Chronic Myelomonocytic Leukemia Patients Research Articles

Loss of ASXL1 in the bone marrow niche dysregulates hematopoietic stem and progenitor cell fates

Somatic or de novo mutations of Additional sex combs-like 1 (ASXL1) frequently occur in patients with myeloid malignancies or Bohring-Opitz syndrome, respectively. We have reported that global loss of Asxl1 leads to the development of myeloid malignancies and impairs bone marrow stromal cell (BMSC) fates in mice. However, the impact of Asxl1 deletion in the BM niche on hematopoiesis remains unclear. Here, we showed that BMSCs derived from chronic myelomonocytic leukemia patients had reduced expression of ASXL1, which impaired the maintaining cord blood CD34+ cell colony-forming capacity with a myeloid differentiation bias. Furthermore, Asxl1 deletion in the mouse BMSCs altered hematopoietic stem and progenitor cell (HSC/HPC) pool and a preferential myeloid lineage increment. Immunoprecipitation and ChIP-seq analyses demonstrated a novel interaction of ASXL1 with the core subunits of RNA polymerase II (RNAPII) complex. Convergent analyses of RNA-seq and ChIP-seq data revealed that loss of Asxl1 deregulated RNAPII transcriptional function and altered the expression of genes critical for HSC/HPC maintenance, such as Vcam1. Altogether, our study provides a mechanistic insight into the function of ASXL1 in the niche to maintain normal hematopoiesis; and ASXL1 alteration in, at least, a subset of the niche cells induces myeloid differentiation bias, thus, contributes the progression of myeloid malignancies.

DNA methylation profile in chronic myelomonocytic leukemia associates with distinct clinical, biological and genetic features

ABSTRACTChromosomal abnormalities are detected in 20–30% of patients with chronic myelomonocytic leukemia (CMML) and correlate with prognosis. On the mutation level, disruptive alterations are particularly frequent in chromatin regulatory genes. However, little is known about the consequential alterations in the epigenetic marking of the genome. Here, we report the analysis of genomic DNA methylation patterns of 64 CMML patients and 10 healthy controls, using a DNA methylation microarray focused on promoter regions. Differential methylation analysis between patients and controls allowed us to identify abnormalities in DNA methylation, including hypermethylation of specific genes and large genome regions with aberrant DNA methylation. Unsupervised hierarchical cluster analysis identified two main clusters that associated with the clinical, biological, and genetic features of patients. Group 1 was enriched in patients with adverse clinical and biological characteristics and poorer overall and progression-free survival. In addition, significant differences in DNA methylation were observed between patients with low risk and intermediate/high risk karyotypes and between TET2 mutant and wild type patients. Taken together, our results demonstrate that altered DNA methylation patterns reflect the CMML disease state and allow to identify patient groups with distinct clinical features.

Azacitidine With or Without Lenalidomide in Higher Risk Myelodysplastic Syndrome & Low Blast Acute Myeloid Leukemia

Standard treatment for higher risk myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML) and low blast acute myeloid leukemia (AML) is azacitidine. In single arm studies, the addition of lenalidomide had been suggested to improve outcomes. The ALLG MDS4 phase II clinical trial randomized such patients to standard azacitidine or combination azacitidine (75mg/m2/d days 1 to 5) with lenalidomide (10mg days 1 to 21 of 28-day cycle from cycle 3) to assess clinical benefit (alive without progressive disease) at 12 months. A total of 160 patients were enrolled; median age 70.7 years (range 42.5-87.2), 31.3% female with 14% CMML, 12% AML and 74% MDS. Adverse events were similar in both arms. There was excellent delivery of protocol therapy (median aza cycles 11 both arms) with few dose reductions, delays or early cessations. At median follow up 33.1 months (range 0.7-59.5), the rate of clinical benefit at 12 months was 65% AZA arm and 54% LEN AZA arm (p=0.2). There was no difference in clinical benefit between each arm according to WHO diagnostic subgroup (MDS, AML or CMML) or IPSS-R. ORR was 57% in AZA arm and 69% in LEN AZA (p=0.14). There was no difference in progression free or overall survival between the arms (each p > 0.12). Although the combination of lenalidomide and azacitidine was tolerable, there was no improvement in clinical benefit, response rates or overall survival in higher risk MDS, CMML or low blast AML patients compared to treatment with azacitidine alone. This trial was registered at www.anzctr.org.au as ACTRN12610000271000. Funding: This study was supported by Celgene with grant funding for study conduct, Snowdome Foundation and the Victorian Epigenetic Group for funding support for molecular studies. Conflict of Interest Disclosure: MK, DH, JFS received research funding from Celgene MK and JFS are advisory board members for Celgene JFS is a member of speakers bureau for Celgene MH is an advisory board member for Takeda, Roche, MSD, Gilead, Janssen WS receives honoraria from Amgen and Novartis The remaining authors declare no conflict of interest; IC, KT, DZant, MW, MD, RE, SJ, TG, DZann, PB, WB. Ethics Approval Statement: Study design and treatment ALLG MDS4 was an open-label, multi-centre study conducted across 30 Australian sites. The study was reviewed and approved by the Human Research Ethics Committees of each centre and conducted according to the Declaration of Helsinki. All patients provided written informed consent prior to participation.

Cooperative Epigenetic Remodeling by TET2 Loss and NRAS Mutation Drives Myeloid Transformation and MEK Inhibitor Sensitivity

Mutations in epigenetic modifiers and signaling factors often co-occur in myeloid malignancies, including TET2 and NRAS mutations. Concurrent Tet2 loss and NrasG12D expression in hematopoietic cells induced myeloid transformation, with a fully penetrant, lethal chronic myelomonocytic leukemia (CMML), which was serially transplantable. Tet2 loss and Nras mutation cooperatively led to decrease in negative regulators of mitogen-activated protein kinase (MAPK) activation, including Spry2, thereby causing synergistic activation of MAPK signaling by epigenetic silencing. Tet2/Nras double-mutant leukemia showed preferential sensitivity to MAPK kinase (MEK) inhibition in both mouse model and patient samples. These data provide insights into how epigenetic and signaling mutations cooperate in myeloid transformation and provide a rationale for mechanism-based therapy in CMML patients with these high-risk genetic lesions.

WHO2016 Vs. WHO2008 Criteria for the Diagnosis of Chronic Myelomonocytic Leukemia

Abstract The new WHO2016 diagnostic criteria for chronic myelomonocytic leukemia (CMML) require both absolute and relative monocytosis (≥1×109/L and ≥10% of the WBC count) in peripheral blood (PB). In addition, they say that the presence of myeloproliferative neoplasms (MPN) features in the bone marrow (BM) and/or MPN-associated mutations (JAK2, CALR, or MPL) tend to support MPN with monocytosis rather than CMML. This study was performed to predict impact of the WHO2016 criteria in the diagnosis of CMML compared to the WHO2008 criteria. We retrospectively reviewed clinical and laboratory data of 38 patients diagnosed of CMML (32 CMML-1, 6 CMML-2) using the WHO2008 criteria in 7 university hospitals from Jan 2012 to Jul 2016. The laboratory data included complete blood cell count with WBC differentials, BM morphology, cytogenetic study, and molecular genetic study such as BCR-ABL1 and mutational status of JAK2 V617F, JAK2 exon12, CALR, and MPL . Male to female ratio was 2.8:1 and the mean age was 71±12 years. The medians of WBC count, hemoglobin, and platelet count were 22×109/L, 90 g/L and 154×109/L, respectively. The medians of monocyte, absolute monocyte count, blast, and nucleated red cell in PB were 20 (range 6~44)%, 4.1 (range 1.1~41.8)×109/L, 1 (range 0~10)%, and 0 (range 0~5)%, respectively. The medians of cellularity, M:E ratio, blast, and monocyte in BM were 90 (range 25~100)%, 5 (range 2~26), 3 (range 0~17)%, and 7 (range 1~26)%, respectively. MPN-associated mutations were present in 8 (7 JAK2 V617F, 1 MPL) out of 21 patients tested and BCR-ABL1 was negative in all 27 patients tested. Coefficient of utilization for MPN-associated mutations at diagnosis, was very low (3~11%) except for JAK2 V617F (50%). WHO2008 criteria classified the patients into 32 CMML-1 and 6 CMML-2. WHO2016 criteria excluded 3 patients for not fulfilling relative monocytosis and 8 patients for having a MPN-associated mutation, and classified the 27 CMML patients into 17 CMML-0, 5 CMML-1, and 5 CMML-2. Sixteen was proliferative type (WBC count≥13×109/L) of CMML and 11 dysplastic type (WBC count The relative monocytosis (≥10% of the WBC count) was not met in 3 (8%) patients and MPN-associated mutations were found in 8 (21%) patients. The WHO2008 criteria might over diagnose CMML. More aggressive use of molecular tests including MPN-associated mutation analyses and more sophisticated clinical or morphologic evaluation are necessary in order to make an accurate diagnosis of CMML. Disclosures No relevant conflicts of interest to declare.

Overexpression of Arginase 1 is linked to DNMT3A and TET2 mutations in lower-grade myelodysplastic syndromes and chronic myelomonocytic leukemia

Immune dysregulation is a common feature of myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML), particularly in early stages. However, the genetic basis remains poorly understood. We recently reported that macrophages from mice deficient in tet methylcytosine dioxygenase 2 (Tet2), a model of MDS/CMML, are hyperinflammatory and have increased expression of arginase 1 (Arg1). In macrophages and myeloid derived suppressor cells (MDSCs) expression of Arg1 contributes to T-cell suppression and immune evasion by L-arginine depletion, in the setting of chronic inflammation and cancer. Since human MDS and CMML are driven by TET2 mutations and associated with chronic inflammation, we hypothesized that arginase enzymatic activity and ARG1 expression would be increased in human MDS/CMML bone marrow. Elevated arginase activity was observed in bone marrow mononuclear cells of MDS and CMML patients with lower-grade features. Immunohistochemical studies confirmed that myelomonocytic cells overexpress ARG1. Additionally, mutations in the epigenetic regulators TET2 and DNMT3A corresponded to high ARG1 expression and activity. These findings suggest ARG1 is a biomarker of immune dysregulation in early MDS and CMML. Recent murine findings have implicated Tet2 and Dnmt3a in regulation of innate immunity. Our study suggests similar changes may be driven by human TET2 and DNMT3A mutations.

Therapy related-chronic myelomonocytic leukemia (CMML): Molecular, cytogenetic, and clinical distinctions from de novo CMML.

Therapy related myeloid neoplasms (t-MN) including therapy related myelodysplastic syndromes (t-MDS) and acute myeloid leukemia (t-AML) are associated with aggressive disease biologies and poor outcomes. In this large (n = 497) and informative (inclusive of molecular and cytogenetic information) chronic myelomonocytic leukemia (CMML) patient cohort, we demonstrate key biological insights and an independent prognostic impact for t-CMML. T-CMML was diagnosed in 9% of patients and occurred approximately 7 years after exposure to prior chemotherapy and/or radiation therapy. In comparison to de novo CMML, t-CMML patients had higher LDH levels, higher frequency of karyotypic abnormalities and had higher risk cytogenetic stratification. There were no differences in the distribution of gene mutations and unlike t-MDS/AML, balanced chromosomal translocations, abnormalities of chromosome 11q23 (1%) and Tp53 mutations (<2%) were uncommon. Molecularly integrated CMML prognostic models were not effective in risk stratifying t-CMML patients and responses to hypomethylating agents were dismal with no complete responses. Median overall (OS) and leukemia free survival (LFS) was shorter for t-CMML in comparison to d-CMML (Median OS 10.9 vs 26 months and median LFS 50 vs 127 months) and t-CMML independently and adversely impacted OS (P = .0001 HR 2.1 95% CI 1.4-3.0). This prognostic impact was retained in the context of the Mayo Molecular Model (P = .001, HR 2.4, 95% CI 1.5-3.7) and the GFM prognostic model (P < .0001, HR 2.15, 95% CI 1.5-3.7). In summary, we highlight the unique genetics and independent prognostic impact of t-CMML, warranting its inclusion as a separate entity in the classification schema for both CMML and t-MN.

Tet2 restrains inflammatory gene expression in macrophages

Tet methylcytosine dioxygenase 2 (TET2) is one of the earliest and most frequently mutated genes in clonal hematopoiesis of indeterminate potential (CHIP) and myeloid cancers, including myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). TET2 catalyzes the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, leading to DNA demethylation, and also affects transcription by recruiting histone modifiers. Inactivating TET2 mutations cause epigenetic dysregulation, clonal hematopoietic stem cell (HSC) dominance, and monocytic lineage skewing. Here, we found that Tet2 was the most highly expressed Tet enzyme in murine macrophage (MΦ) differentiation. Tet2 transcription was further induced by lipopolysaccharide (LPS), but not interleukin (IL)-4, stimulation, potentially in a nuclear factor κβ-dependent manner. Tet2 loss did not affect early LPS gene responses in vitro, but increased Il-1b, Il-6, and Arginase 1 (Arg1) mRNA expression at later stages of stimulation in bone-marrow-derived MΦs (BMMΦs). Tet2-deficient peritoneal MΦs, however, demonstrated profound, constitutive expression of LPS-induced genes associated with an inflammatory state in vivo. In contrast, Tet2 deficiency did not affect alternative MΦ gene expression significantly in response to IL-4. These results suggested impaired resolution of inflammation in the absence of Tet2 both in vitro and in vivo. For the first time, we also detected TET2 mutations in BMMΦs from MDS and CMML patients and assayed their effects on LPS responses, including their potential influence on human IL-6 expression. Our results show that Tet2 restrains inflammation in murine MΦs and mice, raising the possibility that loss of TET2 function in MΦs may alter the immune environment in the large elderly population with TET2-mutant CHIP and in TET2-mutant myeloid cancer patients.

Hypomethylating agent therapy use and survival in older patients with chronic myelomonocytic leukemia in the United States: A large population-based study.

Despite the approval of azacitidine in 2004 and the approval of decitabine in 2006 in the United States for chronic myelomonocytic leukemia (CMML), the overall survival (OS) benefit with hypomethylating agent (HMA) therapy is unclear. Older adults (age ≥ 66 years) who had been diagnosed with CMML from 2001 to 2011 were selected from the Surveillance, Epidemiology, and End Results-Medicare database, and propensity score matching was used to match patients who had been diagnosed after HMA approval (2007-2011) and had received HMA treatment with patients diagnosed before HMA approval (2001-2003). Cox proportional hazards models with the matched sample were used to assess the change in OS. A second matched cohort of patients who did not receive HMA after approval and patients diagnosed before HMA approval was used to evaluate survival change attributable to other potential differences between the 2 time periods, such as improved supportive care. Among 1378 older adults diagnosed with CMML, the median OS was 13 months, and 18.8% received HMAs. In the primary matched analysis, with 225 HMA users diagnosed in 2007-2011 and 395 patients diagnosed in 2001-2003, the median OS times were 17 and 11 months, respectively (hazard ratio, 0.72; 95% confidence interval [CI], 0.58-0.91; P = .005). In a secondary analysis, the risk of death did not differ between 395 propensity score-matched HMA nonusers diagnosed in 2007-2011 and 484 patients diagnosed in 2001-2003 (hazard ratio, 1.09; 95% CI, 0.91-1.32; P = .34). Despite limited evidence, HMAs are commonly used to treat older CMML patients. The use of HMAs was associated with a 28% reduction in the risk of death in adjusted analyses. Improvements in supportive care do not appear to account for temporal improvements in OS. Cancer 2017;123:3754-3762. © 2017 American Cancer Society.

A phase II, multicentre trial of decitabine in higher-risk chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a complex clonal hematological disorder classified among myelodysplastic (MDS)/myeloproliferative neoplasms. Prognosis is poor and there is a lack of effective treatments. The hypomethylating agent decitabine has shown activity against MDS and elderly acute myeloid leukemia, but there is little data focusing specifically on its efficacy in CMML. In this prospective, phase 2 Italian study, CMML patients received intravenous decitabine 20 mg/m2 per day on Days 1–5 of a 28-day treatment cycle. Response was evaluated after four and six cycles; patients responding at the end of six cycles could continue treatment with decitabine. Forty-three patients were enrolled; >50% were high-risk according to four CMML-specific scoring systems. In the intent-to-treat population (n=42), the overall response rate after six cycles was 47.6%, with seven complete responses (16.6%), eight marrow responses (19%), one partial response (2.4%) and four hematological improvements (9.5%). After a median follow-up of 51.5 months (range: 44.4–57.2), median overall survival was 17 months, with responders having a significantly longer survival than non-responders (P=0.02). Grade 3/4 anemia, neutropenia and thrombocytopenia occurred in 28.6%, 50% and 38% of patients, respectively. Decitabine appears to be an effective and well-tolerated treatment for patients with high-risk CMML.

Genomic determinants of chronic myelomonocytic leukemia.

The biology, clinical phenotype and progression rate of chronic myelomonocytic leukemia (CMML) are highly variable due to diverse initiating and secondary clonal genetic events. To determine the effects of molecular features including clonal hierarchy in CMML, we studied whole-exome and targeted next-generation sequencing data from 150 patients with robust clinical and molecular annotation assessed cross-sectionally and at serial time points of disease evolution. To identify molecular lesions unique to CMML, we compared it to the related myeloid neoplasms (N=586), including juvenile myelomonocytic leukemia, myelodysplastic syndromes (MDS) and primary monocytic acute myeloid leukemia and discerned distinct molecular profiles despite similar pathomorphological features. Within CMML, mutations in certain pathways correlated with clinical classification, for example, proliferative vs dysplastic features. While most CMML patients (59%) had ancestral (dominant/co-dominant) mutations involving TET2, SRSF2 or ASXL1 genes, secondary subclonal hierarchy correlated with clinical phenotypes or outcomes. For example, progression was associated with acquisition of new expanding clones carrying biallelic TET2 mutations or RAS family, or spliceosomal gene mutations. In contrast, dysplastic features correlated with mutations usually encountered in MDS (for example, SF3B1 and U2AF1). Classification of CMML based on hierarchies of ancestral and subclonal mutational events may correlate strongly with clinical features and prognosis.

Clinical significance of CSF3R, SRSF2 and SETBP1 mutations in chronic neutrophilic leukemia and chronic myelomonocytic leukemia.

Chronic neutrophilic leukemia (CNL) and chronic myelomonocytic leukemia (CMML) are rare hematologic neoplasms. We performed CSF3R, SRSF2 and SETBP1 mutational analyses in 10 CNL and 56 CMML patients. In this sample cohort, 80% of CNL patients harbored CSF3R mutations, of which the CSF3R T618I mutation was dominant. Mutations in CSF3R and SETBP1 were found in 7.1% and 5.3% CMML patients respectively, while 25% of CMML patients carried SRSF2 mutations. Strikingly, we identified that all of the CSF3R mutations detected in CMML patients were represented by a P733T mutation. The CSF3R P733T mutation represents a novel CSF3R mutation. In addition, none of the four CSF3R P733T mutated patients carried SRSF2 mutations [0/14 (0%) patients with combined CSF3R P733T and SRSF2 mutations vs. 4/42 (9.5%) with CSF3R P733T and wt SRSF2, P < 0.001]. Both mut SRSF2 and mut SETBP1 patients had shorter overall survival (OS) and progression-free survival (PFS) compared to patients with wt SRSF2 (P < 0.001 both) and wt SETBP1 (P < 0.001 and P = 0.02, respectively). While we found no significant differences in OS and PFS as a consequence of CSF3R mutation status, our work suggest that the CSF3R T618I mutation is a diagnostic marker with good specificity and sensitivity for CNL. In conclusion, our study highlights effective diagnostic and prognostic markers of CNL and CMML patients in the Chinese population.

Prognostic significance of SETBP1 mutations in myelodysplastic syndromes, chronic myelomonocytic leukemia, and chronic neutrophilic leukemia: A meta-analysis.

ObjectivesThis meta-analysis investigates the prognostic effect of SET binding protein 1 (SETBP1) mutations in patients with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), or chronic neutrophilic leukemia (CNL).MethodsEligible studies from Pubmed, Embase, and Web of Science were searched from database inception through April 2016. Hazard ratios (HRs) and 95% confidence interval (CI) of overall survival (OS) were pooled to calculate the prognostic significance of SETBP1 mutation in patients.ResultsA total of 12 studies with 2321 patients were included in this meta-analysis; 4 studies for MDS, 5 studies for CMML, and 3 studies for CNL. Pooled results suggested that MDS and CMML patients with SETBP1 mutations had a significantly poorer prognosis when compared with patients with wild-type SETBP1 (MDS: HR = 1.808, 95% CI (1.218–2.685), P = 0.001; CMML: HR = 2.223, 95% CI (1.493–3.308), P<0.001). SETBP1 mutations in CNL patients however, showed no significant effect on the overall survival (HR = 1.773, 95% CI (0.877–3.582), P = 0.111). The Begg’s and Egger’s tests did not show significant publication bias in any groups.ConclusionsCurrent evidence shows that SETBP1 mutation is associated with a poor prognosis in patients with MDS and CMML, but not in patients with CNL.

DNMT3A mutations are associated with inferior overall and leukemia-free survival in chronic myelomonocytic leukemia.

DNMT3A mutations are seen in ∼5% of patients with chronic myelomonocytic leukemia (CMML) and thus far, have had an indeterminate prognostic impact on survival. We carried out this study to assess the prognostic impact of DNMT3A mutations on a larger informative cohort of CMML patients (n=261). DNMT3A mutations were seen in 6% (n=16); 56% (n=9) male, with a median age of 64years. Eighty-one % of DNMT3A mutations were missense, with the Arg882 mutational hot spot accounting for 63% of all changes. Five (31%) patients had an abnormal karyotype whereas concurrent gene mutations (SF3B1/SRSF2/U2AF1-56%, TET2-50%, and ASXL1-25%) were seen in all patients. Apart from a higher frequency of SF3B1 (P=0.0001) and PTPN11 (P=0.005) mutations and a lower frequency of SRSF2 (P=0.004) mutations, there were no significant differences between DNMT3A mutated patients and their wildtype counterparts. In univariate analysis, survival was shorter in DNMT3A mutated (median 8months) versus wildtype (median 27months) patients (P=0.0007; HR 2.9, 95% CI 1.5-5.7); with other variables of significance including lower hemoglobin (P=0.002), higher leukocyte count (P=0.0009), higher monocyte count (P=0.0012), circulating blast % (P=0.001), circulating immature myeloid cells (P=0.01), bone marrow blast % (P=0.045), abnormal karyotype (P=0.02), and ASXL1 (P=0.01) mutations. In a multivariable model that included the aforementioned variables, when both DNMT3A and ASXL1 mutations were added, only DNMT3A (P<0.0001) and ASXL1 (P=0.004) mutations remained significant. DNMT3A mutations were also predictive of a shortened leukemia-free survival. These findings warrant inclusion of DNMT3A mutations in molecularly integrated CMML prognostic models. Am. J. Hematol. 92:56-61, 2017. © 2016 Wiley Periodicals, Inc.

High Spontaneous In Vitro Myeloid Colony Formation in Chronic Myelomonocytic Leukemia is Associated with Mutations in Rasopathy Genes, Myeloproliferation and Inferior Prognosis

Background: Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy with features of both a myelodysplastic syndrome and a myeloproliferative neoplasm.The pathogenesis of CMML is incompletely understood due to the large heterogeneity of molecular aberrations in genes involved in epigenetic regulation, RNA-splicing and signal transduction including components of RAS and JAK2 signaling. Functional tests may be important to better estimate the contribution of a particular molecular aberration in the pathogenesis of the malignancy. We have originally demonstrated extensive in vitro formation of myeloid colonies (CFU-GM) without addition of exogenous growth factors in a subset of patients with CMML (Geissler et al, Leuk Res 1988). We reported that this spontaneous CFU-GM colony formation in CMML is a GM-CSF dependent in vitro phenomenon (Geissler et al, J Exp Med 1996) and could also show in a small retrospective study that CMML patients with high spontaneous CFU-GM growth (>100/105 PBMNC) have a worse prognosis compared to patients with low myeloid colony formation (Sagaster et al, Ann Hematol 2004) suggesting a clinical significance of our observation. In juvenile myelomonocytic leukemia, in which molecular aberrations are mainly restricted to the RASopathy genes including NRAS, KRAS, NF1, CBL and PTPN11, spontaneous formation of CFU-GM due to GM-CSF-specific hypersensitivity is a hallmark feature of disease, which has been included in the diagnostic criteria. We therefore speculated that high spontaneous myeloid colony formation in CMML might also reflect hyperactivation of the RAS signaling pathway.Aim: Our aim was to study the correlation between spontaneous myeloid colony formation and the presence of mutations in RASopathy genes in patients with CMML. Moreover the relationship of high autonomous CFU-GM formation with phenotypic features of CMML and its clinical outcome was investigated.Patients and Methods: In this study we included 137 CMML patients of the "Austrian Biodatabase for CMML (ABCMML)" in whom CFU-GM data and/or molecular data were available. CFU-GM growth in the absence of exogenous cytokines was assessed in a central laboratory using semisolid cultures as previously described (Geissler et al, J Exp Med 1996). Molecular characterization was also performed in a central laboratory using NGS with amplicon-based target enrichment of 39 CMML associated genes. Assuming that clones that are too small are unlikely to significantly impact hematopoiesis only mutations with an allele burden of ≥20% were considered positive in this analysis. Clinical and hematological data were obtained from patients records.Results:High spontaneous CFU-GM growth (≥100/105 PBMNC) was found in 38/135 (28%) CMML patients, of whom 3 were already transformed into secondary AML at the time of in vitro culture testing. There was a significant correlation between high CFU-GM formation in vitro and the presence of mutations in genes involved in the RAS signaling pathway. The incidence of RAS pathway mutations was 72% in CMML patients with high colony growth and 31% in patients with low spontaneous CFU-GM formation (p<0.0001). As shown in Table 1 high spontaneous myeloid colony formation was associated with increased WBC counts, increased blast cells, increased LDH, more pronounced splenomegaly and inferior survival (Fig. 1). There was no significant difference regarding autonomous CFU-GM growth in CMML patients with molecular aberrations in genes of epigenetic regulation and RNA-splicing, respectively. High spontaneous CFU-GM was never observed in CMML patients in whom the JAK2 V617F mutation was the only molecular aberration in signaling pathways (0/8 patients). Furthermore the in vitro conversion from a growth factor dependent to a growth factor independent phenotype by RAS but not by JAK2 could be demonstrated in BaF3 cells (Fig. 2).Conclusion: Our findings indicate that high spontaneous in vitro myeloid colony formation is associated with the presence of RAS pathway mutations, leukocytosis, splenomegaly and reduced survival. These results suggest that CMML with high spontaneous colony growth is a mainly RAS pathway driven malignancy resulting in myeloproliferation and inferior outcome. This may have clinical implications concerning therapeutic strategies aimed at targeting the hyperactive RAS signaling pathway in these patients. [Display omitted] [Display omitted] [Display omitted] DisclosuresGeissler:Novartis: Honoraria. Pfeilstöcker:Novartis: Consultancy, Speakers Bureau. Burgstaller:Novartis: Consultancy, Honoraria. Zach:Novartis: Other: Honoraria for Advisory Board. Hörmann:Novartis: Other: Honoraria for Advisory Board. Jäger:Roche: Other: Personal fees, Research Funding. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Kusec:Novartis: Other: Honoraria for lectures. Valent:Amgen: Honoraria; Novartis: Honoraria, Research Funding; Celegene: Honoraria, Research Funding.

Clinical, Hematological, and Biologic Characteristics in Chronic Myelomonocytic Leukemia Patients with a JAK2 V617F Mutation

▪Background:Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy of the elderly with a heterogenous molecular pathophysiology. Whereas mutations in components of the RAS pathways are among the most common somatic mutations in CMML the JAK2 V617F mutation which is a typical finding in polycythemia vera and around 50% of patients with essential thrombocythemia and primary myelofibrosis, respectively, is by far less frequently detected in CMML but can be consistently found in a subgroup of patients in larger series. Due to the fact that JAK2 V617F-positive CMML is a rare disease the clinical, hematological and in vitro growth characteristics of this entity are poorly investigated. In the "Austrian Biodatabase for Chronic Myelomonocytic Leukemia (ABCMML)" we retrospectively and prospectively collect clinical, biologic, and molecular information of patients with CMML from different centers in a real life setting.Aims:Our aim was to characterize the clinical, hematological, molecular and biologic features of CMML patients harboring a JAK2 V617F mutation.Methods:The diagnosis of CMML was established according to diagnostic criteria of the World Health Organization (WHO) classification of 2008 (Vardiman et al, Blood 2009). Clinical and hematological data were obtained from patients records. For molecular characterization we used next-generation sequencing with amplicon-based target enrichment of 39 CMML associated genes. Only mutations with an allele burden of >10% were considered positive in this analysis. Autonomous colony-forming units granulocyte/macrophage (CFU-GM) growth in the absence of exogenous cytokines was assessed using semisolid cultures as previously described (Geissler et al, J Exp Med 1996).Results:Up to now targeted NGS data are available in 116 patients and in vitro culture data in 75 patients respectively. We identified 13 CMML patients who had a JAK2 V617F mutation with an allele frequency >10%. Clinical, hematological, and biologic characteristics in these patients were compared with 103 patients who had NGS sequencing and were negative for the JAK2 V617F mutation. As shown in Table 1 JAK2 V617F-positive CMML patients had significantly higher WBC counts, higher hemoglobin values, higher platelet counts and more pronounced splenomegaly as compared to JAK2 V617F-negative patients. On the other hand the percentage on monocytes in peripheral blood and the numbers of CFU-GM growing in vitro without addition of exogenous growth factors were lower in CMML patients with the JAK2 V617F mutation as compared to patients without this mutation. The majority of JAK2 V617F-positive patients had additional mutations that can be also found in JAK2 V617F-negative patients, in particular mutations in genes of epigenetic regulation and RNA-splicing, respectively. As shown in Figure 1 there was a trend towards a better survival of patients with the JAK2 V617F mutation as compared to JAK2 V617F-negative patients (p=0.05). In a JAK2 V617F-positive CMML patient with splenomegaly, who was treated with the JAK1/2 inhibitor ruxolitinib off label, we were able to demonstrate the disappearance of constitutional symptoms and a durable spleen response lasting for over 56 months (Fig. 2).Conclusion:Out data show that CMML patients with the JAK2 V617F mutation have hematological, biologic and clinical characteristics different from JAK2 V617F-negative CMML patients. These findings suggest that JAK2 V617F-positive CMML patients should be regarded as a distinct subgroup which may benefit from specific targeted treatments. [Display omitted] [Display omitted] [Display omitted] DisclosuresGeissler:Novartis: Honoraria. Pfeilstöcker:Novartis: Consultancy, Speakers Bureau. Burgstaller:Novartis: Consultancy, Honoraria. Zach:Novartis: Other: Honoraria for Advisory Board. Hörmann:Novartis: Other: Honoraria for Advisory Board. Jäger:Roche: Other: Personal fees, Research Funding. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Kusec:Novartis: Other: Honoraria for lectures. Valent:Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celegene: Honoraria, Research Funding.

Time-to-Treatment in Chronic Myelomonocytic Leukemia - a Novel Prediction Model

IntroductionFor chronic myelomonocytic leukemia (CMML) several scores exist which prognosticate overall survival (OS) based on different clinical and genetic parameters. The time-to-treatment (TTT) among CMML patients is highly variable, and a predictive model to specifically estimate TTT in CMML has not been described so far. The aims of this single-center retrospective study were (a) to test and validate established myelodysplastic syndrome (MDS)-specific and CMML-specific prognostic scores in our patient cohort, (b) to evaluate which baseline factors were relevant to the time point of treatment initiation with either hydroxyurea or azacitidine, and (c) to propose a prediction model for TTT in CMML.MethodsThis retrospective analysis was based on the data of 55 unselected, consecutive CMML patients diagnosed and/or treated at our tertiary center between 2004 and 2015. We applied the following published prognostic models to our CMML cohort, using both OS and TTT as endpoints: the MD Anderson Prognostic Score (MDAPS), the modified MDAPS (MDAPS M1), the CMML-specific Prognostic Scoring System (CPSS), the Mayo Prognostic Model, the Düsseldorf Score, the International Prognostic Scoring System (IPSS), and the Revised International Prognostic Scoring System (IPSS-R).ResultsAccording to the CMML-specific MDAPS, 27% of our patients were classified as "higher-risk" (23% intermediate-2, 4% high-risk) (Figure 1). At the time of data analysis, 38% and 24% of patients had received azacitidine and hydroxyurea as first-line treatment. A total of 40 (73%) patients had died at the time point of data analysis. The median time of follow-up was 24.8 months (range 1.7-74.8 months).All applied MDS-specific (Düsseldorf Score, IPSS, IPSS-R) and CMML-specific (MDAPS, MDAPS M1, CPSS, Mayo Prognostic Model) prediction scores were able to significantly discriminate patient cohorts with different OS probabilities. The following variables were associated with a shorter TTT in the univariate analysis: the presence of immature myeloid cells in the peripheral blood, white blood cell count ≥14.5 G/L, platelet count <55 G/L, absolute neutrophil count ≥6 G/L, absolute lymphocyte count ≥2.3 G/L, absolute monocyte count ≥2.8 G/L, serum lactate dehydrogenase ≥223 G/L, peripheral blood blasts >0%, bone marrow blast percentage ≥7.5%, red blood cell transfusion-dependence, palpable spleen and/or symptomatic splenomegaly, and the presence of B-symptoms at the time of initial diagnosis. In multivariate analysis, the following factors remained independently associated with TTT: lactate dehydrogenase (HR 5.428; p = 0.008), bone marrow blast count (HR 4.570; p = 0.001), and platelet count (HR 2.660; p = 0.027).These three clinical parameters were included in the TTT prediction model and CMML patients were stratified into three subgroups: low-risk, intermediate-risk and high-risk. Median TTT was not reached for low-risk patients, 16.5 months for intermediate-risk patients, and almost immediate treatment initiation (0.6 months) was observed in the high-risk group (Figure 2).ConclusionsWe validated seven existing MDS-specific and CMML-specific prognostic scores in 55 CMML patients treated at the center in Salzburg. We were able to demonstrate that lactate dehydrogenase, bone marrow blast percentage and platelet count at initial diagnosis were the most relevant parameters for predicting time to treatment initiation in our CMML cohort. Based on these three parameters, we propose the first TTT prediction score for treatment-naïve CMML patients. Clinical implications of this score include the identification of CMML patients for early investigational trials, as well as the tailoring of individual follow-up intervals. [Display omitted] [Display omitted] DisclosuresHuemer:Roche: Other: Travel funding; Merck: Other: Travel funding. Egle:Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: travel support; Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel support. Greil:Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Eisai: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Merck: Honoraria; Janssen-Cilag: Honoraria; Genentech: Honoraria, Research Funding; Novartis: Honoraria; AstraZeneca: Honoraria; Roche: Honoraria, Research Funding; Sanofi Aventis: Honoraria; GSK: Research Funding; Ratiopharm: Research Funding; Cephalon: Consultancy, Honoraria, Research Funding; Bristol-Myers-Squibb: Consultancy, Honoraria. Pleyer:Celgene: Consultancy, Honoraria; Bristol-Myers-Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AOP Orphan Pharmaceuticals: Honoraria.

MIR150 Is Involved in the Monocyte Subset Differentiation and Its Down-Regulation Leads to Classical Monocyte Accumulation in Chronic Myelomonocytic Leukemia

Background. Monocytes are a heterogeneous population of peripheral blood leukocytes. The expression of CD14 and CD16 distinguishes CD14+/CD16- classical from CD14+/CD16+ intermediate and CD14low/CD16+ non-classical monocytes. We have shown (Selimoglu-Buet D et al, Blood 2015) that monocytes that accumulate in the peripheral blood of patients with chronic myelomonocytic leukemia (CMML) are predominantly CD14+/CD16- classical monocytes that typically represent more than 94% of total blood monocytes. Strikingly, this phenotypic signature efficiently distinguishes CMML from a reactive monocytosis. Importantly, the CMML-associated increase in classical monocyte fraction disappears in patients who respond to hypomethylating drugs. Whereas in the mouse, the transcription factor Nr4a1 is required for the development of the Ly6Clowmonocytes, the molecular mechanisms that regulate the formation of the three human monocyte populations remain poorly understood. Analysis of the classical monocytes accumulation in CMML may provide insights into the regulation of monocyte subset differentiation.Methods. A microarray screen of miRNA expression was performed in monocytes sorted from 33 CMML and 5 healthy donor blood samples. Validation was performed by qRT-PCR, in monocytes of a cohort of 160 CMML patients and 20 controls, and in CD34+ cells from 44 CMML patients and 19 controls. A mouse model of MIR150-knock-out (Mir150-/-) was used to examine the consequences of the miRNA down-regulation. Multi-color flow cytometry assays were designed to explore mouse and human monocyte subsets.Results. Microarray analyses and validation experiments identified a decreased expression of miR150 in monocytes and CD34+cells from CMML patients compared to controls.Mir150-/- mouse model does not generate monocytosis even in ageing animals. However, an increase in Ly6Chigh inflammatory monocyte fraction at the expense of Ly6Clowpatrolling monocytes was observed in the bone marrow and peripheral blood, leading to further explore the link between MIR150 and monocyte populations.The abnormal repartition of monocyte populations in Mir150-/- mice is a cell-autonomous phenotype as wild-type (WT) mice receiving bone marrow from Mir150-/- mice demonstrated a reduced fraction of Ly6Clow monocytes. This phenotype was rescued by re-expression of MIR150 in LIN- cells of Mir150-/- mice before engraftment. The number of myeloid progenitors was normal in Mir150-/- mice, and the remaining Ly6Clow monocytes did not demonstrate an increased sensitivity to apoptosis. Competitive reconstitution experiments combining WT and Mir150-/- LIN- cells did not identify any significant fitness advantage to Mir150-/- cells, but Mir150-/- donor cells developed reduced numbers of Ly6Clow monocytes than cells from WT donors. These data suggest that MIR150 is involved during late stages of monocyte development and has a key role in the generation of Ly6Clowmonocytes.Finally, TET2 is the main gene mutated in CMML, and Tet2-/- animals develop a monocytosis. Mir150-/- crossed with Tet2-/-mice developed a CMML-like phenotype.In human, the expression of MIR150 decreases along myeloid differentiation and is low in classical compared to intermediate and non-classical monocytes. Depletion or overexpression of MIR150 in human CD34+ cells alters the repartition of CD14+/CD16- and CD14+/CD16+ cells generated in culture. In CMML patients who respond to hypomethylating agents, the normalization of monocyte subset repartition correlates with an increased expression of MIR150, suggesting an epigenetic regulation. MIR150 has several promoters. By combining ChIP-Seq and DNA methylation analyses, a differentially methylated region was detected in one of the MIR150 promoters in CMML patients compared to controls. Using monocyte differentiation conditions, RNA Sequencing performed in CD34+cells overexpressing MIR150, identified ID1 gene as a potential MIR150 target.Conclusion: We demonstrate a role for MIR150 in the generation of intermediate and non-classical monocyte subsets, and its down-regulation in CMML accounts for the characteristic accumulation of classical monocytes. DisclosuresFenaux:Celgene, Janssen,Novartis, Astex, Teva: Honoraria, Research Funding.

Patient-Derived Induced Pluripotent Stem Cells Identified SLITRK4 As a Causative Gene of Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML), the most frequent disease entity of myelodysplastic syndrome/myeloproliferative neoplasm is a clonal hematopoietic malignancy that is characterized by persistent monocytosis, morphologic myeloid dysplasia, and progression to acute myeloid leukemia. The pathogenesis of CMML remains entirely elusive because of the lack of suitable mouse models and the difficulties in the establishment of CMML cell lines.We have previously reported that we established induced pluripotent stem cells (iPSC) from CMML CD34 positive leukemic cells (CMML-iPSC) as a new disease model. Co-cultured with C3H10T1/2 stromal cells in the presence of vascular endothelial growth factor, CMML-iPSC generated CD34 CD43 double-positive hematopoietic progenitor cells (CMML-HPC). CMML-HPC have recapitulated important disease features of parental CMML cells in terms of genetic abnormalities, acceleration of cell proliferation, and aberrant surface markers expression. In addition, a novel human CMML xenograft mouse model has been established through secondary transplantation of human HPCs from CMML-iPSC-derived teratomas. This model produced HPCs that mimicked the properties of CMML in vivo.To identify key molecular abnormalities that contribute to the pathophysiology of CMML, we conducted comprehensive gene expression and DNA methylation profiling analyses of normal and CMML parental CD34 positive cells, iPSC, and their hematopoietic progenies, respectively. Correlation analysis revealed that gene expression and DNA methylation status between normal and CMML iPSC-derived HPC exhibited similar pattern (R2 = 0.92 and 0.96, respectively), although normal and CMML parental CD34 positive cells were quite different (R2 = 0.72 and 0.90, respectively), indicating that reprogramming followed by re-differentiation may enable to obtain more homogenous population of normal and CMML cells that reside in almost the same differentiation stage. These results allowed us to determine the difference in the genetic and epigenetic status between normal and CMML iPSC-derived HPC, which remained through reprogramming and re-differentiation, in order to find out causative genes in the pathogenesis of CMML.Using these combined omics platforms, we identified SLIT and NTRK like family member 4 (SLITRK4) as a candidate gene involving in pathogenesis of CMML, whose expression was enhanced and whose promoters were hypo-methylated in CMML-HPC. In other CMML patients' CD34 positive leukemic cells, the expression of SLITRK4 was up-regulated compared to healthy CD34 positive bone marrow cells and other leukemia cells. In addition, we revealed SLITRK4 had pro-proliferative activity as the knockdown of SLITRK4 inhibited proliferation of leukemic cell lines OCI-AML3. To elucidate whether SLITRK4 exert any biological functions in CMML, we established CMML-iPSC clones harboring hetero-knockout (wt/-) or homo-knockout (-/-) of SLITRK4 gene by CRISPR/Cas9 system. Although SLITRK4 (wt/-) and (-/-) clones did not exhibit any morphological and proliferative difference in CMML-iPSC, the production of HPC from CMML-iPSC was dramatically attenuated in SLITRK4-dependent manner. Therefore, while little has been known about the roles of SLITRK molecules in tumorigenesis, we demonstrated SLITRK4 was indispensable for generation of CMML leukemic cells and suggested the possibility of novel molecular therapy targeting SLITRK4, based on the findings obtained from our combined omics platforms.In summary, we identified SLITRK4 as a novel candidate gene responsible for the pathogenesis of CMML through our combined omics platform using patient-derived iPSC. This platform may provide a potential to trace causative genes in a variety of diseases. DisclosuresKataoka:Kyowa Hakko Kirin: Honoraria; Boehringer Ingelheim: Honoraria; Yakult: Honoraria.

Cooperative Epigenetic Remodeling By TET2 Loss and NRAS Mutation Drives Myeloid Transformation and MEK Inhibitor Sensitivity

Epigenetic modifiers and signaling factors are frequently mutated and often co-occur in various myeloid malignancies. However, precisely how these mutations cooperate to cause myeloid leukemia is not fully understood. Here, we show that cells with concurrent Ten-eleven-translocation 2 (Tet2) loss and Nras mutation can cause lethal chronic myelomonocytic leukemia (CMML) like disease in vivo and synergistically activate Ras signaling through epigenetic silencing of Sprouty 2 (Spry2), thereby making cells with both disease alleles dependent on MAPK signaling and highly sensitive to MEK inhibition.To assess if Tet2 loss and Nras mutation cooperate in myeloid transformation, we crossed Tet2 conditional knockout mice (Mx1-Cre+Tet2f/f) and Nras mutant mice (Mx1-Cre+Nras+/G12D) to generate Mx1-Cre+Tet2f/fNras+/G12D mice (Tet2Δ/ΔNras+/G12D). These mice, compared to single mutant mice with either allele alone, had more significant monocytosis, expansion of Lineage- Sca-1+ c-Kit+ (LSK) and myeloid progenitors in both bone marrow (BM) and spleen and development of lethal CMML-like disease (median survival 264 days). Moreover, serial transplantation of splenic cells derived from leukemic Tet2Δ/ΔNras+/G12D mice caused similar CMML-like disease in recipients, which emanates from LSK-positive stem/progenitor cells as the disease propagating population.To delineate how Tet2 loss and Nras mutation synergize in leukemic transformation, we next performed western blot and phospho-flow analysis of MAPK and PI3K signaling in primary hematopoietic cells. Interestingly, pErk, pAkt and pS6 expression were significantly higher in Tet2Δ/ΔNras+/G12D cells compared to WT or single mutant cells, indicating that Tet2 loss and Nras mutation cooperates to further activate Ras signaling (Figure 1). Consistent with our murine model, TET2 silencing in NRAS mutant human leukemia cells increased MAPK output, consistent with augmentation of signaling by concurrent TET2/NRAS alterations in human leukemia cells.To unravel the molecular mechanism of Ras signaling activation, we assessed mRNA / protein expression and performed bisulfite sequencing of known regulators of MAPK signaling, including Sprouty family members. We observed significant decrease of Spry2 expression, stepwise and specific hyper-methylation of CpG islands in the Spry2 promoter region in Tet2Δ/ΔNras+/G12D cells compared to WT or single mutant cells, consistent with progressive epigenetic remodeling in these leukemia cells in vivo (Figure 2). Genome wide methylation profiling of WT, single mutant and Tet2Δ/ΔNras+/G12D LSK cells using enhanced reduced representation bisulfite sequencing demonstrated clear separation of leukemic Tet2Δ/ΔNras+/G12D LSKs from WT or single mutant LSKs. Most importantly, restoration of Spry2 expression in Tet2Δ/ΔNras+/G12D cells led to decrease in pErk / pAkt level and significantly reduced colony formation, which functionally validates Spry2 as a key epigenetic target in Tet2/Nras mutant leukemia cells.We next assessed whether the increased MAPK signaling seen in Tet2Δ/ΔNras+/G12D cells leads to differential sensitivity to MEK inhibition by performing studies with the clinical MEK inhibitor binimetinib (ARRY162). Tet2Δ/ΔNras+/G12D cells showed significantly higher sensitivity to binimetinib compared to Nras+/G12D cells in vitro (IC50, 6.948nM vs. 690.4nM). Moreover, in vivo treatment of Tet2Δ/ΔNras+/G12D leukemic recipients with binimetinib restored splenomegaly, significantly reduced disease burden in BM and spleen and improved overall survival compared to vehicle treatment (median survival 24.5 days vs. 44.5 days, p=0.0018, Figure 3). Of note, knockdown of human TET2 in NRAS mutant human leukemia cells sensitized to MEK inhibition in a similar manner demonstrating this approach may have value in leukemia patients with concurrent TET2 and NRAS mutations.These data clearly indicate that Tet2 loss and Nras mutation synergize in myeloid transformation and cooperatively remodel DNA methylation, which leads to epigenetic silencing of Spry2 and synergistic activation of MAPK signaling, which can be leveraged through therapeutic MEK inhibition. Our studies provide novel insights into how signaling and epigenetic mutations cooperate in leukemic transformation and provide a rationale for mechanism based therapy in CMML patients with these high risk genetic lesions. [Display omitted] [Display omitted] [Display omitted] DisclosuresMelnick:Janssen: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.