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Mutations in five canonical Ras pathway genes (NF1, NRAS, KRAS, PTPN11 and CBL) are detected in nearly 90% of patients with juvenile myelomonocytic leukemia (JMML), a frequently fatal malignant neoplasm of early childhood. In this report, we describe seven patients diagnosed with SH2B3-mutated JMML, including five patients who were found to have initiating, loss-of-function mutations in the gene. SH2B3 encodes the adaptor protein LNK, a negative regulator of normal hematopoiesis upstream of the Ras pathway. These mutations were identified to be germline, somatic or a combination of both. Loss of function of LNK, which has been observed in other myeloid malignancies, results in abnormal proliferation of hematopoietic cells due to cytokine hypersensitivity and activation of the JAK/STAT signaling pathway. In vitro studies of induced pluripotent stem cell-derived JMML-like hematopoietic progenitor cells also demonstrated sensitivity of SH2B3-mutated hematopoietic progenitor cells to JAK inhibition. Lastly, we describe two patients with JMML and SH2B3 mutations who were treated with the JAK1/2 inhibitor ruxolitinib. This report expands the spectrum of initiating mutations in JMML and raises the possibility of targeting the JAK/STAT pathway in patients with SH2B3 mutations.

Purpose: Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric malignancy with myelodysplastic and myeloproliferative features. Curative treatment is restricted to hematopoietic stem-cell transplantation. Fludarabine combined with cytarabine (FLA) and 5-azacitidine (AZA) monotherapy are commonly used pre-transplant therapies. Here, we present a drug screening strategy using a flow cytometry-based precision medicine platform to identify potential additional therapeutic vulnerabilities., Methods: We screened 120 dual- and 10 triple-drug combinations (DCs) on peripheral blood (n = 21) or bone marrow (n = 6) samples from 27 children with JMML to identify DCs more effectively reducing leukemic cells than the DCs' components on their own. If fewer leukemic cells survived a DC ex vivo treatment compared with that DC's most effective component alone, the drug effect was referred to as cooperative. The difference between the two resistant fractions is the effect size., Results: We identified 26 dual- and one triple-DC more effective than their components. The differentiation agent tretinoin (TRET; all-trans retinoic acid) reduced the resistant fraction of FLA in 19/21 (90%) samples (decrease from 15% [2%-61%] to 11% [2%-50%] with a mean effect size of 3.8% [0.5%-11%]), and of AZA in 19/25 (76%) samples (decrease from 69% [34%-100+%] to 47% [17%-83%] with a mean effect size of 16% [0.3%-40%]). Among the resistant fractions, the mean proportion of CD38 + cells increased from 7% (0.03%-25%; FLA) to 17% (0.3%-38%; FLA + TRET) or from 10% (0.2%-31%; AZA) to 51% (0.8%-88%; AZA + TRET)., Conclusion: TRET enhanced the effects of FLA and AZA in ex vivo assays with primary JMML samples.

Juvenile myelomonocytic leukemia (JMML) is a rare, aggressive pediatric disorder characterized by pathologic myeloproliferation because of alterations in RAS pathway genes. NRAS -mutated JMML encompasses a broad range of clinical severity. Herein we describe 4 unique cases of NRAS -mutated JMML and JMML-like myeloproliferation, 2 with somatic mutations and 2 with germline mutations. These cases illustrate the diverse clinical and hematologic presentation of this subtype of JMML, including a very unusual example presenting with Auer rods. Lastly, this is the first report of patients with phenotypic Costello syndrome presenting with JMML-like myeloproliferation, highlighting an important clinical phenomenon that has not been previously described., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Juvenile myelomonocytic leukemia (JMML) is a rare heterogeneous hematological malignancy of early childhood characterized by causative RAS pathway mutations. Classifying patients with JMML using global DNA methylation profiles is useful for risk stratification. We implemented machine learning algorithms (decision tree, support vector machine, and naïve Bayes) to produce a DNA methylation-based classification according to recent international consensus definitions using a well-characterized pooled cohort of patients with JMML (n = 128). DNA methylation was originally categorized into three subgroups: high methylation (HM), intermediate methylation (IM), and low methylation (LM), which is a trichotomized classification. We also dichotomized the subgroups as HM/IM and LM. The decision tree model showed high concordances with 450k-based methylation [82.3% (106/128) for the dichotomized and 83.6% (107/128) for the trichotomized subgroups, respectively]. With an independent cohort (n = 72), we confirmed that these models using both the dichotomized and trichotomized classifications were highly predictive of survival. Our study demonstrates that machine learning algorithms can generate clinical parameter-based models that predict the survival outcomes of patients with JMML and high accuracy. These models enabled us to rapidly and effectively identify candidates for augmented treatment following diagnosis., (© 2022. The Author(s).)

Development of normal blood cells is often suppressed in juvenile myelomonocytic leukemia (JMML), a myeloproliferative neoplasm (MPN) of childhood, causing complications and impacting therapeutic outcomes. However, the mechanism underlying this phenomenon remains uncharacterized. To address this question, we induced the most common mutation identified in JMML (Ptpn11E76K) specifically in the myeloid lineage with hematopoietic stem cells (HSCs) spared. These mice uniformly developed a JMML-like MPN. Importantly, HSCs in the same bone marrow (BM) microenvironment were aberrantly activated and differentiated at the expense of self-renewal. As a result, HSCs lost quiescence and became exhausted. A similar result was observed in wild-type (WT) donor HSCs when co-transplanted with Ptpn11E76K/+ BM cells into WT mice. Co-culture testing demonstrated that JMML/MPN cells robustly accelerated differentiation in mouse and human normal hematopoietic stem/progenitor cells. Cytokine profiling revealed that Ptpn11E76K/+ MPN cells produced excessive IL-1β, but not IL-6, T NF-α, IFN-γ, IL-1α, or other inflammatory cytokines. Depletion of the IL-1β receptor effectively restored HSC quiescence, normalized their pool size, and rescued them from exhaustion in Ptpn11E76K/+/IL-1R-/- double mutant mice. These findings suggest IL-1β signaling as a potential therapeutic target for preserving normal hematopoietic development in JMML., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Juvenile myelomonocytic leukemia (JMML) is a malignant myeloproliferative disorder arising in infants and young children. The origin of this neoplasm is attributed to an early deregulation of the Ras signaling pathway in multipotent hematopoietic stem/progenitor cells. Since JMML is notoriously refractory to conventional cytostatic therapy, allogeneic hematopoietic stem cell transplantation remains the mainstay of curative therapy for most cases. However, alternative therapeutic approaches with small epigenetic molecules have recently entered the stage and show surprising efficacy at least in specific subsets of patients. Hence, the establishment of preclinical models to test novel agents is a priority. Induced pluripotent stem cells (IPSCs) offer an opportunity to imitate JMML ex vivo, after attempts to generate immortalized cell lines from primary JMML material have largely failed in the past. Several research groups have previously generated patient-derived JMML IPSCs and successfully differentiated these into myeloid cells with extensive phenotypic similarities to primary JMML cells. With infinite self-renewal and the capability to differentiate into multiple cell types, JMML IPSCs are a promising resource to advance the development of treatment modalities targeting specific vulnerabilities. This review discusses current reprogramming techniques for JMML stem/progenitor cells, related clinical applications, and the challenges involved.

Neurofibromatosis type 1 is a common multisystem autosomal dominant syndrome caused by pathogenic heterozygous variants in the neurofibromin gene (NF1). It is associated with a substantially increased cancer risk. Mosaicism for NF1 has been clinically well-established for "second hit" variants in skin lesions and tumor tissues. Here, we report on a 3-month-old boy with multiple café au lait macules (CAMs) and juvenile myelomonocytic leukemia (JMML) who was found to carry a previously established pathogenic NF1 variant (c.586+5G>A), as revealed by whole-exome sequencing. Surprisingly, however, this variant was detected in the homozygous state in the patient and was absent in the parents and siblings. Deep sequencing of this variant using blood, buccal swabs and skin samples was performed. As expected for an NF1 gene mutation promoting JMML, the variant was detected in 90.6% of the blood DNA reads, in sharp contrast to the mere 5% and 0.74% of reads in the saliva- and skin fibroblast-derived DNA, respectively. Our analysis, therefore, confirmed postzygotic origin of the variant followed by a mitotic event resulting in its homozygosity, although we could not differentiate between the possibilities of a gene conversion and mitotic crossover. Apparently de novo homozygous variants should trigger a careful investigation into mosaicism to achieve accurate interpretation., (Copyright © 2021. Published by Elsevier Masson SAS.)

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) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin-CD34+CD38-CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a "first hit," (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease., Competing Interests: Disclosures: S. O’Byrne reports personal fees from Becton, Dickinson and Company (employee as of August 2020) outside the submitted work. M. Salio reports personal fees from Nucleome Therapeutics outside the submitted work. N. Sousos reports expenses reimbursement from Pfizer Limited for attending the Pfizer-organized meeting titled "Leaders in Leukaemia," July 12-13, 2019, in London, UK. N. Sousos reports expenses reimbursement from Constellation Pharmaceuticals for attending the Constellation 0610-02 MANIFEST Study EU Investigator Meeting, January 31-February 1, 2019, in Rome, Italy. N. Sousos reports an educational travel grant from AOP Orphan Pharmaceuticals AG for attending the European Hematology Association meeting "Diagnosis and Management of Myeloproliferative Neoplasms," October 12-14, 2017, in Budapest, Hungary. No other disclosures were reported., (© 2021 Louka et al.)

Myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap syndromes are unique myeloid neoplasms, with overlapping features of MDS and MPN. They consist of four adult onset entities including chronic myelomonocytic leukemia (CMML), MDS/MPN-ring sideroblasts-thrombocytosis (MDS/MPN-RS-T), BCR-ABL1 negative atypical chronic myeloid leukemia (aCML) and MDS/MPN-unclassifiable (MDS/MPN-U); with juvenile myelomonocytic leukemia (JMML) being the only pediatric onset entity. Among these overlap neoplasms, CMML is the most frequent and is hallmarked by the presence of sustained peripheral blood monocytosis with recurrent mutations involving TET2 (60%), SRSF2 (50%) and ASXL1 (40%); with RAS pathway mutations and JAK2V617F being relatively enriched in proliferative CMML subtypes (WBC ≥13 × 109/L). CMML usually presents in the 7th decade of life, with a male preponderance and is associated with a median overall survival of <36 months. Adverse prognosticators in CMML include increasing age, high WBC, presence of circulating immature myeloid cells, anemia, thrombocytopenia and truncating ASXL1 mutations. While allogeneic stem cell transplantation remains the only curative option, given the late onset of this neoplasm and high frequency of comorbidities, most patients remain ineligible. Hypomethylating agents such as azacitidine, decitabine and oral decitabine/cedazuridine have been US FDA approved for the management of CMML, with overall response rates of 40-50% and complete remission rates of <20%. While these agents epigenetically restore hematopoiesis in a subset of responding patients, they do not impact mutational allele burdens and eventual disease progression to AML remains inevitable. Newer treatment modalities exploiting epigenetic, signaling and splicing abnormalities commonly seen in CMML are much needed., Competing Interests: Conflict-of-interest disclosure: M.M.P. has served on advisory boards for Kura Oncology and Stemline Therapeutics. T.L. declares no competing financial interests., (© 2020 by The American Society of Hematology.)

Juvenile myelomonocytic leukaemia (JMML) is a rare clonal disorder of early childhood. Constitutive activation of the RAS pathway is the initial event in JMML. Around 90% of patients diagnosed with JMML carry a mutation in the PTPN11, NRAS, KRAS, NF1 or CBL genes. It has been demonstrated that after this first genetic event, an additional somatic mutation or epigenetic modification is involved in disease progression. The available genetic and clinical data have enabled researchers to establish relationships between JMML and several clinical conditions, including Noonan syndrome, Ras-associated lymphoproliferative disease, and Moyamoya disease. Despite scientific progress and the development of more effective treatments, JMML is still a deadly disease: the 5-year survival rate is ~50%. Here, we report on recent research having led to a better understanding of the genetic and molecular mechanisms involved in JMML., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Monocyte subset analysis by flow cytometry has been shown to be a useful diagnostic tool in chronic myelomonocytic leukemia in adults. An increase in the classical monocyte fraction (CD14++/CD16-) greater than 94.0% of total monocytes is considered highly sensitive and specific in distinguishing chronic myelomonocytic leukemia from other myeloproliferative disorders. In a pilot study of juvenile myelomonocytic leukemia cases, we noted that CD14++/CD16- monocyte fraction was >95% in de novo juvenile myelomonocytic leukemia (JMML) with somatic PTPN11 mutations but normal in those with monosomy 7 or Noonan syndrome. Monocyte subgroup profiling by itself is not diagnostic of JMML but may distinguish molecular subgroups within JMML., (© 2020 Wiley Periodicals LLC.)

Patients with juvenile myelomonocytic leukemia due to germline CBL mutation (10% to 15%) may have a subacute course occasionally associated with autoimmune disorders, which may resemble RAS-associated autoimmune lymphoproliferative disorder. In both conditions, prognosis and standard treatment for autoimmune phenomena remain poorly understood. We report the case of a 7-year-old boy with juvenile myelomonocytic leukemia with severe steroid-dependent uveitis, who did not respond to several therapeutic attempts with immunosuppressant agents, including sirolimus, and was finally successfully treated with adalimumab. This case offers further insight into the management of autoimmune disorders in the context of predisposing genetic conditions.

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare clonal myelodysplastic/myeloproliferative neoplasm of early childhood. Historically, it was difficult to diagnose clinically, as patients present with manifestations shared with other hematologic malignancies or viral infections. It is now clear that JMML is a disease of hyperactive RAS signaling., Patients and Methods: We examined the bone marrow of 41 Egyptian children with JMML by direct sequencing for mutations in the RAS pathway genes., Results: Mutations were detected in 33 (80%) of 41 patients. We identified 12 (29%) of 41 patients with PTPN11 mutation; 18 (44%) of 41 with RAS mutation; 9 (22%) of 41 with NRAS mutation; 9 (22%) of 41 with KRAS mutation; and 3 (7%) of 41 with CBL mutation. Eleven (92%) of the PTPN11 mutations were detected in exon 3 and 1 (8%) in exon 13. Seven of the NRAS mutations were in exon 2, and 2 were in exon 3. All KRAS mutations were in exon 2. The 3 cases with CBL mutation were homozygous mutations in exon 8. All the mutations detected in PTPN11, NRAS/KRAS, and the CBL genes were previously reported missense mutations in JMML., Conclusion: Our results demonstrate that Egyptian children diagnosed with JMML have high frequency of NRAS/KRAS mutations and lower frequency of PTPN11 mutations as compared with previous studies. The concept of mutually exclusive RAS pathway mutations was clearly observed in our patients. All cancer centers in our region should start implementing molecular diagnostic methods before confirming the diagnosis of JMML and before offering hematopoietic stem cell transplantation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches.

Objective: To investigate clinical features, diagnosis, treatment strategies and prognosis of juvenile myelomonocytic leukemia (JMML). Methods: The clinical data of 21 patients with JMML who were diagnosed in our hospital from January 2013 to May 2018 were retrospectively analyzed. Results: Among the 21 children with JMML, 16 were male and 5 were female. Out of the 21 children who were diagnosed with JMML, 7 were lost after treatment while the remaining 14 received A-3V chemotherapy regimen of South Korea. The effective response rate was 78.5%. The three-year overall survival (OS) rate and three-year disease-free survival (DFS) rate were (76.2 ± 14.8)% and (66.2 ± 14)%, respectively. Single factor analysis showed that PLT count ≤33×109/L, LDH level >500 U/L and HbF level >10% and chemotherapy only were the significant factors that lead to poor prognosis in children. Cox multivariate analysis showed that the choice of treatment options affected the prognosis of JMML children. By taking prognostic factors for long-term efficacy into account, patients with treatment strategy of chemotherapy alongside hematopoietic stem cell transplantation (HSCT) have a better prognosis. Conclusion: The PLT count, LDH level, HbF level and choice of treatment plan are important for the evaluation of prognosis for children with JMML. Although there is a lack of consistency in terms of donors but the A-3V scheme is relatively stable, so HSCT should be preferred for children with poor prognostic factors.

Juvenile myelomonocytic leukemia (JMML) has a poor prognosis in general, with hematopoietic stem cell transplant (HSCT) remaining the standard of care for cure. The hypomethylating agent, azacitidine, has been used as a bridging therapy to transplant. However, no patients have been treated with azacitidine without an HSCT post azacitidine. We report on an infant with JMML with somatic KRAS G12A mutation and monosomy 7 who achieved sustained remission following azacitidine monotherapy. He also developed an aberrant B-lymphoblast population which declined with similar kinetics as his JMML-associated abnormalities, suggesting that a B-lymphoblast population in JMML does not always progress to acute leukemia., (© 2019 Wiley Periodicals, Inc.)

We established mutated and non-mutated induced pluripotent stem cell (iPSC) clones from a patient with PTPN11 (c.226G>A)-mutated juvenile myelomonocytic leukaemia (JMML). Both types of iPSCs fulfilled the quality criteria. Mutated iPSC colonies generated significantly more CD34 + and CD34 + CD45 + cells compared to non-mutated iPSC colonies in a culture coated with irradiated AGM-S3 cells to which four growth factors were added sequentially or simultaneously. The haematopoietic differentiation potential of non-mutated JMML iPSC colonies was similar to or lower than that of iPSC colonies from a healthy individual. The PTPN11 mutation coexisted with the OSBP2 c.389C>T mutation. Zinc-finger nuclease-mediated homologous recombination revealed that correction of PTPN11 mutation in iPSCs with PTPN11 and OSBP2 mutations resulted in reduced CD34 + cell generation to a level similar to that obtained with JMML iPSC colonies with the wild-type of both genes, and interestingly, to that obtained with normal iPSC colonies. Transduction of the PTPN11 mutation into JMML iPSCs with the wild-type of both genes increased CD34 + cell production to a level comparable to that obtained with JMML iPSC colonies harbouring the two genetic mutations. Thus, PTPN11 mutation may be the most essential abnormality to confer an aberrant haematopoietic differentiation potential in this disorder., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

Cell surface molecules aberrantly expressed or overexpressed by myeloid leukemic cells represent potential disease-specific therapeutic targets for antibodies. MUC1 is a polymorphic glycoprotein, the cleavage of which yields two unequal chains: a large extracellular α subunit containing a tandem repeat array bound in a strong noncovalent interaction to a smaller β subunit containing the transmembrane and cytoplasmic domains. Because the α-chain can be released from the cell-bound domains of MUC1, agents directed against the α-chain will not effectively target MUC1 + cells. The MUC1 SEA (a highly conserved protein module so called from its initial identification in a sea urchin sperm protein, in enterokinase, and in agrin) domain formed by the binding of the α and β chains  represents a stable structure fixed to the cell surface at all times. DMB-5F3, a partially humanized murine anti-MUC1 SEA domain monoclonal antibody, was used to examine MUC1 expression in acute myeloid leukemia (AML) and was found to bind acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5) cell lines. We also examined monocytic neoplasms freshly obtained from patients including chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia, which were found to uniformly express MUC1. CD34 + /lin - /CD38 - or CD38 + presumed leukemic stem cell populations from CD34 + AML and CD34 - CD38 - or CD38 + populations from CD34 - AML were also found to express MUC1, although at low percentages. Based on these studies, we generated an anti-MUC1 immunotoxin to directly gauge the cytotoxic efficacy of targeting AML-bound MUC1. Using single-chain DMB-5F3 fused to recombinant gelonin toxin, the degree of AML cytotoxicity was found to correlate with MUC1 expression. Our data support the use of an anti-MUC1 SEA module-drug conjugates to selectively target and inhibit MUC1-expressing myelomonocytic leukemic cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Juvenile myelomonocytic leukemia (JMML) is an uncommon myeloproliferative neoplasm driven by Ras pathway mutations and hyperactive Ras/MAPK signaling. Outcomes for many children with JMML remain dismal with current standard-of-care cytoreductive chemotherapy and hematopoietic stem cell transplantation. We used patient-derived induced pluripotent stem cells (iPSCs) to characterize the signaling profiles and potential therapeutic vulnerabilities of PTPN11-mutant and CBL-mutant JMML. We assessed whether MEK, JAK, and PI3K/mTOR kinase inhibitors (i) could inhibit myeloproliferation and aberrant signaling in iPSC-derived hematopoietic progenitors with PTPN11 E76K or CBL Y371H mutations. We detected constitutive Ras/MAPK and PI3K/mTOR signaling in PTPN11 and CBL iPSC-derived myeloid cells. Activated signaling and growth of PTPN11 iPSCs were preferentially inhibited in vitro by the MEKi PD0325901 and trametinib. Conversely, JAK/STAT signaling was selectively activated in CBL iPSCs and abrogated by the JAKi momelotinib and ruxolitinib. The PI3Kδi idelalisib and mTORi rapamycin inhibited signaling and myeloproliferation in both PTPN11 and CBL iPSCs. These findings demonstrate differential sensitivity of PTPN11 iPSCs to MEKi and of CBL iPSCs to JAKi, but similar sensitivity to PI3Ki and mTORi. Clinical investigation of mutation-specific kinase inhibitor therapies in children with JMML may be warranted.

Juvenile myelomonocytic leukemia (JMML) is a unique clonal hematopoietic disorder of early childhood characterized by hyperactivation of the RAS signal transduction pathway. Approximately 90% of patients harbor molecular alteration in 1 of 5 genes ( PTPN11, NRAS, KRAS, NF1, CBL ), which define genetically and clinically distinct JMML subtypes. Three subtypes, PTPN11- , NRAS- , and KRAS -mutated JMML, are characterized by heterozygous somatic gain-of-function mutations in non syndromic children, while two subtypes, JMML in neurofibromatosis type 1 and in JMML in children with CBL syndrome, are characterized by germ line RAS disease and acquired biallelic inactivation of the respective tumor suppressor genes in hematopoietic cells. In addition to the initiating RAS pathway lesion, secondary genetic alterations within and outside of the RAS pathway are detected in about half the patients. Most recently, genome-wide DNA methylation profiles identified distinct methylation signatures correlating with clinical and genetic features and highly predictive of outcome. JMML is a stem cell disorder, and most JMML patients require allogeneic stem cell transplantation for long-term survival. However, spontaneous disease regression is noted in the majority of children with CBL -mutated JMML and in some NRAS -mutated cases. In the absence of 1 of the 5 canonical RAS pathway alteration, rare mutations in other RAS genes and non-JMML myeloproliferative disorders need to be excluded. Understanding the genetic basis of myeloproliferative disorders in early childhood will greatly improve clinical decision making., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests., (© 2018 by The American Society of Hematology. All rights reserved.)

Myelodysplastic syndrome (MDS) is a clonal bone marrow disorder, typically of older adults, which is characterized by ineffective hematopoiesis, peripheral blood cytopenias and risk of progression to acute myeloid leukemia. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm occurring in young children. The common denominator of these malignant myeloid disorders is the limited benefit of conventional chemotherapy and a particular responsiveness to epigenetic therapy with the DNA-hypomethylating agents 5-azacytidine (azacitidine) or decitabine. However, hypomethylating therapy does not eradicate the malignant clone in MDS or JMML and allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment option. An emerging concept with intriguing potential is the combination of hypomethylating therapy and HSCT. Possible advantages include disease control with good tolerability during donor search and HSCT preparation, improved antitumoral alloimmunity, and reduced risk of relapse even with non-myeloablative regimens. Herein we review the current role of pre- and post-transplant therapy with hypomethylating agents in MDS and JMML., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ras mutations are commonly observed in juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). JMML and CMML transform into acute myeloid leukemia (AML) in about 10% and 50% of patients, respectively. However, how additional events cooperate with Ras to promote this transformation are largely unknown. We show that absence of the ubiquitin-specific peptidase 22 (USP22), a component of the Spt-Ada-GCN5-acetyltransferase chromatin-remodeling complex that is linked to cancer progression, unexpectedly promotes AML transformation in mice expressing oncogenic Kras G12D/+ USP22 deficiency in Kras G12D/+ mice resulted in shorter survival compared with control mice. This was due to a block in myeloid cell differentiation leading to the generation of AML. This effect was cell autonomous because mice transplanted with USP22-deficient Kras G12D/+ cells developed an aggressive disease and died rapidly. The transcriptome profile of USP22-deficient Kras G12D/+ progenitors resembled leukemic stem cells and was highly correlated with genes associated with poor prognosis in AML. We show that USP22 functions as a PU.1 deubiquitylase by positively regulating its protein stability and promoting the expression of PU.1 target genes. Reconstitution of PU.1 overexpression in USP22-deficient Kras G12D/+ progenitors rescued their differentiation. Our findings uncovered an unexpected role for USP22 in Ras-induced leukemogenesis and provide further insights into the function of USP22 in carcinogenesis., (© 2018 by The American Society of Hematology.)

The protein tyrosine phosphatase PTPN11 is implicated in the pathogenesis of juvenile myelomonocytic leukemia (JMML), acute myeloid leukemia (AML), and other malignancies. Activating mutations in PTPN11 increase downstream proliferative signaling and cell survival. We investigated the signaling upstream of PTPN11 in JMML and AML cells and found that PTPN11 was activated by the nonreceptor tyrosine/serine/threonine kinase TNK2 and that PTPN11-mutant JMML and AML cells were sensitive to TNK2 inhibition. In cultured human cell-based assays, PTPN11 and TNK2 interacted directly, enabling TNK2 to phosphorylate PTPN11, which subsequently dephosphorylated TNK2 in a negative feedback loop. Mutations in PTPN11 did not affect this physical interaction but increased the basal activity of PTPN11 such that TNK2-mediated activation was additive. Consequently, coexpression of TNK2 and mutant PTPN11 synergistically increased mitogen-activated protein kinase (MAPK) signaling and enhanced colony formation in bone marrow cells from mice. Chemical inhibition of TNK2 blocked MAPK signaling and colony formation in vitro and decreased disease burden in a patient with PTPN11-mutant JMML who was treated with the multikinase (including TNK2) inhibitor dasatinib. Together, these data suggest that TNK2 is a promising therapeutic target for PTPN11-mutant leukemias., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Lymphoid interstitial pneumonia (LIP) is a rare disease characterized by benign reactive polyclonal proliferation of bronchus-associated lymphoid tissue after exposure to inhaled or circulating antigen(s), leading to a disease symptomatology similar to idiopathic interstitial pneumonia. Its association with diseases that are caused due to immune dysregulation (autoimmune diseases, congenital/acquired immunodeficiency, and allogeneic bone marrow transplant) and response to immunomodulatory/suppressive medications suggests an immunologic pathophysiology. Although LIP has been reported in association with lymphoproliferative diseases like Castleman disease, it has never been described in patients with leukemia. We report the first case of LIP in a patient with juvenile myelomonocytic leukemia (JMML) who was found to have a novel germline mutation of unknown significance in additional sex combs-like-1 (ASXL1) gene and a pathogenic somatic mutation of protein tyrosine phosphatase, nonreceptor type 11 (PTPN11) gene at diagnosis. The patient underwent a matched unrelated bone marrow transplant for JMML with complete resolution of JMML and LIP with no recurrence to date. We also emphasize the importance of considering LIP in differential diagnosis of pulmonary lesions seen in conjunction with hematologic malignancies and distinguishing it from malignant infiltration of the lung.

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive childhood leukemia characterized by an excess proliferation of cells of granulocytic and monocytic lineages. The WHO classifies JMML with the myelodysplastic/myeloproliferative neoplasms. Myelodysplasia in JMML is usually minimal to mild. Auer rods have never been reported in JMML. We present a 2-year-old boy with splenomegaly, leukocytosis, thrombocytopenia, anemia, and excess myeloblasts with easily seen Auer rods, and marked dysgranulopoiesis and dyserythropoiesis. Conventional cytogenetic analysis showed a sole abnormality of t(3;5)(q25;q35). Microarray analysis showed a terminal 21 Mb region of copy-neutral loss of heterozygosity on 19q. Disease-related somatic NRAS mutation was detected. This case represents an unusual JMML with Auer rods and marked myelodysplasia. These unusual histopathologic features may be related to the t(3;5)(q25;q35). A t(3;5) with variable breakpoints has been reported in a small proportion of acute myeloid leukemias and myelodysplastic syndromes. To our knowledge, this is the first JMML case reported with this translocation., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by mutations activating RAS signaling. Established clinical and genetic markers fail to fully recapitulate the clinical and biological heterogeneity of this disease. Here we report DNA methylome analysis and mutation profiling of 167 JMML samples. We identify three JMML subgroups with unique molecular and clinical characteristics. The high methylation group (HM) is characterized by somatic PTPN11 mutations and poor clinical outcome. The low methylation group is enriched for somatic NRAS and CBL mutations, as well as for Noonan patients, and has a good prognosis. The intermediate methylation group (IM) shows enrichment for monosomy 7 and somatic KRAS mutations. Hypermethylation is associated with repressed chromatin, genes regulated by RAS signaling, frequent co-occurrence of RAS pathway mutations and upregulation of DNMT1 and DNMT3B, suggesting a link between activation of the DNA methylation machinery and mutational patterns in JMML.

Background: Accumulating evidence suggests the origin of juvenile myelomonocytic leukemia (JMML) is closely associated with fetal development. Nevertheless, the contribution of embryonic progenitors to JMML pathogenesis remains unexplored. We hypothesized that expression of JMML-initiating PTPN11 mutations in HSC-independent yolk sac erythromyeloid progenitors (YS EMPs) would result in a mouse model of pediatric myeloproliferative neoplasm (MPN)., Results: E9.5 YS EMPs from VavCre+;PTPN11 D61Y embryos demonstrated growth hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) and hyperactive RAS-ERK signaling. Mutant EMPs engrafted the spleens of neonatal recipients, but did not cause disease. To assess MPN development during unperturbed hematopoiesis we generated CSF1R-MCM+;PTPN11 E76K ;ROSA YFP mice in which oncogene expression was restricted to EMPs. Yellow fluorescent protein-positive progeny of mutant EMPs persisted in tissues one year after birth and demonstrated hyperactive RAS-ERK signaling. Nevertheless, these mice had normal survival and did not demonstrate features of MPN., Conclusions: YS EMPs expressing mutant PTPN11 demonstrate functional and molecular features of JMML but do not cause disease following transplantation nor following unperturbed development. Developmental Dynamics 246:1001-1014, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that bears distinct characteristics associated with abnormal fetal development. JMML has been extensively modeled in mice expressing the oncogenic KrasG12D mutation. However, these models have struggled to recapitulate the defining features of JMML due to in utero lethality, nonhematopoietic expression, and the pervasive emergence of T cell acute lymphoblastic leukemia. Here, we have developed a model of JMML using mice that express KrasG12D in multipotent progenitor cells (Flt3Cre+ KrasG12D mice). These mice express KrasG12D in utero, are born at normal Mendelian ratios, develop hepatosplenomegaly, anemia, and thrombocytopenia, and succumb to a rapidly progressing and fully penetrant neonatal myeloid disease. Mutant mice have altered hematopoietic stem and progenitor cell populations in the BM and spleen that are hypersensitive to granulocyte macrophage-CSF due to hyperactive RAS/ERK signaling. Biased differentiation in these progenitors results in an expansion of neutrophils and DCs and a concomitant decrease in T lymphocytes. Flt3Cre+ KrasG12D fetal liver hematopoietic progenitors give rise to a myeloid disease upon transplantation. In summary, we describe a KrasG12D mouse model that reproducibly develops JMML-like disease. This model will prove useful for preclinical drug studies and for elucidating the developmental origins of pediatric neoplasms.

Myeloid malignancies showing CD141+ myeloid dendritic cell (MDC) differentiation have not been documented. Here, we describe a patient with juvenile myelomonocytic leukemia in which a prominent CD141+ cell population was identified most consistent with CD141+ MDCs based on phenotypic similarity with normal CD141+ MDCs. Molecular studies demonstrated a KRAS mutation. The findings from the spleen and bone marrow are described. This is the first well-documented demonstration of CD141+ MDC differentiation of a hematopoietic neoplasm., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) are myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap disorders characterized by monocytosis, myelodysplasia, and a characteristic hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Currently, there are no available disease-modifying therapies for CMML, nor are there preclinical models that fully recapitulate the unique features of CMML. Through use of immunocompromised mice with transgenic expression of human GM-CSF, interleukin-3, and stem cell factor in a NOD/SCID-IL2Rγ null background (NSGS mice), we demonstrate remarkable engraftment of CMML and JMML providing the first examples of serially transplantable and genetically accurate models of CMML. Xenotransplantation of CD34 + cells (n = 8 patients) or unfractionated bone marrow (BM) or peripheral blood mononuclear cells (n = 10) resulted in robust engraftment of CMML in BM, spleen, liver, and lung of recipients (n = 82 total mice). Engrafted cells were myeloid-restricted and matched the immunophenotype, morphology, and genetic mutations of the corresponding patient. Similar levels of engraftment were seen upon serial transplantation of human CD34 + cells in secondary NSGS recipients (2/5 patients, 6/11 mice), demonstrating the durability of CMML grafts and functionally validating CD34 + cells as harboring the disease-initiating compartment in vivo. Successful engraftments of JMML primary samples were also achieved in all NSGS recipients (n = 4 patients, n = 12 mice). Engraftment of CMML and JMML resulted in overt phenotypic abnormalities and lethality in recipients, which facilitated evaluation of the JAK2/FLT3 inhibitor pacritinib in vivo. These data reveal that NSGS mice support the development of CMML and JMML disease-initiating and mature leukemic cells in vivo, allowing creation of genetically accurate preclinical models of these disorders., (© 2017 by The American Society of Hematology.)

Background: Immunophenotyping of bone marrow (BM) hemopoietic precursors is useful for diagnosis of adult myelodysplastic syndrome (MDS), but data concerning pediatric patients are limited. We analyzed immunophenotypic features of BM cells at diagnosis of children who were referred to the Brazilian Pediatric Cooperative Group of Myelodysplastic Syndromes., Methods: Diagnosis was based on clinical information, peripheral blood counts, BM cytology and cytogenetics. Patients with Down syndrome were excluded. Children with deficiency anemias or transitory neutropenias were used as controls (CTRLs). Immunophenotyping was performed on an eight-color antibody platform evaluating myelomonocytic maturation and progenitor cells., Results: A total of 32 patients were examined: 6 refractory cytopenia of childhood [RCC]; 5 refractory anemia with excess of blasts [RAEB]; 8 refractory anemia with excess of blasts in transformation [RAEB-t]; 13 juvenile myelomonocytic leukemia [JMML] and 10 CTRLs. Median age was 66 months (RCC), 68 months (RAEB/RAEB-t), 29 months (JMML) and 70 months (CTRLs). Median number of phenotypic alterations was 4 (range 1-6) in RCC; 6 (range 2-11) in RAEB/RAEB-t and 6 (range 2-11) in JMML (P = 0.004). The percentage of CD34 + /CD117 + /CD13 + cells was 0.5% (range 0.1-2.8) in RCC; 4.2% (range 0.3-10.1) in RAEB/RAEB-t and 3.7 % (range 0.5-8.6) in JMML cases, compared with 0.7% (0.5-1.2) in CTRLs (P < 0.0005). Aberrancies in antigen expression of myeloid progenitors were seen in 63% of JMML and in 45% of RAEB/RAEB-t. CD34 + /CD19 + /CD10 + cells were decreased or absent in patients compared with age-matched controls. T lymphocytes were decreased in JMML., Conclusions: Phenotypic abnormalities were similar to those found in adult MDS. A decrease in B-cell precursors was observed especially in RAEB/RAEB-t. JMML and RAEB showed a similar pattern., (© 2016 Wiley Periodicals, Inc.)

Background: Juvenile myelomonocytic leukaemia (JMML) and chronic myelomonocytic leukaemia (CMML) are myelodysplastic myeloproliferative (MDS/MPN) neoplasms with unfavourable prognosis and without effective chemotherapy treatment. Trabectedin is a DNA minor groove binder acting as a modulator of transcription and interfering with DNA repair mechanisms; it causes selective depletion of cells of the myelomonocytic lineage. We hypothesised that trabectedin might have an antitumour effect on MDS/MPN., Methods: Malignant CD14+ monocytes and CD34+ haematopoietic progenitor cells were isolated from peripheral blood/bone marrow mononuclear cells. The inhibition of CFU-GM colonies and the apoptotic effect on CD14+ and CD34+ induced by trabectedin were evaluated. Trabectedin's effects were also investigated in vitro on THP-1, and in vitro and in vivo on MV-4-11 cell lines., Results: On CMML/JMML cells, obtained from 20 patients with CMML and 13 patients with JMML, trabectedin - at concentration pharmacologically reasonable, 1-5 nM - strongly induced apoptosis and inhibition of growth of haematopoietic progenitors (CFU-GM). In these leukaemic cells, trabectedin downregulated the expression of genes belonging to the Rho GTPases pathway (RAS superfamily) having a critical role in cell growth and cytoskeletal dynamics. Its selective activity on myelomonocytic malignant cells was confirmed also on in vitro THP-1 cell line and on in vitro and in vivo MV-4-11 cell line models., Conclusions: Trabectedin could be good candidate for clinical studies in JMML/CMML patients., Competing Interests: Maurizio D'Incalci has received honorarium to participate in a scientific board of PhamaMar. Carlos M Galmarini is an employee of PharmaMar, which produces trabectedin.

Juvenile myelomonocytic leukemia (JMML), previously known as juvenile chronic myeloid leukemia (JCML), is a rare, myelodysplastic-myeloproliferative disease typically presenting in early childhood. This disorder is difficult to distinguish from other myeloproliferative syndromes such as chronic myeloid leukemia (CML) because of the similarities in their clinical and bone marrow findings. However, because of its unique biological characteristics such as absolute monocytosis with dysplasia, absence of Philadelphia chromosome or BCR-ABL fusion protein, hypergammaglobulinemia, and raised fetal hemoglobin level, this disorder does not satisfy the criteria for inclusion in the CML or chronic myelomonocytic leukemia (CMML) group, as seen in adult patients. We describe three cases of JMML, who had very similar clinical and laboratory findings., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Germline activating mutations of the protein tyrosine phosphatase SHP2 (encoded by PTPN11), a positive regulator of the RAS signalling pathway, are found in 50% of patients with Noonan syndrome. These patients have an increased risk of developing leukaemia, especially juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative neoplasm (MPN). Previous studies have demonstrated that mutations in Ptpn11 induce a JMML-like MPN through cell-autonomous mechanisms that are dependent on Shp2 catalytic activity. However, the effect of these mutations in the bone marrow microenvironment remains unclear. Here we report that Ptpn11 activating mutations in the mouse bone marrow microenvironment promote the development and progression of MPN through profound detrimental effects on haematopoietic stem cells (HSCs). Ptpn11 mutations in mesenchymal stem/progenitor cells and osteoprogenitors, but not in differentiated osteoblasts or endothelial cells, cause excessive production of the CC chemokine CCL3 (also known as MIP-1α), which recruits monocytes to the area in which HSCs also reside. Consequently, HSCs are hyperactivated by interleukin-1β and possibly other proinflammatory cytokines produced by monocytes, leading to exacerbated MPN and to donor-cell-derived MPN following stem cell transplantation. Remarkably, administration of CCL3 receptor antagonists effectively reverses MPN development induced by the Ptpn11-mutated bone marrow microenvironment. This study reveals the critical contribution of Ptpn11 mutations in the bone marrow microenvironment to leukaemogenesis and identifies CCL3 as a potential therapeutic target for controlling leukaemic progression in Noonan syndrome and for improving stem cell transplantation therapy in Noonan-syndrome-associated leukaemias.

Noonan syndrome is a heterogeneous autosomal dominant disorder caused by mutations in at least eight genes involved in the RAS/MAPK signaling pathway. Recently, RIT1 (Ras-like without CAAX 1) has been shown to be involved in the pathogenesis of some patients. We report a series of 44 patients from 30 pedigrees (including nine multiplex families) with mutations in RIT1. These patients display a typical Noonan gestalt and facial phenotype. Among the probands, 8.7% showed postnatal growth retardation, 90% had congenital heart defects, 36% had hypertrophic cardiomyopathy (a lower incidence compared with previous report), 50% displayed speech delay and 52% had learning difficulties, but only 22% required special education. None had major skin anomalies. One child died perinatally of juvenile myelomonocytic leukemia. Compared with the canonical Noonan phenotype linked to PTPN11 mutations, patients with RIT1 mutations appear to be less severely growth retarded and more frequently affected by cardiomyopathy. Based on our experience, we estimate that RIT1 could be the cause of 5% of Noonan syndrome patients. Because mutations found constitutionally in Noonan syndrome are also found in several tumors in adulthood, we evaluated the potential contribution of RIT1 to leukemogenesis in Noonan syndrome. We screened 192 pediatric cases of acute lymphoblastic leukemias (96 B-ALL and 96 T-ALL) and 110 cases of juvenile myelomonocytic leukemias (JMML), but detected no variation in these tumoral samples, suggesting that Noonan patients with germline RIT1 mutations are not at high risk to developing JMML or ALL, and that RIT1 has at most a marginal role in these sporadic malignancies.

Background: Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm of childhood whose clinical heterogeneity is only poorly represented by gene sequence alterations. It was previously shown that aberrant DNA methylation of distinct target genes defines a more aggressive variant of JMML, but only few significant targets are known so far. To get a broader picture of disturbed CpG methylation patterns in JMML, we carried out a methylation screen of 34 candidate genes in 45 patients using quantitative mass spectrometry., Findings: Five of 34 candidate genes analyzed showed recurrent hypermethylation in JMML. cAMP-responsive element-binding protein-binding protein (CREBBP) was the most frequent target of epigenetic modification (77 % of cases). However, no pathogenic mutations of CREBBP were identified in a genetic analysis of 64 patients. CREBBP hypermethylation correlated with clinical parameters known to predict poor outcome., Conclusions: This study supports the relevance of epigenetic aberrations in JMML pathophysiology. Our data confirm that DNA hypermethylation in JMML is highly target-specific and associated with higher-risk features. These findings encourage the development of prognostic markers based on epigenetic alterations, which will be helpful in the difficult clinical management of this heterogeneous disease.

Juvenile myelomonocytic leukemia is a clonal malignant disease affecting young children. Current cure rates, even with allogeneic hematopoietic stem cell transplantation, are no better than 50%-60%. Pre-clinical research on juvenile myelomonocytic leukemia is urgently needed for the identification of novel therapies but is hampered by the unavailability of culture systems. Here we report a xenotransplantation model that allows long-term in vivo propagation of primary juvenile myelomonocytic leukemia cells. Persistent engraftment of leukemic cells was achieved by intrahepatic injection of 1×10(6) cells into newborn Rag2(-/-)γc(-/-) mice or intravenous injection of 5×10(6) cells into 5-week old mice. Key characteristics of juvenile myelomonocytic leukemia were reproduced, including cachexia and clonal expansion of myelomonocytic progenitor cells that infiltrated bone marrow, spleen, liver and, notably, lung. Xenografted leukemia cells led to reduced survival of recipient mice. The stem cell character of juvenile myelomonocytic leukemia was confirmed by successful serial transplantation that resulted in leukemia cell propagation for more than one year. Independence of exogenous cytokines, low donor cell number and slowly progressing leukemia are advantages of the model, which will serve as an important tool to research the pathophysiology of juvenile myelomonocytic leukemia and test novel pharmaceutical strategies such as DNA methyltransferase inhibition., (Copyright© Ferrata Storti Foundation.)

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative disorder that occurs during infancy and early childhood; this disorder is characterized by hypersensitivity of the myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. JMML usually involves somatic and/or germline mutations in the genes of the RAS pathway, including PTPN11, NRAS, KRAS, NF1, and CBL, in the leukemic cells. Recently, additional genetic and/or epigenetic alterations have been identified in JMML, and these alterations appear to be prognostic indicators. Moreover, analyses of JMML stem cells and induced pluripotent stem cells (iPSCs) technology are expected to identify new targets for therapeutic interventions. Almost all patients with JMML experience an aggressive clinical course, and hematopoietic stem cell transplantation (HSCT) is the only curative treatment. The most suitable therapeutic regimen after diagnosis and the optimal conditioning regimen prior to HSCT have yet to be identified, though several clinical trials have been initiated worldwide. Taken together, these new findings indicate that genetic and/or epigenetic alteration-specific risk management may be introduced, and that suitable pre- or post-allogeneic HSCT treatments which are less toxic and can improve outcomes will be developed in the near future.

After allogeneic stem-cell transplantation, nonhematopoietic tissues contain donor-derived cells; however, whether cells from malignant hematological disease can also be found in nonhematopoietic tissues is unclear. This report describes a juvenile myelomonocytic leukemia (JMML) case with a typical PTPN11 mutation (p.E76K) at different allele frequencies in the bone marrow mononuclear cells, buccal smear cells, and fingernails at diagnosis, which was suggestive of PTPN11 somatic mosaicism; however, the PTPN11 mutation in the buccal smear cells and fingernails was lost after unrelated cord blood transplantation. These results suggest that JMML-derived cells may migrate into and reside in nonhematopoietic tissues and furthermore that these cells can be eradicated by cord blood transplantation.

An aberrant interaction between hematopoietic stem cells and mesenchymal stromal cells has been linked to disease and shown to contribute to the pathophysiology of hematologic malignancies in murine models. Juvenile myelomonocytic leukemia is an aggressive malignant disease affecting young infants. Here we investigated the impact of juvenile myelomonocytic leukemia on mesenchymal stromal cells. Mesenchymal stromal cells were expanded from bone marrow samples of patients at diagnosis (n=9) and after hematopoietic stem cell transplantation (n=7; from 5 patients) and from healthy children (n=10). Cells were characterized by phenotyping, differentiation, gene expression analysis (of controls and samples obtained at diagnosis) and in vitro functional studies assessing immunomodulation and hematopoietic support. Mesenchymal stromal cells from patients did not differ from controls in differentiation capacity nor did they differ in their capacity to support in vitro hematopoiesis. Deep-SAGE sequencing revealed differential mRNA expression in patient-derived samples, including genes encoding proteins involved in immunomodulation and cell-cell interaction. Selected gene expression normalized during remission after successful hematopoietic stem cell transplantation. Whereas natural killer cell activation and peripheral blood mononuclear cell proliferation were not differentially affected, the suppressive effect on monocyte to dendritic cell differentiation was increased by mesenchymal stromal cells obtained at diagnosis, but not at time of remission. This study shows that active juvenile myelomonocytic leukemia affects the immune response-related gene expression and function of mesenchymal stromal cells. In contrast, the differential gene expression of hematopoiesis-related genes could not be supported by functional data. Decreased immune surveillance might contribute to the therapy resistance and progression in juvenile myelomonocytic leukemia., (Copyright© Ferrata Storti Foundation.)