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Supplementary Figure S1 and Tables S1-3. Figure S1. Frequencies and distribution of co-operating mutations in RUNX1 mutationpositive patients. Table S1. Comparison of clinicohematological characteristics according to the biological activities of RUNX1 in MDS and CMML. Table S2. Correlation of risk of sAML transformation with RUNX1activities or co-existing gene mutations in MDS and CMML patients carrying RUNX1 mutations. Table S3. Correlation of sAML-free survival with cytogenetics or additional co-existing gene mutations in MDS and CMML patients carrying RUNX1 mutations. Supplemental methods and References

Purpose: Transcription factor RUNX1 is essential for normal hematopoiesis. High mutation frequencies of RUNX1 gene in chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS) have been described, whereas the biologic significances of the mutations were not investigated. Here, we aimed to correlate the biologic activities of the RUNX1 mutants with the clinical outcomes of patients.Experimental Design: We examined the mutational status of RUNX1 in 143 MDS and 84 CMML patients. Then, we studied the DNA and CBFβ binding abilities of all the RUNX1 mutants identified by using electrophoretic mobility shift assay and co-immunoprecipitation assay, and also determined their activities on target C-FMS gene induction by Western blotting and luciferase reporter assay. Using luciferase reporter assay, the relative biologic activities of each RUNX1 mutant could be quantified and correlated with the patient outcomes by statistical analyses.Results: We observed that most RUNX1 mutants had reduced abilities in DNA binding, CBFβ heterodimerization, and C-FMS gene induction. The relative biologic activities of RUNX1 mutants were grouped into high- and low-activity mutations. Correlation of the activities of RUNX1 mutants with the clinical outcomes revealed that patients harboring lower activities of RUNX1 mutants had a higher risk and shorter time to secondary acute myeloid leukemia transformation in MDS and CMML. In multivariate analysis, low RUNX1 activity remained an independent predictor for secondary acute myeloid leukemia–free survival in MDS patients.Conclusions: The biologic activity rather than the mutational status of RUNX1 might be an indicator in predicting outcome of patients with MDS and CMML. Clin Cancer Res; 21(15); 3541–51. ©2015 AACR.

Gene mutations involving epigenetic regulators recently have been described in adult acute myeloid leukemia (AML). Similar studies are limited in children. We analyzed gene mutations and cooperation in pediatric AML with special reference on mutated epigenetic regulators. Nineteen gene mutations, including 8 class I genes, 4 class II genes, WT1 and TP53 (class III), and 5 epigenetic regulator genes (class IV), were analyzed in 206 children with de novo AML. Mutational analysis was performed with polymerase chain reaction-based assay followed by direct sequencing. One hundred seventeen of 206 patients (56.8%) had at least one mutation: 51% class I, 13% class II, 6.8% class III, and 5.6% class IV. FLT3-internal tandem duplication was most frequent, and 29% of patients had more than one gene mutation. Two patients carried ASXL1 mutations, both with t(8;21), 2 had DNMT3A mutations, 2 had IDH1 mutations, 1 had IDH2 mutation, and 3 had TET2 mutations. Both patients with IDH1 mutations had AML-M0 subtype and MLL-partial tandem duplication. Cooperating mutations with mutated epigenetic regulators were observed in 8 of 10 patients. We conclude that mutated epigenetic regulators were much less than those in adult AML but with frequent cooperating mutations. ASXL1, TET2, and IDH1 mutations were associated with specific genetic subtypes.

// Hsiao-Wen Kao 1, 2 , Der-Cherng Liang 3 , Ming-Chung Kuo 1, 4 , Jin-Hou Wu 1 , Po Dunn 1, 4 , Po-Nan Wang 1 , Tung-Liang Lin 1 , Yu-Shu Shih 1 , Sung-Tzu Liang 3 , Tung-Huei Lin 1 , Chen-Yu Lai 4 , Chun-Hui Lin 3 , Lee-Yung Shih 1, 4 1 Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan 2 Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan 3 Division of Pediatric Hematology-Oncology, Mackay Memorial Hospital, Taipei, Taiwan 4 College of Medicine, Chang Gung University, Taoyuan, Taiwan Correspondence to: Lee-Yung Shih, e-mail: sly7012@adm.cgmh.org.tw Keywords: MLL-PTD, acute myeloid leukemia, gene mutation, DNMT3A mutation Received: July 30, 2015 Accepted: August 28, 2015 Published: September 08, 2015 ABSTRACT The mutational profiles of acute myeloid leukemia (AML) with partial tandem duplication of mixed-lineage leukemia gene ( MLL -PTD) have not been comprehensively studied. We studied 19 gene mutations for 98 patients with MLL -PTD AML to determine the mutation frequency and clinical correlations. MLL -PTD was screened by reverse-transcriptase PCR and confirmed by real-time quantitative PCR. The mutational analyses were performed with PCR-based assays followed by direct sequencing. Gene mutations of signaling pathways occurred in 63.3% of patients, with FLT3 -ITD (44.9%) and FLT3 -TKD (13.3%) being the most frequent. 66% of patients had gene mutations involving epigenetic regulation, and DNMT3A (32.7%), IDH2 (18.4%), TET2 (18.4%), and IDH1 (10.2%) mutations were most common. Genes of transcription pathways and tumor suppressors accounted for 23.5% and 10.2% of patients. RUNX1 mutation occurred in 23.5% of patients, while none had NPM1 or double CEBPA mutation. 90.8% of MLL -PTD AML patients had at least one additional gene mutation. Of 55 MLL -PTD AML patients who received standard chemotherapy, age older than 50 years and DNMT3A mutation were associated with inferior outcome. In conclusion, gene mutations involving DNA methylation and activated signaling pathway were common co-existed gene mutations. DNMT3A mutation was a poor prognostic factor in MLL -PTD AML.

OBJECTIVE Although the nutritional value of human milk has been thoroughly studied, few reports describing its free amino acid (FAA) content have been published. Although infant formulas are designed to approximate the nutrient composition of human milk, the content and concentration of free amino acids are unknown. We compared the FAA concentrations of milk from mothers of preterm and full-term infants with those in several infant formulas. METHOD Human milk was obtained during three different stages of lactation (colostral, transitional and mature milk). Sixty-seven samples were collected from 44 healthy mothers of term infants and 23 mothers of premature infants 29 to 36 weeks gestation (mean 33 weeks). Two brands of powdered term formula (TF-A and TF-B) and two brands designed for preterm infants (PTF-A and PTF-B )were also studied. Ion exchange chromatography was used for free amino acid analysis. RESULTS The mean concentration of total FAA in human milk was significantly higher than any of the infant formulas (8139 micromol/L for pre-term human milk; 3462 micromol/L for full term human milk; TF-A, 720 micromol/L; TF-B, 697 micromol/L; PTF-A, 820 micromol/L; PTF-B, 789 micromol/L) (P

Purpose: Transcription factor RUNX1 is essential for normal hematopoiesis. High mutation frequencies of RUNX1 gene in chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS) have been described, whereas the biologic significances of the mutations were not investigated. Here, we aimed to correlate the biologic activities of the RUNX1 mutants with the clinical outcomes of patients. Experimental Design: We examined the mutational status of RUNX1 in 143 MDS and 84 CMML patients. Then, we studied the DNA and CBFβ binding abilities of all the RUNX1 mutants identified by using electrophoretic mobility shift assay and co-immunoprecipitation assay, and also determined their activities on target C-FMS gene induction by Western blotting and luciferase reporter assay. Using luciferase reporter assay, the relative biologic activities of each RUNX1 mutant could be quantified and correlated with the patient outcomes by statistical analyses. Results: We observed that most RUNX1 mutants had reduced abilities in DNA binding, CBFβ heterodimerization, and C-FMS gene induction. The relative biologic activities of RUNX1 mutants were grouped into high- and low-activity mutations. Correlation of the activities of RUNX1 mutants with the clinical outcomes revealed that patients harboring lower activities of RUNX1 mutants had a higher risk and shorter time to secondary acute myeloid leukemia transformation in MDS and CMML. In multivariate analysis, low RUNX1 activity remained an independent predictor for secondary acute myeloid leukemia–free survival in MDS patients. Conclusions: The biologic activity rather than the mutational status of RUNX1 might be an indicator in predicting outcome of patients with MDS and CMML. Clin Cancer Res; 21(15); 3541–51. ©2015 AACR.

Runt-related transcription factor 1 (RUNX1) is essential for normal hematopoiesis. RUNX1 mutations have rarely been reported in chronic myelomonocytic leukemia (CMML). We examined RUNX1 mutations in 81 patients with CMML at initial diagnosis. Mutational analysis was performed on bone marrow samples by direct sequencing of all reverse transcription PCR products amplified with three primer pairs that cover the entire coding sequences of RUNX1b. Thirty-two RUNX1 mutations were detected in 30 patients (37%); 23 mutants were located in the N-terminal part and 9 in the C-terminal region. The mutations consisted of 9 missense, 1 silent, 7 nonsense and 15 frameshift mutations. Two patients had biallelic heterozygous mutations. There was no difference in overall survival between patients with and without RUNX1 mutations, but a trend of higher risk of acute myeloid leukemia (AML) progression was observed in mutation-positive patients (16/30 vs 17/51, P=0.102), especially in patients with C-terminal mutations (P=0.023). The median time to AML progression was 6.8 months in patients with C-terminal mutations compared with 28.3 months in those without mutations (P=0.022). This study showed for the first time a high frequency of RUNX1 mutations in CMML. C-terminal mutations might be associated with a more frequent and rapid AML transformation.

Background and Aim: The molecular pathogenesis of progression of chronic myelomonocytic leukemia (CMML) to secondary acute myeloid leukemia (sAML) remains incompletely understood. Genemutations involving DNA methylation in the transformation of CMML to sAML were investigated in matched paired CMML/sAML bone marrow samples to determine the roles of TET2, DNMT3A, IDH1 and IDH2 mutations in the evolution of CMML to sAML. Material and Methods: 106 CMML (63 CMML-1 and 43 CMML-2) patients were analyzed for TET2, DNMT3A, IDH1, and IDH2 mutations at the initial diagnosis. 33 patients had paired CMML/sAML bone marrow samples for comparative analyses. Mutational analysis of TET2 was performed by PCR followed by direct sequencing for PCR products amplified with primer pairs covering the whole coding sequences (exons 3-11). DNMT3A mutations (exons 2-23) were screened by denaturing high-performance liquid chromatography (DHPLC) with adding GC-clamps to the primers to facilitate mutation detection.Samples with abnormal DHPLC profile were then directly sequenced. The hot spots of IDH1 and IDH2 genes on exon 4 were PCR-amplified from gDNA and subjected for direct sequencing. Additional gene mutations were analyzed by PCR-based assays with direct sequencing. The allele burden of gene mutations was measured at both CMML and sAML phases by pyrosequencing with a detection sensitivity of 5%. Results: The frequencies of TET2, DNMT3A, IDH1 and IDH2 in 106 CMML patients were 39.8% (41/103), 8.5% (9/106), 0% (0/106), and 7.5% (8/106), respectively. These epigenetic gene mutations were mostly mutually exclusive. Of the 31 paired CMML/sAML samples examined for DNMT3A, 4 had DNMT3A mutations at diagnosis; the mutation status, patterns and allele burden remained unchanged at sAML phases. None of 33 patients acquired IDH1 mutation and one acquired IDH2 mutation during sAML progression. Of the 30 patients with paired samples analyzed for TET2 mutation, 12 patients had TET2 mutations at both CMML and sAML phases; 10 patients retained the same TET2 mutations with stable allele burden, one patient had clonal expansion of TET2 mutation, and the other patient acquired 3rdTET2 mutation at sAML progression along with expansion of a preexisted TET2 mutant clone and one stable TET2 mutant subclone. Another patient harboring TET2 mutation at CMML diagnosis lost themutation at sAML progression. Acquisition of additional gene mutations during sAML evolution was detected in 5 TET2-mutated patients, including RUNX1, CEBPA, FLT3- ITD, JAK2 V617F, NPM1, SRSF2, and CSF3R, either alone or in combination. Conclusions: Our results showed that TET2 and DNMT3A mutational status and allele burden remained unchanged during the progression of CMML to sAML except that rare patients might have expansion or emergence of TET2 subclone at sAML phase. Acquisition of additional gene mutations occurred in half of TET2-mutated patients during the progression of CMML to sAML. Grant support: NHRI- EX103-10003NI and MOHW103-TD-B-111-09 Disclosures No relevant conflicts of interest to declare.

214 U2AF1 mutation in de novo myelodysplastic syndromes (MDS) and its genetic evolution profile of secondary acute myeloid leukemia (sAML): a comparative analysis of 68 paired matched samples Che-Wei Ou, Ming-Chung Kuo, Tung-Huei Lin, Yu-Shu Shih, Po Dunn, Tung-Liang Lin, Po-Nan Wang, Jin-Hou Wu, Chen-Yu Lai, Der-Cherng Liang and Lee-Yung Shih Chang Gung Memorial Hospital, Chang Gung University and Mackay Memorial Hospital, Taiwan Objectives: We aimed to investigate (1) the clinical relevance of U2AF1 gene mutation and its co-existing mutations in MDS, and (2) the genetic evolution profile of co-existing mutations in sAML transformation from MDS. Materials and Methods: Mutational analysis of U2AF1 (exons 2 and 6) was performed in 68 paired MDS/sAML bone marrow samples and in non-paired diagnosis samples in another 80 cases with de novo MDS. Additional 24 mutated genes were also analyzed at both MDS and sAML phases in U2AF1-mutated patients by using PCR-based assays followed by direct sequencing. The allelic burden of mutated genes was measured by pyrosequencing. Results: Twenty of 148 MDS patients (13.6%) had U2AF1 mutations (12 Ser34Phe, 3 Ser34Tyr, and 5 Gln157Pro). Co-existing mutations of U2AF1 with epigenetic regulators occurred in 10 patients (4 ASXL, two each for DNMT3A, TET2, and EZH2). U2AF1 mutation was significantly associated with younger age (P=0.032). There was no correlation between U2AF1 mutation status and gender, blood counts, cytogenetics, or IPSS-R risk groups whereas RAEB-2 was associated with a lower frequency of U2AF1 mutation (P=0.049). U2AF1 mutation had no impact on risk of sAML transformation (P=0.807), sAML-free survival (P=0.998) or overall survival (P=0.516). Of the 68 paired MDS/sAML samples, 12 had U2AF1 mutations at both phases and none acquired or lost U2AF1 mutation during sAML progression. There was no difference in the U2AF1 mutant allelic burden between MDS and sAML phases (P=0.165). In the 6 patients also carrying epigenetic regulator mutations, the mutation status and mutant levels remained stable in both MDS/sAML phases. In U2AF1-mutated patients, progression to sAML was accompanied by evolution of new mutant clones (2 RUNX1 and one each for PTPN11 and CBL) or clonal expansion of a pre-existing subclones (one each for NRAS and RUNX1). Conclusions: Our results showed that mutations of epigenetic regulators occurred in half of U2AF1-mutated patients. The two mutations remained no changes in sAML transformation whereas acquisition of additional genetic alterations or expansion of small pre-existing subclones was detected in a subset of U2AF1-mutated patients. Grant support: NHRI-EX103-10003NI, DOH-102-TD-C-111-006, MMHE-101-09 and OMRPG3C0021, Taiwan.

Background and purpose: Chronic myeloproliferative neoplasms (MPN) may transform into secondary acute myeloid leukemia (sAML). The genetic evolution pattern is important in understanding the pathogenesis of sAML transformation. We aimed to determine the profile of genetic evolution by examining a cohort of paired samples collected at both MPN and sAML phases using a panel of commonly mutated genes involved in myeloid neoplasms. Methods: Mutational analyses of 14 genes (JAK2V617F, CALR, ASXL1, IDH1, IDH2, TET2, DNMT3A, EZH2, KRAS, RUNX1, TP53, WT1, CBL, and SRSF2) were performed on the paired matched samples at diagnosis and at sAML transformation from 22 patients with MPN (6 polycythemia vera, 5 essential thrombocythemia, and 11 primary myelofibrosis). The samples were analyzed by polymerase chain reaction (PCR)-based assays followed by direct sequencing. For CALRmutational analysis, the PCR products of exon 9 were analyzed by GeneScan for screening deletions and insertions, then validated by Sanger sequencing. The allele frequencies of all mutated genes were determined by pyrosequencing assays in samples harboring any detectable mutations. Results: At the MPN phase, JAK2V617F was the most common mutation (N=12), followed by CALR (N=9). At sAML phase, JAK2V617F was not detected in 4 patients who had JAK2V617F at initial diagnosis while all 9 patients retained CALR mutations. Mutations of epigenetic modifier genes were detected in 13 patients (59%) at MPN phase, including TET2 (N=5), EZH2 (N=4), ASXL1 (N=2), DNMT3A (N=2), IDH1 and IDH2 (N=1 for each). One MPN sample was also found carrying RUNX1 mutation. Acquisition of 12 mutations was found in 11 patients at the sAML phase, including RUNX1 (N=4), KRAS (N=3), IDH2 (N=2), and one each of CBL, ASXL1 and TP53. Except JAK2V617F and TET2 mutations, the mutations present in the MPN phase remained detectable at sAML transformation. The allele frequencies of IDH2 and EZH2 mutant clones were apparently increased at sAML phase. Of note, two patients had double TET2 mutations at the MPN phase, clonal selection of TET2 mutants was explicitly demonstrated in both cases, with disappearance of one clone and expansion of the other during sAML transformation. The median time from MPN to sAML was 71.6 months and the median overall survival was 73.2 months for the cohort of MPN patients who had sAML transformation later on. Female patients had a trend towards lower incidence of CALR mutation (2/11 vs. 7/11, P=0.08) and significantly shorter overall survival (43.3 vs. 129.2 months, P Conclusions: Our results showed that in addition to JAK2V617F and CALR mutations, mutations of epigenetic modifiers were present in more than half of MPN patients at diagnosis who later on had a sAML transformation. During sAML transformation, non-JAK2V617F clone evolved in one third of patients and acquisition of RUNX1, KRAS and IDH2 mutations recurrently emerged. Clonal selection and expansion of TET2 mutations might play a vital role in leukemogenesis of MPN. Grant support: NSC96-2314-B-182-003, CMRPG330303, OMRPG3C0021 and DOH102-TD-C-111-006 Disclosures No relevant conflicts of interest to declare.

Background and purpose: The Mixed-lineage leukemia (MLL) gene located at 11q23 can be fused to a variety of partner genes through chromosomal translocations (MLL-T) in acute leukemia. The co-existence of commonly known mutated genes has not been comprehensively studied in MLL-T AML. We aimed to determine (1) the prevalence and clinical relevance of gene mutations at diagnosis, and (2) the genetic evolution profile at relapse in de novo AML with MLL-T. Materials and methods: Bone marrow samples from 80 de novo AML with MLL-T patients were analyzed on 20 gene mutations involving activating signaling pathway (class I), transcription pathway (class II), epigenetic regulators (class III), and tumor suppressors, TP53 and WT1 (class IV). MLL-T was all confirmed by FISH analysis. The common MLL fusion transcripts were detected by RT-PCR, including 26 MLLT3, 13 MLLT10, 12 MLLT4, 11 ELL, 3 MLLT1, 3 AFF1, and 2 EPS1. Another 6 infrequent or rare partner genes (one each of MLLT6,CBL, ARHGEF12, TET1, SEPT6, and SEPT9) were identified by cDNA panhandle PCR. In the remaining 4 cases, the partner genes were not identified. Mutational analyses were performed with PCR-based assays followed by direct sequencing. Twenty-two of 26 patients who relapsed had relapse samples for comparative analysis. Results: The frequencies of gene mutations of class I, II, III, and IV, in 80 de novo MLL-T AML patients were 49.4%, 3.8%, 10.3% and 1.3%, respectively. Together, 53.8% of patients with MLL-T had at least one mutated gene. KRAS (17.7%), FLT3-TKD (11.5%), and NRAS (11.4%) mutations occurred most frequently. Among the epigenetic regulators, 7.9% of patients had DNMT3A, 2.7% TET2, 1.4% ASXL1, and none had IDH1/2 or EZH2 mutations. Three patients had 3 co-existing mutations and 9 patients had two mutations, with 6 of the 12 carrying FLT3-TKD mutations. Gene mutations of transcription pathway and tumor suppressors rarely occurred, only involving NPM1 in two cases and one each for RUNX1 and WT1 mutation. The patients with DNMT3A mutations were significantly associated with older age (P=0.005), FAB AML-M4 (P Conclusions: Our results showed that co-existing mutations of RAS and FLT3-TKD were most frequently detected at diagnosis in de novo MLL-T AML. Gene mutations, except FLT3-ITD, had no impact on outcomes in MLL-T AML. FLT3-TKD mutations were absent in all relapse samples while clonal evolution of WT1, TP53 or KRAS mutations might contribute to the relapse of leukemia in a subset of MLL-T AML patients. Grant support: NHRI-EX93-9011SL, NHRI-EX96-9434SI, NSC95-2314-B-195-001, NSC96-2314-B-195-006-MY3, NSC97-2314-B-182-011-MY3, MMH-E-101-09 and OMRPG3C0021. Disclosures No relevant conflicts of interest to declare.

Background and purpose Abnormalities of genes regulating DNA methylation have been described in acute myeloid leukemia (AML). MLL protein is a transcriptional regulator and governs proper hematopoiesis through its histone methyltransferase activity. AML with partial tandem duplication of MLL (MLL-PTD) was associated with an unfavorable prognosis. The cooperating roles of MLL-PTD with other mutated genes regulating DNA methylation have not been comprehensively studied in AML. We aimed to determine the prevalence and clinical impact of mutations of DNA methylation regulators in AML with MLL-PTD. Materials and methods Bone marrow samples from 98 AML patients with MLL-PTD were analyzed for gene mutations of TET2, DNMT3A, IDH1 and IDH2. MLL-PTD was screened by RT-PCR and confirmed by real-time quantitative PCR assays. The mutational analysis was performed with PCR assays followed by direct sequencing for TET2 (whole coding exons 3–11) and IDH1/2 (hotspots exon 4). For the detection of DNMT3A mutations, the PCR products amplified for entire coding exons 2 to 23 were first screened with denaturing high-performance liquid chromatography followed by direct sequencing for the abnormal profiles. Results The frequency of TET2, IDH1, IDH2 and DNMT3A mutations in AML patients with MLL-PTD was 17.0% (16/94), 10.2% (10/98), 18.4% (18/98), and 31.6% (31/98), respectively. Taken together, 61.1% of patients with MLL-PTD had at least one mutated gene of DNA methylation regulators. TET2, IDH1 and IDH2 mutations were mutually exclusive with each other whereas DNMT3A mutations frequently co-existed with other DNA methylation modifiers:TET2 (n=8), IDH1 (n=5) and IDH2 (n=4). No differences were observed between the mutation status of the DNA methylation modifiers and clinico-hematologic features of patients with MLL-PTD except that TET2 (P=0.012) and DNMT3A (P=0.024) mutations were associated with older age. Of the 55 MLL-PTD patients who received standard chemotherapy, IDH2 mutation was associated with a lower complete remission rate (25.0% vs 67.8%, P=0.018), while DNMT3A mutations conferred an inferior event-free survival (0.0 vs 6.8 months, P=0.027) and overall survival (6.0 vs 11.5 months, P=0.032). In multivariate analysis, older age (P=0.008) and DNMT3A mutations (P=0.049) were independent adverse factors for overall survival. The crosstalk between MLL-PTD and genes involving DNA methylation in the leukemogenesis of AML warrants further investigation. Conclusions Gene mutations involving DNA methylation frequently co-existed in AML patients with MLL-PTD, especially DNMT3A mutations which conferred a poor outcome. Our study demonstrated the importance of genetic alterations involving DNA methylation in the pathogenesis of MLL-PTD AML and provided potential epigenetic-targeted therapy. Grant support The work was supported by NHRI-EX93-9011SL, NSC95-2314-B-195-001, NSC96-2314-B-195-006-MY3, NSC97-2314-B-182-011-MY3 and MMH-E-101-09. Disclosures: No relevant conflicts of interest to declare.

Background and purpose ASXL1 and EZH2 are histone modifiers. Mutations of ASXL1 and EZH2 genes have been described in both myelodyspolastic syndromes (MDS) and acute myeloid leukemia (AML). The role of ASXL1 or EZH2 mutations in the progression from MDS to secondary AML (sAML) is unclear. We aimed to determine the clinical relevance of ASXL1 and EZH2 mutations in patients with MDS and to investigate the role of ASXL1 or EZH2 mutation with its cooperating mutated genes in sAML progression. Methods ASXL1 and EZH2 mutations were analyzed on bone marrow samples from 126 patients with de novo MDS (45 RAEB1, 53 RAEB2, 24 RCMD and 4 RARS). Paired matched MDS/sAML samples were available for ASXL1 mutational analysis in 58 patients and for EZH2 in 54 patients. Mutational analysis of ASXL1 exon 12 was performed by PCR assays followed by direct sequencing, EZH2 mutations were first screened with denaturing high-performance liquid chromatography on amplified PCR fragments covering the whole coding sequencing from exons 2 to 20 of EZH2 gene followed by sequencing of the abnormal profile. Additional 20 known gene mutations in myeloid neoplasms were also examined in patients carrying ASXL1 or EZH2 mutations. Relative allele frequency was determined by pyrosequencing. Results Among the 126 patients, ASXL1 mutations were detected in 18 (14.3%) patients and EZH2 in 11 patients (8.7%), 3 of them had both mutations. Taken together, 20.6% of patients carried mutations of ASXL1 and/or EZH2. ASXL1-mutated patients had male predominance (17 out of 18 patients, P=0.012) and fewer circulating blasts (P=0.007). ASXL1-mutated and -unmutated patients had no difference in hemoglobin levels, white blood cell counts, platelet counts, cytogenetics, WHO subtypes, bone marrow blasts, IPSS-R or risk to sAML. ASXL1 mutations had no impact on overall survival (P=0.765) or time to sAML transformation (P=0.605). No significant difference was observed between EZH2 mutation status and clinicohematologic features or outcomes. Of the 58 paired samples, ASXL1 mutations were detected in 8 cases at diagnosis of MDS, all were also present at the time of sAML progression with no difference in the mutant allele burden (P=0.614), 2 patients acquired ASXL1 mutations. Five had EZH2 mutations at both phases of disease which exhibited a similar allele frequency (P= 0.434), none lost and one acquired EZH2 mutation at sAML phase. Progression to sAML was accompanied by additional gene mutations including RUNX1 (n=4), TET2 (n=3), CEBPα (n=2), PTPN11 (n=2), and one each for FLT3-ITD, CBL, MLL-PTD, DNMT3A, IDH2 and EZH2 in ASXL1-mutated patients; TET2 (n=4), RUNX1 (n=3), N-RAS (n=2) and single cases for DNMT3A, IDH1, and ASXL1 in EZH2-mutated patients. Cooperating mutations were either detected in both MDS and sAML samples or newly appeared in sAML samples except one who lost JAK2V617F mutation while acquired EZH2 and N-RAS mutations during sAML progression. Conclusions Our study on a large cohort of paired MDS and sAML samples demonstrated that ASXL1 or EZH2 mutations remained stable at both phases of disease in most patients. Clonal evolution can occur and cooperation of additional gene mutations is frequently detected in patients harboring ASXL1 or EZH2 mutations in the progression of MDS to sAML. Grant support This work was supported by NHRI-EX102-10003NI and DOH102-TD-C-111-006, Taiwan. Disclosures: No relevant conflicts of interest to declare.

Abstract 2785 Background and Objectives: RUNX1 is an essential transcription factor for normal hematopoiesis, and also the most frequent mutation target in leukemia. RUNX1 mutations have been described in myelodysplastic syndrome (MDS), and related to the clinicohematologic features. However, the biological activities of the RUNX1 mutants were not thoroughly investigated or even correlated to the progression of the disease. Therefore, we analyzed RUNX1 gene mutations in de novo MDS and studied the biological functions of the mutants. We sought to quantify the biological activities of the RUNX1 mutants and correlate the activities with the disease outcome. Methods: Bone marrow samples from 134 patients with high risk de novo MDS (54 RAEB1, 52 RAEB2, 24 RCMD and 4 others) were analyzed for RUNX1 mutations. Mutational analysis of RUNX1 was performed by RT-PCR amplification of whole coding region of RUNX1 followed by sequencing of the PCR products. Each mutation was reconfirmed by PCR with alternative primers and genomic DNA as template. Expression plasmids of wild-type RUNX1 and the mutants were constructed and applied for functional study. Immunopreciptation followed by Western blotting and electromobility shift assay (EMSA) were performed to examine the association with co-factor and DNA of the mutants. Transactivities of the mutants were quantified by luciferase reporter assay and statistically correlated to the outcome of the disease. Results: Among the 134 patients, 23 (17.2%) harbored RUNX1 mutations at MDS stage. In total, 21 RUNX1 mutations were identified in 23 patients, including 3 silent mutations (I87I, S195S and L445L), 5 nonsense mutations (R139X, Q245X, M283X, R293X and S383X), 9 missense mutations (S73F, H78Y, R135K, R139G, R139Q, A160T, R174Q, V425G and M439L) and 4 frameshift mutations (Q186fsX209, T219fsX233, Ser329fsX572 and D424fsX572). The mutant, R174Q, lost DNA binding ability but interacted with co-factor, CBFβ, exhibited very low transactivities and served as dominant-negative inhibitors for wt-RUNX1. R139 was demonstrated an important site in RUNX1 transactivity; therefore, R139X, R139G and R139Q mutants exhibited low activities in our assay system. H78Y near the DNA binding site R80 of RUNX1 hindered DNA binding and showed low transactivities. The mutants of Q186fsX209, T219fsX233, Q245X, M283X and R293X lacked tansactivation domain (TAD) exhibited less than 50% transactivities compared to wt-RUNX1. A160T and S73F mutants did not interact with CBFβ but showed comparable transactivity to wt-RUNX1. The C-terminal frameshift mutant, D424fsX572, and the 4 mutants, R135K, S383X, V425G and M439L, neither affected DNA and CBFβ binding nor TAD, displayed similar transactivities to wt-RUNX1. S329fsX572 mutant lost part of TAD, also showed similar transactivities to wt-RUNX1. Seventeen of 23 (73.9%) RUNX1 mutation-positive patients progressed to sAML compared with 66 of 111 (59.5%) RUNX1 mutation-negative patients (P=0.242), indicating mutation of RUNX1 may not be a crucial factor in disease progression. RUNX1 mutants were subsequently divided to high activity (> 50% transactivity compared to wt-RUNX1) and low activity ( Conclusions: Our study determined the biological activities of the RUNX1 mutants and showed that RUNX1 mutants with lower transactivities conferred a higher risk and more rapid progression to sAML in high risk MDS patients. Supported by grants NHRI-EX99-9711SI, DOH100-TD-C-111-006, BMRPG380031 and MMH-E-100-09. Disclosures: No relevant conflicts of interest to declare.

Abstract 4025 Background. Myelodysplastic syndrome (MDS) is a heterogeneous disease group characterized by ineffective hematopoiesis, cytopenias, and various probability of acute myeloid leukemia (sAML) transformation. The molecular genetic changes from MDS to sAML are not clear. We aimed to determine the roles of gene mutations involving receptor tyrosine kinases (RTKs: FLT3/ITD, FLT3/TKD, c-KIT, or c-FMS), Ras pathways (NRAS, KRAS, or PTPN11) and JAK2V617F in the progression of MDS by analyzing matched paired high risk MDS and sAML samples. Patients and Methods. A total of 122 patients (50 RAEB1, 52 RAEB2, and 20 RCMD) were examined; 82 had sAML evolution and 68 of them had matched paired bone marrow samples (22 RAEB1, 29 RAEB2 and 17 RCMD) available for comparative analysis for at least one gene mutation. Mutational analyses were performed by DNA/cDNA PCR with GeneScan analysis for FLT3/ITD, PCR-RFLP followed by direct sequencing for FLT3/TKD, DNA/cDNA PCR and direct sequencing for c-KIT, c-FMS, NRAS, KRAS, and PTPN11 mutations, and allele-specific PCR for JAK2V617F mutation. Results. Of the 122 cases, 3 had FLT3 mutations (2 FLT3/ITD, 1 FLT3/TKD), none had c-KIT or KRAS mutations, one had c-FMS mutation, 3 each had JAK2V617F and NRAS mutations, and 2 had PTPN11 mutations. Patients harboring mutations of RTK or Ras pathways at diagnosis were not at increased risk of sAML (P=0.5501 and 1.0000, respectively). Presence of any RTK mutations at diagnosis (N=3) did not influence outcome in terms of time to sAML (P=0.6711) or overall survival (OS) (P=0.7162) compared to those who did not have any RTK mutation (N=119). Time to sAML (P=0.6093) or OS (P=0.4515) were not different between patients carrying Ras pathway mutations (N=5) and those without any mutations (N=117). The results of the mutation status for each gene in the 68 paired samples are shown in Table 1. Seven patients (10.3%) gained mutations of RTK pathways at sAML. Patients with mutations of RTKs at diagnosis had shorter time to sAML than those who did not have any mutations (3.4±2.0 vs 10.9±1.1 months, P=0.0004), but the OS was not different between these two groups (P=0.2015). For the Ras pathways, 12 patients (17.6%) acquired mutations at sAML, one of them also acquired FLT3/TKD mutation. Additional one patient gained JAK2V617F mutation. Time to sAML (P=0.2578) and OS (P=0.2637) were not different between patients with Ras pathway mutations at diagnosis and those without mutations. Taken together, 8 patients (11.8%) had mutations at diagnosis and 24 (35.3%) at sAML, one lost PTPN11 mutation and 19 patients (27.9%) acquired mutations which drive proliferation and survival of malignant clone during the progression to sAML from MDS. Conclusions. Our results showed that acquisition of mutations involving RTK, JAK2, or Ras pathways played important roles in the progression of MDS to sAML. Patients with mutations of RTKs progressed to sAML more rapidly. Supported by grants NHRI-EX99-9711SI, NSC97-2314-B-182-011-MY3 and DOH99-TD-C-111-006. Disclosures: No relevant conflicts of interest to declare.

Abstract 2713 Background. The cooperation of gene mutations, especially their impacts on survivals of childhood acute myeloid leukemia (AML) has not been well known. Aims. Our aims were (1) to study the frequency of each gene mutation in childhood AML, (2) to study the impact of each gene mutation on the treatment outcome, and (3) to examine the cooperativity of gene mutations. Materials and Methods. From Feb. 1996 to Jan. 2010, bone marrow samples at diagnosis from 198 children with AML at Chang Gung Children's Hospital, Taoyuan and Mackay Memorial Hospital, Taipei, were analyzed for gene mutations including FLT3-ITD, FLT3-TKD (D835), c-KIT, cFMS, JAK 2V617F, NRAS, KRAS, PTPN11 (Class I mutations), RUNX1, CEBPα, NPM1 (Class II mutations), WT1 and P53 (tumor suppressor genes). The subtypes included: t(8;21) 19.9%, inv(16) 8.9%, t(15;17) 8.4%, t(9;11) 5.2%, t(10;11) 2.6%, trisomy 21 4.2%, intermediate-risk group 40.3% (including 13 patients with other MLL translocations), and poor-risk group 11.0% (including 7 patients with complex chromosomal abnormalities and 4 patients with MLL-PTD). Results. FLT3-ITD occurred in 15.0% of patients, FLT3-TKD 7.2%, c-KIT 11.5%, c-FMS 2.9%, JAK2V617F 3.3%, NRAS 9.1%, KRAS 7.7%, PTPN11 3.3%, RUNX1 2.7%, CEBPα 7.9%, NPM1 4.1%, WT1 3.9% and P53 1.7%. Taken together, 52.5% of patients had Class I gene mutations, 13.1% had Class II gene mutations, and 5.1% had WT1 or P53 mutations. In all, 59.1% of patients had Class I, Class II or tumor suppressor gene mutations. Only one patient (0.5 %) had gene mutations involving all Class I, Class II and tumor suppressor genes. Ninety-eight patients, who were treated with Taiwan Pediatric Oncology Group (TPOG) APL protocols (for acute promyelocytic leukemia) and TPOG 97A protocol (for other AML) (Liang et al, Leukemia 2006), were analyzed for survivals. In patients with t(8;21), the 5-year event-free survival (EFS) was 66±12%; 71±17% for patients with c-KIT mutations and 50±35% for the 2 patients with JAK2V617F. In patients with inv(16), the EFS of 70±15% seemed to be compromised (60±22%) for those with c-KIT mutations. In patients with t(15;17), the EFS of 78±11% was not compromised by FLT3-ITD or FLT3-TKD mutations. In patients with t(9;11), the EFS of 64% seemed to be compromised (50±35%) in the 2 patients with FLT3-TKD mutations. In 3 patients with t(10;11), no gene mutations were found. In trisomy 21, the EFS of 75±22% seemed to be compromised (50±35%) in the 2 patients with CEBPα mutations. Of the 5 patients with complex chromosomal abnormalities, the only one patient carrying RUNX1 survived. Of the 3 patients with MLL-PTD having an EFS of 33±27%, one each patient with c-FMS or WT1 mutation died. The only one patient who had all Class I, Class II and tumor suppressor gene mutations (FLT3-TKD+ CEBPα+ WT1) died in induction therapy. Two of the other 4 patients who had 3 mutations acrossing 2 classes had EFS of 6 and 10 months, respectively. Conclusions. Our study on a large cohort of pediatric AML patients revealed that 59.1% patients had at least one gene mutation. That 3 of 5 patients who had 3 gene mutations soon failed suggested that gene mutations, especially in 3 combinations, might compromise the survival. Further study on more patients is warranted to explore more of the prognostic significance of cooperating gene mutations in pediatric AML. (Supported by grants MMH-E-98009, NSC 96–2314-B-195-006-MY3, NHRI-EX-96-9434SI and DOH99-TD-C-111-006.) Disclosures: No relevant conflicts of interest to declare.

Abstract 4019 Background. The molecular pathogenesis of myelodysplastic syndrome (MDS) and its role in the progression to secondary acute myeloid leukemia (sAML) remain to be further explored. Somatic TET2 and C-CBL mutations have recently been described in myeloid neoplasms including MDS and sAML. Most studies of TET2 or C-CBL mutations were carried out separately either at MDS or at sAML phases. There was also a discrepancy in the results of the impact of TET2 and C-CBL mutations on outcome of MDS patients. Aims. We aimed (1) to correlate the TET2 and C-CBL mutations with clinicohematological features and outcome, and (2) to determine the role of TET2 or C-CBL gene mutations in sAML derived from MDS. Materials and Methods. Bone marrow (BM) samples from 161 MDS patients (3 RCUD, 1 RARS, 36 RCMD, 118 RAEB, 1 MDS5q-, 2 MDS-U) at initial diagnosis were analyzed for both TET2 and C-CBL mutations; 49 of them had matched paired sAML BM samples available for comparative analysis. Mutational analysis was performed by DHPLC and/or direct sequencing of all RT-PCR or DNA-PCR products amplified with different primer pairs covering the whole coding sequences of TET2 and exons 7 to 9 of C-CBL. Results. The frequency of TET2 and C-CBL mutations in 161 MDS patients was 17.9 % and 1.9 %, respectively. Seven patients with polymorphism/germ line mutations or missense mutations outside of BOX 1 or 2 of TET2 gene were excluded. Of the 26 patients with 38 TET2 mutations, 14 had nonsense, 11 missense, 11 frameshift, 1 deletion, and 1 splice site mutations; 12 of them had double mutations. Three patients had C-CBL mutations (Y371S, F418S and G415S) at MDS phase. No patient had coexistence of TET2 and C-CBL mutations. TET2 mutations were significantly associated with increased risk of AML transformation (P= 0.037), whereas C-CBL mutations did not influence the risk of AML transformation (P=0.335). Patients carrying TET2 mutations had a trend of shorter time to sAML compared with those without TET2 mutations (estimated median time to sAML 8.7 months vs 15.0 months, P=0.067). Except for lower platelet count in patients with TET2 mutations (P=0.038), there were no significant differences in clinicohematological features including age, sex, hemoglobin level, white blood cell count, percentage of blasts in BM or peripheral blood, cytogenetics or IPSS (≤1.5 vs ≥2.0) between patients with and without TET2 or C-CBL mutations. No significant differences were found in overall survival with respect to mutation status of TET2 (P= 0.244) or C-CBL (P= 0.646). Of the 49 paired BM samples, 3 patients harboring C-CBL mutations at MDS phase retained the identical mutations and another 3 acquired C-CBL mutations (L370_Y371 ins L, L399V and C416W) during sAML evolution. There was an increased risk of acquiring C-CBL mutations during AML transformation (odds ratio=4.16, P=0.041), whereas TET2 mutation patterns remained unchanged and none acquired or lost TET2 mutations in sAML progression. Conclusions. Our results showed an increased risk of acquiring C-CBL mutations during sAML transformation. TET2 mutations played a role as an initial event in the development of MDS in a subset of patients and were associated with more frequent and rapid sAML evolution. Supported by grants NHRI-EX99-9711SI, NSC97-2314-B-182-011-MY3 and DOH99-TD-C-111-006. Disclosures: No relevant conflicts of interest to declare.

Abstract 3468 Poster Board III-356 Background Transcription factor RUNX1 is essential for normal hematopoiesis. RUNX1 mutations, mainly at Runt homology domain (RHD), have been described in patients with AML-M0 and were rarely found in non-M0 AML. Aim We aimed to analyze the RUNX1 mutations in AML patients with partial tandem duplication of MLL (MLL-PTD) and to investigate the biological functions of the mutants detected. Patients and methods Bone marrow samples from 93 patients with MLL-PTD were analyzed for RUNX1 mutations. MLL-PTD was screened by Southern-blot analysis followed by RT-PCR or detected by real-time quantitative PCR. Mutational analysis of RUNX1 gene was performed by sequencing of all RT-PCR products amplified from exon 3 through exon 8. Each mutation was reconfirmed with alternative primers. The wild-type, all mutants of RUNX1 (except those truncated at RHD or silent) and pcDNA3.1 were transiently transfected into Cos-7 cells. Immunoblot analysis after immunoprecipitation with anti-FLAG RUNX1 antibody and electrophoretic mobility shift assay were used to determine the interaction with CBFβ and DNA-binding ability of the RUNX1 mutants. Dual luciferase assay system was used to analyze the transactivation potential of RUNX1 mutants in K562 cells. Results RUNX1 mutations were detected in 23 patients (24.7%) at diagnosis, with 3 patients carrying double mutations; 14 mutations were located in RHD (exons 3-5) and 12 at C-terminal region (exons 6-8). In addition, one patient acquired a C-terminal mutation at relapse. The patterns of 27 mutations consisted of 6 missense mutations, 3 nonsense mutations, 17 frame-shift mutations, and 1 silent mutation; all were heterozygous. Of the 3 patients with double mutations, clonal analysis showed that one patient had combined missense and frame-shift mutations on the same allele, the other patient had two missense mutations on different alleles, and another patient had a missense mutation and a silent mutation on the same allele. Functional analyses revealed significant difference among mutants. Two missense RUNX1 mutants at RHD (G108D and R174L) and all of the frame-shift mutants in the transactivation domain (TAD) ( S287fsX571, S295fsX571, L300fsX570, V333fsX574, I339fsX569 and P355fsX572) exhibited lack of DNA-binding ability and inhibited transactivation activity of wild-type RUNX1 in a dominant-negative effect. All frame-shift mutants distal to the TAD generated termination codons within the 3'-untranslated region (H377fsX565, Q388fsX572, L414fsX569, L414fsX567 and V425fsX576), they all retained the normal transactivation activity as the wild-type. R177X and R205W retained the ability of heterodimerization with CBFβ but they had markedly reduced DNA-binding and no transactivation potential without inhibitory effect on wild-type RUNX1. L183fsX185 could bind DNA but lacked transactivation activity. S114P and Q370R had normal transactivation activity. Conclusions Our results showed that patients with de novo AML with MLL-PTD had a high frequency of frame-shift mutations at C-terminal region of RUNX1; those within TAD had dominant-negative effects whereas those distal to TAD retained the normal transactivation potential. Supported by grants NHRI-EX96-9434SI, NSC97-2314-B-182 -011-MY3 and MMH-E-96009. Disclosures No relevant conflicts of interest to declare.

Two-hit model of leukemogenesis has been proposed for AML. In AML with MLL rearrangements ( MLL -R), MLL gene is fused to a variety of partner genes through reciprocal chromosomal translocations ( MLL /t11q23), or is rearranged to generate a partial tandem duplication ( MLL -PTD). The cooperating mutations of AML with MLL -R have not been systematically analyzed. We aimed to determine the cooperating mutations, including receptor tyrosine kinase (RTK) /Ras signaling pathway, NPM1 and myeloid transcription factors in de novo AML with MLL -R. MLL -R was screened by Southern blot analysis. RT-PCR was used to detect common MLL fusion transcripts. cDNA panhandle PCR was used to identify the infrequent or unknown MLL partner genes. Mutational analysis was peformed by DNA/cDNA PCR-GeneScan analysis for FLT3 /ITD, by PCR-RFLP followed by direct sequencing for FLT3 /TKD, by DNA/cDNA PCR and direct sequencing for N- Ras , K- Ras , c-KIT , c-FMS , PTPN11 , NPM1 , AML1 and CEBP α. Of the 131 patients with MLL -R, 77 had MLL -PTD and 54 had MLL /t11q23. None of the 131 patients with MLL -R had c-FMS mutations and c-KIT mutation was present in only one patient with MLL /t11q23. NPM1 mutations occurred in one with MLL -PTD and 2 with MLL /t11q23. The frequencies of other cooperating mutations are shown in Table 1. Taken together, cooperating mutations involving RTK/Ras pathway, NPM1, and/or myeloid transcription factors occurred in 71.4% (55/77) of patients with MLL -PTD and 59.3% (32/54) of patients with MLL /t11q23. In MLL -PTD group, coexistence of two mutations occurred in 23 patients. In MLL /t11q23 group, 6 patients had two mutations. Of the 18 patients with MLL -PTD and AML1 mutations, 8 mutations were located in the Runt homology domain (RHD) and 10 in the non-RHD, 15 were frameshift or nonsense mutations and 3 were missense mutations. Fourteen patients with MLL -PTD and AML1 mutations also had mutations of RTK/Ras singling pathway. Three patients with MLL /t11q23 and AML1 mutations, one in the RHD and 2 in the non-RHD, all were missense mutations. Of the 5 patients with MLL -PTD and CEBP α mutations, 3 harbored FLT3 /ITD. Patients with MLL -PTD had a significantly higher frequency of cooperating mutations with myeloid transcription factors than patients with MLL /t11q23 (20/77 vs. 3/54, P =0.002), whereas there was no difference in the frequency of mutations involving RTK/Ras pathway between MLL -PTD and MLL /t11q23 groups (51/77 vs. 29/54, P =0.202). Our results showed that patients with de novo AML and MLL -R had a high frequency of cooperating mutations with RTK/Ras signaling pathway, NPM1 or myeloid transcription factors, and the mutation patterns were different between MLL -PTD and MLL /t11q23 groups. | | FLT3 /ITD | FLT3 /TKD | N- Ras | K- Ras | PTPN11 | AML1 | CEBP α | |:----------- | --------- | --------- | ------ | ------- | ------ | ----- | ------ | | MLL -PTD | 35/77 | 11/77 | 5/77 | 0/77 | 3/77 | 18/77 | 5/77 | | MLL /t11q23 | 2/54 | 7/54 | 9/54 | 13/54 | 1/53 | 3/54 | 0/54 | | P value

Transcription factor AML1 is essential for normal hematopoiesis. AML1 mutations have been described in myelodysplastic syndrome (MDS), especially in therapy-related MDS but were rarely examined in patients with chronic myelomonocytic leukemia (CMML). We sought to determine the frequency of AML1 mutations in CMML and to assess their role in the progression of CMML to AML. Mutation analysis of AML1 was performed on bone marrow samples from 63 patients with CMML by direct sequencing of all RT-PCR products amplified with 3 overlapping primer pairs which cover the coding sequences of AML1 gene from exon 3 through exon 8. AML1 mutations were detected in 24 of 63 patients (38%) at initial diagnosis, 16 patients had mutations located in Runt homology domain (RHD) (exons 3–5), and 8 had mutations located in the C-terminal region (exons 6–8). One patient had 2 missense mutations in RHD and another one had 2 frameshift mutations in RHD. Taken together, the patterns of 26 mutations consisted of 7 missense mutations, 5 nonsense mutations, 2 silent mutations and 12 frameshift mutations. Ten of 24 patients (41.7%) with mutations progressed to AML compared to 13 of 39 patients (33.3%) without mutations, no difference was observed in the risk of AML transformation between the two groups (P=0.593). Of the 23 patients who progressed to AML, 17 also had bone marrow samples available at AML transformation for mutation analysis. AML1 mutations were detected in 8 of 17 patients at both diagnosis of CMML and AML transformation, all exhibited identical patterns at both phases, with no loss or gain of mutations during AML progression. There were no differences in age, sex, blood counts, percentage of blasts in bone marrow or peripheral blood, subtype of CMML (1 or 2), and cytogenetic risk group between patients with AML1 mutations and those without mutations. Time to AML transformation and overall survival of patients with AML1 mutations did not differ from those without AML1 mutations. Our study showed that AML1 mutations were frequently detected in CMML, with 70% located in RHD. All mutant clones remained identical at initial diagnosis and AML transformation, suggesting that AML1 mutations play an important role in the leukemogenesis in this subset of patients with CMML.