Search

Background: Azacitidine (AZA) is approved for the treatment of high-risk chronic myelomonocytic leukemia (CMML) of myelodysplastic (MD) subtype. Data of response rates using the specific response criteria for this disease are scarce. The aim of this study was to evaluate the response to AZA in patients diagnosed with CMML from the Spanish Registry of Myelodysplastic Syndromes (MDS) applying the overlap myelodysplastic/myeloproliferative neoplasms (MDS/MPN) response criteria. Methods: We retrospectively studied 91 patients with CMML treated with at least one cycle of AZA from the Spanish Registry of MDS. As it was a real-world study, the response rate was evaluated between cycle 4 and 6, applying the MDS/MPN response criteria Findings: The overall response rate at cycle 4-6 was 58%. Almost half of the patients achieved transfusion independence and one quarter showed clinical benefit, regardless of the CMML French-American-British (FAB) and World Health Organization (WHO) subtypes and CMML Specific Prognosis Scoring (CPSS) risk groups. Toxicity was higher in the MD-CMML subtype. Interpretation: In our series, most CMML patients achieved an overall response rate with AZA according to the overlap-MDS/MPN response criteria regardless of the CMML FAB and WHO subtypes and CPSS risk groups. Thus, AZA may also be a treatment option for patients with the myeloproliferative CMML subtype and those with a lower-risk CPSS, but symptomatic.

Hypomethylating agent (HMA) failure myelodysplastic syndrome (MDS) patients have poor outcomes and urgent need for novel therapies. Hedgehog pathway signaling upregulation plays a central role in myeloid neoplasm pathogenesis and leukemia stem cell survival. We evaluated the efficacy and safety of the smoothened inhibitor glasdegib in HMA-failure MDS (n = 35, median age 73 years). According to the International Prognostic Scoring System and the MD Anderson Global Risk Model, 54% and 77% had higher risk disease, respectively. Overall response was 6% (n = 2), and best response was marrow complete remission with hematologic improvement in both patients. Median OS and median follow-up were 10.4 and 42.8 months, respectively. Drug response/stable disease (SD) resulted in better OS than treatment failure (20.6 [95% CI, 10.4-] vs 3.9 months [95% CI, 0.7–9.1]; P < .0001). Response/SD was confirmed to be an independent covariate for improved OS (P < .0001). Grade 3 or higher infections occurred in 11% of patients (n = 4); non-hematologic toxicities were rare. Early mortality (< 30 days) occurred in 11% of patients (n = 4). Glasdegib was well tolerated among HMA-failure MDS patients, although single-agent activity was limited. SD or better resulted in notably superior OS. These results support further investigation of glasdegib, potentially in novel drug combinations, in MDS patients.

Chronic myelomonocytic leukemia (CMML) is characterized by myelomonocytic bias and monocytic proliferation. Whether cell-intrinsic innate immune or inflammatory upregulation mediate disease pathogenesis and phenotype or whether the degree of aberrant monocytic differentiation influences outcomes remains unclear. We compared the transcriptomic features of bone marrow CD34+ cells from 19 patients with CMML and compared to healthy individuals. A total of 1495 genes had significantly differential expression in CMML (q0.05, fold change2), including 1271 genes that were significantly upregulated and 224 that were significantly downregulated in CMML. Top upregulated genes were associated with interferon (IFN) alpha and beta signaling, chemokine receptors, IFN gamma, G protein-coupled receptor ligand signaling, and genes involved in immunomodulatory interactions between lymphoid and non-lymphoid cells. Additionally, 6 gene sets were differentially upregulated and 139 were significantly downregulated in patients with myeloproliferative compared to myelodysplastic CMML. A total of 23 genes involved in regulation of monopoiesis were upregulated in CMML compared to healthy controls. We developed a prediction model using Cox regression including 3 of these genes, which differentiated patients into two prognostic subsets with distinct survival outcomes. This data warrants further evaluation of the roles and therapeutic potential of type I IFN signaling and monopoiesis in CMML.

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.

International audience; We wanted to describe the characteristics, treatment and outcome of autoimmune and inflammatory diseases (SAIDs) associated with chronic myelomonocytic leukemia (CMML), and conducted a French multicenter retrospective study and a literature review. We included 26 cases of CMML (median age 75 years, 54% female), 80% with CMML-1. CPSS score was low (0 or 1) in 75% of cases. SAIDS was systemic vasculitis in 54%. Diagnosis of the 2 diseases was concomitant in 31% cases, and CMML was diagnosed before SAIDs in 12 cases (46%). First line treatment for SAIDs consisted mostly of steroid, with 85% of response. Second-line treatment was needed in 40% cases. Six patients received hypomethylating agents, with 66% response on SAIDs. A literature review found 49 cases of CMML-associated SAIDs, in whom SAIDs was systemic vasculitis in 29% cases.Hence, vasculitis is the most frequent SAIDs associated with CMML. After initial response to steroids, recurrence and steroid-dependence were frequent. Hypomethylating agents may be interesting in this context. © 2016 Elsevier Ltd.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a therapeutic option that may lead to improved outcomes in patients with chronic myelomonocytic leukemia (CMML). However, few studies have examined the impact of the grade of graft-versus-host disease (GVHD) on post-transplant outcomes for CMML. We retrospectively analyzed the outcomes of 141 patients with CMML who underwent allo-HSCT between 1987 and 2010, and achieved neutrophil engraftment. The effects of acute GVHD (aGVHD) or chronic GVHD (cGVHD) on overall survival (OS), leukemia-associated mortality (LAM), and transplant-related mortality were evaluated by hazards regression models, in which the onset date of aGVHD or cGVHD was treated as a time-dependent covariate. Grade I aGVHD was associated with better OS and lower LAM (P=0.042, P=0.033, respectively) than no GVHD in univariate analyses, but not in the multivariate analyses. The multivariate analyses demonstrated that extensive cGVHD significantly associated with better OS (Hazard Ratio [HR] 0.35 [95% confidence intervals (CI), 0.16-0.74]; P=0.007) and lower LAM (HR 0.36 [95% CI, 0.14-0.92]; P=0.033) in patients who were not in complete remission at transplantation. In conclusion, the occurrence of cGVHD may be an important factor affecting the outcomes of CMML patients who received transplantation.

3q26.2/EVI1 rearrangements resulting in EVI1 overexpression play an important role in leukemogenesis and are associated with treatment resistance and a poorer prognosis in patients with acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia and BCR-ABL negative myeloproliferative neoplasms. In this study, we aim to explore the clinicopathological features of myelodysplastic/myeloproliferative (MDS/MPN) neoplasms with 3q26.2/EVI1 rearrangements and determine the potential impact of these cytogenetic abnormalities on treatment response and survival. The study group included 12 cases of MDS/MPN with 3q26.2 rearrangements detected by conventional karyotyping. There were 7 men and 5 women with a median age of 67 years (range, 51–79 years) at time of initial MDS/MPN diagnosis. Ten cases were classified as chronic myelomonocytic leukemia (CMML) and 2 were MDS/MPN, unclassifiable. Among CMML cases, 5 (50%) were proliferative type and 5 (50%) were dysplastic type. Based on blast counts, these 10 CMML were: CMML-0 (n = 2), CMML-1 (n = 3), and CMML-2 (n = 5). Eleven (92%) patients had 3q26 rearrangements at the initial diagnosis. Inv(3)(q21q26.2) was most common, identified in 7(58%) patients, followed by t(3;21)(q26.2;q22) in 2 patients and 1 patient each with t(3;3)(q21;q26.2), t(2;3)(p21;q26-27), and t(3;6)(q26.2;q26). Six (50%) patients had 3q26.2 rearrangements as a sole cytogenetic abnormality and 6 (50%) patients had additional cytogenetic abnormalities. Molecular studies revealed DNMT3A mutations in all 3 patients assessed and RAS mutations in 2 of 8 (25%) patients. No mutations in ASXL1 (n = 3), TET2 (n = 3), FLT3 ITD/D835 (n = 10), and CEBPA (n = 7) were detected. Most patients received hypomethylating agent based chemotherapy. The median follow-up was 11.5 months (range, 1.5–24 months) and at time of last follow-up, 11 (92%) died with a median survival of 13.4 months (range, 1.5–24 months). The only patient alive had a relatively short follow-up of 2.4 months and showed disease progression at the last visit. In conclusion, 3q26.2/EVI1 rearrangements are a rare event and usually present at time of initial diagnosis in MDS/MPN. The presence of 3q26.2/EVI1 rearrangements in MDS/MPN is associated with rapid disease progression, poor response to treatment, and a poor prognosis.

MDS patients may present with monocytic marrow proliferation not fulfilling criteria for CMML. We analyzed MDS patients with or without a marrow monocytic proliferation by following up the amount of monocytic proliferation and characterizing their molecular profile. 315 MDS patients of Duesseldorf MDS registry were divided into two groups: A) 183 patients with monocytic esterase positive cells in marrow and monocytes between 101 and 900/μl in blood and B) 132 patients without monocytic esterase positive cells in marrow and monocytes in blood ≤100/μl. Twenty patients of each group were screened with regard to ASXL1, TET2, RUNX1, SETBP1, NRAS, and SRSF2 using Illumina myeloid panel. Group A patients were older, had significantly higher WBC, hemoglobin levels, neutrophils and platelets. CMML evolution rates were 4.9% and 1.5%, respectively (p = n.s.). TET2, NRAS and SRFS2 mutation frequencies were higher in group A and four patients had coexisting TET2 and SRFS2 mutation, which was shown to be characteristic but not specific for CMML. MDS patients with marrow monocytic proliferation have a more CMML-like pheno- and genotype and develop CMML more often. Those patients could potentially be very early stages of CMML or represent a CMML-like myeloid neoplasma with marrow adherence of the monocytic cell population.

Metronomic, low dose chemotherapy may have anti-angiogenic effects and augment the effects of lenalidomide in MDS and CMML. We evaluated the clinical efficacy, tolerability and anti-angiogenic effects of melphalan 2mg and lenalidomide 10mg for 21 days/28 in CMML (n=12) and higher risk MDS (n=8) patients in a prospective phase II study. The primary endpoint was overall response and secondary endpoints included survival, progression-free survival, toxicity and biomarkers of angiogenesis. The median age was 73 years, 55% were pretreated and transfusion dependent. The overall response rate was 3(15%) of 19 evaluable patients but 25% in CMML and 33% in pCMML. Dose reductions and/or delays were common due to myelosuppression. Transient spikes in circulating endothelial cells that declined below baseline were seen in responders and patients with CMML, suggesting anti-angiogenic activity. In conclusion, lenalidomide and metronomic low dose melphalan demonstrate signals of clinical and possible anti-angiogenic activity in patients with pCMML that require future validation. This trial was registered at clinicaltrial.gov under # NCT00744536.

Sole del(7p) is a rare finding in myeloid neoplasms and its clinical significance is largely unknown. Here we report 10 patients with isolated del(7p), 4 had acute myeloid leukemia (AML), 2 myelodysplastic syndromes (MDS), 1 chronic myelomonocytic leukemia (CMML), 1 primary myelofibrosis (PMF), and 2 AML in remission. Seven patients had large and 3 had small del(7p) clone. For patients with AML, 3 acquired del(7p) either at disease relapse or disease progression, then became refractory to therapy and died shortly thereafter (median 5 months). Detection of del(7p) in patients with MDS, CMML, or PMF appeared to predict poorer prognosis as all 4 patients experienced disease progression or transformation to AML after 5-24 months. In the remaining 3 patients (1 AML and 2 AML in remission), del(7p) was only detected in 10% to 30% of metaphases and was a transient finding that did not appear to have any clinical impact. We conclude that detection of del(7p) in myeloid neoplasms, when presents as a major clone, often poses a high risk for disease progression and refractoriness to therapy; whereas when del(7p) presents as a small clone, it may not carry any clinical significance.

Chronic myelomonocytic leukemia (CMML) typically shows monocytosis in the peripheral blood (PB), which must be differentiated from reactive monocytosis. To determine the clonality of CMML, we performed molecular and cytogenetic analysis in Korean patients. To investigate whether monocytes in the PB harbored clonal mutational changes, we performed single-cell sequencing after selecting monocytes, neutrophils, and lymphocytes by morphology-aided laser microdissection. Targeted sequencing was performed in 35 patients with CMML with 41 bone marrow samples. Single-cell analysis was performed in two cases. Most (94.3%) patients harbored at least one variant, in genes considered as potential therapeutic targets, while cytogenetic aberrations occurred in only 28.6% of cases. ASXL1 (54.3%), SRSF2 (37.1%), NRAS (31.4%), and TET2 (25.7%) were frequently mutated, with lower frequencies of TET2 mutation and higher frequencies of NRAS, DNMT3A (17.1%), and NPM1 (11.4%) mutations compared to in previous studies of Caucasians. Patients with SETBP1 mutation and those with more than two variants showed poorer survival than those without mutation (P0.001 and P = 0.007, respectively). Most (70.8%) variants were detected at diagnosis and follow-up with no significant differences in variant allele frequency, warranting sequencing during follow-up if diagnostic samples were unavailable. Single-cell analysis revealed clonal monocytes with mutations, and the same mutations were also identified in lymphocytes and neutrophils. Targeted sequencing aided in clonality detection in most patients with CMML and single-cell sequencing facilitated identification of clonal monocytes and the co-existence of mutations in non-myeloid cells, suggesting that certain mutations are acquired by pluripotent stem cells.

Our retrospective analysis explored the role of leukemia cutis (LC) in disease progression of chronic myelomonocytic leukemia (CMML). Of 108 patients with CMML, 11 patients (10.2%) had LC including its equivalent (2 patients). Four of these patients developed acute myeloid leukemia (AML) within 0-4 months. The remaining 7 patients demonstrated increased monocytes (

To investigate clinical characteristic and prognostic factors for chronic myelomonocytic leukemia (CMML).A retrospective cohort study was used in the research. We investigated clinical and laboratory characteristics of CMML patients and survival status. Patients were followed up regularly through out the course of the research.Forty-one cases were diagnosed as CMML, including 27 male and 14 female patients. Median WBC was 13.7 x 10(9)/L. Five patients had leukocytopenia (1.92-3.46 x 10(9)/L). Median monocyte count in the peripheral blood was 2.13 x 10(9)/L. All patients presented with bone marrow dysplasia, and most showed hyperplasia, except 3 cases. Abnormal chromosome was detected in 34% cases. Median survival time for CMML-1 and CMML-2 was 20 and 12 months, respectively, but there were no statistical significance of survival duration between them. Univariable analysis showed that age (60 years), neutrophil count (2.0 x 10(9)/L), lymphocyte count (1.0 x 10(9)/L), mature monocyte count (or=5 x 10(9)/L) and anemia (Hb60 g/L) were associated with poor prognosis for CMML. There was no statistical significance in LDH, gender, and abnormal chromosome for survival time. Only lymphocyte count and neutrophil count in peripheral blood were independent prognostic factors for CMML after multivariate analysis.CMML mainly occurs in elderly patients. Although most patients have leukocytosis and monocytosis at diagnosis, few cases show leucopenia and monocytopenia. Age, neutrophil, lymphopenia, monocytosis, and severe anemia are associated with inferior prognosis of CMML. Lymphocyte1.0 x 10(9)/L and neutrophil count2.0 x 10(9)/L are adversely independent prognostic factors for CMML.

The functional behavior of hematopoietic stem cell (HSC) and progenitors in chronic myelomonocytic leukemia (CMML) and on disease progression is little known. We performed cell proliferation, apoptosis, hematopoietic colony forming/replating and differentiation potential studies in the purified subpopulations of Lin(-)CD34(+)CD38(-) and Lin(-)CD34(+)CD38(+) cells from 16 CMML with 6 cases after acute myeloid leukemia transformation (AML-t). We observed an expansion of the hematopoietic progenitor pool (Lin(-)CD34(+) cells) in AML-t comprising mainly Lin(-)CD34(+)CD38(+) cells. The Lin(-)CD34(+)CD38(+) cells in AML-t displayed high proliferative activity, resistance to apoptosis, enhanced myeloid colony formation/replating ability and a complete dendritic cell (DC) differentiation block. Our findings suggest Lin(-)CD34(+)CD38(+) cells instead of Lin(-)CD34(+)CD38(-) cells could be the target(s) of secondary genetic lesions underpinning progression from CMML to AML, which have implications for the further study of the biology of leukemic transformation and the design of new strategies for the effective treatment of CMML.

Distinct morphologic and clinical features associated with specific chromosomal abnormalities have been described in subgroups of myelodysplastic syndromes (MDS), which often are losses or gains and only rarely translocations. Among 103 consecutive MDS patients diagnosed and karyotyped at the Albert-Ludwigs University of Freiburg (ALU) between 1993 and 1999, two chronic myelomonocytic leukemias (CMMoL) displayed trisomy 19 (+19) as the sole chromosomal abnormality. Three further CMMoL cases with +19 as the single abnormality, two of which previously reported, were collected from other centers. Four of the five patients presented with leukocytosis and splenomegaly, and an increased number of ringed sideroblasts was observed in two cases. Treatment was low-dose Decitabine (cases 1 and 2), oral steroids (case 3), hydroxyurea (case 4), and daunorubicin/Ara-C (case 5). Transformation to acute myeloid leukemias (AML) occurred in three/five patients (cases 1, 2, and 4) 26, 12, and 22 months after diagnosis of CMMoL, respectively. We conclude that +19 as the sole anomaly is a rare but recurrent change in CMMoL, in particular of the proliferative type. It is at present unclear which gene(s) located on chromosome 19 might have a functional role for the development of this phenotype.

Therapy strategies for myelodysplastic syndromes (MDS) and juvenile myelomonocytic leukemia (JMML) vary considerably.To review the treatment of Brazilian children who were diagnosed with MDS or JMML in the past decade and reported to the Brazilian Cooperative Group on Pediatric Myelodysplastic Syndromes (BCG-MDS-PED).Of 173 children reported to the BCG-MDS-PED from January 1997 to January 2003 with a suspected diagnosis of MDS or JMML, 91 had the diagnosis confirmed after central review of the bone marrow aspirate and biopsy. Information on previous treatments was available for 78 MDS/JMML patients. Treatment varied from different schedules of low-dose (14%) and standard-dose chemotherapy (50%), granulocyte-colony-stimulating factor (G-CSF 7%), interferon (5%), steroids (2%) and erythropoietin (2%) to allogeneic stem-cell transplantation (SCT) (14%). No survival advantage could be demonstrated based on Hasle's classification or based on treatment.This report reflects the current practice in treating Brazilian children with MDS/JMML without specific Cooperative Group guidelines. Treatment modalities were very heterogeneous. The strategies for implementing a national protocol should consider international guidelines and focus on local experience and available resources.

Myeloid malignancies involve a clonal proliferation of abnormal stem cells and show various phenotypes consisting of acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) including various overlap and transition forms of these diseases1. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloid neoplasm of early childhood that is clinically characterized by overproduction of monocytic cells that can infiltrate organs, including the spleen, liver, gastrointestinal tract, and lung. JMML is categorized as an overlap MPN/MDS by the World Health Organization (WHO) and also shares some clinical and molecular features with chronic myelomonocytic leukemia (CMML), a pathomorphologically related disease in adults. Or both CMML and JMML have the following clinical characteristics: 1) persistent peripheral blood monocytosis >1 × 106/ml; 2) no Philadelphia chromosome; 3) no eosinophilia; 4) fewer than 20% blasts in the blood and bone marrow; and 5) dysplasia in one or more myeloid lineages. Recently, we have applied whole exome sequencing to a group of MDS patients followed by a targeted screen of specific genes for putatively recurrent mutations identified in the initial whole exome sequencing screen. In a systematic analysis of somatic events tested for recurrence and potential functional significance, we noted several somatic mutations affecting genes involved in the splicing machinery, consisting of SF3B1 (splicing factor 3b, subunit 1), U2AF1 (U2 small nuclear RNA auxiliary factor 1) and SRSF2 (serine/arginine-rich splicing factor 2) 2, 3, which Yoshida K and Papaemmanuil E reported to be frequently mutated in adult MDS, AML, and in particular chronic myelomonocytic leukemia (CMML) patients4, 5. These findings suggested that mutations of the genes involved in splicing machinery were a common pathogenetic pathway in adult myeloid malignancies. However, while it is likely that spliceosomal genes are also involved in the pathogenesis of childhood myeloid malignancies, including JMML, to date such a screen has been I performed by only one institute6. In the past, several similarities (e.g., CBL mutations in CMML and JMML) but also significant differences (e.g., TET2 mutations in CMML and MDS but not in JMML) were found in the mutational spectrum of patients with seemingly related disorders of adults and children. Consequently, we have designed this study to establish whether spliceosomal mutations contribute to the pathogenesis of pediatric myeloid malignancies, in particular JMML. We have studied 206 children, aged 0–18 years with myeloid malignancies, consisting of 102 with AML, 94 with JMML, and 10 with MDS. Patients with AML and MDS were classified according to the FAB classification. A total of 102 AML patients were studied [M0=1, M1=7, M2=16, M3 variant=1, M4=9, M5=14, M7=44 (including 21 patients with Down syndrome), and 10 with unknown subtypes]. All of the 10 children with MDS were classified as refractory anemia with excess blasts (RAEB) or RAEB in transformation (RAEB-T). JMML was defined as described in the 2008 WHO classification. Four of 94 JMML patients had clinical evidence of neurofibromatosis type 1. The results in children were contrasted with those obtained in an additional series of adult CMML tested for the presence of somatic mutations in the same set of genes. Previously we performed mutation analyses of PTPN11, NRAS, KRAS and CBL genes in 94 JMML patients, and detect genetic mutations in 29 (31%) for PTPN11, 12 (13%) for NRAS, 11 (12%) for KRAS and 7 (7%) for CBL genes7. Moreover, EZH2, RUNX1, ASXL1 and TET2 mutations were found in 0%, 0%, 4% and 0% of patients with JMML, respectively7,8. In contrast, our previous studies in adult CMML have shown the presence of TET2, ASXL1, CBL, EZH2, NRAS and KRAS, in 49%, 43%, 14%, 6%, 7% and 4% of patients, respectively6–8. In the current study, we analyzed genomic DNA derived from patients’ bone marrow or blood samples obtained before therapy according to appropriate protocol and consent approved by the intitutional review board of the two academic research centers. According to our initial screen in 50 adult patients with CMML, we have detected 2/50 mutations in exon 14 and 15 of SF3B1, 8/50 in exon 2 and 6 of U2AF1 and 18/50 in exon 1 of SRSF2 genes. The screening of mutations in these exons were performed by polymerase chain reaction (PCR) and direct sequencing. These exons were amplified from genomic DNA using the Platinum® Taq DNA Polymerase (Life Technologies, Carlsbad, CA, USA) and the following forward and reverse primers: SF3B1_ex13-14-F (5′-TCCCTTGATTAACAAAAGTCCTG-3′), SF3B1_ex13-14-R (5′-TGAGTCCAGTCTGGGCAAC-3′), SF3B1_ex15-16-F (5′-GTTGATATATTGAGAGAATCTGGATG-3′), SF3B1_ex15-16-R (5′-TTTAAAATTCTGTTAGAACCATGAAAC-3′), U2AF1_ex2-F (5′-GCTGCTGACATATTCCATGTG-3′), U2AF1_ex2-R (5′-AAGTCGATCACCTGCCTCAC-3′), U2AF1_ex6-F (5′-CATTTGGCAAAATCTTGGAC-3′), U2AF1_ex6-R (5′-TGGATAATGAAGTTACCTGTGTGC-3′), U2AF1_ex6-FS (5′-CGTGGATGGCAAGCACTTCTGTTT-3′), SRSF2_ex1-F (5′-AAGGCAACTGCCTGAGAGG-3′), SRSF2_ex1-R (5′-CGGACCTTTGTGAGGTCG-3′). In addition, we evaluated sequence analysis of spliceosome genes ZRSR2 (zinc finger, RNA-binding motif and serine/arginine rich 2) and LUC7L2 (LUC7-like 2) which we had identified as somatic mutations in adult MDS patients by whole exome sequencing3. The forward and reverse primers for ZRSR2 and LUC7L2 were summarized in supplemental table 1. The purified PCR products were directly sequenced using Big Dye Terminator V3.1 (Life Technologies) and analyzed using the ABI-3130 Genetic Analyzer. The most recurrent mutations in adult CMML cohort were S34F in U2AF1 and P95H in SRSF2. However, when children with myeloid malignancies including JMML were screened, no mutations in any of the genes were found in the 206 cases. Thus if present, spliceosomal mutations must occur at a frequency of

Chronic myelomonocytic leukemia (CMML) is a clinically heterogeneous disease, with no standard treatment. We present the outcome of ten patients diagnosed with CMML and treated with AZA in our institutions between 2005 and 2010. All patients were transfusion dependent at the time of initiation of therapy. The overall response rate was 60%. Responses were obtained in 2/3 of the patients with proliferative CMML. The median survival from start of therapy was 20 months. AZA treatment was well-tolerated and associated with a significant response rate in all forms of the disease.

Infections are a major cause of morbidity and mortality in patients with myelodysplastic syndrome (MDS) due to quantitative and qualitative granulocytic defects. We evaluated the in vitro bactericidal and fungicidal activities of neutrophils isolated from MDS. With comparison to those from healthy individuals, MDS neutrophils following infection showed a significantly reduced killing activity against Escherichia coli at 8 (p=0.002), 24 (p

The c-kit mutation Asp-816-->Val is detectable not only in neoplastic mast cells (MCs) in patients with systemic mastocytosis (SM) but also in most associated hematologic non-MC lineage disease (AHNMD). In order to prove a monoclonal disease evolution we investigated DNA of pooled microdissected single cells for the presence of the mutation in a patient with SM and concomitant chronic myelomonocytic leukemia (CMML). LightCycler melting curve analysis and direct sequencing of nested polymerase chain reaction (PCR) products revealed the c-kit mutation in tryptase-positive MC and in leukemic CD15-positive cells in bone marrow infiltrates, but not in colonic epithelial cells, thus, suggesting a monoclonal evolution of SM and concurrent CMML on the basis of a somatic mutation in a common hematologic progenitor.

The clinical records of 70 patients seen at our hospital between 1976 and 1998 and diagnosed as suffering from chronic myelomonocytic leukaemia (CMML) were reviewed in order to confirm the validity of the classification into two forms of disease that the French-American-British Co-operative Leukaemia Group (FAB) proposed in 1994: myelodysplastic (MD) and myeloproliferative (MP), depending on the peripheral white blood cell count (WBC) (less or more than 13 x 10(9)/l, respectively). After the rejection of incomplete records and lost to follow up patients, our study population consisted of 49 records. Our results confirm that, even though this classification is useful in order to separate two classes of patients, it is not enough to predict the prognosis in an accurate manner. A lot of studies have tried to find some prognostic factors, but the results have been discordant. The multivariate analysis of our group of patients showed three prognostic factors: serum lactate dehydrogenase (LDH)1.5 times normal level, blasts in bone marrow5%, and peripheral blood leukocytes10 x 10(9)/l. A second multivariate analysis led us to distinguish two groups: high risk (2-3 risk factors) and low risk (0-1 risk factors) (median survival 7 and 44 months, respectively) with a very high statistic significance (P0.0001). This score should be applied to other series of CMML patients in order to confirm its validity.

Outcome of patients with high risk MDS and CMML who failed treatment with azacitidine remains poor with a median survival of 6 months, without established therapy available except allogeneic hematopoietic stem cell transplantation. The objective of our study was to evaluate efficacy of decitabine after azacitidine failure in a relatively large patient cohort based on conflicting results with 0-28% response rates (RR) in this setting in small patient series. Thirty-six consecutive high risk MDS and CMML patients who received decitabine after azacitidine failure were retrospectively reviewed. Response was based on IWG 2006 criteria for MDS and CMML with WBC

In chronic myelomonocytic leukemia (CMML) segregation of two subtypes has been suggested depending on WBC count-myelodysplastic (MD-CMML) and myeloproliferative (MP-CMML). In a retrospective analysis of 91 (60/31) previously untreated CMML patients, we compared the presenting clinical, haematological, laboratory and bone marrow features and examined the clinical impact of this reclassification. LDH values and bone marrow cellularity were significantly increased in MP-CMML. Median survival was significantly longer for patients with MD-CMML, progression rate was higher for MP-CMML. Patients with MD-CMML had longer median preleukemic duration; after transition to AML, MP-CMML patients had longer median survival. In MDS phase anemia was more common in MP-CMML and thrombocytopenia more common in MD-CMML whereas transfusion rates showed no difference. Evaluation of prognostic scoring systems for both groups confirmed that patients' characteristics and outcome could be well compared. Our data suggest that segregation into MD-CMML and MP-CMML is justified.

Several authors have tried to solve the problems in the classification of CMML. A fully suitable classification does not exist. The goal of our study was to determine common and different signs of MD and MP type of CMML and to observe frequency of shifts from MD to MP-CMML. Sixty nine CMML patients were divided according to FAB proposal into two groups: 31 patients into the MD group (WBC < or = 13 x 10(9)/l) and 38 patients into the MP group (WBC < or = 13 x 10(9)/l). Presenting features and the course of the disease in both groups were evaluated. The median age of patients was not different in both groups (71.5 and 74 years, respectively), male/female ratio was 1.1 and 2.4, respectively. The median follow-up time was 15.5 months (1-58.8) in MP group and 24 months (2-118) in MD group. In MP group splenomegaly, hepatomegaly, lymphadenopathy, abnormal karyotype and skin involvement were found more often than in MD group. Median LDH value was higher in MP group. Probability of survival was higher in the MD group than in MP group (median 30 and 11 months, respectively). Leukaemia transformation frequency was similar in both groups. In 12 out of 24 (50%) MD group patients WBC increased during the course of the disease over 13 x 10(9)/l. Oscillation of WBC values below and over 13 x 10(9)/l was observed in three patients. During the follow-up time number of patients with splenomegaly and/or immature granulocytes in the PB increased. After inclusion of 12 patients who shifted from MD to MP group a new CMML group resulted characterised by longer median survival (17 months) due to a higher number of patients in an earlier stage of the disease. Failure of evolution of myeloproliferative signs and lower frequency of AL in the remaining group might be explained by an early stage of CMML, untimely deaths due to unrelated causes and/or by patients suffering of RA with monocytosis rather than of CMML. In summary, our data suggest, that evolution from MD-CMML to MP-CMML is a frequent event and that MD-CMML could be the early stage of CMML in most of cases. The WBC at diagnosis as the single criterion for subclassification of CMML does not seem to be fully justified. We propose that CMML should not be divided in MD and MP types and that monitoring of patients and search for other signs of myeloproliferation such as PB immature granulocytes, splenomegaly, lymphadenopathy, skin involvement, pleural or peritoneal effusions, spontaneous growth of CFU-GM in vitro should be taken in consideration for a better classification of CMML, which would have an impact on the therapeutic approach.

Pericardial effusion is a rare but potentially life-threatening complication of chronic myelomonocytic leukemia. A case of a 70-year-old female with CMML is reported, who developed a severe pericardial effusion after an initially stable course of disease. She underwent pericardiocentesis and subsequent intracardial instillation of mitoxantrone with poor response. Subxiphoid pericardiotomy was successfully performed. Leukocytosis did not respond to hydroxyurea. Because of decreasing performance status of the patient systemic chemotherapy was not applied and the patient died within 2 weeks. A review of the literature shows that there is no effective treatment for pericardial effusion in CMML, and some common features may be noted.

The skin involvement of the myelodysplastic syndrome (MDS) can take the form of either a neoplastic infiltration or various non specific lesions. The occurrence of these lesions may be the presenting feature of the disease (MDS) or may herald its progression to acute leukemia. Recognition and early diagnosis have therapeutic and prognostic significance.

Treatment of CMML remains a clinical challenge, with no drug demonstrating clear clinical benefit. Even if azacitidine is approved in the treatment of CMML, its role remains disputed. We report a cohort of 76 CMML patients (according to WHO classification) treated with azacitidine in 3 programs (French AZA compassionate program, Cleveland Clinic Foundation and H. Lee Moffitt Cancer Center). 45% had CMML2, and 55% had splenomegaly and/or WBC counts >13 G/L, which are known to be poor prognostic factors in CMML. All patients received AZA for at least one cycle, and the median number of cycles administered was 6. Thirty-three patients (43%) achieved a response according to IWG 2006 criteria, including 13 complete remissions (17%). Median survival was 29 months. Increased bone marrow blast percentage and proliferative features of the disease, including splenomegaly and high WBC counts, were significantly associated with shorter survival. By multivariate analysis, only marrow blasts >10% and palpable splenomegaly had prognostic impact on survival. Although promising, the efficacy of azacitidine in advanced CMML needs to be confirmed in a randomized prospective study.

Chronic myelomonocytic leukemia is a very rare blood cancer observed mostly in the elderly. Here we report the incidence trends and survival of patients with chronic myelomonocytic leukemia over a 15-year period (1993-2007). Cases were provided by the population-based Girona Cancer Registry. The crude incidence rate was 0.72/100,000 inhabitants/year. No statistically significant increase in trends was detected over the 15 years. Median overall survival was 28 months although survival markedly decreased with advancing age. The 5-years observed and relative survivals were 20% and 29%, respectively. This is the first population-based study that reports the incidence and survival of chronic myelomonocytic leukemia in Spain.

The plasma soluble interleukin 2 receptor (sIL-2R) level and its relationships with haematologic and immunologic data were examined in 40 patients with myelodysplastic syndromes (MDS). The plasma sIL-2R level was significantly higher in the high-risk MDS group (refractory anaemia with excess blasts (RAEB), RAEB in transformation and chronic myelomonocytic leukaemia) than in the low-risk MDS group (refractory anaemia (RA) and RA with ringed sideroblasts) or in normal subjects, although there was considerable variation in the plasma sIL-2R level within each MDS group. The plasma sIL-2R level correlated positively with the bone marrow cellularity and bone marrow blast mass, but not with the absolute number of CD25+ lymphocytes. This may support the idea that plasma sIL-2R is derived from malignant MDS cells in the bone marrow. The plasma sIL-2R level correlated negatively with the absolute numbers of the CD8+, CD3-CD16+, and CD3-CD56+ cell populations in freshly isolated lymphocytes, the percentage of CD3-CD56+ cells in lymphokine (interleukin 2)-activated killer (LAK) cells, and the cytotoxicity of LAK cells. We conclude that MDS patients having a high plasma sIL-2R level often have a defect in natural killer and CD8+ T-cells.

Clonality, in MDS, can only be assessed in patients with chromosomal rearrangements or in females heterozygote for X chromosome restricted polymorphisms. "Illegitimate" rearrangements of the immunoglobulin heavy chain (IgH) gene and incomplete rearrangements involving V delta 2 and D delta 3 segments of the T-cell receptor delta (TcR delta) gene are seen in some cases of AML, and AML post-SMD, and can be detected by a sensitive PCR method. In order to analyse clonality in additional cases in MDS, we looked for Ig H and TcR delta gene rearrangement by PCR in 95 cases of MDS. A rearrangement of the Ig H gene was seen in 2 of the 95 patients: in the circulating blood of 2 of the 36 cases of chronic myelomonocytic leukaemia (CMML) and in none of the marrow samples of the other 59 MDS. A rearrangement of the TcR delta gene (involving V delta 2 and D delta 3 segments) was seen in three cases (in the circulating blood of two other CMLL patients, and in the bone marrow of another MDS patient). Twenty-five of the 90 cases of MDS with negative PCR findings, in addition to the five cases with positive PCR findings underwent Southern blot analysis of Ig H and TcR delta genes, and PCR analysis of V delta 1 and J delta 1 segments of the TcR delta gene. Those examinations were normal in all the cases tested. In patients with positive PCR findings for Ig H or V delta 2 D delta 3 rearrangements, the proportion of rearranged cells was evaluated at 1-5% in four cases, and 5-10% in the remaining patient. Because the analysis was performed on total circulating leukocytes or total nucleated marrow cells, the nature of the clonal population in positive cases (lymphoid cells? myeloid cells? blasts?) could not be determined. From a practical point of view, Ig H and TcR delta gene rearrangements seem to very rare in MDS, and cannot be used as clonality markers in most cases.

Alterations of the ten-eleven translocation-2 (TET2) gene have been recently identified in patients with myeloid malignancies using molecular, comparative genomic hybridization and single nucleotide polymorphism array techniques. We have performed TET2 fluorescence in situ hybridization analysis in a cohort of patients with myeloid disorders including myeloid malignancies and chronic idiopathic neutropenia, aiming to determine the usefulness of the technique in the identification of TET2 gene alterations. A TET2 deletion was found in one patient with chronic myelomonocytic leukemia suggesting that fluorescence in situ hybridization may have a role in identification of TET2 deletions, at least in this group of patients.

The majority of patients suffering from myelodysplastic syndromes (MDS) die of complications due to cytopenia. Clinical trials have demonstrated that in an essential number of MDS patients cytopenia can be ameliorated by exogenously supplied growth factors. We investigated endogenous serum levels of GM-CSF and IL-3 in 15 healthy individuals and 34 patients suffering from MDS. No circulating growth factors were detected in the serum of healthy controls, nor was IL-3 measurable in MDS patients. GM-CSF serum levels, however, were elevated in a significant number of patients (26.5%). Levels did not correlate with FAB classification, leukocyte count or chromosomal abnormalities. No significant differences in GM-CSF or IL-3 receptor expression were detected between healthy individuals and MDS patients. One patient with increased endogenous GM-CSF serum level and normal surface receptor expression responded to exogenously applied GM-CSF. In the light of these results, a funetional alteration of growth factor receptors or disturbances of signal transduction pathways must be discussed for MDS.

MDS is primarily a disease of the elderly. Cases who give a history of exposure to X-rays, cytotoxic drugs or leukaemogenic chemicals may be younger. Many cases of MDS present because of an incidental blood count. The most prominent clinical features are those of anaemia, neutropenia, thrombocytopenia. Because haemopoietic tissue is also dysfunctional the pathological effect is often greater than the figures would suggest, even leading to infection of bleeding with normal neutrophil or platelet counts. Occult abscesses are a particular feature. Despite documented abnormalities of the lymphoid system, neither infections characteristic of T-cell immunodeficiency nor autoimmunity is a problem. The proliferation of monocytes in CMML leads to organomegaly, leukaemia cutis, serous effusions and vasculitic lesions caused by the mishandling of circulating immune complexes. Cancer is no commoner than in age-matched controls, but coincident lymphoid tumours do occur. Many patients require long-term blood transfusion and will run into problems of iron overload unless precautions are taken.

CMML is a preleukemia that results in cytopenias, dysplastic morphology of bone marrow and peripheral blood cells, produces large numbers of ineffective monocytes, and is notoriously hard to treat. The recent revision of the WHO classification excludes CMML from the myelodysplastic syndromes (MDS), and reclassifies it as a myelodysplastic/myeloproliferative disease. Decitabine (Dacogen™) is a cytosine analog that reverses aberrant DNA hypermethylation, leading to re-expression of silenced tumor suppressor genes. The reclassification of CMML has led to a review of CMML patients treated with decitabine. Overall response rates (ORR) (complete response [CR] + partial response [PR]) from one pivotal phase 3 trial (D-0007) and two phase 2 trials (PCH 95-11, PCH 97-19) in the subset of patients with CMML receiving decitabine were reviewed. The two phase 2 trials were open-label and single-arm, with a recommended minimum of 4 treatment cycles and a maximum of 8 cycles. The phase 3 trial used a 1:1 randomized comparison of decitabine plus supportive care (SC) vs SC alone with a maximum of 10 cycles of therapy. The phase 3 trial study design dictated that patients be removed from therapy following 8 cycles of decitabine if CR was not achieved, and following 6 cycles in the absence of PR. Patients who maintained a CR for 2 cycles were removed from therapy. For consistency across trials, all decitabine-treated patients were evaluated using the phase 2 response criteria (CR was defined by normocellular bone marrow with 1.5 mmol/L, WBC count by >1000, and platelet count by >50,000). A total of 28 patients diagnosed with CMML are included in this review. Similar demographics and disease characteristics were observed in all 3 studies, with an average age of 70.2 years and 71% of patients male. A baseline WBC of >20,000 was observed in 8/28 (29%) patients and baseline bone marrow blasts >5% were observed in 11/28 (39%) patients. All clinical responses were centrally reviewed. The ORR was 25% (14% CR + 11% PR). Hematologic improvement was observed in 11% of patients and stable disease in 39% of patients. The decitabine adverse event profile seen in CMML patients was similar to observations in other hematologic patient populations, with myelosuppression and related infectious complications. These data demonstrate encouraging activity for decitabine in CMML, and suggest that studies in other myeloproliferative diseases may be warranted.

Myelodysplastic syndromes (MDS) are comprised of a heterogeneous group of stem cell disorders characterized by ineffective hematopoiesis and susceptibility to transform to acute myeloid leukemia. The molecular pathways underlying disease initiation and evolution are still largely unknown. We recently demonstrated that acquired mutations in PTPN11 are a major event in JMML and occur with variable prevalence in children with other hematologic malignancies, including MDS. Here, we investigated contribution of PTPN11 mutations to adult MDS and CMML pathogenesis. Our results indicate that PTPN11 lesions might play a role in adult MDS/CMML pathogenesis but do not represent a major molecular event.

The effective treatment for juvenile myelomonocytic leukemia (JMML) patients lacking access to stem cell transplantation remains unavailable. Here, we describe a promising result obtained with novel regimen comprised of a combination of chemotherapy and differentiation therapy. Five patients diagnosed as JMML were treated with a standard regimen (cytosine arabinoside (Ara-C) 100mg/m(2) per day continuous infusion (days 0-6), etoposide 100mg/m(2) per day (days 0-4), vincristine 1.5mg/m(2) per d (day 9) and isotretinoin 75-100mg/m(2) per day (days 10-20)). All patients responded to the standard regimen. Three of the five were later treated with salvage a regimen (Ara-C 100mg/m(2) per day continuous infusion (days 0-4), etoposide 100mg/m(2) per day (days 0-4) and Ara-C 15mg/m(2) per day SC (days 6-15)) when immature myeloid cells reappeared in the peripheral blood, the spleen increased or blast crisis occurred. Immature myeloid cells disappeared again after one cycle of salvage regimen in all patients. All patients are alive now with a median follow up duration of 27 months (8-69 months). Although the number of patients enrolled was limited, the standard and salvage regimens were found to be safe and effective alternatives for JMML patients without a matched donor. These regimens also could be used safely before stem cell transplantation.

The syndrome of chronic myelomonocytic leukemia (CMML) includes a heterogeneous group of patients who exhibit both myelodysplastic and myeloproliferative clinicopathological features. Troublesome splenomegaly is uncommon in myelodysplastic syndrome (MDS), but when organomegaly occurs, this complication is more likely to be associated with myelodysplastic-myeloproliferative overlap syndromes such as CMML rather than "dysplasia-only" MDS types such as refractory anemia. We report a single-institution experience with splenectomy in CMML patients, including a detailed review of splenic histopathology. Twelve patients with CMML underwent splenectomy at the Mayo Clinic, primarily because of refractory thrombocytopenia and/or mechanical complications related to splenomegaly. Three of the 12 patients (25%) died as a direct result of surgery, and significant postoperative morbidity was seen in another 4 patients (33%). Thrombocytopenia improved in 4 of the 11 patients (36%) with low platelet counts before surgery. Three of the four responders had an abundance of CD68 (PGM1)-positive foamy histiocytes in the marginal zone surrounding the splenic white pulp-a pattern which can be seen in immune thrombocytopenia-and two of these three patients had thrombocytopenia out of proportion to the degree of anemia pre-operatively, suggestive of peripheral destruction of platelets. More consistent splenic pathological findings in the 12 patients included trilineage extramedullary hematopoiesis in splenic red pulp and expansion of splenic cords by a myelomonocytic infiltrate. This study underscores both the uniformity and diversity of splenic findings in CMML, highlights the potential dangers and benefits of splenectomy in this group, and suggests peripheral destruction of platelets as a mechanism contributing to thrombocytopenia in a subset of CMML patients.

This report describes the case of an 8-month-old infant with a diagnosis of juvenile myelomonocytic leukemia (JMML) and type 1 neurofibromatosis that presented progression to B lineage acute lymphoid leukemia (ALL). The same rearrangement of gene T-cell receptor gamma (TCR gamma) was detected upon diagnosis of JMML and ALL, suggesting that both neoplasias may have evolved from the same clone. Our results support the theory that JMML may derive from pluripotential cells and that the occurrence of monosomy of chromosome 7 within a clone of cells having an aberrant neurofibromatosis type 1 (NF1) gene may be the cause of JMML and acute leukemia.

Dendritic cells (DCs) are professional antigen presenting cells derived from myeloid or lymphoid precursors. Functional DCs have been generated from the malignant counterpart of these precursor cells. Herein, we describe the generation of DCs from different leukemias and determine the optimal culture conditions with minimal manipulation. Primary leukemic cells were cultured for 1, 3, and 5 days in 11 different cytokine combinations and analyzed for the expression of a mature DC phenotype. Optimal growth and DC characteristics were obtained with GM-CSF, FL, and SCF in 3-5 day cultures, suggesting a practical strategy for the immunotherapy of leukemia.

The third exon of the c-myc gene contains a CpG site which has been implicated as a regulatory region. When this site is methylated it has protein binding properties and binds a different set of proteins in normal and neoplastic cells. Recent work using myeloma cell lines indicates a correlation between hypomethylation at this site and enhanced expression of the myc protein. We investigated the methylation of this site in 10 cases of myeloma but found that there was no change from the high degree of methylation found in normal cells. Therefore, methylation status at this site is unlikely to serve as a prognosticator in myelomatosis. However, methylation changes at this site were observed in DNA from two cases of CMML, in which hypomethylation was observed and in three AML cases, which were completely methylated at this site.

To understand the underlying mechanisms in myelodysplastic syndromes (MDS) by identifying target tumor suppressor genes, we performed a detailed deletional mapping of the short arm of chromosome 1 in 38 paired samples of bone marrow and peripheral blood obtained from individuals with MDS by PCR amplification of a total of 23 highly informative microsatellite sequences. We identified the commonly deleted region between D1S508 and D1S244. LOH of this region was found in five patients (13%). In addition, LOH at 1p was associated with a poor clinical outcome, suggesting that the deletion of a gene in this region may be involved in the course of this disease. By analyzing the chromosomal map of this region, we found TNFRSF12 as a candidate tumor suppressor gene. However, our search for mutations in this gene did not identify somatic mutations in MDS. Our findings are consistent with the possible existence of an as-yet unknown tumor suppressor gene in this region that is altered in MDS.