Pediatric Myeloproliferative Neoplasms Research Articles

A predictive classifier of poor prognosis in transplanted patients with juvenile myelomonocytic leukemia: a study on behalf of the Société Francophone de Greffe de Moelle et de Thérapie Cellulaire.

Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric myeloproliferative neoplasm requiring hematopoietic stem cell transplantation (HSCT) in most cases. We retrospectively analyzed 119 JMML patients who underwent first allogeneic HSCT between 2002 and 2021. The majority (97%) carried a RAS-pathway mutation, and 62% exhibited karyotypic alterations or additional mutations in SETBP1, ASXL1, JAK3 and/or the RAS pathway. Relapse was the primary cause of death, with a 5-year cumulative incidence of 24.6% (95% CI: 17.1-32.9). Toxic deaths occurred in 12 patients, resulting in treatment-related mortality (TRM) of 9.0% (95% CI: 4.6-15.3). The 5-year overall (OS) and event-free survival were 73.6% (95% CI: 65.7-82.4) and 66.4% (95% CI: 58.2-75.8), respectively. Four independent adverse prognostic factors for OS were identified: age at diagnosis >2 years, time from diagnosis to HSCT ≥6 months, monocyte count at diagnosis >7.2x109/L, and the presence of additional genetic alterations. Based on these factors, we proposed a predictive classifier. Patients with 3 or more predictors (21% of the cohort) had a 5-year OS of 34.2%, whereas those with none (7%) had a 5-year OS of 100%. Our study demonstrates improved transplant outcomes compared to prior published data, which can be attributed to the synergistic impacts of a low TRM and a reduced, yet still substantial, relapse incidence. By integrating genetic information with clinical and hematologic features, we have devised a predictive classifier. This classifier effectively identifies a subgroup of patients who are at a heightened risk of unfavorable post-transplant outcomes who would benefit from novel therapeutic agents and post-transplant strategies.

Mutational Profile of Children with Myeloproliferative Neoplasms

Introduction Myeloproliferative neoplasms (MPNs) are clonal bone marrow disorders of over proliferation driven largely by mutations in the JAK2, CALR, and MPL genes. Mutational profiles including driver and secondary mutations in adult subjects with MPNs have been well studied and recent work has determined that driver mutations occur as early as in utero. There is little described about mutational profiles in pediatric patients with MPNs. Given the importance of clonal evolution and the relevance of MPN-associated mutations for risk assessment, clinical features, and outcomes, it is important to develop an understanding of the mutational spectrum in young subjects. Therefore, we sought to evaluate a pediatric MPN cohort for potential MPN-associated mutations. Methods We isolated mononuclear cells from the blood and/or bone marrow of 29 pediatric patients (median age 10 years, range 2-21) with MPNs. DNA was extracted and sequenced on the HEMEPACT platform, a gene panel including almost 600 genes focused on myeloid malignancies. Variants were called using a combination of variant callers. For single-nucleotide variants (SNV), we used CaVEMan, Mutect, and Strelka, and for small insertions and deletions also used Pindel. VAF was uniformly reported across all called variants using a realignment procedure. Likely artifact variants were filtered out based on: (i) The number of callers calling a given variant and the combination of filters from the triple callers. (ii) Variants with VAF <2% in all samples or less than 3 mutant supporting reads were excluded. Results In this cohort, 18 subjects had essential thrombocytosis (ET), 8 subjects had polycythemia vera (PV), 2 had pre-fibrotic myelofibrosis, and 1 had myelofibrosis. Clinical sequencing identified 9 subjects with ET with a JAK2V617F mutation, 3 ET subjects with CALR mutations, and 6 ET subjects who were "triple negative" (TN) for driver mutations. Six subjects with PV were found to have a JAK2V617F mutation, while 2 had JAK2 exon 12 mutations. Both subjects with pre-fibrotic myelofibrosis had CALR mutations, and the subject with myelofibrosis had a MPL mutation. HEMEPACT identified mutations in 10 additional cancer-associated genes (Figure 1A). After JAK2 and CALR, the most frequently mutated genes were TET2 and SETBP1. Two subjects had TET2 mutations that co-occurred with JAK2V617F (Figure 1B), and both had very low TET2 variant allele frequencies (VAFs). Two subjects had SETBP1 mutations, one co-occurring with JAK2V617F, and 1 with CALR mutation (Figure 1B), both of whom had high SETBP1 VAFs, possibly implying a germline mutation. No mutations in ASXL1, EZH2, or DNMT3A were identified. Of the 6 TN subjects, 3 had mutations detected on HEMEPACT (in CHEK2, CUX1, and RB1). Eight of the 29 subjects (28%) had more than 1 mutation detected, and the average number of mutations was 1.2 per subject. In contrast, in a small cohort of adult (median age 41 years, range 21-82) subjects with ET (n=2) and PV (n=7), 6 of 9 subjects (67%) had more than 1 mutation identified, and the average number of mutations per subject was 2. JAK2V617F variant allele frequency of the adult cohort was 4-times higher than in the pediatric cohort (median=0.16 vs. median=0.04). Conclusion This is one of the largest analyses to date of the mutational spectrum of pediatric MPNs. TET2 and SETBP1 mutations co-occurred with driver mutations, and no DNMT3A or ASXL1 mutations were seen in these young patients. While fewer pediatric than adult subjects had multiple mutations, there were some young patients who had already developed multiple mutations in childhood and it is important to better understand the role clonal hematopoiesis may play in pediatric MPNs. Buccal samples in our pediatric patients showed evidence of contamination and identifying the best way to sample the germline from children with these disorders is crucial for the study of MPNs in young patients. There is much to learn about the mutational spectrum of pediatric MPNs, including the role of germline mutations in disease pathogenesis, drivers of MPN development in triple-negative MPNs, mutation acquisition over time, and role of various mutations in MPN progression and outcomes. With this initial evaluation we have begun the process of answering some of these questions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pediatric Myeloproliferative Neoplasm in Era of Who Classification Systematic Review

Introduction Philadelphia-negative MPNs are most prevalent in the older population (median age at the diagnosis above 60 years) and are rarely diagnosed in pediatrics. It is estimated that the overall incidence of MPNs in pediatric patients is around 0.82 per 100,000 patients yearly, and MPNs are approximately 100 times more common in adults than children. Methods: We performed a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. English literature (PubMed, SCOPUS, and Google Scholar) was searched for studies, reviews, case series, and case reports of patients with Philadelphia negative MPNs (including essential thrombocythemia, polycythemia vera, primary myelofibrosis) in the pediatric age group (< 18 years). Only studies that fulfilled WHO-2008 or 2016 criteria for MPN were included. Results: We reviewed 33 articles of available published literature from 2008 to 2022 and collected data on a total of 196 patients in the pediatric population. Among the cohort of patients, 139 had essential thrombocythemia (ET), 20 had polycythemia vera (PV), and 37 had myelofibrosis (MF). Patients' clinical and biological characteristics are addressed in Table 1. The median age at the time of diagnosis for each disease varied, with 8.8 years for ET, 10 years for PV, and 3.6 years for MF. There was a variety in the blood counts at the time of diagnosis between all three diseases (Table 1). Discussion: Based on our review, the presence of splenomegaly pediatrics with PV was not mentioned in most cases, yet around a third of the reported data have splenomegaly, matching the percentage found in adults. On the other hand, hemorrhage was not observed in pediatrics with PV, thrombosis complicated in 2 (10%) cases, and leukemic transformation was documented in 2 (10%) cases. The number of PV cases in this review is small, with only 20 reported cases, so it is challenging to get conclusions, yet the presentation and complications between adults and pediatrics are comparable, apart from a possibly higher chance for AML transformation in the pediatric population. In adults with PV, around 98% of the patients were positive for either JAK2V617F in 95% or other JAK2 mutations in 3%. While in this pediatrics review, JAK2V617F mutation was found in only 45% of the cases and JAK2 exon 12 mutation in 15%. 36% of the ET cases were asymptomatic at presentation, a number lower than that observed in adults (45%). However, thrombosis is less common in pediatrics than in adults (9% versus 21%), and bleeding as a complication from ET in pediatrics was equal to adults (9%). JAK2V617F was the most common mutation (20% of the ET patients). Based on our review, MF cases in pediatrics are more common than PV cases in comparison with adults, in which MF is the least common. Data for pediatric patients with PMF is scarce; based on this sample of 37 patients, the percentage of symptomatic patients is higher than adults, but unlike adults, most pediatrics are presented with symptoms of cytopenia rather than constitutional symptoms, same for palpable splenomegaly where it was more reported compared to adults. The pediatric population with PMF has an entirely different mutation profile, with none of the patients in our data having JAK2V617F or MPL mutations. Interestingly in our collected data, none of the patients with PMF developed thrombosis or leukemic transformation, compared with 7% of adult patients who have documented leukemic transformation. Conclusion: Given the rarity of MPNs in pediatrics and different characteristics compared with adults, international collaborative efforts to study MPNs in this age group are needed to understand these disorders better. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transcriptome Profiling of Pediatric Myeloproliferative Neoplasms Demonstrates Dysregulation of Platelet-Relevant Genes and Enrichment of Inflammatory and JAK2 Mediated Complement Pathways

Introduction : Myeloproliferative neoplasms (MPNs) are rare clonal bone marrow disorders in children characterized by high blood counts, predisposition to clotting events, and the potential to transform to myelofibrosis or acute myeloid leukemia (AML). Children with MPNs have lower rates of the known driver mutations (in JAK2, MPL, and CALR) than adult patients, and the underlying pathways and molecular derangements in young patients remain unknown. Given the lack of knowledge about pediatric MPNs, it is critical that we gain a better understanding of the dysregulated pathways in these diseases, which is necessary for improving disease understanding and broadening treatment options in children. Therefore, the objective of this work was to identify differentially expressed genes and pathways between children with MPNs and healthy controls, as well as children with AML, to guide further study. Methods : Mononuclear cells were extracted from peripheral blood of pediatric MPN patients (n=20) and pediatric and young adult AML patients (n=1410), and bone marrow of normal controls (NC, n=68). AML patient samples were being evaluated as part of a Children's Oncology Group planned analysis. To identify an expression profile unique to MPNs, transcriptome data from MPN patients was contrasted against NC and AML patients. All samples were ribodepleted and underwent Illumina RNA-Seq to generate transcriptome expression data. All analyses were performed in R. Differentially expressed genes were identified using the voom function from the limma package (v. 3.38.3), and enriched pathways were identified using the pathfindR package (v. 1.3.1). Unsupervised hierarchical clustering and heatmap generation was performed using the ComplexHeatmap package (v. 1.20.0). Results : MPN patient samples showed a unique expression signature, distinct from both AML patients and normal controls. Unsupervised PCA plot (Figure 1A) and heatmaps (Figure 1B) show that MPN samples cluster together. There were 4,012 differentially expressed (DE) genes in MPNs compared to NC and 6,743 DE genes in MPNs compared to AML patients. There were 2,493 shared genes between the 2 groups (Figure 1C.) Significantly DE genes between MPNs and other groups included multiple platelet-relevant genes including PF4 (CXCL4), PF4V1, P2RY12, and PPBP (CXCL7). Interestingly, PF4V1 was the most DE gene in MPNs compared to AML, and third highest versus NC. Dysregulation of some of these genes has been seen in adult MPNs, as well as thrombosis. Further comparison of transcriptome profiles between children with (n=13) and without (n=7)JAK2 mutations showed upregulation of three genes, CFB, C2, and SERPING1, which are all known complement genes, implicating complement activation in JAK2-mutated MPN patients. Complement activation has previously been reported in adult MPNs. Pathway enrichment analysis shows a number of immune and inflammatory pathways as enriched in MPN patients compared to both AML and NC. There were 179 enriched pathways in MPNs compared to AML and 142 compared to NC, with 134 common pathways (Figure 1D.) The systemic lupus erythematosus pathway was the most heavily enriched pathway in MPNs compared to both AML and NC. Additional pathways with significant enrichment include hematopoietic cell lineage, cytokine-cytokine interactions, DNA replication, and various infection-relevant pathways. The JAK-STAT signaling pathway was also enriched in MPNs compared to both AML and NC, as was the platelet activation pathway. Conclusion: Transcriptome evaluation of childhood MPNs shows enrichment of numerous inflammatory and immune pathways, highlighting that, as in adult MPNs, inflammation is implicated in pediatric MPNs. Furthermore, specific complement genes were upregulated in JAK2-mutant MPN. Upregulation of platelet-specific genes implies potential insights into disease mechanisms and warrants more study. Variations in the cell populations may account for some of the differences seen, however all samples were largely mononuclear cells, making their comparisons reasonable. Further analysis of this early data is needed to better assess inflammatory changes and platelet activation in pediatric MPNs, as are larger sample sizes. Individual cells may have differential expression of various genes, and future experiments with single-cell RNA-seq would be helpful to further elucidate differences. Disclosures Levine: Novartis: Consultancy; Loxo: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Roche: Consultancy, Research Funding; Lilly: Honoraria; Amgen: Honoraria; Qiagen: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees.

Clinical Features of Children with Polycythemia Vera, Essential Thrombocythemia, and Primary Myelofibrosis in Japan: Retrospective Nationwide Survey

Background: Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders characterized by overproduction of differentiated hematopoietic cells. Among MPNs, chronic myeloid leukemia (CML) with BCR-ABL1 is the best studied subtype and is sometimes diagnosed during childhood. "Ph-negative" MPNs are typically found in older adults and are exceedingly rare during childhood. These classical Ph-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Because of this rarity, clinical features of pediatric MPNs are largely unknown, except for CML. Method: We administered questionnaires to institutions registered with the Japanese Society of Pediatric Hematology/Oncology (JSPHO). We collected clinical information of pediatric and young adult patients aged younger than 30 years who had been diagnosed with Ph-negative MPNs during 2000-2016. This study was conducted as a project of the Leukemia and Lymphoma Committee of the JSPHO. Results: We received responses from 145 institutions, which included all 107 board-certificated educational institutions of the JSPHO. A summary of the clinical features of included patients is shown in Table 1. From 2000 to 2016, 5 patients with PV, 42 with ET, and 2 with PMF were newly diagnosed with Ph-negative MPNs. Among them, 1 patient with PMF was excluded from subsequent analysis because of insufficient clinical information. The male-female ratio was almost balanced. The median age at diagnosis was 14 years for patients with PV, 9 years for patients with ET, and 1 patient with PMF was 10 months old. Only 1 patient with PV had a karyotype abnormality. The median follow-up period was 51 months since diagnosis. The JAK2V617F mutation was screened in almost all patients, and no patients with PV and 9 patients with ET had this mutation. With regard to treatment, 3 of 5 patients with PV received phlebotomy. Among patients with ET, aspirin was used most frequently (21 patients), followed by anagrelide (11 patients). However, 2 patients with PV and 8 patients with ET received no therapeutic approach. With regard to adverse events, hemorrhage and thrombosis were observed in 4 and 3 patients with ET, respectively, but these events were not found in patients with PV or PMF. Transformation to leukemia or myelofibrosis was found in 1 and 1 patient with PV, and 1 and 3 patients with ET, respectively. Among the patients who received no therapeutic approach, only 1 patient with PV experienced a hemorrhagic event and no other patients experienced any adverse events. Only 1 child with PV died owing to subsequent development of myelofibrosis and acute myeloid leukemia approximately 11 years after the initial diagnosis of PV. Conclusion: To the best of our knowledge, this is the first nationally-representative survey to clarify the clinical aspects of pediatric patients with Ph-negative MPNs. Although the methodology was not standardized, the frequency of the JAK2V617F mutation appears to be lower than that previously reported for adult patients. Moreover, the incidence of adverse events, such as thrombosis, hemorrhage, leukemia development, and transformation to myelofibrosis, was much lower than that of adult patients. As a result, although at relatively short durations of follow-up, the prognosis of pediatric patients with Ph-negative MPNs appears to be comparatively good. Further study with genetic analysis is warranted to determine the clinical and genetic features of pediatric patients with Ph-negative MPNs. Disclosures Mitsui: Astellas pharmaceutica: Research Funding; Daiichi Sankyo pharmaceutical: Research Funding; Chugai pharmaceutical: Research Funding; Teijin pharmaceutical: Research Funding; JCR pharmaceutical: Research Funding; Maruho pharmaceutical: Research Funding; MSD pharmaceutical: Research Funding; Shionogi pharmaceutical: Research Funding.

Use of Pegylated Interferon in Six Pediatric Patients with Myeloproliferative Neoplasms

Use of Pegylated Interferon in Six Pediatric Patients with Myeloproliferative Neoplasms

Hematopoietic-restricted Ptpn11E76K reveals indolent MPN progression in mice.

Juvenile Myelomonocytic Leukemia (JMML) is a pediatric myeloproliferative neoplasm (MPN) that has a poor prognosis. Somatic mutations in Ptpn11 are the most frequent cause of JMML and they commonly occur in utero. Animal models of mutant Ptpn11 have probed the signaling pathways that contribute to JMML. However, existing models may inappropriately exacerbate MPN features by relying on non-hematopoietic-restricted Cre-loxP strains or transplantations into irradiated recipients. In this study we generate hematopoietic-restricted models of Ptpn11E76K-mediated disease using Csf1r-MCM and Flt3Cre. We show that these animals have indolent MPN progression despite robust GM-CSF hypersensitivity and Ras-Erk hyperactivation. Rather, the dominant pathology is pronounced thrombocytopenia with expanded extramedullary hematopoiesis. Furthermore, we demonstrate that the timing of tamoxifen administration in Csf1r-MCM mice can specifically induce recombinase activity in either fetal or adult hematopoietic progenitors. We take advantage of this technique to show more rapid monocytosis following Ptpn11E76K expression in fetal progenitors compared with adult progenitors. Finally, we demonstrate that Ptpn11E76K results in the progressive reduction of T cells, most notably of CD4+ and naïve T cells. This corresponds to an increased frequency of T cell progenitors in the thymus and may help explain the occasional emergence of T-cell leukemias in JMML patients. Overall, our study is the first to describe the consequences of hematopoietic-restricted Ptpn11E76K expression in the absence of irradiation. Our techniques can be readily adapted by other researchers studying somatically-acquired blood disorders.

Yolk sac erythromyeloid progenitors expressing gain of function PTPN11 have functional features of JMML but are not sufficient to cause disease in mice.

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). E9.5 YS EMPs from VavCre+;PTPN11D61Y 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+;PTPN11E76K ;ROSAYFP 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. 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.

Mice expressing KrasG12D in hematopoietic multipotent progenitor cells develop neonatal myeloid leukemia.

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.

RUNX1 Is a Candidate Transcriptional Effector in Juvenile Myelomonocytic Leukemia

Juvenile Myelomonocytic Leukemia (JMML) is an aggressive pediatric myeloproliferative neoplasm. Mutations leading to constitutive activation of the non-receptor tyrosine phosphatase Shp2 (PTPN11) and/or RAS signaling are driver events. Yet, the key downstream transcriptional effectors of this aberrant signaling have not been defined. We previously found that Shp2 dephosphorylates the hematopoietic transcription factor RUNX1, leading to its activation. Overexpression of a mutant RUNX1 molecule that mimics constitutive RUNX1 tyrosine dephosphorylation in murine hematopoietic stem/progenitor cells led to massive expansion of myelomonocytic cells and growth under low cytokine conditions. Likewise, RUNX1 is known to be activated by extracellular signal-related kinase (ERK) mediated phosphorylation, which is downstream of activated RAS. To further investigate the role of RUNX1 in JMML, we analyzed gene expression datasets derived from CD34+ cells from JMML patients versus healthy controls. Thirty-four genes were down regulated and 13 were up regulated in the JMML versus control CD34+ cells using a cut-off of a log2-fold change > 0 and false-discovery rate (FDR) adjusted p-value ≤ 0.1. H3K27Ac and K3K4Me1 peaks located between -50 kb 5’ of the transcriptional start site (TSS) to +50 kb 3’ of the transcriptional end site (TES) of the differentially expressed genes were used to identify putative enhancer elements. DNA-binding factor motif enrichment analysis was then performed at these regions. This revealed a significant enrichment for RUNX DNA consensus binding sequences in the putative enhancer elements (p-value 8.05e-03). There was also significant enrichment for ETS family transcription factors, including Spi1 (PU.1) (p-values 1.94e-06 to 1.21e-03) and for Serum Response Factor (SRF) (p-value 1.11e-04). ETS factors frequently physically and functionally cooperate with RUNX transcription factors. Analysis of the proximal promoters showed only enrichment for ETS factors (p-value 2.42e-04 to 4.02e-03) and BATF::JUN (4.64e-03). Examination of ChIP-seq datasets for RUNX1 and PU.1 in CD34+ cells from healthy controls demonstrated that many of these putative sites are occupied by RUNX1, and some with PU.1. Overall, this data supports a model in which RUNX1 (and likely ETS factors) are important transcriptional effectors of aberrant cell signaling in JMML. DisclosuresLoh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

Illuminating the mutational spectrum of pediatric myeloproliferative neoplasms

Illuminating the mutational spectrum of pediatric myeloproliferative neoplasms

HSC-Independent Yolk Sac Progenitors Bear Hallmarks of JMML in a PTPN11D61Y Mouse Model

Juvenile Myelomonocytic Leukemia is the most common pediatric myeloproliferative neoplasm (MPN). JMML is characterized by myeloid populations with mutually-exclusive mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. JMML is notable among pediatric MPNs as being refractory to chemotherapy and having a 50% relapse rate following allogeneic hematopoietic stem cell (HSC) transplantation. As such, there is an urgent need for novel JMML therapies. The recent discovery of yolk sac myeloid lineages that persist into adulthood independently of bone marrow HSC contributions suggests a mechanism for JMML relapse following HSC transplantation. In this study, we sought to determine whether yolk sac HSC-independent myeloid progenitors bear hallmarks of MPN in a mouse model of JMML.Using the Vav1 promoter-directed Cre recombinase, we generated a mouse model of JMML that expresses the PTPN11D61Y gain of function mutation in all waves of embryonic and adult hematopoiesis, including yolk sac myeloid progenitors that emerge prior to and independently from HSCs. PTPN11D61Y/+; VavCre+ mice are viable, born at expected Mendelian ratios, and develop peripheral blood monocytosis as early as 4 weeks of age. Given this early onset, we hypothesized MPN may develop in these mice during embryonic development. E14.5 fetal liver progenitors from PTPN11D61Y/+; VavCre+ embryos displayed marked GM-CSF hypersensitivity in methylcellulose colony forming assays (Figure-1A), possessed hyperactive Ras-Erk pathway signaling (Figure-2), and had a skewed progenitor distribution with a greater proportion of megakaryocyte-erythroid progenitors (63.5% vs. 50.1%, p<0.01) and fewer common myeloid progenitors (9.2% vs. 19.3%, p<0.01) than littermate controls. Since the E14.5 fetal liver contains both HSC-dependent and HSC-independent myeloid progenitors, we repeated these experiments using E9.5 yolk sac samples to restrict our analysis to HSC-independent hematopoiesis. Compared to littermate controls, PTPN11D61Y/+; VavCre+ E9.5 embryos had no difference in overall number of yolk sac myeloid progenitors (Ter119-; cKit+, CD41Dim), and in the number and distribution of CFU-E/GM/GEMM colonies in methylcellulose assay. However, PTPN11D61Y/+; VavCre+ yolk sac progenitors demonstrated marked GM-CSF hypersensitivity in colony forming assay (Figure-1B) as well as hyperactive Ras-Erk signalling by western blot analysis (Figure-2) and intracellular flow cytometry with antibodies against pSTAT5 and pERK (Figure-3).We have demonstrated that HSC-independent myeloid lineages from the murine yolk sac possess GM-CSF hypersensitivity and Ras-Erk pathway hyperactivation in a mouse model of JMML. These findings suggest that HSC-independent hematopoietic populations may be involved in the development of JMML. Our study highlights the need to further assess the role of bone marrow-independent myeloid lineages in pediatric MPN, and to identify innovative therapies that can specifically target HSC-independent hematopoietic lineages. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Issues in Categorization and Management of Pediatric Patients with Myeloproliferative Neoplasms

Myeloproliferative Neoplasms (MPN) are well characterized in adults, with symptoms including thrombotic episodes, severe bleeding episodes, splenomegaly, and transformation to AML. Accepted treatment algorithms are available for management and multiple clinical trials are being done in adult patients. Much has been done to understand the pathogenesis in adults, and mutations in JAK2, MPL, and most recently CALR are established as causative lesions in these disorders. In contrast, knowledge of these disorders in children is more rudimentary, with limited information available regarding long-term clinical outcomes in children. Children have a lower frequency of the known mutations; studies to identify alternative driver mutations in children are ongoing.Eight pediatric patients with MPN have been seen at Cornell during their illness. Seven were diagnosed with Essential Thrombocytosis (ET) and the eighth had an initial diagnosis of ET but later was diagnosed with myelofibrosis (MF). One patient did not have bone marrow testing but was diagnosed by her primary hematologist on the basis of extreme thrombocytosis, and JAK2V617F positive testing on peripheral blood when no cause of reactive thrombocytosis was identified. Table 1 summarizes key features of these patients. The average age of diagnosis for these children is 10 years and 3 months. The oldest patient was diagnosed in 2006 and has had the longest consecutive period of thrombocytosis, 8 years.These patients presented with a variety of symptoms, including headache, abdominal pain, fatigue, and bleeding. All the children experienced extreme thrombocytosis (platelet count > 1,000 x 109/L) during their illness. Despite the presence of acquired von Willebrand’s disease in at least three patients, none have experienced severe bleeding symptoms. There have been no reported severe thrombotic episodes, and any evaluations done for complaints of headache, chest pain, or palpitations have shown no CNS infarcts or cardiac abnormalities. One child developed pseudo tumor cerebri with no cerebral venous sinus thrombosis or CNS lesion detected. Four children have been treated with aspirin with no significant bleeding complications. Six of the eight had been treated with cytoreductive therapy (Hydroxyurea) by their primary hematologist at some point in their course, for various findings including extreme thrombocytosis, fatigue, erythromelalgia, and recurrent headache. All showed a good response in platelet count to Hydroxyurea treatment, none developed dose-limiting side effects, and the highest dose that was needed to relieve symptoms at any point was 35mg/kg. Four patients remain on therapy with Hydroxyurea.Genetic sequencing was positive for JAK2V617F in three of eight patients. All of these patients have received Hydroxyurea treatment, and the child with pseudotumor cerebri was JAK2V617F positive. Of the five patients who are negative for JAK2 mutation, four were tested for MPLW515K/L and all are negative. Two of these patients have had sequencing sent for CALR mutations, and one patient is negative for CALR mutation as well (one patient is pending.) Figure 1 summarizes the sequencing completed to date on these patients.This cohort of pediatric patients shows a lower frequency of the known causative mutations than their adult counterparts. There is variation in how these patients were managed by their primary hematologists, likely due to the lack of treatment guidelines in this patient population (Kucine et al, 2014.) With the apparent differences in clinical course and genetic drivers from adults with MPN, there is a clear need for further research on the outcomes and pathogenesis in pediatric patients with MPN. Our group is currently working to answer some of the questions regarding pathogenesis and outcomes in pediatric patients with MPN to allow for individualization of treatment recommendations and family guidance in these children.Table 1Summary of Eight Pediatric MPN PatientsPatientAge at Diagnosis (years)DiagnosisInitial SymptomsA9ETWeakness, ParesthesiasB7ETHeadacheC10ETHeadache, FatigueD7ETHeadacheE8ETBleedingF13MFAbdominal Pain, SplenomegalyG10ETHeadacheH19ETFatigue, Itching [Display omitted] ReferencesKucine N, Chastain KM, Mahler MB, Bussel JB. Primary thrombocytosis in children. Haematologica. 2014;99(4)620-28. DisclosuresOff Label Use: Hydroxyurea use in certain children with MPN is accepted practice but is technically off-label.

The JAK2 V617F Mutation in Pediatric Myeloproliferative Neoplasms: How and When?

The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are uncommon in children w...