Phenotypic Correlates and Prognostic Outcomes of TET2 Mutations in Myelodysplastic Syndrome/Myeloproliferative Neoplasm Overlap Syndromes: A Comprehensive Study of 504 Patients

Introduction: Myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) overlap syndromes consist of 5 distinct WHO-defined entities; namely chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia, BCR/ABL1- (aCML), juvenile myelomonocytic leukemia (JMML), MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), and MDS/MPN, unclassifiable (MDS/MPN-U) (Arber et al., Blood 2016). With the notable exception of JMML, a bona fide RASopathy, the other entities are characterized by clinical heterogeneity and molecular diversity. Loss of function TET2 mutations (TET2MT) are common in myeloid neoplasms, especially CMML (60%), and are known leukemogenic drivers. We carried out this study to assess the TET2 mutational landscape and phenotypic correlates in patients with MDS/MPN overlap syndromes. Methods: After approval by the institutional review board, adult patients with WHO defined MDS/MPN overlap syndromes were included; with the exception of JMML. The BM morphology, cytogenetics and 2016, WHO-diagnoses were retrospectively reviewed and all patients underwent targeted next generation sequencing for 29 myeloid-relevant genes, obtained on BM mononuclear cells, at diagnosis, or at first referral, by previously described methods (Patnaik et al., BCJ 2016). Results: Five hundred and four patients were included in the study; including 387 (77%) with CMML, 48 (10%) with MDS/MPN-RS-T, 17 (3%) with aCML and 52 (10%) with MDS/MPN-U. The median age at diagnosis was 71 (range, 18-99) years, and 333 (66%) were male. TET2MT were seen in 212 (42%) patients, with the frequency of other mutations being: ASXL1 45%, SRSF2 40%, NRAS 15%, SF3B1 13%, CBL, RUNX1 and SETBP1 12% each, and JAK2 V617F 11% (Figure B). Among the MDS/MPN overlap syndromes, TET2 was more frequently mutated in CMML (49%) and aCML (47%) compared to MDS/MPN-RS-T (10%) and MDS/MPN-U (15%). The prevalence of patients with TET2MT increased with age, a finding consistent across all MDS/MPN subtypes (Figure C). Overall, 341 TET2MT were identified in 212 patients (mean 1.6 variants/patient, range 0-5): 120 (24%) had >1 TET2MT, while 113 (22%), 5 (1%) and 2 (0.4%) had 2, 3 and 5 mutations, respectively. CMML and aCML patients were more likely to have an age-independent increase in multiple TET2MT (28% and 24%), in comparison to MDS/MPN-RS-T (4%) and MDS/MPN-U (8%). TET2 MT spanned the entire coding sequence and were mostly truncating (78%, Figure A): 59 (17%) were missense, 14 (4%) involved the splice-donor/acceptor sites, 2 (0.5%) were in-frame deletions, 129 (38%) were nonsense, and 137 (40%) were frameshift mutations. Overall, the distribution of TET2MT was superimposable across CMML, aCML, and MDS/MPN-U; the only exception being the absence of splice site mutations in the latter two. One hundred and eighty-seven (55%) TET2MT were secondary to pathogenic single nucleotide variants (SNV), while the remainders were secondary to deletions (25%) and insertions (15%). Transitions comprised the most frequent type of SNV (65%), with the C:G>T:A being the most common (56%). Patients with MDS/MPN overlap syndrome and TET2MT were more likely to have additional gene mutations compared to wild type patients (mean mutation number 3.1 vs 2.1, p<0.0001), with common co-mutations being SRSF2 (51%), ASXL1 (42%), and CBL (17%). The median overall survival (OS) of the entire cohort was 29 (range, 0-170) months; 29 months for CMML, 63 months for MDS/MPN-RS-T, 14 months for aCML, and 25 months for MDS/MPN-U. On univariate analysis, OS was superior in CMML patients with TET2MT (35 months) compared to wild type cases (21 months, p<0.0001, Figure D), and in CMML patients with >1 TET2MT (41 months) in comparison to wild type (21 months, p<0.0001) and single TET2MT (29 months, p=0.0476) cases (Figure E). These observations were not seen in patients with aCML, MDS/MPN-RS-T, and MDS/MPN-U. Conclusion: Our study demonstrates that TET2MT are among the most frequent mutations in patients with MDS/MPN overlap syndromes (42%), especially CMML (49%), with an age-dependent increase in the frequency and number of TET2MT. Mutations in TET2 were found to span the entire coding sequence, with truncating mutations being more common (78%). Importantly, in CMML, TET2MT, including number of TET2MT, were associated with favorable survival outcomes. Figure Disclosures Al-Kali: Astex Pharmaceuticals, Inc.: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

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Evidence-Based Minireview: Myelodysplastic syndrome/myeloproliferative neoplasm overlap syndromes: a focused review

Hematology ◽

10.1182/hematology.2020000163 ◽

2020 ◽

Vol 2020 (1) ◽

pp. 460-464

Author(s):

Mrinal M. Patnaik ◽

Terra Lasho

Keyword(s):

Myelodysplastic Syndrome ◽

Late Onset ◽

Myeloproliferative Neoplasm ◽

Treatment Modalities ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Pathway ◽

Overlap Syndromes ◽

Recurrent Mutations ◽

Male Preponderance ◽

Myelomonocytic Leukemia

Abstract 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.

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Genomics of myelodysplastic syndrome/myeloproliferative neoplasm overlap syndromes

Hematology ◽

10.1182/hematology.2020000130 ◽

2020 ◽

Vol 2020 (1) ◽

pp. 450-459

Author(s):

Mrinal M. Patnaik ◽

Terra L. Lasho

Keyword(s):

Myelodysplastic Syndrome ◽

Single Gene ◽

Myeloproliferative Neoplasm ◽

Copy Number Variations ◽

Driver Mutations ◽

Juvenile Myelomonocytic Leukemia ◽

Prognostic Information ◽

Overlap Syndromes ◽

Ring Sideroblasts ◽

Myelomonocytic Leukemia

Abstract Myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap syndromes are uniquely classified neoplasms occurring in both children and adults. This category consists of 5 neoplastic subtypes: chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), BCR-ABL1–negative atypical chronic myeloid leukemia (aCML), MDS/MPN-ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), and MDS/MPN-unclassifiable (U). Cytogenetic abnormalities and somatic copy number variations are uncommon; however, >90% patients harbor gene mutations. Although no single gene mutation is specific to a disease subtype, certain mutational signatures in the context of appropriate clinical and morphological features can be used to establish a diagnosis. In CMML, mutated coexpression of TET2 and SRSF2 results in clonal hematopoiesis skewed toward monocytosis, and the ensuing acquisition of driver mutations including ASXL1, NRAS, and CBL results in overt disease. MDS/MPN-RS-T demonstrates features of SF3B1-mutant MDS with ring sideroblasts (MDS-RS), with the development of thrombocytosis secondary to the acquisition of signaling mutations, most commonly JAK2V617F. JMML, the only pediatric entity, is a bona fide RASopathy, with germline and somatic mutations occurring in the oncogenic RAS pathway giving rise to disease. BCR-ABL1–negative aCML is characterized by dysplastic neutrophilia and is enriched in SETBP1 and ETNK1 mutations, whereas MDS/MPN-U is the least defined and lacks a characteristic mutational signature. Molecular profiling also provides prognostic information, with truncating ASXL1 mutations being universally detrimental and germline CBL mutations in JMML showing spontaneous regression. Sequencing information in certain cases can help identify potential targeted therapies (IDH1, IDH2, and splicing mutations) and should be a mainstay in the diagnosis and management of these neoplasms.

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Secondary Mutations of JAK3 and SETBP1 in Juvenile Myelomonocytic Leukemia and Their Propagating Capacity; A Report from the AIEOP Study Group

Blood ◽

10.1182/blood.v124.21.4625.4625 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4625-4625

Author(s):

Silvia Bresolin ◽

Paola De Filippi ◽

Francesca Vendemini ◽

Riccardo Masetti ◽

Franco Locatelli ◽

...

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Hot Spot ◽

Univariate Analysis ◽

Driver Mutations ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Signaling ◽

Nsg Mice ◽

Myelomonocytic Leukemia

Abstract INTRODUCTION Juvenile myelomonocytic leukemia is a rare early childhood leukemia, characterized by excessive proliferation of granulocytic and monocytic cells. About 95% of JMML patients harbor driver mutations in the RAS signaling pathway. Recently, secondary hits in SETBP1 and JAK3 have been reported in a Japanese cohort of JMML patients showing an adverse clinical outcome of patients carrying these mutations. Here we report the mutational analysis of SETBP1 and JAK3 and clinical implications in a cohort of Italian JMML patients. METHODS Samples collected at diagnosis of 65 patients with JMML were analyzed by Sanger sequencing. Mutations were found in RAS (NRAS-KRAS) 31%, PTPN11 35%, CBL 5%, whereas in 29% of patients none of the above cited mutations was present. Mutation hot spot regions of SETBP1 (SKI domain) and of JAK3 (PTK domains) were sequenced. A xenografted murine model was used to assess the in vivo competitive repopulation advantage of clones carrying mutations of JAK3 and SETBP1. Mononuclear cells from a patient with JMML at diagnosis harboring PTPN11, SETBP1 and JAK3 mutations were transplanted in NSG mice and assessed for mutational status in the bone marrow and spleen after engraftment of JMML cells. RESULTS Screening for JAK3 and SETBP1 mutations in patients revealed 9 mutations in 8 out of 65 patients at diagnosis of JMML. All of the identified secondary mutations were associated with known driver mutations, more frequent with mutated PTPN11 and RAS (p=0.036 and p= 0.01 respectively) than with CBL or in cases without known driver mutations. Seventy-five percent of secondary mutations were found in SETBP1 and only 1 patient harbored a mutation in JAK3. Remarkably one patient carried mutations in JAK3 (L857P and L857Q, both predicted to damaging protein function), PTPN11 (G503A) and SETBP1 (D868N). All variants were identified as heterozygous mutations, confirmed bi-allelic expression at the transcriptome level. The only patient carrying JAK3 as secondary mutation at E958K showed wild-type expression of JAK3 pointing to absence of a functional role at the protein level. Univariate analysis revealed association between the presence of secondary mutations and patient’s age at diagnosis, with older patients carrying JAK3 and SETBP1 mutations (p=0.0067); no other clinical and biological characteristics (i.e. WBC count, percentage of monocyte, HbF level and platelet count) being significantly associated with the presence of secondary hits in bone marrow of JMML cases. Patients with secondary mutations showed a trend to shorter survival compared to those without secondary events in JAK3 and SETBP1 (5-years OS= 0% vs 54.01%, SE=8.1; p=0.41, respectively). Interestingly, the in vivo assay using xenografted mice revealed a different propagating capacity of JAK3 clones of patients carrying JAK3 (2 different clones), SETBP1 and PTPN11 mutations. Indeed, for JAK3 only the clone with the L857Q mutation engrafted in BM and spleen of the mouse, together with SETBP1 and PTPN11 mutations. Moreover, a second mouse engrafted with mononuclear cells of the same patients showed that only cells carrying the PTPN11 mutation had engrafted. CONCLUSIONS In conclusion we identified secondary mutations in JAK3 and SETBP1 in 12% of patients of a representative cohort of Italian JMML patients, showing a trend of adverse outcome for patients carrying these mutations. These secondary events in JMML patients showed to have distinct propagating capacities upon engraftment in NSG mice pointing to a different functional impact of these mutations. Disclosures No relevant conflicts of interest to declare.

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Myelodysplastic Syndrome Caused By Activating Kras Mutation in the Non-Hematopoietic Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v126.23.359.359 ◽

2015 ◽

Vol 126 (23) ◽

pp. 359-359

Author(s):

Lena Osswald ◽

Cornelius Miething ◽

Justus Duyster ◽

Tilman Brummer ◽

Robert Zeiser

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

B Cell ◽

Hematopoietic Cells ◽

Tamoxifen Treatment ◽

Juvenile Myelomonocytic Leukemia ◽

Single Mutation ◽

Wild Type ◽

Hematologic Disorder ◽

Myelomonocytic Leukemia

Abstract Oncogenic Ras mutations occur frequently in myelodysplastic and myeloproliferative syndromes as juvenile myelomonocytic leukemia (JMML) and the myeloproliferative variant of chronic myelomonocytic leukemia (MP-CMML) as well as in acute myeloid leukemia. However in these reports the mutations were in the hematopoietic cells. Here, we show that an activating mutation of Kras in the non-hematopoietic system leads to hematologic disorder resembling human myelodysplastic syndrome (MDS). Rosa26CreERT2;LSL-KrasG12D mice (CD45.2) were lethally irradiated and transplanted with wild-type bone marrow (CD45.1). After control of engraftment efficiency (above 99.6%), the mice were treated with Tamoxifen to induce the expression of KrasG12D in non-hematopoietic cells. 6-8 weeks after Tamoxifen treatment, the mice developed anemia, leukocytopenia and thrombocytopenia and had a highly increased percentage of myeloid cells in peripheral blood, spleen and bone marrow. FACS-analysis confirmed that these cells were donor-derived and therefore of wild-type origin. The frequency of immature myeloid progenitors (CD11b+ c-kit+) was increased in bone marrow of Rosa26CreERT2;LSL-KrasG12D mice compared to littermate controls suggesting a disturbed differentiation. Morphological analysis of blood smears and bone marrow revealed a high number of dysplastic hypersegmented neutrophils as well as the occurrence of myeloid blasts. Additionally, a significant decrease of B-lymphocytes was observed in the bone marrow of KrasG12D recipient mice which has also been described in human MDS. Osteoblasts have been shown to contribute to B-cell lymphopoiesis which implicates that decreased B-cell lymphopoiesis in this study may be a result of oncogenic Kras expression in osteoblasts. All these data indicate that a single mutation in the hematopoietic microenvironment can initiate a severe hematologic disorder. The expression of oncogenic Kras in bone marrow stroma cells leads to a shift to myeloid differentiation, severe anemia and thrombocytopenia as well as reduced B-cell counts recapitulating main signs of human myelodysplastic syndrome. Disclosures No relevant conflicts of interest to declare.

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BRAF Mutations in Juvenile Myelomonocytic Leukemia.

Blood ◽

10.1182/blood.v110.11.4602.4602 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4602-4602

Author(s):

Andrica C.H. de Vries ◽

Ronald W. Stam ◽

Christian Kratz ◽

Martin Zenker ◽

Oskar A. Haas ◽

...

Keyword(s):

Mononuclear Cells ◽

Clinical Signs ◽

Juvenile Myelomonocytic Leukemia ◽

Reaction Products ◽

Braf Gene ◽

Braf Mutations ◽

Ras Pathway ◽

Coding Sequence ◽

Ras Genes ◽

Myelomonocytic Leukemia

Abstract Approximately 75% of patients with juvenile myelomonocytic leukemia (JMML) harbour mutations in PTPN11, NF1 and RAS genes. The remaining cases presumably carry somatic mutations in other genes in the RAS pathway. BRAF plays a central role in this pathway between RAS and downstream molecules including MEK and ERK. BRAF mutations frequently occur in cancer. Recently, BRAF mutations were found in leukemia. Besides that, germline BRAF mutations cause cardio-facio-cutaneous syndrome, which shares many features with Noonan syndrome (NS). NS predisposes to a myeloproliferative disease resembling JMML. In 65 JMML patients screening for V600E mutations in exon 15 of the BRAF gene was performed from mononuclear cells. In a subset of patients, without RAS or PTPN11 mutations, and no clinical signs of NF1, the entire coding sequence of BRAF was analyzed. Sequence analysis was performed by direct, bidirectional sequencing of purified polymerase chain reaction products. In none of the 65 cases a V600E mutation of the BRAF gene was found. In a subset of patients in which the entire coding sequence of BRAF was analyzed, no mutations were identified either. Mutant proteins of the RAS-RAF-MEK-ERK pathway play an important role in the pathogenesis of JMML, resulting in GM-CSF hypersensitivity. In about 75% of the JMML cases these mutations affect RAS, NF1 or PTPN11 genes. The hypothesis for this study was that BRAF might play an important role in JMML as it is an important downstream effector of RAS. Our data show that apparently BRAF mutations do not play a role in JMML. Therefore, additional analysis of genes of the RAS pathway will be necessary to identify genetic aberrations in cases without known mutations.

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Korszakváltás a gyermekkori szerzett csontvelő-elégtelenséggel járó kórképek kezelésében Magyarországon

Orvosi Hetilap ◽

10.1556/650.2018.31171 ◽

2018 ◽

Vol 159 (42) ◽

pp. 1710-1719

Author(s):

Krisztián Kállay ◽

Judit Csomor ◽

Emma Ádám ◽

Csaba Bödör ◽

Csaba Kassa ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Survival Rate ◽

Myelodysplastic Syndrome ◽

Aplastic Anemia ◽

Severe Aplastic Anemia ◽

Juvenile Myelomonocytic Leukemia ◽

Reduced Intensity Conditioning ◽

Myelomonocytic Leukemia ◽

Reduced Intensity

Abstract: Introduction: Acquired bone marrow failures are rare but fatal diseases in childhood. Since 2013, Hungary has been participating as a full member in the work of the European Working Group on uniform diagnostics and therapy in patients with acquired bone marrow failure syndromes. Hypocellular refractory cytopenia of childhood has been emphasized as a frequent entity, transplanted by reduced intensity conditioning with excellent outcomes. Aim: To analyse and compare the results of treatment before and after our joining. Method: A total of 55 patients have been treated in the 8 centres of the Hungarian Pediatric Oncology Network during 5 years between 2013 and 2017 (severe aplastic anemia: 9, myelodysplastic syndrome: 41, juvenile myelomonocytic leukemia: 5 patients). Allogeneic hematopoietic stem cell transplantation was performed in severe aplastic anemia in 7 cases, while antithymocyte globulin was administered in one case and one patient died before diagnosis. In patients with myelodysplastic syndromes, watch and wait strategy was applied in 4, while transplantation in 37 cases. Reduced intensity conditioning was used in 54 percent of these cases. Transplantation was the treatment of choice in all 5 patients with juvenile myelomonocytic leukemia. Results: In the whole patient cohort, the time from diagnosis to treatment was median 92 (3–393) days, while in severe aplastic anemia median 28 (3–327) days only. Grade II–IV acute graft versus host disease occurred in 22.6%, grade III–IV in 6.8% and chronic in 11.2%. All the patients treated with severe aplastic anemia are alive and in complete remission (100%). The overall estimated survival rate is 85.1% in myelodysplastic syndrome, while 75% in juvenile myelomonocytic leukemia. The median follow-up was 30.4 (1.1–62.5) months. There was a remarkable increase in overall survival comparing the data before (1992–2012) and after (2013) joining the international group, 70% vs. 100% (p = 0.133) in severe aplastic anemia and 31.3% vs. 85.1% (p = 0.000026) in myelodysplastic syndrome. Conclusion: Due to a change in the paradigm of the conditioning regimen in hypocellular refractory cytopenia of childhood, the overall survival rate has significantly increased. Orv Hetil. 2018; 159(42): 1710–1719.

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A simple and robust methylation test for risk stratification of patients with juvenile myelomonocytic leukemia

Blood Advances ◽

10.1182/bloodadvances.2021005080 ◽

2021 ◽

Author(s):

Hironobu Kitazawa ◽

Yusuke Okuno ◽

Hideki Muramatsu ◽

Kosuke Aoki ◽

Norihiro Murakami ◽

...

Keyword(s):

Myeloproliferative Neoplasm ◽

Enzyme Analysis ◽

Support Vector ◽

Juvenile Myelomonocytic Leukemia ◽

Restriction Enzyme Analysis ◽

Clinical Test ◽

Consensus Clustering ◽

International Consensus ◽

Consensus Definition ◽

Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that develops during infancy and early childhood. The array-based international consensus definition of DNA methylation has recently classified patients with JMML into the following three groups: high methylation (HM), intermediate methylation (IM), and low methylation (LM). To develop a simple and robust methylation clinical test, 137 patients with JMML have been analyzed using the Digital Restriction Enzyme Analysis of Methylation (DREAM), which is a next-generation sequencing based methylation analysis. Unsupervised consensus clustering of the discovery cohort (n=99) using the DREAM data has identified HM and LM subgroups (HM_DREAM, n=35; LM_DREAM; n=64). Of the 98 cases that could be compared with the international consensus classification, 90 cases of HM (n=30) and LM (n=60) had 100% concordance with the DREAM clustering results. For the remaining eight cases classified as the IM group, four cases were classified into the HM_DREAM group and four cases into the LM_DREAM group. A machine-learning classifier has been successfully constructed using a Support Vector Machine (SVM), which divided the validation cohort (n=38) into HM (HM_SVM; n=18) and LM (LM_SVM; n=20) groups. Patients with the HM_SVM profile had a significantly poorer 5-year overall survival rate than those with the LM_SVM profile. In conclusion, a robust methylation test has been developed using the DREAM analysis for patients with JMML. This simple and straightforward test can be easily incorporated in diagnosis to generate a methylation classification for patients so that they can receive risk-adapted treatment in the context of future clinical trials.

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Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment

Leukemia ◽

10.1038/s41375-019-0662-y ◽

2019 ◽

Vol 34 (6) ◽

pp. 1658-1668

Author(s):

Aurélie Caye ◽

Kevin Rouault-Pierre ◽

Marion Strullu ◽

Elodie Lainey ◽

Ander Abarrategi ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Myeloproliferative Neoplasm ◽

Juvenile Myelomonocytic Leukemia ◽

Integrated Analysis ◽

Cell Phenotype ◽

Hematopoietic Stem ◽

Activating Mutations ◽

Colony Forming Assay ◽

Myelomonocytic Leukemia

AbstractJuvenile 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.

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