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The current study was inspired by observations from exploratory analyses of an institutional cohort with chronic myelomonocytic leukemia (CMML; N = 398) that revealed no instances of blast transformation in the seven patients with plant homeodomain finger protein 6 (PHF6) mutation (PHF6 MUT ). A subsequent Mayo Clinic enterprise-wide database search identified 28 more cases with PHF6 MUT . Compared with their wild-type PHF6 counterparts (PHF6 WT ; N = 391), PHF6 MUT cases (N = 35) were more likely to co-express TET2 (89% vs. 45%; p < .01), RUNX1 (29% vs. 14%; p = .03), CBL (14% vs. 2%; p < .01), and U2AF1 (17% vs. 6%; p = .04) and less likely SRSF2 (23% vs. 45%; p < .01) mutation. They were also more likely to display loss of Y chromosome (LoY; 21% vs. 2%; p < .01) and platelets <100 × 10 9 /L (83% vs. 51%; p < .01). Multivariable analysis identified PHF6 MUT (HR 0.28, 95% CI 0.15-0.50) and DNMT3A MUT (HR 5.8, 95% CI 3.3-10.5) as the strongest molecular predictors of overall survival. The same was true for blast transformation-free survival with corresponding HR (95% CI) of 0.08 (0.01-0.6) and 9.5 (3.8-23.5). At median 20 months follow-up, blast transformation was documented in none of the 33 patients with PHF6 MUT /DNMT3A WT but in 6 (32%) of 19 with DNMT3A MUT and 74 (20%) of 374 with PHF6 WT /DNMT3A WT (p < .01). The specific molecular signatures sustained their significant predictive performance in the context of the CMML-specific molecular prognostic model (CPSS-mol). PHF6 MUT identifies a unique subset of patients with CMML characterized by thrombocytopenia, higher prevalence of LoY, and superior prognosis., (© 2024 The Author(s). American Journal of Hematology published by Wiley Periodicals LLC.)

Purpose: The prognostic role of TET2 and/or ASXL1 mutations which are common gene mutations in chronic myelomonocytic leukemia (CMML) remains controversial. Therefore, we conducted this meta-analysis to evaluate the prognostic efficacy of ASXL1 and TET2 mutations in CMML population., Methods: PubMed, Cochrane and Embase for relevant research were employed to identify 16 studies. Overall survival rate (OS) with hazard ratios (HRs) was used for analysis, and each individual HR was applied to calculate the combined HR., Results: The total HR of OS was 0.74, 95% CI = 0.61 - 0.91, P  = 0.005, compared with CMML patients without TET2 mutations (TET2 MT ), and the total HR of OS was 1.56, 95% CI = 1.34 - 1.80, P  = 0.000, compared with CMML patients without ASXL1 mutation (ASXL1 WT ), indicating that TET2 MT and ASXL1 WT were favorable for prognosis of CMML. According to whether the gene is mutated or not, the acute transformation rate of disease and mortality rate were further considered for assessment. Compared with the CMML patients with TET2 MT and ASXL1 WT , the HR of patients with in both TET2 MT and ASXL1 MT was 1.51 (95% CI = 1.14 - 1.99; P  = 0.004), the HR of patients with neither TET2 MT nor ASXL1 MT was 1.49 (95%CI = 1.12 - 1.98; P  = 0.007), and the HR of TET2 WT and ASXL1 MT patients was 1.88 (95%CI = 1.21 - 2.94; P  = 0.005)., Conclusion: Presence of TET2 MT and ASXL1 WT genotype was the most beneficial for the survival of CMML patients.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations., (© 2022 by The American Society of Hematology.)

We evaluated the mutational landscape of chronic myelomonocytic leukemia (CMML) and its potential clinical significance. We analyzed 47 samples with a panel of 112 genes using next-generation sequencing. Forty-five of the 47 patients (95.74%) had at least one mutation identified, with an average of 3.7 (range 0-9) per patient. The most common mutation was NRAS, followed by ASXL1, TET2, SRSF2, RUNX1, KRAS, and SETBP1. Patients 60 years and older more frequently had mutations in TET2 (56% vs. 9.09%, P = 0.001) and ASXL1 (48% vs. 18.18%, P = 0.031) than patients younger than 60 years. Median overall survival (OS) in patients with CMML was 22.0 months (95% CI 19.7-24.3 months). ASXL1 (18 vs. 22 months, P = 0.012), RUNX1 (17 vs. 22 months, P = 0.001), and SETBP1 (20 vs. 27 months, P = 0.032) mutations predicted inferior OS. However, only RUNX1 mutation was significantly associated with inferior acute myeloid leukemia (AML)-free survival. Our data showed that mutation profile differed significantly between CMML patients aged 60 years and older versus those younger than 60 years, and some of these mutations impact the progression and prognosis of the disease to a certain extent., (© 2021. Japanese Society of Hematology.)

ASXL1 is one of the most frequently mutated genes in malignant myeloid diseases. In patients with myeloid malignancies, ASXL1 mutations are usually heterozygous frameshift or nonsense mutations leading to C-terminal truncation. Current disease models have predominantly total loss of ASXL1 or overexpressed C-terminal truncations. These models cannot fully recapitulate leukemogenesis and disease progression. We generated an endogenous C-terminal-truncated Asxl1 mutant in zebrafish that mimics human myeloid malignancies. At the embryonic stage, neutrophil differentiation was explicitly blocked. At 6 months, mutants initially exhibited a myelodysplastic syndrome-like phenotype with neutrophilic dysplasia. At 1 year, about 13% of mutants further acquired the phenotype of monocytosis, which mimics chronic myelomonocytic leukemia, or increased progenitors, which mimics acute myeloid leukemia. These features are comparable to myeloid malignancy progression in humans. Furthermore, transcriptome analysis, inhibitor treatment, and rescue assays indicated that asxl1-induced neutrophilic dysplasia was associated with reduced expression of bmi1a, a subunit of polycomb repressive complex 1 and a reported myeloid leukemia-associated gene. Our model demonstrated that neutrophilic dysplasia caused by asxl1 mutation is a foundation for the progression of myeloid malignancies, and illustrated a possible effect of the Asxl1-Bmi1a axis on regulating neutrophil development., (© 2021. The Author(s).)

While oncogenes promote tumorigenesis, they also induce deleterious cellular stresses, such as apoptosis, that cancer cells must combat by coopting adaptive responses. Whether tumor suppressor gene haploinsufficiency leads to such phenomena and their mechanistic basis is unclear. Here, we demonstrate that elevated levels of the anti-apoptotic factor, CASP8 and FADD-like apoptosis regulator (CFLAR), promotes apoptosis evasion in acute myeloid leukemia (AML) cells haploinsufficient for the cut-like homeobox 1 (CUX1) transcription factor, whose loss is associated with dismal clinical prognosis. Genome-wide CRISPR/Cas9 screening identifies CFLAR as a selective, acquired vulnerability in CUX1-deficient AML, which can be mimicked therapeutically using inhibitor of apoptosis (IAP) antagonists in murine and human AML cells. Mechanistically, CUX1 deficiency directly alleviates CUX1 repression of the CFLAR promoter to drive CFLAR expression and leukemia survival. These data establish how haploinsufficiency of a tumor suppressor is sufficient to induce advantageous anti-apoptosis cell survival pathways and concurrently nominate CFLAR as potential therapeutic target in these poor-prognosis leukemias.

Many prognostic scoring systems have been developed for chronic myelomonocytic leukemia (CMML). Although these efforts have been informative, no single model has been considered the consensus for CMML prognostication and all models are only moderately prognostic. CMML clinical models utilize mainly hematology and morphology parameters to estimate risk. A better understanding of cytogenetics and the genomic landscape of CMML have resulted in integrated risk models such as CMML Prognostic Scoring System (CPSS)-Mol and Mayo Molecular that may provide better prognostic accuracy for an individual patient. For example, frameshift/nonsense ASXL1 mutations have been consistently shown to confer inferior outcomes leading to its incorporation into some of the major risk classification systems. Prognostication in the setting of therapeutic interventions such as hypomethylating agents and allogeneic hematopoietic cell transplantation have also garnered considerable interest. Despite having many validated risk models available, not a single system is universally adopted. Herein, we will provide an overview of how these systems evolved and progress toward a uniform system., Competing Interests: Declaration of competing interest The authors have no relevant conflicts of interest to disclose., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Additional sex combs-like 1 (ASXL1) mutations have been described in all forms of myeloid neoplasms including chronic myelomonocytic leukemia (CMML) and associated with inferior outcomes, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) remains poorly understood. Transformation of CMML to secondary AML (sAML) is one of the leading causes of death in CMML patients. Previously, we observed that transcription factor RUNX1 mutations (RUNX1-MT) coexisted with ASXL1-MT in CMML and at myeloid blast phase of chronic myeloid leukemia. The contribution of RUNX1 mutations in the pathogenesis of myeloid transformation in ASXL1-mutated leukemia, however, remains unclear., Methods: To evaluate the leukemogenic role of RUNX1-MT in ASXL1-mutated cells, we co-expressed RUNX1-MT (R135T) and ASXL1-MT (R693X) in different cell lines and performed immunoblot, co-immunoprecipitation, gene expression microarray, quantitative RT-PCR, cell proliferation, differentiation, and clonogenic assays for in vitro functional analyses. The in vivo effect was investigated using the C57BL/6 mouse bone marrow transplantation (BMT) model., Results: Co-expression of two mutant genes increased myeloid stem cells in animal model, suggesting that cooperation of RUNX1 and ASXL1 mutations played a critical role in leukemia transformation. The expression of RUNX1 mutant in ASXL1-mutated myeloid cells augmented proliferation, blocked differentiation, and increased self-renewal activity. At 9 months post-BMT, mice harboring combined RUNX1 and ASXL1 mutations developed disease characterized by marked splenomegaly, hepatomegaly, and leukocytosis with a shorter latency. Mice transduced with both ASXL1 and RUNX1 mutations enhanced inhibitor of DNA binding 1 (ID1) expression in the spleen, liver, and bone marrow cells. Bone marrow samples from CMML showed that ID1 overexpressed in coexisted mutations of RUNX1 and ASXL1 compared to normal control and either RUNX1-MT or ASXL1-MT samples. Moreover, the RUNX1 mutant protein was more stable than WT and increased HIF1-α and its target ID1 gene expression in ASXL1 mutant cells., Conclusion: The present study demonstrated the biological and functional evidence for the critical role of RUNX1-MT in ASXL1-mutated leukemia in the pathogenesis of myeloid malignancies.

Background: Molecular genetic abnormalities are observed in over 90% of chronic myelomonocytic leukemia (CMML) cases. Recently, several studies have demonstrated the negative prognostic impact of ASXL1 mutations in CMML patients. We evaluated the prognostic impact of ASXL1 mutations and compared five CMML prognostic models in Korean patients with CMML., Methods: We analyzed data from 36 of 57 patients diagnosed as having CMML from January 2000 to March 2016. ASXL1 mutation analysis was performed by direct sequencing, and the clinical and laboratory features of patients were compared according to ASXL1 mutation status., Results: ASXL1 mutations were detected in 18 patients (50%). There were no significant differences between the clinical and laboratory characteristics of ASXL1-mutated (ASXL1⁺) CMML and ASXL1-nonmutated (ASXL1⁻) CMML patients (all P>0.05). During the median follow-up of 14 months (range, 0-111 months), the overall survival (OS) of ASXL1⁺ CMML patients was significantly inferior to that of ASXL1⁻ CMML patients with a median survival of 11 months and 19 months, respectively (log-rank P=0.049). An evaluation of OS according to the prognostic models demonstrated inferior survival in patients with a higher risk category according to the Mayo molecular model (log-rank P=0.001); the other scoring systems did not demonstrate a significant association with survival., Conclusions: We demonstrated that ASXL1 mutations, occurring in half of the Korean CMML patients examined, were associated with inferior survival. ASXL1 mutation status needs to be determined for risk stratification in CMML., Competing Interests: No potential conflicts of interest relevant to this article were reported., (© The Korean Society for Laboratory Medicine.)

Additional sex combs-like 1 (ASXL1) is a well‑known tumor suppressor gene and epigenetic modifier. ASXL1 mutations are frequent in myeloid malignances; these mutations are risk factors for the development of myelodysplasia and also appear as small clones during normal aging. ASXL1 appears to act as an epigenetic regulator of cell survival and myeloid differentiation; however, the molecular mechanisms underlying the malignant transformation of cells with ASXL1 mutations are not well defined. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing, heterozygous and homozygous ASXL1 mutations were introduced into human U937 leukemic cells. Comparable cell growth and cell cycle progression were observed between wild-type (WT) and ASXL1-mutated U937 cells. Drug-induced cytotoxicity, as measured by growth inhibition and apoptosis in the presence of the cell-cycle active agent 5-fluorouracil, was variable among the mutated clones but was not significantly different from WT cells. In addition, ASXL1-mutated cells exhibited defects in monocyte/macrophage differentiation. Transcriptome analysis revealed that ASXL1 mutations altered differentiation of U937 cells by disturbing genes involved in myeloid differentiation, including cytochrome B-245 β chain and C-type lectin domain family 5, member A. Dysregulation of numerous gene sets associated with cell death and survival were also observed in ASXL1-mutated cells. These data provide evidence regarding the underlying molecular mechanisms induced by mutated ASXL1 in leukemogenesis.

Objectives: Although additional sex comb-like 1 (ASXL1) gene mutations have long been reported in myelodysplastic syndromes (MDSs) and chronic myelomonocytic leukemia (CMML), the prognostic significance has been controversial. Therefore, a meta-analysis to study the impact of ASXL1 mutations on patients with MDS and CMML is useful., Methods: The identified articles were retrieved from some common databases. We extracted hazard ratios (HRs) for overall survival (OS) and leukemic-free survival (LFS) and P-value of some clinical parameters, which compared AXSL1 mutations to those without from the available studies. Each individual HR and P-value was used to calculate the pooled HR and P-value., Results: Six studies covering 1689 patients were selected for this meta-analysis. The pooled HRs for OS and LFS were 1.45 (95% confidential interval (CI), 1.24-1.70) and 2.20 (95% CI, 1.53-3.17), respectively. When considering CMML patients alone the HR for OS was 1.50 (95% CI, 1.18-1.90). Additionally, ASXL1 mutations were more frequently found in male (P = 0.008), older (P = 0.019), and patients with lower platelets (P = 0.009) or hemoglobin level (P = 0.0015) and associated with other mutations such as EZH2, IDH1/2, RUNX1, and TET2., Discussion: Although our analysis has its limitation, it showed that ASXL1 mutations had significant inferior impact on OS and LFS for French-American-British-defined MDS patients. However, the influence of different types of ASXL1 mutations on patients with MDS still needs illustrating., Conclusion: ASXL1 mutations were associated with poor prognosis in MDS, which may contribute to risk stratification and prognostic assessment in the disease.

Chronic myelomonocytic leukemia (CMML) is a clonal stem cell disorder with features that overlap those of myelodysplastic syndromes (MDSs) and myeloproliferative neoplasms (MPNs). Chronic myelomonocytic leukemia often results in peripheral blood monocytosis and has an inherent tendency to transform to acute myeloid leukemia. Clonal cytogenetic changes are seen in approximately 30% of patients, and molecular abnormalities are seen in more than 90%. Gene mutations involving TET2 (∼60%), SRSF2 (∼50%), ASXL1 (∼40%), and RAS (∼30%) are frequent, with nonsense and frameshift ASXL1 mutations being the only mutations identified thus far to have an independent negative prognostic effect on overall survival. Contemporary molecularly integrated prognostic models (inclusive of ASXL1 mutations) include the Molecular Mayo Model and the Groupe Français des Myélodysplasies model. Given the lack of formal treatment and response criteria, management of CMML is often extrapolated from MDS and MPN, with allogeneic stem cell transplant being the only curative option. Hydroxyurea and other cytoreductive agents have been used to control MPN-like features, while epigenetic modifiers such as hypomethylating agents have been used for MDS-like features. Given the relatively poor response to these agents and the inherent risks associated with hematopoietic stem cell transplant, newer drugs exploiting molecular and epigenetic abnormalities in CMML are being developed. The creation of CMML-specific response criteria is a much needed step in order to improve clinical outcomes., (Copyright © 2016 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

Mutations involving epigenetic regulators (TET2~60% and ASXL1~40%) and splicing components (SRSF2~50%) are frequent in chronic myelomonocytic leukemia (CMML). On a 27-gene targeted capture panel performed on 175 CMML patients (66% males, median age 70 years), common mutations included: TET2 46%, ASXL1 47%, SRSF2 45% and SETBP1 19%. A total of 172 (98%) patients had at least one mutation, 21 (12%) had 2, 24 (14%) had 3 and 30 (17%) had >3 mutations. In a univariate analysis, the presence of ASXL1 mutations (P=0.02) and the absence of TET2 mutations (P=0.03), adversely impacted survival; while the number of concurrent mutations had no impact (P=0.3). In a multivariable analysis that included hemoglobin, platelet count, absolute monocyte count and circulating immature myeloid cells (Mayo model), the presence of ASXL1 mutations (P=0.01) and absence of TET2 mutations (P=0.003) retained prognostic significance. Patients were stratified into four categories: ASXL1wt/TET2wt (n=56), ASXL1mut/TET2wt (n=31), ASXL1mut/TET2mut (n=50) and ASXL1wt/TET2mut (n=38). Survival data demonstrated a significant difference in favor of ASXL1wt/TET2mut (38 months; P=0.016), compared with those with ASXL1wt/TET2wt (19 months), ASXL1mut/TET2wt (21 months) and ASXL1mut/TET2mut (16 months) (P=0.3). We confirm the negative prognostic impact imparted by ASXL1 mutations and suggest a favorable impact from TET2 mutations in the absence of ASXL1 mutations.

Colony stimulating factor 3 receptor gene (CSF3R) mutations have recently been associated with chronic neutrophilic leukemia (CNL). Fourteen patients with CSF3R-mutated CNL (median age 67 years; 57% males) were screened for additional mutations; 8 (57%) and 5 (38%) harbored an ASXL1 and/or SETBP1 mutation (two patients expressed both), respectively. Two patients developed blastic transformation, both SETBP1-mutated and ASXL1-unmutated, whereas two other cases evolved into chronic myelomonocytic leukemia (CMML), both ASXL1-mutated and SETBP1-unmutated. Median survival was 23.2 months (10 deaths documented). On multivariable analysis mutated ASXL1 (P = 0.009; HR 19.6, 95% CI 2.1-184.1) and thrombocytopenia (P = 0.005; HR 28.8, 95% CI 2.8-298.2) were independently predictive of shortened survival. This study provides information on the natural history of CSF3R-mutated CNL and identifies mutant ASXL1 and thrombocytopenia as risk factors for survival. The study also suggests pathogenetic roles for SETBP1 and ASXL1 mutations in disease evolution into blast phase disease and CMML, respectively., (© 2015 Wiley Periodicals, Inc.)

In a cohort of 466 patients, we sought to clarify the prognostic relevance of ASXL1 and SETBP1 mutations, among others, in World Health Organization-defined chronic myelomonocytic leukemia (CMML) and its added value to the Mayo prognostic model. In univariate analysis, survival was adversely affected by ASXL1 (nonsense and frameshift) but not SETBP1 mutations. In multivariable analysis, ASXL1 mutations, absolute monocyte count >10 × 10(9)/l, hemoglobin <10 g/dl, platelets <100 × 10(9)/l and circulating immature myeloid cells were independently predictive of shortened survival: hazard ratio (95% confidence interval (CI)) values were 1.5 (1.1-2.0), 2.2 (1.6-3.1), 2.0 (1.6-2.6), 1.5 (1.2-1.9) and 2.0 (1.4-2.7), respectively. A regression coefficient-based prognostic model based on these five risk factors delineated high (≥3 risk factors; HR 6.2, 95% CI 3.7-10.4) intermediate-2 (2 risk factors; HR 3.4, 95% CI 2.0-5.6) intermediate-1 (one risk factor; HR 1.9, 95% CI 1.1-3.3) and low (no risk factors) risk categories with median survivals of 16, 31, 59 and 97 months, respectively. Neither ASXL1 nor SETBP1 mutations predicted leukemic transformation. The current study confirms the independent prognostic value of nonsense/frameshift ASXL1 mutations in CMML and signifies its added value to the Mayo prognostic model, as had been shown previously in the French consortium model.

Additional sex comb-like 1 ( ASXL1) is an enhancer of Trithorax and Polycomb family, which are necessary for the maintenance of stable repression of homeotic and other loci. Recently, alterations of ASXL1 gene were identified in the hematopoietic cells from patients with a variety of myeloid malignancies, including chronic myelomonocytic leukemia (CMML, 43% of cases), myelodysplastic syndrome (MDS, 20%), myeloproliferative neoplasms (MPN, 10%) and acute myeloid leukemia (AML, 20%). The majority of ASXL1 mutations are frameshift and nonsense mutations. These clinical data suggest an important role of ASXL1 in the pathogenesis and/or transformation of myeloid malignancies. However, the role of ASXL1 in the pathogenesis of myeloid malignancies and in normal hematopoiesis in vivo, as well as the underlying mechanisms remains unknown. This article reviews the structure and function of ASXL1, the clinical characteristic and prognostic significance of ASXL1 mutation, the association of ASXL1 with other gene mutation, as well as ASXL1 knock-down or silence in vitro and in vivo models.

Inherited or sporadic heterozygous mutations in the transcription factor GATA2 lead to a clinical syndrome characterized by non-tuberculous mycobacterial and other opportunistic infections, a severe deficiency in monocytes, B cells and natural killer cells, and progression from a hypocellular myelodysplastic syndrome to myeloid leukemias. To identify acquired somatic mutations associated with myeloid transformation in patients with GATA2 mutations, we sequenced the region of the ASXL1 gene previously associated with transformation from myelodysplasia to myeloid leukemia. Somatic, heterozygous ASXL1 mutations were identified in 14/48 (29%) of patients with GATA2 deficiency, including four out of five patients who developed a proliferative chronic myelomonocytic leukemia. Although patients with GATA2 mutations had a similarly high incidence of myeloid transformation when compared to previously described patients with ASXL1 mutations, GATA2 deficiency patients with acquired ASXL1 mutation were considerably younger, almost exclusively female, and had a high incidence of transformation to a proliferative chronic myelomonocytic leukemia. These patients may benefit from allogeneic hematopoietic stem cell transplantation before the development of acute myeloid leukemia or chronic myelomonocytic leukemia. (ClinicalTrials.gov identifier NCT00018044, NCT00404560, NCT00001467, NCT00923364.).

Patients with low-risk myelodysplastic syndromes (MDS) that rapidly progress to acute myeloid leukemia (AML) remain a challenge in disease management. Using whole-exome sequencing of an MDS patient, we identified a somatic mutation in the BCOR gene also mutated in AML. Sequencing of BCOR and related BCORL1 genes in a cohort of 354 MDS patients identified 4.2% and 0.8% of mutations respectively. BCOR mutations were associated with RUNX1 (P = .002) and DNMT3A mutations (P = .015). BCOR is also mutated in chronic myelomonocytic leukemia patients (7.4%) and BCORL1 in AML patients with myelodysplasia-related changes (9.1%). Using deep sequencing, we show that BCOR mutations arise after mutations affecting genes involved in splicing machinery or epigenetic regulation. In univariate analysis, BCOR mutations were associated with poor prognosis in MDS (overall survival [OS]: P = .013; cumulative incidence of AML transformation: P = .005). Multivariate analysis including age, International Prognostic Scoring System, transfusion dependency, and mutational status confirmed a significant inferior OS to patients with a BCOR mutation (hazard ratio, 3.3; 95% confidence interval, 1.4-8.1; P = .008). These data suggest that BCOR mutations define the clinical course rather than disease initiation. Despite infrequent mutations, BCOR analyses should be considered in risk stratification.

We evaluated the prognostic relevance of several clinical and laboratory parameters in 226 Mayo Clinic patients with chronic myelomonocytic leukemia (CMML): 152 (67%) males and median age 71 years. At a median follow-up of 15 months, 166 (73%) deaths and 33 (14.5%) leukemic transformations were documented. In univariate analysis, significant risk factors for survival included anemia, thrombocytopenia, increased levels of white blood cells, absolute neutrophils, absolute monocyte count (AMC), absolute lymphocytes, peripheral blood and bone marrow blasts, and presence of circulating immature myeloid cells (IMCs). Spliceosome component (P=0.4) and ASXL1 mutations (P=0.37) had no impact survival. On multivariable analysis, increased AMC (>10 × 10(9)/l, relative risk (RR) 2.5, 95% confidence interval (CI) 1.7-3.8), presence of circulating IMC (RR 2.0, 95% CI 1.4-2.7), decreased hemoglobin (<10 g/dl, RR 1.6, 99% CI 1.2-2.2) and decreased platelet count (<100 × 10(9)/l, RR 1.4, 99% CI 1.0-1.9) remained significant. Using these four risk factors, a new prognostic model for overall (high risk, RR 4.4, 95% CI 2.9-6.7; intermediate risk, RR 2.0, 95% CI 1.4-2.9) and leukemia-free survival (high risk, RR 4.9, 95% CI 1.9-12.8; intermediate risk, RR 2.6, 95% CI 1.1-5.9) performed better than other conventional risk models and was validated in an independent cohort of 268 CMML patients.

Purpose: Several prognostic scoring systems have been proposed for chronic myelomonocytic leukemia (CMML), a disease in which some gene mutations-including ASXL1-have been associated with poor prognosis in univariable analyses. We developed and validated a prognostic score for overall survival (OS) based on mutational status and standard clinical variables., Patients and Methods: We genotyped ASXL1 and up to 18 other genes including epigenetic (TET2, EZH2, IDH1, IDH2, DNMT3A), splicing (SF3B1, SRSF2, ZRSF2, U2AF1), transcription (RUNX1, NPM1, TP53), and signaling (NRAS, KRAS, CBL, JAK2, FLT3) regulators in 312 patients with CMML. Genotypes and clinical variables were included in a multivariable Cox model of OS validated by bootstrapping. A scoring system was developed using regression coefficients from this model., Results: ASXL1 mutations (P < .0001) and, to a lesser extent, SRSF2 (P = .03), CBL (P = .003), and IDH2 (P = .03) mutations predicted inferior OS in univariable analysis. The retained independent prognostic factors included ASXL1 mutations, age older than 65 years, WBC count greater than 15 ×10(9)/L, platelet count less than 100 ×10(9)/L, and anemia (hemoglobin < 10 g/dL in female patients, < 11g/dL in male patients). The resulting five-parameter prognostic score delineated three groups of patients with median OS not reached, 38.5 months, and 14.4 months, respectively (P < .0001), and was validated in an independent cohort of 165 patients (P < .0001)., Conclusion: A new prognostic score including ASXL1 status, age, hemoglobin, WBC, and platelet counts defines three groups of CMML patients with distinct outcomes. Based on concordance analysis, this score appears more discriminative than those based solely on clinical parameters.

The ASXL1 gene is one of the most frequently mutated genes in malignant myeloid diseases. The ASXL1 protein belongs to protein complexes involved in the epigenetic regulation of gene expression. ASXL1 mutations are found in myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML). They are generally associated with signs of aggressiveness and poor clinical outcome. Because of this, a systematic determination of ASXL1 mutational status in myeloid malignancies should help in prognosis assessment.

Chronic myelomonocytic leukaemia (CMML) is a haematological disease currently classified in the category of myelodysplastic syndromes/myeloproliferative neoplasm (MDS/MPN) because of its dual clinical and biological presentation. The molecular biology of CMML is poorly characterized. We studied a series of 53 CMML samples including 31 cases of myeloproliferative form (MP-CMML) and 22 cases of myelodysplastic forms (MD-CMML) using array-comparative genomic hybridisation (aCGH) and sequencing of 13 candidate genes including ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, PTPN11, RUNX1, TET2 and WT1. Mutations in ASXL1 and in the genes associated with proliferation (CBL, FLT3, PTPN11, NRAS) were mainly found in MP-CMML cases. Mutations of ASXL1 correlated with an evolution toward an acutely transformed state: all CMMLs that progressed to acute phase were mutated and none of the unmutated patients had evolved to acute leukaemia. The overall survival of ASXL1 mutated patients was lower than that of unmutated patients., (© 2010 Blackwell Publishing Ltd.)

The myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal haematological diseases characterized by ineffective haematopoiesis and predisposition to acute myeloid leukaemia (AML). The pathophysiology of MDSs remains unclear. A definition of the molecular biology of MDSs may lead to a better classification, new prognosis indicators and new treatments. We studied a series of 40 MDS/AML samples by high-density array-comparative genome hybridization (aCGH). The genome of MDSs displayed a few alterations that can point to candidate genes, which potentially regulate histone modifications and WNT pathways (e.g. ASXL1, ASXL2, UTX, CXXC4, CXXC5, TET2, TET3). To validate some of these candidates we studied the sequence of ASXL1. We found mutations in the ASXL1 gene in four out of 35 MDS patients (11%). To extend these results we searched for mutations of ASXL1 in a series of chronic myelomonocytic leukaemias, a disease classified as MDS/Myeloproliferative disorder, and found mutations in 17 out of 39 patients (43%). These results show that ASXL1 might play the role of a tumour suppressor in myeloid malignancies.

The ETV6 gene is a member of the ETS family of transcription factors and the main target of chromosomal rearrangements affecting chromosome band 12p13. To date, more than 15 fusion partners of ETV6 have been characterized at the molecular level. Most of these fusions encode chimeric proteins with oncogenic properties. However, some of the translocations do not produce a functional fusion protein, but may induce ectopic expression of oncogenes located close to the breakpoint. We herein report the characterization and cloning of a novel cryptic translocation, t(12;17)(p13;p12-p13), occurring in a patient with an acute myeloid leukemia evolving from a chronic myelomonocytic leukemia. Cytogenetic analysis suggested the presence of a deletion of the short arm of chromosome 12, del(12)(p13), in three of the five metaphase cells analyzed. However, fluorescence in situ hybridization (FISH) with the ETV6-specific cosmid clones 179A6, 50F4, 163E7, and 148B6 as well as probes hybridizing to the TP53 gene on 17p13 and the subtelomeric region of 17p revealed the presence of a translocation between 12p and 17p. By FISH, the breakpoints could be localized in intron 1 of ETV6 and centromeric to TP53. By 3' rapid amplification of cDNA ends-polymerase chain reaction (3' RACE-PCR), a fusion transcript between exon 1 of ETV6 and the antisense strand of PER1 (period homolog 1, Drosophila), a circadian clock gene, could be identified. This ETV6-PER1 (antisense PER1 strand) fusion transcript does not produce a fusion protein, and no other fusion transcripts could be detected. We hypothesize that in the absence of a fusion protein, the inactivation of PER1 or deregulation of a gene in the neighborhood of PER1 may contribute to the pathogenesis of leukemias with a t(12;17)(p13;p12-p13)., (Copyright 2003 Wiley-Liss, Inc.)

TEL and AML1 genes occur in a markedly high number of different aberrations in haematological malignancies. Besides the AML1, TEL is often fused to genes, which encod thyrosin-kinases. AML1 gene is a part of CBF transcription factor. AML1 can be altered in childhood acute lymphoblastic leukaemia (ALL) and also in a substantial number of acute myeloid leukaemias (most frequently as an AML1/ETO fusion). TEL/AML1 fusion gene (derived from t(12;21)(p13;q22) translocation) became recently one of the most important genetic aberrations in children with ALL. TEL/AML1 act presumably as dominant inhibitors of the second AML1 allele and thus they block transcription of genes dependent on CBF factor. Childhood ALL with TEL/AML1 hybrid gene is very frequent (approximately 22% of overall childhood ALL in the Czech Republic) and patients with this fusion form relatively homogenous group. These children are diagnosed mostly in pre-school age as a B cell precursor leukaemias and they have very good treatment results.

Chronic myelomonocytic leukemia (CMML) is a pre-leukemic syndrome that displays both myelodysplastic and myeloproliferative features. The t(5;12) chromosomal translocation, present in a subset of CMML patients with myeloproliferation fuses the amino terminal portion of the ets family member, Tel, with the transmembrane and tyrosine kinase domains of platelet-derived growth factor receptor beta (PDGFRbeta) gene. To investigate the role of this fusion protein in the pathogenesis of CMML, we expressed the Tel-PDGFRbeta fusion cDNA in hematopoietic cells of transgenic mice under the control of the human CD11a promoter. Transgenic founders and their offspring express the transgene specifically in hematopoietic tissues and develop a myeloproliferative syndrome characterized by: overproduction of mature neutrophils and megakaryocytes in the bone marrow; splenomegaly with effacement of splenic architecture by extramedullary hematopoiesis; an abnormal population of leukocytes co-expressing lymphoid and myeloid markers; and increased numbers of colonies in in vitro bone marrow CFU assays. All mice expressing the transgene exhibited at least one of these features of dysregulated myelopoiesis, and 20% progressed to a myeloid or lymphoid malignancy. This murine model of CMML parallels a myeloproliferative syndrome in humans and implicates the Tel-PDGFRbeta fusion protein in its pathogenesis.

Interferon regulatory factor 1 (IRF-1) is a transcriptional activator in the interferon system and acts as a tumor suppressor. The structurally related IRF-2 represses the effects of IRF-1 by competitive binding to the same DNA sequence elements. Changes in the relative balance between IRF-1 and IRF-2 lead to dysregulation of cell growth and may play a role in the development of neoplasias. The loss of functional IRF-1 has been observed in a number of patients with myelodysplastic syndrome (MDS) and leukemia, suggesting a potentially critical role of IRF-1 in leukemogenesis. We studied the expression of both transcription factors in peripheral blood (PB) and bone marrow (BM) cells of children with juvenile myelomonocytic leukemia (JMML) using RT-PCR and Southern blot hybridization. No significant difference between the expression levels of IRF-1 and IRF-2 could be detected in PB and BM of patients with JMML and normal donors. Although our results are preliminary they suggest that neither the tumor suppressor gene IRF-1 nor the oncogene IRF-2 is involved in the pathogenesis of JMML.

Juvenile myelomonocytic leukemia (JMML) is a rare disorder of early childhood, to which no recurrent chromosome rearrangement has been yet associated. We report a case where leukemic cells harbored a 46,XX,der(12)t(3;12) (q21 approximately 22;p13.33) karyotype, resulting in partial trisomy of 3q. The origin of chromosome material translocated to chromosome 12 was assessed by chromosome painting using a whole chromosome 3-specific probe. The breakpoint regions were defined by FISH using YAC probes from 3q and 12p chromosomal regions. Interestingly, partial trisomy of 3q has been detected in a previously reported JMML case, consequent to the presence of a der(15)t(3;15)(q13.1;q26). The involvement of a similar chromosome 3 rearrangement in these two JMML cases suggests the hypothesis that either the resulting duplication of some gene/s on 3q or the loss of heterozygosity (LOH) of some gene/s on 3p may be involved in one of the steps leading to JMML. On the other hand, it cannot be ruled out that the relevant mutation in our case might be consequent to the particular breakpoints at bands 3q21 approximately 22 and 12p13.3, that may alter the structure and/or expression of the involved gene/s.

The TEL/PDGF beta R fusion protein is the product of the t(5;12) translocation in patients with chronic myelomonocytic leukemia. The TEL/PDGF beta R is an unusual fusion of a putative transcription factor, TEL, to a receptor tyrosine kinase. The translocation fuses the amino terminus of TEL, containing the helix-loop-helix (HLH) domain, to the transmembrane and cytoplasmic domain of the PDGF beta R. We hypothesized that TEL/PDGF beta R self-association, mediated by the HLH domain of TEL, would lead to constitutive activation of the PDGF beta R tyrosine kinase domain and cellular transformation. Analysis of in vitro-translated TEL/ PDGF beta R confirmed that the protein self-associated and that self-association was abrogated by deletion of 51 aa within the TEL HLH domain. In vivo, TEL/PDGF beta R was detected as a 100-kDa protein that was constitutively phosphorylated on tyrosine and transformed the murine hematopoietic cell line Ba/F3 to interleukin 3 growth factor independence. Transformation of Ba/F3 cells required the HLH domain of TEL and the kinase activity of the PDGF beta R portion of the fusion protein. Immunoblotting demonstrated that TEL/PDGF beta R associated with multiple signaling molecules known to associate with the activated PDGF beta R, including phospholipase C gamma 1, SHP2, and phosphoinositol-3-kinase. TEL/PDGF beta R is a novel transforming protein that self-associates and activates PDGF beta R-dependent signaling pathways. Oligomerization of TEL/PDGF beta R that is dependent on the TEL HLH domain provides further evidence that the HLH domain, highly conserved among ETS family members, is a self-association motif.

A t(5;12)(q33;p13) translocation is a recurrent chromosome abnormality in a subgroup of myeloid malignancies with features of both myeloproliferative disorders and myelodysplastic syndromes (MDSs). The molecular consequence of a t(5;12) is a fusion between the platelet-derived growth factor receptor-B gene on chromosome 5 and a novel ETS-like gene, TEL, on chromosome 12. We report on three patients with a t(5;12)(q33;p13) diagnosed as chronic myelomonocytic leukemia, and one case of a t(10;12)(q24;p13) in a progressive MDS, with eosinophilia and monocytosis. Involvement of the TEL gene in these chromosome translocations was investigated by fluorescence in situ hybridization (FISH) with cosmid probes containing selectively the 5' end or 3' end of TEL. Hybridization of these cosmids to the der(5)/der(10) or a der(12), respectively, demonstrated a rearrangement of TEL in both translocations, showing that the t(10;12) is a variant translocation of the t(5;12). Cloning of the fusion cDNA of one case of t(5;12) showed that the breakpoint occurred at the RNA level at exactly the same position as reported by Golub et al (Cell 77:307, 1994). In addition, the TEL gene on chromosome 12 could be localized between two probes previously mapped to 12p13, namely PRB1 and D12S178, leading to a better definition of the position of TEL in this chromosome region. Moreover, in the case involving chromosome 10, the breakpoint occurred between cKTN206 and cKTN312/LYT-10 at 10q24. Clinicohematological data in these studies as well as the restriction mapping of chromosomal breakpoints strongly suggest that (1) common features in MDSs involving the TEL gene are monocytosis and eosinophilia, (2) chromosomes other than no. 5 may be involved and at least a t(10;12)(q24;p13) variant chromosome translocation does exist in these MDSs, and (3) both standard and variant 12p/TEL translocations may be identified by FISH with appropriate probes.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome characterized by abnormal clonal myeloid proliferation and by progression to acute myelogenous leukemia (AML). CMML thus offers an opportunity to study early genetic events in the transition to AML. A recently recognized subgroup of CMML has a t(5;12)(q33;p13) balanced translocation. We report that the consequence of the t(5;12) translocation is expression of a fusion transcript in which the tyrosine kinase domain of the platelet-derived growth factor receptor beta (PDGFR beta) on chromosome 5 is coupled to a novel ets-like gene, tel, on chromosome 12. The tel-PDGFR beta fusion demonstrates the oncogenic potential of PDGFR beta and may provide a paradigm for early events in the pathogenesis of AML.