Acute Monoblastic/Monocytic Leukemia and Chronic Myelomonocytic Leukemia Share Common Immunophenotypic Features but Differ in the Extend of Aberrantly Expressed Antigens and the Amount of Granulocytic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3799-3799
Author(s):  
Wolfgang Kern ◽  
Claudia Haferlach ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Normal Karyotype ◽  
Cell Types ◽  
Chronic Myelomonocytic Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Monocytic Leukemia ◽  
Common Features ◽  
Equity Ownership ◽  
Wbc Count ◽  
Myelomonocytic Leukemia

Abstract 3799 Poster Board III-735 Acute monoblastic/monocytic leukemia (AMoL) is grouped into acute myeloid leukemia and related neoplasms by the 2008 WHO classification while chronic myelomonocytic leukemia (CMML) in grouped into myelodysplastic/myeloproliferative neoplasms (MDS/MPN). However, both entities share common features and sometimes may be difficult to differentiate from each other. In particular, monopoietic cells can be easily identified by multiparameter flow cytometry (MFC) based on their CD45/side-scatter (SSC) signal. However, immunophenotypes of monoblasts and dysplastic monocytes largely overlap. Their differentiation from each other is better accomplished by cytomorphology (CM) and the use of non-specific esterase. We compared cytomorphologic, immunophenotypic and cytogenetic results between 26 patients with AMoL and 139 patients with CMML which were analyzed at diagnosis between 2005 and 2009 in order to identify shared and discriminative characteristics. In AMoL vs. CMML 65.4% vs. 73.4% were male, the median age was 65.4 vs. 72.5 years (range 19.3-82.0 vs. 21.9-87.9 years, p<0.001), and the median WBC count was 38.0 G/L vs. 10.4 G/l (range 0.8-126.0 G/l vs. 0.9-92.0 G/l, p<0.001). Cytogenetics differed in that a normal karyotype was found in 79.1% of CMML cases and in only 38.5% of AMoL cases (p<0.0001). Per definition, a t(9;11) (19.2%) was found only in AMoL (p<0.001) which was almost identical for other translocations involving MLL (15.4% vs. 0.7%, p=0.003). Other cytogenetic abnormalities did not significantly differ between AMoL and CMML. First, immunophenotypes of monopoietic cells were compared between AMoL and CMML. While an aberrant coexpression of CD56 was present in all cases with AMoL this was true in only 82.0% of CMML cases (p=0.015). No other aberrantly expressed antigen was found in AMoL while in CMML a coexpression of CD2 was found in 21.6% (p=0.005), a lack of CD13 expression in 10.8% (n.s.), and a lack of HLA-DR expression in 4.3% (n.s.). Next, we compared the frequencies of different cell types between AMoL and CMML as identified by CM and MFC. In this analysis, granulocytic cells by CM included cells of all maturation grades from promyelocyte to granulocyte, which corresponds to the population identified in MFC by their dim expression of CD45 and their strong SSC signal. As anticipated when considering the definition of the respective entities CM identified higher percentages of blasts (mean±SD 78.7±13.8 vs. 8.6±5.1, p<0.001) and lower percentages of monocytes (1.7±3.9 vs. 13.8±11.9, p<0.001) and granulocytic cells (7.9±7.4 vs. 53.8±15.8, p<0.001) in AMoL vs. CMML. Applying the blast gate in the CD45/side-scatter analysis for the identification of blasts (CD45(+)SSC-) MFC also found higher percentages of blasts in AMoL vs. CMML (18.2±25.1 vs. 4.0±3.2, p<0.001) and monopoietic cells were found also at higher frequencies in AMoL (28.6±27.4 vs. 20.1±12.3, p=0.012). Interestingly, however, MFC identified significantly higher frequencies of granulocytic cells in CMML (52.2±19.3 vs. 26.2±22.6, p<0.001). Thus, while the CD45-SSC analysis is not capable of differentiating between monocytes and monoblasts it may be used to identify high percentages of granulocytic cells which is characteristic for CMML but not for AMoL. Based on these findings, the ratio M:G (monopoietic cells:granulocytic cells) was calculated for each case. The ratio M:G was significantly higher in AMoL as compared to CMML (mean±SD 5.2±10.4 vs. 0.8±2.3, p<0.001). In conclusion, the present data demonstrate that AMoL and CMML after being discriminated according to WHO guidelines for cytomorphology share common features as identified by MFC but differ in the extend of aberrantly expressed antigens and the amount of granulocytic cells. For further studies and to support CM these parameters should be evaluated when using MFC as a diagnostic tool for AMoL and CMML. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Concomitant Analysis of EZH2 and ASXL1 Mutations In Myelofibrosis, Chronic Myelomonocytic Leukemia and Blast-Phase Myeloproliferative Neoplasms

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3070-3070 ◽  
Author(s):  
Omar Abdel-Wahab ◽  
Animesh Pardanani ◽  
Jay Patel ◽  
Terra Lasho ◽  
Adriana Heguy ◽  
...  
Keyword(s):  
Mutation Analysis ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Univariate Analysis ◽  
Multivariable Analysis ◽  
Normal Karyotype ◽  
Chronic Myelomonocytic Leukemia ◽  
Blast Phase ◽  
Idh Mutations ◽  
Myelomonocytic Leukemia

Abstract Abstract 3070 Background: EZH2 and ASXL1 mutations were recently described in a spectrum of myeloid malignancies; mutational analysis of small patient cohorts has suggested the highest mutational frequency in myelofibrosis (MF) and chronic myelomonocytic leukemia (CMML). The current study seeks to determine i) EZH2 and ASXL1 mutational frequencies in WHO-defined subcategories of MF, CMML and blast-phase myeloproliferative neoplasm (MPN), ii) if these mutations are mutually exclusive of TET2, IDH, JAK2 and MPL mutations and iii) clinical correlates of ASXL1 and EZH2 mutations in primary MF (PMF) and CMML. Methods: The study population included 94 patients: 46 PMF, 22 post-polycythemia vera/essential thrombocythemia MF (post-PV/ET MF), 11 blast-phase MPN and 15 CMML (10 CMML-1 and 5 CMML-2). High throughput DNA resequencing was used to screen archived bone marrow for EZH2, ASXL1, TET2, IDH, JAK2 and MPL mutations. Results: ASXL1 mutations were identified in all disease categories, including PMF (13%), post-PV/ET MF (23%), blast phase MPN (18%), and CMML (20%). We identified somatic mutations in TET2 in 15%, 14%, 18%, and 13% of PMF, post-PV/ET MF, blast phase MPN, and CMML, respectively. By contrast, mutations in EZH2 and IDH1/2 were less frequent. EZH2 mutations were seen in 3 out of 46 PMF patients (7%) and were not observed in patients with post-PV/ET MF or blast phase MPN. Mutations in IDH1/2 were restricted to blast-phase MPN (36%) and PMF (7%). No mutations in EZH2 or IDH1/2 were seen in CMML. Although we identified frequent TET2 and ASXL1 mutations, we only identified one patient with concurrent mutations in both genes. Three ASXL1 mutation-positive patients also had mutations in EZH2 or IDH and one patient had concurrent ASXL1, TET2 and IDH mutations. In addition, 7 ASXL1, 7 TET2, and 1 IDH mutated patients were JAK2V617F-positive. MPL mutations were also documented in all three mutation categories. All EZH2- and ASXL1-mutated PMF patients displayed normal karyotype and none underwent leukemic transformation during follow-up. Furthermore, mutated versus unmutated patients, in both instances, were not significantly different in age and sex distribution or clinical characteristics. The 3 EZH2-mutated PMF patients died after 29, 48 and 67 months from the time of mutation analysis. In univariate analysis, the presence of mutant ASXL1 in PMF was associated with worse survival (p=0.06) but the borderline significance was lost during multivariable analysis that included risk stratification according to DIPSS (Passamonti et al. Blood 2010; 115: 1703–1708). The 3 ASXL1 mutated CMML cases were alive after 40, 34 and 12 months from time of mutation analysis and none of them had progressed to acute leukemia; karyotype was normal in two of the patients and showed isolated trisomy 8 in one. Conclusions: ASXL1 mutations are as frequent as TET2 mutations in MF and CMML. In contrast, EZH2 mutations are infrequent and cluster with PMF. ASXL1 and EZH2 mutations are not mutually exclusive events, seem to be associated with normal karyotype and do not appear to be leukemogenic or prognostically detrimental in PMF or CMML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Chronicles of Myelodysplastic/ Myeloproliferative Legacy - Chronic Myelomonocytic Leukemia

2020 ◽  
Vol 7 (11) ◽  
pp. C149-154
Author(s):  
Vinu Sugathan ◽  
Latha K Abraham ◽  
Mobin Paul
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Tertiary Care ◽  
Case Series ◽  
Chronic Myelomonocytic Leukemia ◽  
Trisomy 8 ◽  
Tertiary Care Centre ◽  
Clinical Presentations ◽  
Progression Of Disease ◽  
Wbc Count ◽  
Myelomonocytic Leukemia

Chronic myelomonocytic leukemia is a heterogeneous syndrome with features of both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). The varied clinical presentations add to the distinctiveness of the disease. This heterogeneity should invigorate the search for reliable predictors of evolution and progression of disease. We report a case series from a tertiary care centre in Kerala, South India. This was a retrospective observational study of all cases of CMML, which was diagnosed in the departments of Pathology and Clinical Haematology & Haemato- oncology of our institution between January 2017 to May 2020. The clinical presentation, laboratory investigations, and treatment details were noted. Nine cases of CMML were encountered during the study period. The mean age of study subjects was 70.4 years with a female predilection. Fever and weight loss were the most common clinical presentations. Four patients were classified as CMML- 2, three patients as CMML- 1, and two as CMML- 0. Based on the WBC count, five patients were classified as dysplastic and four as proliferative subtypes. Two patients had grade 1/3 (one case each of CMML- 2 and CMML- 1) and one patient had grade 2/3 fibrosis (a case of CMML- 1) in the bone marrow. Thirty-three percentage patients had clonal cytogenetic abnormalities, the commonest being trisomy 8. Renal function was deranged in three patients and two patients had a deranged liver function and hepatomegaly. Four patients underwent treatment with hypomethylating agents or cytoreduction with hydroxyurea. One of the patients (CMML- 2 with marked leucocytosis) succumbed to disease.

Myelodysplastic/Myeloproliferative Neoplasms

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 264-272 ◽  
Author(s):  
Mario Cazzola ◽  
Luca Malcovati ◽  
Rosangela Invernizzi
Keyword(s):  
Clinical Laboratory ◽  
Myeloproliferative Neoplasms ◽  
Chronic Myelomonocytic Leukemia ◽  
World Health ◽  
Refractory Anemia ◽  
Molecular Diagnostic ◽  
Juvenile Myelomonocytic Leukemia ◽  
Lymphoid Tissues ◽  
Ring Sideroblasts ◽  
Myelomonocytic Leukemia

Abstract According to the World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, myelodysplastic/myeloproliferative neoplasms are clonal myeloid neoplasms that have some clinical, laboratory, or morphologic findings that support a diagnosis of myelodysplastic syndrome, and other findings that are more consistent with myeloproliferative neoplasms. These disorders include chronic myelomonocytic leukemia, atypical chronic myeloid leukemia (BCR-ABL1 negative), juvenile myelomonocytic leukemia, and myelodysplastic/myeloproliferative neoplasms, unclassifiable. The best characterized of these latter unclassifiable conditions is the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis. This article focuses on myelodysplastic/myeloproliferative neoplasms of adulthood, with particular emphasis on chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts associated with marked thrombocytosis. Recent studies have partly clarified the molecular basis of these disorders, laying the groundwork for the development of molecular diagnostic and prognostic tools. It is hoped that these advances will soon translate into improved therapeutic approaches.

27 Does WBC count really define two different subtypes of chronic myelomonocytic leukemia (CMML)? Analysis of a series of 119 patients

1997 ◽  
Vol 21 (1) ◽  
pp. S7 ◽  
Author(s):  
J. Cervera ◽  
G.F. Sanz ◽  
T. Valles'i ◽  
M.C. del Can˜izo ◽  
D. Irriguible ◽  
...  
Keyword(s):  
Chronic Myelomonocytic Leukemia ◽  
Wbc Count ◽  
Myelomonocytic Leukemia

Next-Generation Sequencing Technology Reveals a Characteristic Pattern of Molecular Mutations in 75% of Chronic Myelomonocytic Leukemia (CMML) by Detecting Frequent Alterations in TET2, RUNX1, CBL, and RAS.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 417-417 ◽  
Author(s):  
Alexander Kohlmann ◽  
Vera Grossmann ◽  
Claudia Haferlach ◽  
Beray Kazak ◽  
Sonja Schindela ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Mutation Screening ◽  
Point Mutations ◽  
Chronic Myelomonocytic Leukemia ◽  
Melting Curve Analysis ◽  
Next Generation ◽  
Monosomy 7 ◽  
Equity Ownership ◽  
Myelomonocytic Leukemia ◽  
Generation Sequencing

Abstract Abstract 417 Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. Here, we analyzed 81 CMML cases (45 CMML-1, 36 CMML-2). In chromosome banding analysis 59/76 (77.6%) patients showed a normal karyotype (data not availabel in 5 cases). Recurrent chromosome aberrations were trisomy 8 (n=6; 7.9%), monosomy 7 (n=3; 3.9%), and loss of the Y-chromosome (n=5; 6.6%). Fluorescence in situ hybridization (FISH) detected the deletion of one allele of the TET2 gene in 4/71 cases (5.6%). Thus, the majority of cases can not be genetically characterized by these techniques. Therefore, we applied next-generation sequencing (NGS) technology to investigate 7 candidate genes, represented by 43 PCR-products, at known mutational hotspot regions, i.e. CBL (exons 8 and 9), JAK2 (exons 12 and 14), MPL (exon 10), NRAS (exons 2 and 3), and KRAS (exons 2 and 3). In addition, complete coding regions were analyzed for RUNX1 (beta isoform) and TET2. NGS was performed using 454 FLX amplicon chemistry (Roche Diagnostics Corporation, Branford, CT). The median number of base pairs sequenced per patient was 9.24 Mb. For each target gene a median of 911 reads was generated (coverage range: 736-fold to 1606-fold). This approach allowed a high-sensitive detection of molecular mutations, e.g. detecting the JAK2 V617F mutation down to 1.16% of reads. In total, 146 variances were detected by this comprehensive molecular mutation screening (GS Amplicon Variant Analyzer software version 2.0.01). In 80.4% of variances consistent results were obtained after confirming NGS mutations with melting curve analysis and conventional sequencing. In the remaining discrepant variances (19.6%) NGS deep-sequencing outperformed conventional methods due to the higher sensitivity of the platform. After excluding 19 polymorphisms or silent mutations 127 distinct mutations in 61/81 patients (75.3%) were detected: CBL: n=21 point mutations and one deletion (18 bp) found in 20 cases (24%); JAK2: n=8 mutations (V617F) found in 8 cases (9.8%); MPL: no mutations found; NRAS: n=23 mutations found in 18 cases (22.2%); KRAS: n=12 mutations found in 10 cases (12.3%); RUNX1: n=6 point mutations and one deletion (14 bp) found in 7 cases (8.6%); and TET2: n=49 point mutations and 6 deletions (2-19 bp; 5/6 out-of-frame) found in 41 cases (50.6%). Furthermore, in 21 TET2-mutated cases 11 mutations previously described in the literature were detectable, whereas 28 cases carried novel mutations (n=28). In the cohort of TET2-mutated cases 17/41 (41.3%) patients harbored TET2 abnormalities as sole aberration. Interestingly, CBL mutations were found to be significantly associated with TET2 mutations (Fisher's exact test, p=0.008). In 17 of 20 (85.0%) CBL-mutated cases TET2 abnormalities were concomitantly observed. In contrast, no significant associations were found between any of the point mutations or deletions and the karyotype. There were also no associations observed between molecular aberrations and the diagnostic categories CMML-1 and CMML-2. With respect to clinical data a trend for better outcome was seen for patients that carried either or both TET2 and CBL mutations (median OS 130.4 vs. 17.3 months, alive at 2 yrs: 72.0% vs. 43.9%; p=0.13). In conclusion, 75.3% of CMMLs harbored at least one molecular aberration. In median 2 mutations per case were observed. Compared to limited data from the literature we detected not only a higher frequency of CBL mutations, but also add data on novel TET2 mutations. In particular, comprehensive NGS screening here for the first time has demonstrated its strength to further genetically characterize and delineate prognostic groups within this type of hematological malignancy. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kazak:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Clinical Phenotype of Myeloproliferative Neoplasms with Activating CBL-mutations Resembles Chronic Myelomonocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3895-3895
Author(s):  
Juliana Popa ◽  
Susanne Schnittger ◽  
Philipp Erben ◽  
Tamara Weiss ◽  
Ayalew Tefferi ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Clinical Phenotype ◽  
Myeloproliferative Neoplasms ◽  
Direct Sequencing ◽  
Jak2 V617f ◽  
Chronic Myelomonocytic Leukemia ◽  
A Genome ◽  
Length Mutations ◽  
Myelomonocytic Leukemia

Abstract Abstract 3895 Poster Board III-831 A genome-wide single nucleotide polymorphism (SNP) screen led to the identification of 11q aUPD in patients diagnosed with various subtypes of myeloproliferative neoplasms (MPN), e.g. chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML) and myelofibrosis (MF) (Grand et al., Blood 2009;113:6182). Further molecular analyses revealed acquired activating point and length mutations in CBL exons 8 and 9 in 10% of CMML, 8% of aCML and 6% of MF cases. Most variants were missense substitutions in the RING or linker domains that abrogated CBL ubiquitin ligase activity and conferred a proliferative advantage to 32D cells overexpressing FLT3. In this study, 160 patients with BCR-ABL and JAK2 V617F negative MPNs were screened for CBL mutations by PCR and direct sequencing. Eighteen known (Y371H, L380P [2x], C381R, C381Y [2x], C384Y, C396Y, H398P, H398Q, W408C, P417H, F418L, R420Q [5x]) and four new (F378L, G397V, I423N, V430M) missense mutations affecting fourteen residues were identified in 20 patients. Two patients harbored two different mutations. The clinical phenotype could be characterized more precisely in 17 patients. Median age was 68 years (range 59–85) with a slight female predominance (f, n=10; m, n=7). Striking hematological features were leukocytosis (14/17; 82%; median 29,000/μl, range 4,500-141,000) with continuously left-shifted granulopoiesis (blasts, promyelocytes, myelocytes, metamyelocytes) in 85% and elevated monocytes (median 2,500/μl, range 630-10,656) >1,000/μL in 88% (15/17) of patients. Eosinophilia (>1,500/μL) was rare (3/17, 18%). Anemia (normal values: f, Hb <12g/dL; m, Hb <14g/dL) was present in all 17 patients (f, median 10g/dL, range 8.7-11.8; m, median 11.2g/dL, range 8.6-12.9). Platelets did not exceed 300,000/μL in any patient while 11/17 (65%) patients presented with thrombocytopenia (median 125,000/μL, range 18,000-271,000). Splenomegaly was present in 11/17 patients (65%) and LDH was elevated (median 304U/L, range 189-729) in 9/17 patients (52%). Bone marrow histology and immunohistochemistry were available from 12 patients. Relevant features were hypercellularity, marked granulopoiesis and microlobulated megakaryocytes without clusters in 11/12 patients (92%), respectively. Increased fibres were seen in 8/12 (67%) patients of whom one showed severe fibrosis. Clinical follow-up was available from 17 patients. Thirteen patients (76%) have died because of progression to secondary acute myeloid leukemia/blast phase (n=7), cytopenia-related complications (n=2) or for unknown reasons (n=4) after a median of 23 months (range 3-60) following diagnosis. In conclusion, point mutations of CBL exons 8 and 9 are present in approximately 6-12% of BCR-ABL and JAK2 V617F negative MPNs. They are associated with a distinct clinical and hematological phenotype presenting with myeloproliferative features allowing diagnosis of a proliferative subtype of CMML rather than aCML or MF in the majority of cases. Patients with left-shifted leukocytosis, monocytosis, anemia and lack of thrombocytosis who are negative for BCR-ABL and point or length mutations of JAK2 should be routinely screened for CBL mutations. Disclosures: No relevant conflicts of interest to declare.

Analyses of 81 Chronic Myelomonocytic Leukemia (CMML) for EZH2, TET2, ASXL1, CBL, KRAS, NRAS, RUNX1, IDH1, IDH2, and NPM1 Revealed Mutations In 86.4% of All Patients with TET2 and EZH2 Being of High Prognostic Relevance

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 296-296 ◽  
Author(s):  
Vera Grossmann ◽  
Alexander Kohlmann ◽  
Christiane Eder ◽  
Nicholas C.P. Cross ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Chronic Myelomonocytic Leukemia ◽  
Molecular Heterogeneity ◽  
Cell Renewal ◽  
Wild Type ◽  
Employment Equity ◽  
Ras Pathway ◽  
Equity Ownership ◽  
Exon 4 ◽  
Myelomonocytic Leukemia

Abstract Abstract 296 Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. Recently, we investigated 81 CMML cases (45 CMML-1, 36 CMML-2) diagnosed between 10/2005 - 9/2008, which had been characterized by chromosome banding analysis and mutation analysis in 6 candidate genes: Mutations were detected in TET2 (44.4%), CBL (22.2%), NRAS (22.2%), KRAS (12.3%), JAK2 (9.8%), RUNX1 (8.7%) (Kohlmann et al., J Clin Oncol. 2010 Jul 19). We now applied amplicon-based deep-sequencing using the small volume Titanium chemistry assay (454 Life Sciences, Branford, CT) to investigate additional 4 candidate genes: IDH1 (exon 4), IDH2 (exon 4) and NPM1 (exon 12) (at known mutational hotspot regions) and the complete coding region of EZH2. EZH2 encodes a catalytic subunit of the polycomb repressive complex 2, a highly conserved histone H3 lysine 27 methyltransferase that influences stem cell renewal. Mutations in EZH2 were recently described to play a role in MPN/MDS. The sequencing library preparation for IDH1, IDH2, NPM1, and EZH2, in total 22 amplicons, was performed using 48.48 Access Array technology (Fluidigm, South San Francisco, CA) to cope with the number of amplicons. In median, 498 reads per amplicon were obtained, thus yielding sufficient coverage for detection of mutations with high sensitivity. After excluding polymorphisms and silent mutations aberrations were detected in IDH1 (1/81; 1.2%), IDH2 (3/81; 3.7%), NPM1 (1/81; 1.2%), and EZH2 (10/81; 12.3%). Another gene recently described in hematological diseases is ASXL1 (additional sex combs like 1) on chromosome 20q11.1. Therefore, the hotspot region of ASXL1 exon 12 was additionally investigated by Sanger sequencing in those 20 cases, in which no mutation had been observed thus far. Nine of these 20 cases (45%) harbored a mutation in ASXL1, thus only 11 cases (13.6%) remained in this cohort in which no mutation was detected. Summarizing this data, 86.4% of these CMMLs harbored at least one molecular aberration with a median of two genes mutated (range 1–4). In more detail, we observed 11 novel distinct EZH2 mutations in ten patients: 7 missense, 3 frameshifts (2 deletions, 1 insertion), and one splice site mutation. EZH2 mutations were found to be heterogeneous and were spread over several exons, predominantly located in the four conserved regions (6/11 in the conserved SET domain; e.g. H680R, N659S). No case revealed a Tyr641 of EZH2 mutation as described for follicular and diffuse large B-cell lymphomas. In median, the burden of EZH2 mutations was 42.5% of sequencing reads per patient (range 1.4–98%). Similarly, a high mutation burden was detected in RUNX1 (median 46.7%), TET2 (median 44.6%), and CBL (median 42.5%) whereas the burden was low in RAS pathway alterations, i.e. NRAS (median 11.1%), KRAS (median 27%), or JAK2 V617F mutations (median 6.9%). With respect to associations of distinct mutations no specific pattern was observed, i.e. EZH2 mutations were concomitantly detected with TET2 (4/10), RUNX1 (3/10), CBL (3/10), JAK2 (3/10), NRAS (2/10), KRAS (1/10), and IDH2 (1/10), respectively. Further, EZH2 mutations were associated neither with morphologic CMML subtype or dysplastic or myeloproliferative characteristics nor with age, white blood cell count, thrombocytes count, or hemoglobin. However, with respect to clinical data a very poor outcome was observed for patients that carried EZH2 mutations compared to EZH2 wild-type cases (median OS 4.3 vs. 130.4 months; p<0.001). In contrast, a significantly better outcome was seen for patients who carried TET2 mutations compared to TET2 wild-type cases (median OS 130.4 vs. 53.6 months, p=0.013). Subsequently, we performed a survival analysis taking both EZH2 and TET2 mutations into account. Here, the cohort was significantly separated into three distinct prognostic groups, i.e. EZH2-mutated with a poor median OS of 4.3 months, EZH2/TET2 wild-type with a median OS of 90 months and TET2-mutated cases with a median OS of 130.4 months (p<0.001). In conclusion, our study revealed molecular mutations in 86.4% of 81 CMML patients providing new insights into the molecular heterogeneity of this disease. Besides alterations in TET2, CBL, ASXL1, and the RAS pathway, EZH2 is targeted by various types of frameshift and point mutations and is a novel biomarker with unfavorable prognosis and clinical utility. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

In Myelodysplastic/Myeloproliferative Neoplasms Aberrant Antigen Expression As Assessed by Multiparameter Flow Cytometry Is Related to Molecular Genetic Mutations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5152-5152
Author(s):  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
Tamara Alpermann ◽  
Claudia Haferlach ◽  
Torsten Haferlach
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Molecular Genetic ◽  
Antigen Expression ◽  
Multiparameter Flow Cytometry ◽  
Employment Equity ◽  
Aberrant Expression ◽  
Equity Ownership ◽  
Diagnostic Work ◽  
Work Up ◽  
Diagnostic Work Up

Abstract Abstract 5152 Background: Immunophenotyping by multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of patients with cytopenias and suspected myelodysplastic syndromes (MDS). Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) comprise a group of diseases with some features of MDS and is separately classified in the current WHO system. While the immunophenotype of chronic myelomonocytic leukemia has been described in detail, data is scarce on the use of MFC in myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPNu) as well as on refractory anemia with ring sideroblasts and thrombocytosis (RARS-T), which is a provisional entity in the current WHO classification. Aim: To assess patients with MDS/MPNu and RARS-T for MDS-related aberrant immunophenotypes in the context of a comprehensive diagnostic work-up including cytomorphology, cytogenetics, and molecular genetics. Patients and Methods: A total of 91 patients were analyzed in parallel by cytomorphology, cytogenetics, and MFC applying an antibody panel designed to diagnose MDS. MFC was used to detect expression of mature antigens in myeloid progenitors; abnormal CD13-CD16- and CD11b-CD16-expression patterns, aberrant expression of myeloid markers and reduced side scatter signal in granulocytes; reduced expression of myelomonocytic markers in monocytes; aberrant expression of CD71 in erythroid cells; as well as expression of lymphoid markers in all myeloid cell lines. In 77/91 patients molecular genetic markers were investigated. The median age of the patients was 75.1 years (range, 35.3–87.4). The male/female ratio was 60/31. Six patients had RARS-T and 85 had MDS/MPNu. Results: In 54/91 (59.3%) patients MFC identified an MDS-immunophenotype. This was true in 4/6 (66.7%) RARS-T and in 50/85 (58.8%) MDS/MPNu (n.s.). Cases with MDS-immunophenotype displayed aberrancies significantly more frequently than those without as follows: in myeloid progenitor cells (number of aberrantly expressed antigens, mean±SD: 0.5±0.6 vs. 0.2±0.4, p=0.002), granulocytes (2.7±1.3 vs. 1.2±1.1, p<0.001), and monocytes (1.7±1.2 vs. 0.5±0.7, p<0.001). Accordingly, there was a significant difference in the total number of aberrantly expressed antigens (4.9±2.4 vs. 2.0±1.4, p<0.001). The presence of an aberrant karyotype was not related to an MDS-immunophenotype which was observed in 11/18 (61.1%) cases with aberrant karyotype and in 43/73 (58.9%) with normal karyotype (n.s.). Mutations in RUNX1 and TET2 as well as FLT3-ITD were predominantly present in cases with an MDS-immunophenotype (10/33, 30.3%) and occurred less frequently in cases without (1/7, 9.1%, n.s.). In detail, RUNX1 mutations were present in 4/26 (10.3%) vs. 0/2, TET2 mutations were present in 4/6 (66.7%) vs. 1/2 (50%), and FLT3-ITD was present in 3/29 (10.3%) vs. 0/5. Accordingly, in cases with RUNX1 or TET2 mutations or with FLT3-ITD a significantly higher number of aberrantly expressed antigens was observed as compared to cases with none of these mutations (mean±SD, 6.4±2.0 vs. 4.4±2.5, p=0.024). In contrast, JAK2V617F mutations occurred at identical frequencies in patients with and without MDS-immunophenotype (11/38, 28.9% vs. 9/31, 29.0%). Regarding prognosis, the presence of an MDS-immunophenotype had no impact on overall survival. Conclusions: These data demonstrates that MDS-related aberrant antigen expression is present in the majority of patients with RARS-T and MDS/MPNu. While there is no association between the presence of an MDS-immunophenotype and the detection of JAK2 mutations cases with an MDS-immunophenotype tended to more frequently carry mutations in RUNX1 and TET2 as well as FLT3-ITDs. These data therefore suggests that MDS/MPNu may be subdivided based on molecular genetics and on the immunophenotype into cases with MDS-related features and those without. Further analyses are needed to validate these findings and their potential significance in RARS-T. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Sole Trisomy 8 in AML: Concomitant Molecular Markers, Stability of Genetic Patterns and Impact On Outcome.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2503-2503 ◽  
Author(s):  
Tamara Alpermann ◽  
Claudia Haferlach ◽  
Christiane Eder ◽  
Alexander Kohlmann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Cox Regression ◽  
Normal Karyotype ◽  
Study Groups ◽  
Intermediate Risk ◽  
Trisomy 8 ◽  
Course Of Disease ◽  
Cox Regression Analysis ◽  
Equity Ownership ◽  
Wbc Count

Abstract Abstract 2503 Background: Trisomy 8 belongs to the most frequent cytogenetic aberrations in AML and is classified to intermediate-risk karyotypes if not within favorable or complex karyotypes (Grimwade et al., Blood 2010). Some study groups showed pts with sole trisomy 8 (+8sole) respond poorly to cytarabine-based chemotherapies. In addition, there is dissent whether trisomy 8 is a primary event or a secondary hit in pathogenesis. Aim: Evaluation of pts with +8sole with respect to related molecular markers, stability of cytogenetic and molecular aberrations and impact on outcome. Patients and Methods: 1,181 newly diagnosed adult AML pts with intermediate-risk karyotypes were distributed as follows: +8 (n=117), normal karyotype (NK) (n=801), other intermediate-risk abnormalities (n=263). In detail, 80/117 pts with trisomy 8 (68.4%) showed +8sole. All of these 80 pts were screened for the molecular markers: ASXL1, CEBPA, FLT3- ITD, FLT3- TKD, IDH1, MLL- PTD, NPM1, and RUNX1. WT1 was analyzed in 79 and IDH2 in 78 pts. For comparison we characterized 400 NK pts for all before mentioned molecular markers. Results: Comparing clinical features +8sole pts were older than NK (69.9 vs 63.6 years; p<0.001). No differences were seen for gender, Hb, WBC or PLT counts. Molecular Mutations: +8sole pts harbored more often ASXL1 mut than NK pts (47.5% (38/80) vs 15.0% (60/400); p<0.001) and also more frequently RUNX1 mut (36.3% (29/80) vs. 15.8% (63/400); p<0.001). In contrast, +8sole pts less frequently showed NPM1 mut (16.3% (13/80) vs 49.3% (197/400; p<0.001) or CEBPA mut (5.0% (4/80) vs 14.5% (58/400); p=0.018). All 4 cases with +8sole and CEBPA mut were monoallelic, whereas only 37.7% (23/58) of NK pts with CEBPA mut were monoallelic (p=0.031). Incidences for FLT3- ITD, FLT3- TKD, IDH1, IDH2, MLL- PTD, and WT1 did not differ between +8sole and NK. Genetic Stability: We analyzed relapse samples of 12 pts: 10/12 (83.3%) showed +8sole and their individual molecular marker pattern identical to diagnosis. However, 2/12 did show progress: one with +8sole and NPM1 mut at diagnosis gained FLT3- ITD in relapse. Case 2 habored an ASXL1 mut in addition to +8sole at diagnosis but relapsed with ASXL1 mut and a complex karyotype (including +8). Thus, +8sole as well as the corresponding mutations are stable through course of disease. Furthermore, in 5 pts we analyzed samples from MDS phase preceding AML. Two pts were genetically stable with either ASXL 1mut or MLL- PTD, and +8sole in MDS as well as in s-AML. One patient showed +8sole and ASXL1 mut in MDS and gained RUNX1 mut in s-AML. Interestingly, 2 pts harbored an ASXL1 mut and a NK at diagnosis of MDS, but an ASXL1 mut and +8sole at diagnosis of s-AML. This leads to the hypothesis of ASXL1 mut being first hit, whereas +8sole is gained during course of disease. Comparing overall survival (OS) for all pts with available follow-up data and intensive treatment regimes there was a significantly worse outcome for pts with +8sole (n=38) compared to NK (n=300; 13.0 vs 35.7 mo; p=0.003). Furthermore, we evaluated the total cohort (n=338) for impact of molecular markers. Pts with ASXL1 mut, FLT3-ITD, MLL-PTD, and RUNX1 mut showed worse outcome than pts without the respective marker mutated (p<0.001; <0.001; 0.009; <0.001, respectively). In contrast, pts with NPM1 mut, NPM1 mut/no FLT3-ITD or biallelic CEBPA mut showed better OS (p=0.003; <0.001; 0.044). FLT3- TKD, IDH1, IDH2, WT1, and CEBPA had no impact on OS. Combing markers associated with poor prognosis (ASXL1, FLT3-ITD, MLL- PTD, RUNX1) we detected at least one of these in 75% (60/80) within +8sole but only in 46.3% (185/400) within NK (p<0.001). Therefore we evaluated pts with at least one of these poor prognostic markers (n=147) and found median OS of 12.4 vs 49.6 months for pts with none of these (n=191; p<0.001). For multivariable Cox regression analysis age, WBC count, karyotype, biallelic CEBPA, NPM1 and the combined group of poor prognostic molecular markers were included (all being significant in univariable analysis). Age, WBC count and the combined group of poor prognostic molecular markers were significant (p<0.001, <0.001, 0.026, respectively). +8sole did not show an independent impact on OS. Conclusions: +8sole had poor outcome compared to NK. This is related to the concomitant presence of adverse molecular markers, particularly ASXL1, and RUNX1, or both. Thus, for risk stratification of pts with +8sole ASXL1 and RUNX1 mutation assessment should be considered. Disclosures: Alpermann: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

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