258 MUTATIONAL SPECTRUM AND PROGNOSTIC VALUE OF GENE MUTATIONS IN CHRONIC MYELOMONOCYTIC LEUKEMIA PATIENTS

Abstract Abstract 1402 Chronic myelomonocytic leukemia (CMML) is characterized by monocytic proliferation, cytomorphologic dysplasia and frequent progression to acute myelogeneous leukemia (AML). The molecular basis of CMML is poorly defined, although somatic mutations in a number of genes have recently been identified in a proportion of patients (epigenetic regulatory genes, spliceosomal genes, apoptosis genes, growth signal transducers and others). We performed a comprehensive analysis of molecular lesions, including somatic mutations detected by sequencing and chromosomal abnormalities investigated by metaphase and SNP-array karyotyping. We have selected a cohort of 72 patients (36 CMML1, 16 CMML2 and 20 sAML evolved from CMML). Our mutational screen performed in stages (as new mutations were discovered by our and other groups) and currently reveals mutations in UTX in 8%, DNMT3A in 9%, CBL in 14%, IDH1/2 in 4%, KRAS in 2.7%, NRAS in 4.1%, JAK2 in 1%, TET2 in 48%, ASXL1 in 43%, EZH2 in 5.5%, RUNX1 37%. Based on the discovery of various spliceosomal mutations in myeloid malignancies, novel mutations were also found in CMML, in U2AF1 in 12%, SF3B1 in 14%, SFRS19 in 6 % of cases tested. Chromosomal defects were detected in 60% of patients. In particular, a high frequency of somatic uniparental disomy (sUPD) were identified 71% of patients with abnormal cytogenetics, including UPD1p (N=3), UPD7q (N=8), UPD4q (N=6), UPD2p (N=2), UPD17q (N=2), UPD11q (N=5), UPDX (N=1), UPD21q (N=2). Some of the detected mutations were homozygous through their association with sUPD as for example for 3 EZH2, 1 UTX, 6 TET2, 2 DNMT3A, 5 CBL, 1 NRAS, 1 U2AF1 mutations. Furthermore, UPD17p implies that a P53 mutation is also present in this case as previously LOH17p was shown to be invariably associated with P53 mutations. Similarly, 2 cases of UPD17q imply that homozygous mutation of SRSF2, which is one of the Serine/arginine-rich splicing factor, may be present in this location and the mutation analysis is ongoing. In over 90% of >1 mutation was found but many patients harbored multiple mutations with frequent combinations of TET2/CBL or TET2/ASXL1 as well as RUNX1 and U2AF1 serving as examples. There was an accumulation of mutations from sAML, CMML2 and CMML1 suggesting stepwise accumulation of lesions. In serial studies, some of the mutations were present at the inception (e.g., TET2, ASXL1 and DNMT3A) in some cases originally heterozygous mutations were also while other can occur in the course of disease (e.g. CBL). RAS and DNMT3A mutations were associated with a higher blasts count. In sum, combined analysis of molecular lesions in CMML reveals that similar phenotype may be a result of diverse mutations associated with seemingly unrelated pathways and that clinical phenotype may be a result of a combination of mutations which accumulate as the disease progresses. Survival analyses will require large cohorts to account for various confounding factors including the presence of multiple chromosomal abnormalities and mutations in one patient, however currently EZH2, DNMT3 and CBL mutations appear to convey less favorable prognosis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Gene mutations differently impact the prognosis of the myelodysplastic and myeloproliferative classes of chronic myelomonocytic leukemia

American Journal of Hematology ◽

10.1002/ajh.23702 ◽

2014 ◽

Vol 89 (6) ◽

pp. 604-609 ◽

Cited By ~ 26

Author(s):

Nathalie Cervera ◽

Raphael Itzykson ◽

Emilie Coppin ◽

Thomas Prebet ◽

Anne Murati ◽

...

Keyword(s):

Gene Mutations ◽

Chronic Myelomonocytic Leukemia ◽

Myelomonocytic Leukemia

Download Full-text

SRSF2 is Mutated in 47.2% (77/163) of Chronic Myelomonocytic Leukemia (CMML) and Prognostically Favorable in Cases with Concomitant RUNX1 mutations

Blood ◽

10.1182/blood.v118.21.274.274 ◽

2011 ◽

Vol 118 (21) ◽

pp. 274-274 ◽

Cited By ~ 4

Author(s):

Susanne Schnittger ◽

Manja Meggendorfer ◽

Alexander Kohlmann ◽

Vera Grossmann ◽

Kenichi Yoshida ◽

...

Keyword(s):

Hot Spot ◽

Myeloproliferative Neoplasm ◽

Gene Mutations ◽

Chronic Myelomonocytic Leukemia ◽

Structural Basis ◽

Employment Equity ◽

Cell Counts ◽

Number Of Genes ◽

Equity Ownership ◽

Myelomonocytic Leukemia

Abstract Abstract 274 Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. We previously investigated 81 CMML cases and detected a number of genes frequently mutated (TET2 44.4%, CBL 22.2%, NRAS 22.2%, KRAS 12.3%, JAK2 9.8%, RUNX1 8.7%, EZH2 12.3% (Kohlmann et al., JCO, 2010; Grossmann et al., Leukemia, 2011). Recently, we detected a new candidate gene, SRSF2 (serine/arginine-rich splicing factor 2, also known as SC35) that is a component of the RNA splicing machinery and found it to be frequently mutated in MDS. Aim: As CMML has been increasingly characterized by a growing number of genes during the last years we here analyzed both the frequency of SRSF2 mutations in this entity and the relevance in the context of other previously described gene mutations, as well as to look for a potential prognostic implication. Patients and Methods: In total, 163 cases with CMML (CMML-1 n=105, CMML-2 n=58) were included. The cohort comprised 115 males and 48 females with a median age of 72.8 yrs (range: 21.9 – 88.8 yrs) including all 81 pts that have been published previously. 112 cases (69%) had a normal karyotype and 51 (31%) showed aberrant karyotypes. The mutational hot spot region of SRSF2 around Proline codon 95 (P95) was analyzed by Sanger sequencing in all cases. Data on further mutations were available in respective subcohorts: ASXL1 (n=128), CBL (n=162), EZH2 (n=134), JAK2V617F (n=162), KRAS (n=140), NRAS (n=79), RUNX1 (n=156), TET2 (n=143), TP53 (n=80). Results:SRSF2 mutations of P95 were detected in 77/163 (47.2%) of all cases (49/105, 46.7% in CMML-1, and 28/58, 48.3% in CMML-2). In detail, 74 cases had a missense mutation leading to a change of P95 to P95H (n=33), P95L (n=24), P95R (n=16) or P95A (n=1). In further 3 cases a newly described 24 bp (8 amino acids) deletion starting at P95 was observed. All cases had a mutation load of approximately 50%. The mutations were correlated with higher age (73.3 yrs vs 68.7 yrs in the SRSF2wt cases, p=0.010) and higher hemoglobin levels (11.4 vs 10.5 g/dl in the SRSF2wt cases, p=0.019) whereas white blood cell counts were not different. Further, SRSF2 mutations were mutually exclusive of EZH2 mutations (0/12, 0% vs. 66/122, 54.1% in the EZH2wt, p<0.001) whereas a high coincidence occurred with RUNX1 mutations (22/35, 62.9% vs 52/121, 43% in the RUNX1wt, p=0.054) and TET2 mutations (50/82, 61% vs 18/61, 29.5% in the TET2wt, p<0.001). With respect to associations with all other gene mutations investigated and karyotype no specific pattern was observed. In the total cohort no impact of SRSF2 on survival was observed. Because of the high coincidence of SRSF2mut with RUNX1mut and TET2mut, we performed an analysis in these specific subcohorts. No impact of SRSF2mut in the TET2mut subcohort was found. Whereas in the RUNX1mut subcohort SRSF2mut had a favorable impact on overall survival compared to SRSF2wt (median OS: 108.0 months vs 41.8 months, p=0.05). Conclusions:SRSF2 has recently been described as a new marker in CMML and demonstrated to be useful to delineate further the genetic defects of this disease. This very frequent new mutation is characterized by higher age, higher hemoglobin levels and a high coincidence with TET2 and RUNX1 mutations. It is mutually exclusive of EZH2 mutations. In the subset of RUNX1 mutated CMML SRSF2 mutations demonstrated a favorable impact on outcome. Furthermore, for the first time a 24 bp deletion was observed in three cases that may provide further insight into the structural basis for the abnormal function of SRSF2. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

Mutational Spectrum In Chronic Myelomonocytic Leukemia Includes Genes Associated with Epigenetic Regulation Such as UTX and EZH2

Blood ◽

10.1182/blood.v116.21.611.611 ◽

2010 ◽

Vol 116 (21) ◽

pp. 611-611 ◽

Cited By ~ 4

Author(s):

Anna Jankowska ◽

Hideki Makishima ◽

Ramon V. Tiu ◽

Hadrian Szpurka ◽

Yun Huang ◽

...

Keyword(s):

Epigenetic Regulation ◽

Conflicts Of Interest ◽

Risk Group ◽

Prognostic Significance ◽

Uniparental Disomy ◽

Chronic Myelomonocytic Leukemia ◽

Future Research ◽

Molecular Abnormalities ◽

Mutational Spectrum ◽

Myelomonocytic Leukemia

Abstract Abstract 611 Chronic myelomonocytic leukemia (CMML), a myelodysplastic/myeloproliferative overlap neoplasm, is characterized by monocytic proliferation, cytomorphologic dysplasia and frequent progression to acute myelogeneous leukemia (AML). The molecular basis of CMML is poorly defined, although somatic mutations in a number of genes have recently been identified in a proportion of patients. Single nucleotide polymorphisms array (SNP-A) technologies have improved the definition of shared regions of loss of heterozygosity (LOH), including uniparental disomy (UPD) and facilitated discovery of new mutations c-CBL, TET2, and EZH2 which can occur in a homozygous configuration in the areas of UPD. Other mutations such as ASXL1 have been found in heterozygous form. In myeloid malignancies we have also identified mutations in UTX, which like EZH2 and ASXL1, are involved in modification of histone methylation. Based on these findings we hypothesized that defining the mutational spectrum of CMML would help in the molecular characterization of this disease and have diagnostic and prognostic significance. Within this spectrum, we stipulated that various genes involved in epigenetic regulation may be especially affected by mutations in CMML. Here we present results of broad molecular screen in a group of 63 patients with CMML (32 CMML-1, 15 CMML-2 and 16 CMML-derived sAML) which included SNP-A karyotyping and mutational screen for IDH1/2, RAS, TET2, ASXL1, c-CBL, JAK2, UTX and EZH2. First, we aligned all lesions that were detected by SNP-A. In addition to microdeletions involving 4q24 and 11q23.3, we detected recurrent areas of somatic UPD involving chromosomes 1, 4, 7 and 11 and the corresponding homozygous mutations in RAS (UPD1p, N=1), EZH2 (UPD7q N=3), c-CBL (UPD11q, N=4), TET2 (UPD4q, N=6), and UTX genes (UPDXq, N=1). When all patients were sequenced, TET2, ASXL1, c-CBL, IDH1/2, RAS, JAK2, UTX and EZH2 mutations were found in 48%, 24%, 14%, 5%, 11%, 2%, 6% and 8% of patients, respectively. In 78% of patients, >1 mutation was found. Concomitant second and third mutations were found in 34% and 5% of patients, respectively. The most frequently observed combinations included TET2 and ASXL1 (14%) and TET2 and c-CBL (6%). Only 22% of patients had no alterations in analyzed genes. Novel UTX and EZH2 mutations were present either alone or in combination with other mutations. Study of potential functional consequences of the foregoing gene mutations revealed an association of TET2 mutations with consistently low levels of 5-hydroxymethylcytosine (5-hmC), quantitated by dot blot assay, while c-CBL mutations were associated with aberrant phospho-STAT5 staining. Loss of H3K27-me3 in cases with EZH2 mutations but not controls, and an increase in UTX mutant case was identified as measured by ELISA and western blot. When we tested for association of different mutations with pathomorphologic features, specific clinical features were not identified, except for an association of TET2 and c-CBL mutations with more advanced age (p=.0004 and p=.02, respectively), RAS mutation with increased blasts (p=.03) and UTX with dysplastic megakaryocytes (p=.03). Splenomegaly was noted more frequent in c-CBL mutants than any other patient group. No differences in OS and EFS were observed between mutant and wt cases. There is a trend toward better OS in TET2 mutants compared to WT in the good cytogenetic risk group (17 vs 8 mo, p=.07) but worse outcomes in TET2 mutants in the intermediate cytogenetic risk group (OS 2 vs. 16 mo, p=.001; EFS 2 vs. 9 mo, p=.04). As expected, patients who have accumulated more mutations have a trend toward inferior outcomes compared to those with single mutations but better than those who are WT (>1 mutations vs 1 mutation vs WT, 16 vs 18 vs 9 mo, p=.07 in low risk CMML). In summary, our study identified the presence of a wide spectrum of mutations in CMML with various combinations, including the newly discovered mutations in UTX and EZH2 genes. Our results suggest that molecular abnormalities affecting various pathways can lead to a clinically indistinguishable phenotype. It is possible that these mutations are secondary in nature but work in conjunction with a yet unidentified founder defect. The abundance of mutations in factors known or hypothesized to be involved in epigenetic regulation in CMML provide important implications for future research into the development of effective therapies for this disease. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Validation of the 2017 revision of the WHO chronic myelomonocytic leukemia categories

Blood Advances ◽

10.1182/bloodadvances.2018019224 ◽

2018 ◽

Vol 2 (15) ◽

pp. 1807-1816 ◽

Cited By ~ 13

Author(s):

Sanam Loghavi ◽

Dawen Sui ◽

Peng Wei ◽

Guillermo Garcia-Manero ◽

Sherry Pierce ◽

...

Keyword(s):

Prognostic Value ◽

Chronic Myelomonocytic Leukemia ◽

Key Points ◽

Myelomonocytic Leukemia

Key Points Validation of the WHO 2017 CMML categories supports distinguishing MP-CMML from MDS–CMML. The prognostic value of the new 3-tiered blast-based CMML stratification scheme is limited.

Download Full-text

Prognostic Score Including Gene Mutations in Chronic Myelomonocytic Leukemia

Journal of Clinical Oncology ◽

10.1200/jco.2012.47.3314 ◽

2013 ◽

Vol 31 (19) ◽

pp. 2428-2436 ◽

Cited By ~ 298

Author(s):

Raphaël Itzykson ◽

Olivier Kosmider ◽

Aline Renneville ◽

Véronique Gelsi-Boyer ◽

Manja Meggendorfer ◽

...

Keyword(s):

Cox Model ◽

Prognostic Score ◽

Gene Mutations ◽

Scoring Systems ◽

Chronic Myelomonocytic Leukemia ◽

Clinical Variables ◽

Mutational Status ◽

Wbc Count ◽

Prognostic Scoring Systems ◽

Myelomonocytic Leukemia

Purpose Several prognostic scoring systems have been proposed for chronic myelomonocytic leukemia (CMML), a disease in which some gene mutations—including ASXL1—have been associated with poor prognosis in univariable analyses. We developed and validated a prognostic score for overall survival (OS) based on mutational status and standard clinical variables. Patients and Methods We genotyped ASXL1 and up to 18 other genes including epigenetic (TET2, EZH2, IDH1, IDH2, DNMT3A), splicing (SF3B1, SRSF2, ZRSF2, U2AF1), transcription (RUNX1, NPM1, TP53), and signaling (NRAS, KRAS, CBL, JAK2, FLT3) regulators in 312 patients with CMML. Genotypes and clinical variables were included in a multivariable Cox model of OS validated by bootstrapping. A scoring system was developed using regression coefficients from this model. Results ASXL1 mutations (P < .0001) and, to a lesser extent, SRSF2 (P = .03), CBL (P = .003), and IDH2 (P = .03) mutations predicted inferior OS in univariable analysis. The retained independent prognostic factors included ASXL1 mutations, age older than 65 years, WBC count greater than 15 ×109/L, platelet count less than 100 ×109/L, and anemia (hemoglobin < 10 g/dL in female patients, < 11g/dL in male patients). The resulting five-parameter prognostic score delineated three groups of patients with median OS not reached, 38.5 months, and 14.4 months, respectively (P < .0001), and was validated in an independent cohort of 165 patients (P < .0001). Conclusion A new prognostic score including ASXL1 status, age, hemoglobin, WBC, and platelet counts defines three groups of CMML patients with distinct outcomes. Based on concordance analysis, this score appears more discriminative than those based solely on clinical parameters.

Download Full-text

DNTM3A Mutations and Prognosis in Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood.v128.22.1988.1988 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1988-1988

Author(s):

Mrinal M Patnaik ◽

Terra L. Lasho ◽

Christy Finke ◽

Matthew T Howard ◽

Curtis A. Hanson ◽

...

Keyword(s):

Conflicts Of Interest ◽

Univariate Analysis ◽

Multivariable Analysis ◽

Gene Mutations ◽

Chronic Myelomonocytic Leukemia ◽

World Health ◽

Prognostic Impact ◽

Wild Type ◽

The Impact ◽

Myelomonocytic Leukemia

Abstract Background : DNMT3A mutations result in epigenetic dysregulation and impart a negative prognostic impact in acute myeloid leukemia and myelodysplastic syndromes. In chronic myelomonocytic leukemia (CMML), DNMT3A mutations are seen in 2-5% of patients. In a large Groupe Français des Myélodysplasies (GFM) study (n=312), DNMT3A mutations were seen in 2% and were not included in further survival analyses (Itzykson JCO 2013). In a prior Mayo Clinic study (n=175), DNMT3A mutations were seen in 5% (n=9) and on univariate, but not multivariate analysis (Patnaik Blood C J 2016), were associated with shortened over-all survival (OS). We carried out this study on a larger CMML cohort (n=261), with more (n=15) informative cases to assess the impact of DNMT3A mutations. Methods : 261 patients with World Health Organization (WHO)-defined CMML were included in the study. All patients had bone marrow (BM) biopsies and cytogenetics performed at diagnosis. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for the following genes; TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, KRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1. The 2016 WHO diagnostic criteria were used. Results: Among the 261 study patients, 65% were males and median age was 70 years (range, 28-91). 154 (59%), 64 (25%) and 43 (16%) patients were classified as CMML-0, 1 and 2, respectively. At a median follow-up of 23 months, 174 (67%) deaths and 37 (14%) leukemic transformations (LT) were documented. Mutational frequencies ≥4% were encountered in; TET2 45%, ASXL1 45%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, DNMT3A 6%, U2AF1 6%, SF3B1 5%, ZRSR2 4%, Tp53 4%, and IDH2 4%. i) DNTM3A mutated CMML: phenotypic and molecular correlates DNMT3A mutations were seen in 15 (6%) patients; 64% male with a median age of 64 years. DNMT3A amino acid substitutions included; R882H 50%, R882C 29%, R910P 7%, R598* 7% and R320* 7%. The median variant allele frequency burden was 45%. Concurrent gene mutations were detected in; TET2 43%, ASXL1 21%, SF3B1 21%, U2AF1 14%, RUNX1 14%, SETBP1 14%, NRAS 14%, SRSF2 7%, JAK2 7% and Tp53 7%. There was no difference between DNMT3A mutated and wild-type patients in terms of age and gender distribution, hemoglobin level, leukocyte, monocyte (AMC), and platelet counts, peripheral blood (PB) or BM blast content. Concurrent gene mutations were equally distributed with the exception for a higher prevalence of SF3B1 (p=0.003) and a lower prevalence of SRSF2 (p=0.004) mutations in DNMT3A mutated CMML. Four (29%) patients underwent leukemic transformation. ii) Impact on OS and leukemia-free survival (LFS): Median survival for the entire cohort (n=261) was 24 months. In univariate analysis, survival was shorter in DNMT3A mutated (median 8 months) versus wild-type (median 27 months) patients (p=0.0007; HR 2.9, 95% CI 1.5-5.7; Figure 1A). Other variables of significance, in univariate analysis, included lower hemoglobin (p=0.002), higher leukocyte count (p=0.0009), higher AMC (p=0.0012), PB blast % (p=0.001), circulating immature myeloid cells (IMC, p=0.01), BM blast % (p=0.045), abnormal karyotype (p=0.02), and ASXL1 (p=0.01) mutations. Survival was also adversely affected by the presence of either (n=133) or both (n=3) ASXL1/DNMT3A mutations (0=0.007, Figure 1B). In multivariable analysis (MVA) excluding ASXL1 and DNMT3A mutations, hemoglobin (p=0.03), IMC (p=0.013) and AMC (p=0.02) retained significance. When ASXL1 mutations were added to the MVA, ASXL1 (p=0.01) mutations, AMC (p=0.012) and IMC (p=0.03) retained significance. Similarly, when only DNMT3A mutations were added to the MVA, DNMT3A (p=0.003) mutations, IMC (p=0.01) and AMC (p=0.02) retained significance. When both DNMT3A and ASXL1 mutations were added to the MVA, only DNMT3A (p<0.0001) and ASXL1 (p=0.004) mutations remained significant. DNMT3A mutations predicted shortened OS, independent of the ASXL1 inclusive GFM model (p<0.0001) and Mayo Molecular Model (p=0.002). DNMT3A mutations (p=0.0018), along with low hemoglobin levels (p=0.003) independently predicted for a shorter LFS. Conclusions: DNMT3A mutations are seen in ~5% of patients with CMML and impart a negative prognostic impact on both OS and LFS. This finding warrants inclusion of DNMT3A mutations in molecularly integrated CMML prognostic models. Disclosures No relevant conflicts of interest to declare.

Download Full-text