemic stem cell, shown to be responsible for relapse, is needed to improve treatment options and survival. Recently, it has become clear that non-coding RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), play a role in the development of human diseases, including pediatric cancer. Nevertheless, non-coding RNA expression data in pedAML are scarce. Here, we explored lncRNA (n = 30,168) and miRNA (n = 627) expression in pedAML subpopulations (leukemic stem cells (LSCs) and leukemic blasts (L-blasts)) and their normal counterparts (hematopoietic stem cells and control myeloblasts). The potential regulatory activity of differentially expressed lncRNAs in LSCs (unique or shared with the L-blast comparison) on miRNAs was assessed. Moreover, pre-ranked gene set enrichment analyses of (anti-) correlated protein-coding genes were performed to predict the functional relevance of the differentially upregulated lncRNAs in LSCs (unique or shared with the L-blast comparison). In conclusion, this study provides a catalog of non-coding RNAs with a potential role in the pathogenesis of pedAML, paving the way for further translational research studies. ispartof: CANCERS vol:14 issue:9 ispartof: location:Switzerland status: published

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct., The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 programme 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). TS and LS were supported by a Scientific Grant from the Medical University of Silesia Nr. PCN-1-050/K/0/K.

Outcomes in childhood T-cell acute lymphoblastic leukaemia (T-ALL) are steadily improving due to intensive therapy. Between 1989 and 2008, 599 children with newly diagnosed T-ALL were enrolled in two successive European Organization for Research and Treatment of Cancer - Children's Leukaemia Group trials (58881 and 58951), both based on the Berlin-Frankfurt-Munster protocol and without cranial irradiation. In the latter trial induction chemotherapy was intensified. The most important randomizations were Medac Escherichia coli asparaginase versus Erwinia asparaginase in trial 58881, and dexamethasone (6 mg/m2/day) versus prednisolone (60 mg/m2/day) and prolonged versus conventional asparaginase duration in trial 58951. 8-year event-free survival (EFS) increased from 65·1% to 74·0% in trial 58951. Improvement was most profound for patients with white blood cell (WBC) counts, SCOPUS: ar.j, info:eu-repo/semantics/published

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.

Pediatric myelodysplastic syndrome (PMDS) is a very rare and still poorly characterized disorder. In this work, we identified novel potential targets of PMDS by determining genes with aberrant expression, which can be correlated with PMDS pathogenesis. We identified 291 differentially expressed genes (DEGs) in PMDS patients, comprising genes involved in the regulation of apoptosis and the cell cycle, ribosome biogenesis, inflammation and adaptive immunity. Ten selected DEGs were then validated, confirming the sequencing data. These DEGs will potentially represent new molecular biomarkers and therapeutic targets for PMDS.

Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We evaluated the immunophenotypic profile of 72 AMKL and 114 non-AMKL AML patients using the EuroFlow AML panel. Univariate and multivariate/multidimensional analyses were performed to identify most relevant markers contributing to the diagnosis of AMKL. AMKL patients were subdivided into transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), AML—not otherwise specified with megakaryocytic differentiation (NOS-AMKL), and AMKL—other patients (AML patients with other WHO classification but with flowcytometric features of megakaryocytic differentiation). Flowcytometric analysis showed good discrimination between AMKL and non-AMKL patients based on differential expression of, in particular, CD42a.CD61, CD41, CD42b, HLADR, CD15 and CD13. Combining CD42a.CD61 (positive) and CD13 (negative) resulted in a sensitivity of 71% and a specificity of 99%. Within AMKL patients, TAM and ML-DS patients showed higher frequencies of immature CD34+/CD117+ leukemic cells as compared to NOS-AMKL and AMKL-Other patients. In addition, ML-DS patients showed a significantly higher expression of CD33, CD11b, CD38 and CD7 as compared to the other three subgroups, allowing for good distinction of these patients. Overall, our data show that the EuroFlow AML panel allows for straightforward diagnosis of AMKL and that ML-DS is associated with a unique immunophenotypic profile., 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 HematoOncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA). The work of C.E.P. and E.S.C. was partially supported by FAPERJ (Grant E26/200.840/2021- CNE; E26/210.379/2018 and E26/110.105/2014); CAPES-PROEX; and CNPq (Grant 306258/2019-6 and 303765/2018-6). M.N. and E.M. were supported by the Ministry of Health of the Czech Republic, grant number NU20J-07-00028. S.M. was supported by Acción Estratégica en Salud (AES) (Grant PI21_01115) and the grant of CIBERONC of the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Madrid, Spain and FONDOS FEDER (no. CB16/12/00400).

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.