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

Download Full-text

Genome-Wide Methylation Analysis Using the Digital Restriction Enzyme Analysis of Methylation for Stratification of Patients with Juvenile Myelomonocytic Leukemia

Blood ◽

10.1182/blood-2019-127792 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2973-2973

Author(s):

Hironobu Kitazawa ◽

Hideki Muramatsu ◽

Norihiro Murakami ◽

Yusuke Okuno ◽

Manabu Wakamatsu ◽

...

Keyword(s):

Restriction Enzyme ◽

Methylation Level ◽

Enzyme Analysis ◽

Juvenile Myelomonocytic Leukemia ◽

Restriction Enzyme Analysis ◽

Consensus Clustering ◽

Cpg Sites ◽

Genome Wide ◽

Svm Model ◽

Myelomonocytic Leukemia

Background Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/ myeloproliferative neoplasm that occurs during infancy and early childhood. The clinical course of the disease varies widely. The majority of children require allogenic hematopoietic stem cell transplantation (HSCT) for long term survival, but the disease will eventually resolve spontaneously in ~15% of patients. Previous studies have identified clinical and molecular risk factors in JMML. More recently, three groups independently discovered that genome-wide methylation profiling using 450K Illumina array revealed that the high methylation (HM) subgroup was significantly associated with poor survival compared to the low methylation (LM) subgroup (Murakami 2018 Blood, Stieglitz 2017 Nat. Commun., Lipka 2017 Nat. Commun.). 450K could be a standard assay for stratification of JMML. However, it is now unavailable because the manufacture replaced it with EPIC array. Here, we developed a next-generation sequencing-based clinical test recapitulate 450K clustering results using the digital restriction enzyme analysis of methylation (DREAM) method (Jelinek 2012 Epigenetics). Patients and Methods We studied 99 children (67 boys and 32 girls) with JMML. All the patients were included in our previous publications. First, we assessed JMML samples with DREAM. Briefly, genomic DNA was sequentially cut with two enzymes SmaI and XmaI recognizing the same sequence, CCCGGG sites in DNA. Enzyme-treated DNA was then used to generate sequencing libraries according to the Illumina protocols, and run on an Illumina Hiseq 2500. We assessed 10 JMML samples with reduced representation bisulfite sequencing (RRBS) (Meissner 2005 Nucleic Acids Res.). In brief, purified genomic DNA was digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. The CpG-rich DNA fragments (40-220 bp) were size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome analyzer. Results We analyzed 99 samples using the DREAM with 8.87 (4.09-16.35) million reads (median, [range]), and determined methylation level in 62,525 (52,356-75,185) CpG sites (median [range]). We observed a strong correlation between DREAM methylation ratio and 450K beta-value of overlapping CpG sites (Pearson r2 = 0.95 [0.913-0.962], median [range]). We performed unsupervised consensus clustering with DREAM methylation data of 7,704 CpG sites within ±1 kb from TSS on autosomal chromosomes detected in ≥95% of the samples with imputation of the missing data using the median of each CpG site methylation level. Clustering identified two distinct subgroups, the HM subgroup (n = 35) and the LM subgroup (n = 64), matching 95% (94 of 99) with the 450K clustering results. The HM subgroup patients showed significantly poorer 5-year OS than the LM subgroup patients (41.9% [95% confidence interval {CI}], 25.3%-57.6%) vs. 71.4% [95% CI, 56.2%-82.1%]; P = 0.00345). Discrepancies in the clustering results between DREAM and 450K were observed in only 5 patients (2 survived and 3 died); all 5 patients were reclassified as those with LM with DREAM from being HM with 450K. We also performed RRBS methylation analysis on 10 patients. Unsupervised consensus clustering using promoter-associated 4,971 CpG sites measured with RRBS identified HM (n = 5) and LM (n = 5) subgroups and completely matched with the classification made using DREAM and 450K. Then, we developed a prediction model of the methylation subgroups using a machine-learning program. We selected 85 CpG sites from 7,704 CpG sites used for unsupervised clustering of the DREAM assay that showed a distinct difference in the average methylation level (>0.3) between the HM and LM subgroups of the learning cohort (n = 70) and developed a support vector machine (SVM) model. As a validation cohort, we analyzed the remaining 29 JMML samples with a SVM model and confirmed a high matching rate with 450K clustering results (100%, 29 of 29). Conclusions We could develop a methylation test for JMML using the DREAM assay. Both the unsupervised clustering analysis and SVM model could repeat the result of 450K-based methylation classification, i.e., the HM and LM subgroups. The relatively lower cost of the DREAM assay (US$200/sample) enabled us to incorporate methylation classification in JMML in most settings. Disclosures No relevant conflicts of interest to declare.

Download Full-text

International Consensus Definition of DNA Methylation Subgroups in Juvenile Myelomonocytic Leukemia

Clinical Cancer Research ◽

10.1158/1078-0432.ccr-20-3184 ◽

2020 ◽

Vol 27 (1) ◽

pp. 158-168

Author(s):

Maximilian Schönung ◽

Julia Meyer ◽

Peter Nöllke ◽

Adam B. Olshen ◽

Mark Hartmann ◽

...

Keyword(s):

Dna Methylation ◽

Juvenile Myelomonocytic Leukemia ◽

International Consensus ◽

Consensus Definition ◽

Definition Of ◽

Myelomonocytic Leukemia

Download Full-text

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.

Download Full-text

CREB Deficiency: A Potential Critical Factor for Selective GM-CSF Hypersensitivity in Juvenile Myelomonocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.2604.2604 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2604-2604

Author(s):

Y. Lucy Liu ◽

Priyangi A Malaviarachchi ◽

Shelly Y. Lensing ◽

Robert P. Castleberry ◽

Peter Dean Emanuel

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Signaling ◽

Camp Response Element ◽

Response Element ◽

Creb Protein ◽

Gm Csf ◽

Myelomonocytic Leukemia ◽

Normal Controls

Abstract Abstract 2604 Poster Board II-580 Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative neoplasm (MDS/MPN) of infancy and early childhood. The pathogenesis of JMML has been linked to dysregulated signal transduction through the NF1/RAS signaling pathway and PTPN11. This dysregulation results in JMML cells demonstrating selective hypersensitivity to GM-CSF in in vitro dose-response assays. Since JMML hematopoietic progenitor cells are selectively hypersensitive to (rather than independent of) GM-CSF, it is rational to hypothesize that the function of the GM-CSF receptor in JMML patients is not constitutively over-active unless stimulated by the cytokine. We previously reported that PTEN is deficient in JMML patients. PTEN expression is up-regulated by Egr-1, which is one of the targets of the cAMP-response-element-binding protein (CREB). CREB, as a transcriptional factor, is expressed ubiquitously and bound to the cAMP-response-element (CRE) of the Egr-1 promoter. After phosphorylation at serine 133, CREB selectively activates the transcription of Egr-1 in response to GM-CSF stimulation in hematopoietic cells. We evaluated the CREB protein level in peripheral blood or bone marrow samples collected from 26 JMML patients. Mononuclear cells (MNCs) were isolated and lysed in lysis buffer at a density of 107/100μl. Protein levels of CREB were evaluated by ELISA and Western-blot. We found that 22/26 (85%) of subjects were substantially CREB deficient while they had constitutively high activity of MAP kinase (Erk-1/2). In comparison to normal controls (n=7), the median level of total CREB protein by ELISA was significantly lower in JMML subjects (0.62 vs 8.85 ng/mg BSA in normal controls; p=0.006). The mechanism that causes CREB deficiency in JMML is under further investigation and further results may be available to present at the meeting. This is the first evidence that CREB, a critical component downstream of the GM-CSF receptor, is highly deficient in the majority of JMML cases. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

JMML tumor cells disrupt normal hematopoietic stem cells by imposing inflammatory stress through overproduction of IL-1β

Blood Advances ◽

10.1182/bloodadvances.2021005089 ◽

2021 ◽

Author(s):

Yuhan Yan ◽

Lei Dong ◽

Chao Chen ◽

Kevin D Bunting ◽

Qianjin Li ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Myeloproliferative Neoplasm ◽

Juvenile Myelomonocytic Leukemia ◽

Common Mutation ◽

Hematopoietic Stem ◽

Inflammatory Stress ◽

Therapeutic Outcomes ◽

Tnf Α ◽

Myelomonocytic Leukemia

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, TNF-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.

Download Full-text

MEK Inhibition Demonstrates Activity in Relapsed, Refractory Patients with Juvenile Myelomonocytic Leukemia: Results from COG Study ADVL1521

Blood ◽

10.1182/blood-2021-144850 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3679-3679

Author(s):

Elliot Stieglitz ◽

Mignon L. Loh ◽

Julia Meyer ◽

Chujing Zhang ◽

Donald A Barkauskas ◽

...

Keyword(s):

United States ◽

Clinical Trial ◽

Dna Methylation ◽

Objective Response ◽

The United States ◽

Juvenile Myelomonocytic Leukemia ◽

International Consensus ◽

Molecular Responses ◽

Mek Inhibition ◽

Myelomonocytic Leukemia

Abstract Background: Juvenile myelomonocytic leukemia (JMML) is a hematologic malignancy of infants and toddlers with both myelodysplastic and myeloproliferative features. The prognosis for patients (pts) with relapsed or refractory (r/r) JMML is poor and hematopoietic stem cell transplant (HCT) is the only curative therapy. The molecular hallmark of JMML is activation of the Ras/MAPK pathway. In preclinical studies, MEK inhibition was shown to be effective at reducing spleen sizes, restoring normal hematopoiesis, and extending survival compared to placebo in several genetically engineered mouse models of JMML. Trametinib is an orally bioavailable MEK1/2 inhibitor and is approved for treatment of several malignancies in adults. This Children's Oncology Group study (ADVL1521, NCT03190915) is the first clinical trial for pts with r/r JMML conducted in the United States. Pts are eligible if they have persistent clinical or molecular evidence of JMML after 1 cycle of high dose cytarabine, 2 cycles of a hypomethylating agent or HCT. Pts receive daily trametinib for up to 12 cycles (28 days) in the absence of disease progression or dose-limiting toxicity (DLT). Dosing is age-based with pts less than 6 years of age receiving 0.032mg/kg/day and those 6 years or older receiving 0.025mg/kg/day. An oral suspension is available for pts unable to swallow tablets. Using a Simon 2-stage design (10 pts in each stage), trametinib would be deemed effective if 3 or more pts achieved an objective response. Results: From 2018-2021, 9 pts were enrolled; all 9 were eligible and evaluable for toxicity and response. Each pt had a detectable Ras mutation at the time of enrollment and was monitored for response using clinical and molecular criteria developed by an international consensus panel (Niemeyer et al, 2015). Five pts were less than 2 years of age. Three patients had relapsed post-HCT prior to enrolling and 6 patients were refractory to a median of 1.5 prior therapies (range 1-3). Four pts had an objective response (1 clinical complete response (cCR), 3 clinical partial responses, (cPR); 2 pts had stable disease and 3 had progressive disease (Table 1). Both pts with stable disease completed the maximum 12 cycles permitted on study. Two pts who achieved a cPR proceeded to HCT. One patient who achieved a cCR remains on study. No molecular responses were achieved. There were no dose-limiting toxicities; one pt had grade 4 thrombocytopenia probably related to trametinib. Of the 8 patients who consented to correlative studies, 7 had DNA methylation testing, 6 had kinome profiling, and 5 had RNASeq testing performed on both pre- and post-trametinib paired samples. DNA methylation testing revealed stable intrapatient methylation signatures across diagnostic, relapse and post-trametinib timepoints using the international consensus criteria (Schönung et al, 2020). Integrated kinome and RNASeq analysis revealed downregulation of proteins and genes involved in Ras/MAPK signaling. Conclusions: In the first clinical trial for r/r JMML patients in the United States, 4 objective responses were observed among the initial 9 patients meeting the pre-defined criteria to deem trametinib effective. While clinical responses including resolution of splenomegaly, resolution of monocytosis and increased platelets counts were observed, no molecular responses were noted. The treatment of r/r JMML has historically depended on HCT. Recently, azacitidine has shown promise in treating r/r JMML. This trial demonstrates that trametinib is active in r/r JMML and has a favorable side effect profile. Ongoing analysis of extensive correlative testing results have revealed potential mechanisms of response and resistance to MEK inhibition. Future studies will focus on children with newly diagnosed JMML and combine trametinib with azacitidine with or without HCT. Figure 1 Figure 1. Disclosures Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees. Barkauskas: Genentech: Current Employment.

Download Full-text

Germline Mutations in CBL Cause a Predisposition to Juvenile Myelomonocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.310.310 ◽

2009 ◽

Vol 114 (22) ◽

pp. 310-310 ◽

Cited By ~ 1

Author(s):

Charlotte M Niemeyer ◽

Michelle Kang ◽

Ingrid Furlan ◽

Danielle Shin ◽

Debbie S Sakai ◽

...

Keyword(s):

Autosomal Dominant ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Familial Cancer ◽

Myeloproliferative Neoplasm ◽

Germline Mutations ◽

Clinical Syndrome ◽

Juvenile Myelomonocytic Leukemia ◽

Published Data ◽

Myelomonocytic Leukemia

Abstract Abstract 310 Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm (MPN) of early childhood. Up to 60% of patients harbor activating mutations in either NRAS, KRAS, or PTPN11, while another 15% of children have neurofibromatosis type 1 (NF-1) and demonstrate loss of the wildtype NF1 allele in their hematopoietic cells at diagnosis. We recently described that an additional 10–15% of children with JMML harbor missense homozygous mutations in exons 8 and 9 of CBL (Loh, Blood, 2009). Cbl is a complex protein that functions primarily as an E3 ubiquitin ligase but also serves numerous important adaptor functions. Mutations in CBL have recently been reported in adults with MPNs and the available evidence in adults indicates that these lesions are somatically acquired. We noted that a number of children with JMML and CBL mutations had neurological conditions including developmental delay and dysmorphic stigmata, although these features were not 100% penetrant. Based on these observations, we performed mutational studies in fibroblasts and buccal epithelial cells, which were available from 13 JMML patients with homozygous CBL mutations in their bone marrow at diagnosis. We now show that in all 13 patients the initial CBL mutation occurred as a heterozygous germline event (Table 1). Interestingly, a child with the 1222 T>C later developed a brain tumor with a homozygous CBL lesion. Mutational studies on parental DNA were informative in 11 cases and indicated autosomal inheritance in 6 families. Furthermore, two of these children had extensive family histories in which several young family members died of JMML. There were no known features of NF-1 in either pedigree. Subsequent analysis of these two pedigrees revealed a pattern of autosomal dominant inheritance that spanned 4 generations in 1 family and 3 generations in the other. To rule out normal genetic variation, a cohort of 240 healthy individuals were screened without detection of a CBL abnormality. One hallmark feature of JMML myeloid progenitor cells is their sensitivity to granulocyte-macrophage colony stimulating factor (GM-CSF) in colony-forming assays. Retroviral transduction of the 371 Tyr>His and 384 Cys>Arg mutations into wildtype murine fetal liver cells failed to induce a hypersensitive phenotype. However, recently published data suggest that a full oncogenic phenotype is conferred when the wildtype allele is deleted (Sanada, Nature, 2009), as occurs in the human diseases. Transduction of the most common human JMML mutations into BaF3-EpoR cells that have had murine Cbl knocked down is ongoing, as is further biochemical analysis. In summary, germline mutations in CBL cause a clinical syndrome with a predisposition to JMML, and importantly, can be inherited in an autosomal dominant fashion, thus establishing CBL as a new familial cancer predisposition gene. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Potential Role Of RUNX1 In The Pathogenesis Of Juvenile Myelomonocytic Leukemia (JMML)

Blood ◽

10.1182/blood.v122.21.45.45 ◽

2013 ◽

Vol 122 (21) ◽

pp. 45-45 ◽

Cited By ~ 1

Author(s):

Hui Huang ◽

Daniel E. Bauer ◽

Mignon L. Loh ◽

Govind Bhagat ◽

Alan B. Cantor ◽

...

Keyword(s):

Bone Marrow ◽

Tyrosine Phosphorylation ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Brdu Incorporation ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Signaling ◽

Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children. The only current curative treatment is bone marrow transplantation. Yet even with this aggressive therapy, ∼50% of children still die from their disease. Somatic mutations leading to constitutive activation of the tyrosine phosphatase Shp2 (also called PTPN11) or of RAS signaling occur in ∼90% cases of JMML. However, the transcription factors that act downstream of these aberrant signaling events have not been identified. We recently showed that RUNX1 is a direct interacting partner of Shp2 in megakaryocytic cells (Huang et al. 2012. Genes Dev 26: 1587-1601). Moreover, we showed that RUNX1 is normally negatively regulated by src-family kinase (SFK) mediated tyrosine phosphorylation in megakaryocytes and T-lymphocytes, and that Shp2 contributes to RUNX1 tyrosine dephosphorylation. We now show that overexpression of a mutant RUNX1 (RUNX1Y260F, Y375F, Y378F, Y379F, Y386F, “RUNX1-5F”), which is expected to mimic constitutive dephosphorylation by Shp2 in murine Lin- Sca-1+ c-kit+ (LSK) bone marrow cells is resistant to SFK-mediated tyrosine phosphorylation and leads to a dramatic expansion of CFU-M/CFU-GM and Gr1+Mac1+ cells in vitro and in vivo. In contrast, these effects are not seen when wild type RUNX1 or RUNX1Y260D, Y375D, Y378D, Y379D, Y386D (“RUNX1-5D”; mimicking constitutive RUNX1 tyrosine phosphorylation) are overexpressed. The RUNX1-5F expressing cells also have increased replating activity in serial colony forming assays, increased proliferation (BrdU incorporation), decreased apoptosis, and reduced cytokine dependence. This partially phenocopies conditional knock-in mice that express JMML associated activating Shp2 mutations. Flow sorted Gr1+Mac1+ cells from the RUNX1-5F transduced cultures expressed higher levels of the direct RUNX1 target gene PU.1, which plays a role in myelomonocytic growth, and Cyclin D1. To test whether RUNX1 is required for the myelomonocytic hyperproliferation in JMML, CD34+ peripheral blood cells from a patient with JMML and known activating Shp2 mutation (Shp2E76G) were lentivirally transduced with doxycycline-inducible RUNX1-5D or RUNX1-5F expression constructs and cultured under myeloid growth conditions. Upon doxycycline induction, the RUNX1-5D overexpressing cells (resistant to Shp2) exhibited at 32% reduction in BrdU incorporation. In contrast, the control RUNX1-5F expressing cells had no significant reduction in proliferation. These results are consistent with RUNX1 acting as an essential downstream target of activated Shp2 in JMML. As ERK mediated phosphorylation (downstream of RAS/MEK) is also known to increase RUNX1 activity, we propose that RUNX1 may be a common downstream transcriptional target of both activated Shp2 and RAS signaling in the pathogenesis of JMML. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

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.

Download Full-text

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

Blood ◽

10.1182/blood-2019-124115 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3005-3005

Author(s):

Giacomo Coltro ◽

Guadalupe Belen Antelo ◽

Terra Lasho ◽

Christy Finke ◽

Animesh Pardanani ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Mononuclear Cells ◽

Univariate Analysis ◽

Myeloproliferative Neoplasm ◽

Juvenile Myelomonocytic Leukemia ◽

Wild Type ◽

Single Nucleotide Variants ◽

Overlap Syndromes ◽

Coding Sequence ◽

Myelomonocytic Leukemia

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.

Download Full-text