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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 274-274 ◽  
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.

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.

scholarly journals Clinical, Hematologic, Biologic and Molecular Characteristics of Patients with Myeloproliferative Neoplasms and a Chronic Myelomonocytic Leukemia-Like Phenotype

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1891
Author(s):  
Sonja Heibl ◽  
Bettina Gisslinger ◽  
Eva Jäger ◽  
Agnes Barna ◽  
Michael Gurbisz ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Chronic Myelomonocytic Leukemia ◽  
Molecular Characteristics ◽  
Molecular Features ◽  
Cell Counts ◽  
Myelomonocytic Leukemia

Patients with a myeloproliferative neoplasm (MPN) sometimes show a chronic myelomonocytic leukemia (CMML)-like phenotype but, according to the 2016 WHO classification, a documented history of an MPN excludes the diagnosis of CMML. Forty-one patients with an MPN (35 polycythemia vera (PV), 5 primary myelofibrosis, 1 essential thrombocythemia) and a CMML-like phenotype (MPN/CMML) were comprehensively characterized regarding clinical, hematologic, biologic and molecular features. The white blood cell counts in MPN/CMML patients were not different from CMML patients and PV patients. The hemoglobin values and platelet counts of these patients were higher than in CMML but lower than in PV, respectively. MPN/CMML patients showed myelomonocytic skewing, a typical in vitro feature of CMML but not of PV. The mutational landscape of MPN/CMML was not different from JAK2-mutated CMML. In two MPN/CMML patients, development of a CMML-like phenotype was associated with a decrease in the JAK2 V617F allelic burden. Finally, the prognosis of MPN/CMML (median overall survival (OS) 27 months) was more similar to CMML (JAK2-mutated, 28 months; JAK2-nonmutated 29 months) than to PV (186 months). In conclusion, we show that patients with MPN and a CMML-like phenotype share more characteristics with CMML than with PV, which may be relevant for their classification and clinical management.

GM-CSF Signaling Abnormalities in Chronic Myelomonocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1713-1713 ◽  
Author(s):  
Eric Padron ◽  
Jeffrey S. Painter ◽  
Adam W Mailloux ◽  
Jessica M. McDaniel ◽  
Christopher Bebbington ◽  
...  
Keyword(s):  
Scoring System ◽  
Who Classification ◽  
Chronic Myelomonocytic Leukemia ◽  
Newly Diagnosed ◽  
Employment Equity ◽  
Serum Free ◽  
Gm Csf ◽  
Equity Ownership ◽  
Md Anderson ◽  
Myelomonocytic Leukemia

Abstract Abstract 1713 Background: Chronic Myelomonocytic Leukemia (CMML) and Juvenile Myelomonocytic leukemia (JMML) are classified as MDS/MPN in the WHO classification system. Despite sharing clinical and histological features, CMML is characterized by a heterogeneous collection of molecular lesions while JMML is defined by well-established molecular aberrations clustered along the RAS pathway leading directly to GM-CSF hypersensitivity; a pathognomonic characteristic of JMML. Here we test whether a molecular signature for GM-CSF hypersensitivity in JMML, determined by the pSTAT5 activation assay, is also present in CMML and whether this signature clusters within a specific CMML subgroup. Methods: Cryopreserved bone marrow aspirates from 24 patients with newly diagnosed or relapsed CMML were obtained from the Moffitt Cancer Center Tissue Repository. Cells were thawed and rested in Stem Span H3000 with 10% FBS for 2 hours and then either starved for one hour in serum-free media, serum free group (n=12), or rested in Stem Span for an additional hour, serum group (n=12), prior to stimulation with G-CSF, IL-3, or GM-CSF for 15 minutes and then fixed and permeabilized with formaldehyde and methanol, as previously described. Samples were stained with an anti-pSTAT5(Y-694) antibody and analyzed by flow cytometry (Kotecha, Cancer Cell. 2009). Cells stained with isotype-control antibody were used to establish the threshold for basal STAT5 phosphorylation. Because STAT5 was constitutively phosphorylated in serum, and to a lesser extent in serum-free conditions, inducible cytokine activation was defined as the percentage of pSTAT5 positive cells above untreated samples in both CMML and healthy controls. A retrospective chart review was performed to obtain clinical variables including age, sex, WHO classification, Dusseldorf scoring system, MD Anderson scoring system, WBC, peripheral monocyte count, blast percentage, anemia, platelet count, splenomegaly, and metaphase cytogenetics. Results: The percentage of pSTAT5 responsive cells after G-CSF stimulation with doses up to 10 ng/ml was similar in cases and normal BM controls (p=0.14), whereas, a statistically significant increase in the percentage of inducible pSTAT5 positive cells was observed with GM-CSF 0.1 ng/ml (p=0.04), GM-CSF 1 ng/ml (p=0.02), and GM-CSF 10 ng/ml (p=0.01) in CMML BM cells compared to healthy donor BM cells, as shown in Figure 1. Using one standard deviation below the mean as a cut point, only 5 patients failed to show GM-CSF hypersensitivity in the serum (n=3) and serum-free groups (n=2), respectively. IL-3 and GM-CSF play similar roles in hematopoietic growth through the activation of JAK2/STAT5 and share a common beta-chain required for signaling. Signaling mediated by GM-CSF and IL3 converge to activate RAS and other downstream intermediates that regulate DNA synthesis, cell-cycle progression and suppression of apoptosis. The concentration of IL3 required to induce STAT5 phosphorylation was 10-fold greater than GM-CSF in CMML cells, but the percentage of cells responsive to IL3 was greater in CMML cases compared to controls at 10 ng/ml (p=0.02). Analysis of the percentage of GM-CSF hypersensitive cells and clinical parameters revealed no associations with age at onset, WHO classification, Dusseldorf scoring system, MD Anderson scoring system, blast percentage, anemia, platelet count, splenomegaly, or karyotype. The percentage of pSTAT5 positive cells with GM-CSF 0.1 ng/ml positively correlated with the total leukocyte (p=0.03) and total monocyte (p=0.02) count indicating that the JAK2/STAT5 signaling response is indicative of disease burden. Conclusions: Based on the threshold for cytokine stimulation and percentage of cells that display pSTAT5 induction, CMML appears to preferentially utilize GM-CSF for survival and/or expansion. Although RAS mutations were not assessed, CMML cells were preferentially sensitive to GM-CSF in newly diagnosed cases independent of cytogenetic abnormalities suggesting that JMML and CMML share biological features of GM-CSF hypersensitivity. Disclosures: Padron: KaloBios Pharmaceuticals, Inc.: Research Funding. Bebbington:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Baer:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership.

scholarly journals ETV6 and signaling gene mutations are associated with secondary transformation of myelodysplastic syndromes to chronic myelomonocytic leukemia

Blood ◽  
2014 ◽  
Vol 123 (23) ◽  
pp. 3675-3677 ◽  
Author(s):  
Eric Padron ◽  
Sean Yoder ◽  
Sateesh Kunigal ◽  
Tania Mesa ◽  
Jamie K. Teer ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
Gene Mutations ◽  
Chronic Myelomonocytic Leukemia ◽  
Myelomonocytic Leukemia

scholarly journals Impact of Somatic Gene Mutations on Response to Lenalidomide (LEN) in IPSS Lower-Risk Myelodysplastic Syndromes (MDS) Patients (Pts) without Del(5q) and Ineligible for or Refractory to Erythropoiesis-Stimulating Agents (ESAs)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 225-225 ◽  
Author(s):  
Valeria Santini ◽  
Pierre Fenaux ◽  
Aristoteles Giagounidis ◽  
Uwe Platzbecker ◽  
Alan F List ◽  
...  
Keyword(s):  
Lower Risk ◽  
Research Funding ◽  
Response Rate ◽  
Single Gene ◽  
Gene Mutations ◽  
Advisory Board ◽  
Employment Equity ◽  
Mutation Status ◽  
Somatic Gene ◽  
Equity Ownership

Abstract Background: Somatic gene mutations occur in the majority of MDS pts; specific mutations and high mutation frequency have prognostic relevance (Papaemmanuil et al. Blood. 2013;122:3616-27). Evaluation of somatic mutations may support the diagnosis of MDS and guide treatment (Tx) selection. The phase 3 randomized MDS-005 study compared LEN and placebo (PBO) Tx in red blood cell transfusion-dependent (RBC-TD) non-del(5q) lower-risk MDS pts ineligible for or refractory to ESAs. Deletions in chromosome 5q are associated with a high response rate to LEN in MDS pts; however, no mutations have been definitively associated with a predictable clinical response to LEN in non-del(5q) MDS. Aim:To investigate the relationship between somatic gene mutations detected by targeted next-generation sequencing (NGS) and response and overall survival (OS) in lower-risk non-del(5q) MDS pts treated with LEN in the MDS-005 study. Methods: Eligible pts were: RBC-TD (≥ 2 units packed RBCs/28 days 112 days immediately prior to randomization) with International Prognostic Scoring System defined Low-/Intermediate-1-risk non-del(5q) MDS; ineligible for ESA Tx (serum erythropoietin > 500 mU/mL); or unresponsive or refractory to ESAs (RBC-TD despite ESA Tx with adequate dose and duration). 239 pts were randomized 2:1 to oral LEN 10 mg once daily (5 mg for pts with creatinine clearance 40-60 mL/min) or PBO. DNA was isolated from bone marrow mononuclear cells or whole blood collected at screening from a subset of pts who gave informed consent for this exploratory biomarker analysis and had adequate tissue for analysis. Targeted NGS of 56 genes was performed at Munich Leukemia Laboratory; average sequencing coverage was 2,000-5,000-foldand the variant allele frequency detection cutoff was 3%. Target regions varied by gene, including all exons to hotspots. For association tests, mutant variants (heterozygous or homozygous) were scored as 1 (mutant) or 0 (wildtype) for gene-level analyses. A Fisher exact test was used to test association of mutation status with response. Median OS was calculated by the Kaplan-Meier method. Hazard ratios and 95% confidence intervals were determined by a non-stratified Cox proportional hazards model. A log-rank test was used to test treatment effect with OS for single gene mutation status. Results: The biomarker cohort included 198 of 239 pts (83%; LEN n = 130, PBO n = 68). At least 1 mutation was detected in 30/56 (54%) genes and 173/198 (87%) pts. The most frequently mutated genes were SF3B1 (59%), TET2 (33%), ASXL1 (23%), and DNMT3A (14%); the most frequent co-mutations were SF3B1/TET2 (23%), SF3B1/DNMT3A (10%), SF3B1/ASXL1 (10%), and TET2/ASXL1 (9%) (Figure). Of 116 pts with SF3B1 mutations, 115 (99%) had ≥ 5% ring sideroblasts. The 56-day RBC transfusion-independence (RBC-TI) response rate was significantly lower in LEN-treated ASXL1 mutant pts vs wildtype pts (10% vs 32%, respectively; P = 0.031). At 168 days, the RBC-TI response rate was still lower in LEN-treated ASXL1 mutant pts vs wildtype pts (7% vs 22%); however, the difference was not significant (P = 0.101). LEN-treated DNMT3A mutant pts had a higher 56-day RBC-TI response rate vs wildtype pts (44% vs 25%); however, this difference did not reach significance (P = 0.133) due to the small sample size. RBC-TI response rate with LEN was similar regardless of total number of mutations per pt. Higher numbers of mutations were significantly associated (P = 0.0005) with worse median OS. Mutation in any of the genes associated with a negative prognosis reported by Bejar et al. (N Engl J Med. 2011;346:2496-506) was also significantly associated (P = 0.0003) with worse median OS.However, OS was not significantly different in LEN- vs PBO-treated pts based on any single gene mutation status. Conclusions: In this group of lower-risk RBC-TD non-del(5q) MDS pts, somatic mutations in genes recurrently mutated in myeloid cancers were detected in 87% of pts. SF3B1 mutations (alone or in combination) were most frequent and not associated with response to LEN. ASXL1 mutant pts had a significantly lower LEN response rate vs wildtype pts, whereas DNMT3A mutant pts had a trend for improved LEN response. Median OS was influenced by mutations, but not significantly modified by LEN. Determining predictive clinical markers for Tx response in non-del(5q) MDS pts remains challenging; nevertheless, there is a significant need to identify pt subsets who may be responsive to LEN Tx. Figure. Figure. Disclosures Santini: Novartis: Consultancy, Honoraria; Amgen: Other: advisory board; Onconova: Other: advisory board; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Astex: Other: advisory board. Fenaux:Celgene, Janssen, Novartis, Astex, Teva: Research Funding; Celgene, Novartis, Teva: Honoraria. Giagounidis:Celgene Corporation: Consultancy. Platzbecker:Janssen-Cilag: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; TEVA Pharmaceutical Industries: Honoraria, Research Funding. Zhong:Celgene Corporation: Employment, Equity Ownership. Wu:Celgene Corporation: Employment, Equity Ownership. Mavrommatis:Discitis DX: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Employment, Equity Ownership. Beach:Celgene Corporation: Employment, Equity Ownership. Hoenekopp:Celgene Corporation: Employment, Equity Ownership. MacBeth:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties, Research Funding.

scholarly journals Prognostic Role of Gene Mutations in Chronic Myelomonocytic Leukemia Patients Treated With Hypomethylating Agents

EBioMedicine ◽  
2018 ◽  
Vol 31 ◽  
pp. 174-181 ◽  
Author(s):  
Matthieu Duchmann ◽  
Fevzi F. Yalniz ◽  
Alessandro Sanna ◽  
David Sallman ◽  
Catherine C. Coombs ◽  
...  
Keyword(s):  
Gene Mutations ◽  
Chronic Myelomonocytic Leukemia ◽  
Hypomethylating Agents ◽  
Prognostic Role ◽  
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.

Occurrence of T-Lymphocytes with Aberrant Immunophenotypes Suggestive of Mature T-Cell Neoplasms In Patients with Hairy Cell Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4165-4165
Author(s):  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
Torsten Haferlach
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Hairy Cell Leukemia ◽  
Initial Diagnosis ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership

Abstract Abstract 4165 Mature T-Cell Neoplasms (MTN) comprise a heterogeneous group of diseases with largely varying clinical courses ranging from indolent cases which are asymptomatic for years to aggressive cases requiring immediate therapy. The incidence of MTN is increasing with age, however, particularly due to the large group of cases with very indolent clinical course MTN are considered underdiagnosed to a significant portion. Published data indicates that T-lymphocytes with aberrant immunophenotype, i.e. double-positive cell expressing both CD4 and CD8, are present in healthy subjects, however, their frequency in general amounts to clearly less than one percent of total leukocytes. With increasing age, these T-lymphocytes with aberrant immunophenotype are detected in more subjects and at higher frequencies suggesting a higher incidence of mature T-cell neoplasms or at least of pre-malignant conditions in these cases. The diagnosis of hairy cell leukemia has not yet been linked to a higher frequency of these T-lymphocytes with aberrant immunophenotype or of MTN. Following the identification of various cases with both a diagnosis of hairy cell leukemia and the presence of T-lymphocytes with aberrant immunophenotype in our laboratory we hypothesized that both of these conditions co-occur at a higher rate than would be expected by chance. We therefore retrospectively evaluated multiparameter immunophenotyping results of 338 patients diagnosed with hairy cell leukemia (newly diagnosed or during follow-up) between August 2005 and July 2010 for the presence of an increased percentage (more than 1%) of T-lymphocytes with an aberrant immunophenotype. We identified 31 such patients, i.e. 9.2% of all patients with hairy cell leukemia. 17 were identified at initial diagnosis and 14 during follow-up after therapy for hairy cell leukemia. The patients` ages ranged from 43.4 to 89.3 years (median, 62.1 years), 21 were male. The aberrant immunophenotype comprised the coexpression of CD3, CD4, and CD8 in all cases and in addition of CD56 in 25/31 (80.6%) cases. The median values and ranges for blood cell counts amounted to: WBC, 2.6 ×10e9/l, 1.0–24.6 ×10e9/l; hemoglobin, 12.7 g/dl, 7.3–16.5 g/dl; thrombocytes, 116 ×10e9/l, 24–258 ×10e9/l. The percentage of T-lymphocytes with an aberrant immunophenotype (compared to all leukocytes) ranged from 1% to 22% (median, 4%); the respective concentrations ranged from 0.013 ×10e9/l to 0.984 ×10e9/l (median, 0.122 ×10e9/l). The concentrations of T-lymphocytes with an aberrant immunophenotype tended to be higher in cases at follow-up as compared to those at initial diagnosis, although this difference was not significant (mean±SD, 0.266±0.275 ×10e9/l vs. 0.144±0.119 ×10e9/l). In four of the 31 patients with T-lymphocytes with an aberrant immunophenotype molecular genetic analysis of T-cell receptor (TCR) rearrangement was performed. In 3/4 patients both TCR beta and gamma were found rearranged and in one of these also TCR delta was rearranged, however, in 1/4 patient no TCR rearrangement was present. This data indicates that T-lymphocytes with aberrant immunophenotypes are present in patients with hairy cell leukemia much more often and at higher concentrations than in the general population. This data therefore suggests that the incidence of mature T-cell neoplasms may be higher in hairy cell leukemia patients. Clinical symptoms and findings like cytopenia and splenomegaly therefore may not be attributable to hairy cell leukemia alone which may have significant therapeutic implications. It is suggested to monitor for T-lymphocytes with aberrant immunophenotypes in patients with hairy cell leukemia and to perform analysis for TCR rearrangements in positive cases. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Spliceosome GENE MUTATIONS ARE Also PRESENT In the Diverse Mutational Spectrum of CHRONIC Myelomonocytic LEUKEMIA

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1402-1402
Author(s):  
Hideki Makishima ◽  
Anna M Jankowska ◽  
Valeria Visconte ◽  
Ramon V. Tiu ◽  
Kathryn M Guinta ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Gene Mutations ◽  
P53 Mutation ◽  
Chronic Myelomonocytic Leukemia ◽  
Homozygous Mutation ◽  
Myelomonocytic Leukemia

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.

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