Mutational Screening Of CSF3R, ASXL1, SETBP1, and SRSF2 In Chronic Neutrophilic Leukemia (CNL), Atypical CML and CMML Cases

Abstract Introduction Chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) are rare myeloproliferative and myelodysplastic/myeloproliferative neoplasms. So far, the diagnosis of CNL and aCML has been based on cytomorphology and the absence of JAK2V617F and PDGFR rearrangements. Recently, mutations in CSF3R and SETBP1 were identified and associated with CNL and aCML, respectively. Chronic myelomonocytic leukemia (CMML) and aCML also share several characteristics and need to be discriminated especially by the absolute number of monocytes in the peripheral blood. Aim To determine the frequency of CSF3R mutations (CSF3Rmut) in CNL, aCML, and CMML and to investigate a mutation pattern, cytogenetics and clinical data in all three entities. Patients and Methods To first delineate patients with potential CNL, we investigated blood and bone marrow smears and depicted patients with a white blood cell count >25x109/L, neutrophils >80%, immature granulocytes <10%, <1% myeloblasts and hypercellular bone marrow (according to WHO 2008). BCR-ABL1 fusion transcript, JAK2 and MPL mutations were excluded in all cases by RT-PCR and melting curve analyses. Indication for PDGFR rearrangements was precluded by over-expression analyses of PDGFRA and PDGFRB by quantitative real-time PCR, resulting in a final cohort of 20 cases declared as CNL patients. Additional 60 aCML and 252 CMML patients were included. Cytogenetics was available in 330/332 cases. Mutations in CSF3R exons 14 and 17 (n=332), in ASXL1 exon 13 (n=321), and the mutational hot spots in SETBP1 (n=331) and SRSF2 (n=320) were analyzed by Sanger sequencing. Results In the total cohort of 332 patients we detected CSF3R mutations in 11 cases (3.3%). 8/11 cases showed a p.Thr618Ile mutation in exon 14, four of them carried an additional nonsense/frame-shift mutation in exon 17. One additional patient was mutated in p.Thr615Ala and showed a nonsense mutation in exon 17. Two cases showed a mutation in exon 17 only, one a nonsense the other a frame-shift mutation, respectively. Analyzing the mutation frequencies within the different entities revealed a clustering of CSF3Rmut within CNL cases with 7 of 20 (35%) mutated cases in contrast to 2 of 60 (3.3%; p=0.001) aCML and 2 of 252 (0.8%; p<0.001) CMML cases. Cytogenetics in CSF3Rmut cases showed that 9/11 cases had a normal karyotype and only one aCML patient harbored a del(3q) and one CMML patient a complex karyotype. Mutations in the three other analyzed genes ASXL1, SETBP1 and SRSF2 were detected in the total cohort in 156/321 (49%), 34/331 (10%), and 149/330 (45%) patients, respectively. Analyses regarding concomitant mutations of CSF3R with ASXL1, SETBP1 or SRSF2 revealed no additional mutation in two cases. In 8 of 11 parallel analyzed CSF3Rmut patients an ASXL1mut was identified, SETBP1 as well as SRSF2 were mutated in 3 of the 11 cases. Notably, the 7 CSF3Rmut within the CNL group had no mutation in SETBP1. Analysis of mutational loads in CNL showed that 6/7 CSF3Rmut had a higher mutational load than the second mutated gene (range: 25-50% vs. 10-30%). In one case both mutated genes had equal mutational loads (40%). In contrast, in CMML and aCML 3/4 patients had lower mutational loads in CSF3Rmut than in the additional mutated genes (20-50% vs. 40-50%), while also one case showed equal mutational loads in the mutated genes (50%). Combining the mutational results of these four genes indicate a specific and individual molecular pattern for these three different entities. While ASXL1 is frequently mutated in all entities (CNL: 8/11 (73%); aCML: 38/59 (64%); CMML: 110/251 (44%)), SRSF2 shows the highest mutation frequency in CMML cases (121/251; 48%), followed by aCML (24/60; 40%) and CNL (4/19; 21%). In contrast, SETBP1 is often mutated in aCML (19/60; 32%) and rarely in CMML (13/252; 5%) and CNL (2/19; 10.5%) patients. In addition, CSF3R is much more associated with the CNL cases (35%) and less frequently found in aCML (2%) and CMML (1%). Conclusion 1) CNL, aCML and CMML are related diseases and difficult to distinguish by cytomorphology alone and therefore require additional diagnostic criteria, i.e. molecular mutations. 2) ASXL1 is the most frequently mutated gene in these entities and thus can help to prove clonality. 3) SETBP1 much more closely relates to aCML and SRSF2 to CMML. 4) Mutations in the novel marker CSF3R are closely related to CNL and thus qualify as a new molecular marker for diagnosis of CNL. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schrauder:MLL Munich Leukemia Laboratory: Employment. Konietschke:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Comprehensive Analysis Of U2AF1 Mutations In 843 Patients With Myeloid Neoplasms With Respect To Other Genetic Alterations

Blood ◽

10.1182/blood.v122.21.4973.4973 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4973-4973

Author(s):

Manja Meggendorfer ◽

Christiane Eder ◽

Sabine Jeromin ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hot Spots ◽

Melting Curve ◽

Gene Mutations ◽

Next Generation ◽

Employment Equity ◽

Total Cohort ◽

Equity Ownership ◽

Generation Sequencing ◽

Splicing Machinery

Abstract Introduction Genes affecting the splicing machinery have been found to be frequently mutated in MDS patients. U2AF1 codes for one of these splicing components, showing two distinct mutational hot spots at amino acids Ser34 and Gln157. Mutations in U2AF1 induce global abnormalities in RNA splicing, producing intron containing unspliced RNAs. U2AF1 has been shown to be most frequently mutated in MDS cases (7-11%), but was so far investigated only in small subsets of AML and MPN and was found rarely mutated. Aim To determine the frequency of U2AF1 mutations (U2AF1mut) in different myeloid entities and to evaluate the correlation of U2AF1mut with other gene mutations, cytogenetics and clinical features. Patients and Methods The total cohort consisted of 843 patients, whereof 74 were diagnosed as AML, 201 as MDS, 243 as MPN, and 325 as MDS/MPN overlap. 331 patients were female, 512 male. Cytogenetics was available in 830 patients and these were grouped by the following karyotypes: normal karyotype (n=561), +8 (n=39), -7 (n=15), del(20q) (n=95), -Y (n=29), other aberrations (n=59), and complex karyotype (n=32). Based on the previously described association of U2AF1mut with del(20q) there was an intended selection bias to this abnormality. Mutational analyses for U2AF1 were performed by either melting curve analyses or next generation sequencing. In subcohorts we investigated mutations in ASXL1 (n=505), CBL (n=647), CEBPA (n=68), CSF3R (n=213), DNMT3A (n=260), ETV6 (n=129), EZH2 (n=355), FLT3-ITD (n=352), FLT3-TKD (n=239), IDH1/2 (n=367 and 286, respectively), JAK2 (n=681), KITD816 (n=244), KRAS (n=393), MLL-PTD (n=384), MPLW515 (n=612), NPM1 (n=477), NRAS (n=509), RUNX1 (n=516), SETBP1 (n=336), SF3B1 (n=839), SRSF2 (n=784), TET2 (n=428), and TP53 (n=239) by Sanger sequencing, next generation sequencing, gene scan, or melting curve analysis. Results In the total cohort we detected U2AF1 mutations in 55/843 (6.5%) patients, the two mutational hot spots were equally affected with 29 p.Ser34 and 26 p.Gln157 mutations, respectively. Mutation frequencies were 10.9% in MDS, 9.5% in AML, 7.1% in MDS/MPN overlap and 1.2% in MPN. U2AF1mut patients were older (median: 72.6 vs. 71.8 years; p=0.012), the mutation was more frequent in males (42/512 (8.2%) vs. 13/331 (3.9%) in females; p=0.015) and associated with lower hemoglobin levels (median: 9.5 vs. 11.0g/dL; p<0.001), and platelet counts (median: 78x109/L vs. 179x109/L; p=0.002). Regarding cytogenetics we found a high association of U2AF1mut to del(20q): in 18 of 95 cases (18.9%) with del(20q) a U2AF1 mutation was detected compared to 37 U2AF1mut in 735 cases (5.0%) with any other karyotype (p<0.001). This was true for AML (5/16 vs. 2/56; p=0.005), MDS (11/49 vs. 11/150; p=0.007) and MDS/MPN overlap cases (1/8 vs. 21/309; p=0.441). In contrast in MPN none of the 21 del(20q) patients showed a U2AF1 mutation compared to 18/74 in all other entities (p=0.01). Mutations in the two other genes of the splicing machinery, SF3B1 and SRSF2, occurred in 122/839 (14.5%) and 198/784 (25.3%) cases and were mutually exclusive with U2AF1mut. Only one case each showed an U2AF1mut and a SF3B1 (p=0.002) or SRSF2 (p<0.001) mutation. We furthermore analyzed a number of other gene mutations frequently mutated in myeloid entities and their association to U2AF1mut. There was no correlation to mutations in NPM1, FLT3-ITD and FLT3-TKD, MLL-PTD, and CEBPA in AML patients. In MDS patients there was also no correlation to mutations in ASXL1,ETV6, EZH2, TP53, RUNX1, NRAS, and KRAS. This was also true for JAK2, MPL, CBL, and TET2 mutations in MPN. However in MDS/MPN overlap patients U2AF1mut were more frequently found in cases with ASXL1mut (14/115 (12.2%) in ASXL1mut vs. 7/179 (3.9%) in ASXL1wt; p=0.01) and together with KITD816mut (3/10 (30%) in KITD816mut vs. 15/212 (7%) in KITD816wt; p=0.038). Conclusion 1) U2AF1 is most frequently mutated in MDS, followed by AML and MDS/MPN overlap and in contrast rarely mutated in MPN. 2) U2AF1mut highly correlates with del(20q) in MDS, AML and MDS/MPN overlap but not in MPN cases. 3) In MDS/MPN overlap U2AF1mut associates significantly with ASXL1mut and KITD816mut. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Co-Occurrence of Separate BCR-ABL1-Positve and JAK2V617F-Positive Clones in 23 Patients Reveals Biologic and Clinical Importance

Blood ◽

10.1182/blood.v124.21.3175.3175 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3175-3175

Author(s):

Pedro Martin-Cabrera ◽

Claudia Haferlach ◽

Torsten Haferlach ◽

Wolfgang Kern ◽

Susanne Schnittger

Keyword(s):

Limit Of Detection ◽

Myeloproliferative Neoplasms ◽

Melting Curve ◽

Rare Event ◽

Gene Mutations ◽

Jak2v617f Mutation ◽

Positive Clone ◽

Employment Equity ◽

Equity Ownership

Abstract Background: The simultaneous detection of a BCR-ABL1 rearrangement and a JAK2V617F mutation in the same patient is a very rare event and has previously been described in case reports or very small series of cases only. Aim: 1) To establish the incidence of cases with concurrent BCR-ABL1 rearrangement and JAK2V617F mutation. 2) Evaluate whether one clone harbours both mutations or whether there are two independent clones. 3) Establish whether these patients have additional concurrent gene mutations and how they influence the evolution of both diseases. Patients and Methods: A total of 27,907 patients with suspected myeloproliferative neoplasms (MPN) where studied in parallel for BCR-ABL1 and JAK2V617F mutation from May 2005 to June 2014 at our institution. BCR-ABL1 analysis was performed by multiplex RT-PCR and JAK2V617F mutation analysis by melting curve based LightCycler assay. A total of 23 patients (0.08%) were positive for both mutations. Eleven patients were male and 12 were female with a median age at diagnosis of 72 years (range 46-80 years). Of fifteen patients 2 or more sample time points were available for follow-up analyses (median follow-up: 4 years, range: 5 months - 9 years). Both BCR-ABL1 and JAK2V617F mutation loads were assessed by quantitative real time PCR. In addition, 22/23 cases were analyzed upon detection of co-occurrence of both clones with a pan-myeloid gene panel consisting of 25 genes: TET2, RUNX1, PHF6, ASXL1, CBL, DNMT3A, SF3B1, TP53, BCOR, BRAF, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, KIT, KRAS, MPL, NPM1, NRAS, SRSF2, U2AF1, and WT1. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Lexington, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). RUNX1 was sequenced on the 454 Life Sequence NGS platform (Roche 454, Branford, CT). The median coverage per amplicon was 2,215 reads (range 100-24,716). The lower limit of detection was set at a cut-off of 1.5%. Results: At the time point of detection of both mutations morphological assessment was available in 12 patients. The remaining 5 showed features typical for CML. Bone marrow blast count was <5% in all cases. Cytogenetics was available in 18/23 cases (78.3%). The classical t(9;22)(q34;q11) was identifiable in 16/18 patients. Two patients had a normal karyotype as they were in complete cytogenetic remission of their CML (due to TKI treatment) at diagnosis of the JAK2 V617F positive clone. In the majority of patients (n=16) the JAK2V617F mutation predated the BCR-ABL1 clone, in 4 patients CML was known before the detection of the JAK2V617 positive clone, in 1 patient both were diagnosed simultaneously and in another 2 patients information in this regard was lacking. BCR-ABL1 transcript types distributed as follows: b2a2 and/or b3a2 (n=18), and e1a2 (n=5). The continuous quantitative assessment of BCR-ABL1 and JAK2V617F mutational loads in 15 patients showed asynchronous patterns of courses in all cases giving proof of these aberrations representing two different clones in these cases. When treatment with TKI was initiated, the BCR-ABL1 clone decreased while the JAK2V617F clone either remained stable or increased in all 15 cases. Next generation sequencing revealed further mutations in 13/22 analyzed patients (56.5%). One mutation was detected in 8 patients, 4 patients revealed 2, and one patient even 3 different additional mutations. In detail, mutations in the following genes were detected: TET2 (n=8), ASXL1 (n=4), RUNX1 (n=2), CBL (n=1), DNMT3A (n=1), PHF6 (n=1) SF3B1 (n=1) and TP53 (n=1). These mutations were traced and quantified retrospectively. Data suggests that they were most probably present in the JAK2V617F positive clone. This again supports the theory of both clones being independent of each other. Conclusions: 1) Co-occurrence of BCR-ABL1 and JAK2V617F is a very rare event (0.08%). 2) BCR-ABL1 and JAK2V617F represent two different clones. 3) Additional gene mutations are detected in 56% of these cases and all seem to be within the JAK2V617F positive clone. 4) Clinically, the BCR-ABL1 clone is easily controlled with TKI, however, the combined management of the JAK2V617F clone is more challenging especially when a fibrotic phase of the disease takes over. The long-term effect of JAK2-inhibitors in the management of these patients is yet to be established. Disclosures Martin-Cabrera: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Astarabine, a Novel Leukemia-Targeted Cytarabine Composition Allows, for the First Time, the Delivery of High Cytarabine Doses for Older or Unfit Patients with Acute Leukemia. Results of an Ongoing Phase I/IIa Study

Blood ◽

10.1182/blood.v128.22.1650.1650 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1650-1650

Author(s):

Tsila Zuckerman ◽

Stela Gengrinovitch ◽

Ruth Ben-Yakar ◽

Ron Hoffman ◽

Israel Henig ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

De Novo ◽

Intensive Therapy ◽

High Dose ◽

Newly Diagnosed ◽

Employment Equity ◽

Unfit Patients ◽

Equity Ownership ◽

High Doses

Abstract Introduction: Therapy of acute myeloid leukemia (AML) has not changed significantly during several decades. High-dose cytarabine, although used as the first-line treatment for AML since 1970s and as a second-line treatment for acute lymphoblastic leukemia (ALL), is associated with severe side effects, such as cerebellar toxicity and bone marrow suppression. Hence, while the incidence of AML increases with age, doses of cytarabine are significantly attenuated or the drug is entirely excluded from the regimen used in older adults due to its potential toxicities, particularly in individuals with hepatic or renal dysfunction. Astarabine is a new composition of cytarabine covalently bound to asparagine. It is designed to target cytarabine to leukemic blasts, thus avoiding extramedullary toxicity. Leukemic cells, which are dependent on an external source of amino acids in general and asparagine in particular, due to their high metabolic rate, have a relatively increased uptake of Astarabine. Inside the blasts, Astarabine is cleaved to cytarabine, enabling targeted killing and relative sparing of normal hematopoiesis. As such, Astarabine may serve as an ideal therapy for leukemia, particularly for delivering high doses of cytarabine to medically unfit or older adults who otherwise can be given supportive therapy only. The aim of this study was to evaluate the safety and optimal dose of Astarabine in refractory/relapsed or medically unfit patients with acute leukemia. Methods: This Phase I/IIa prospective open label study enrolled patients aged ≥18 years with relapsed/refractory or newly-diagnosed acute leukemia unfit for intensive therapy, as judged by the treating physician. The study was approved by the Rambam IRB (approval #0384-11). Patients were enrolled into 6 Astarabine escalating-dose cohorts, each composed of 3-6 patients. Treatment was administered as a 1-hour single daily infusion for 6 days. For cohorts 1-4, Astarabine doses for each infusion were 0.5g/m2, 1.5g/m2, 3g/m2 and 4.5g/m2. The doses were reduced by 50% for patients >50 years. Since dose limiting toxicity (DLT) was not reached in cohorts 1-4, the study was extended to include cohorts 5 and 6 with daily Astarabine doses of 4.5g/m2 and 6g/m2, respectively, with no dose reduction for patients >50 years old. Results: The outcome of 15 patients is reported herein. Six patients with a median age of 64 years (range 27-81) had refractory/relapsed AML, 9 patients with a median age of 80 years (range 70-90) were newly diagnosed (secondary AML - 6, de-novo AML - 2, de-novo ALL - 1) and unfit for intensive therapy. Astarabine treatment was well-tolerated. Two patients died (one from pneumonia and one from sudden death 2 weeks from end of treatment) before completing 30 days post-treatment and hence were excluded from the outcome analysis. Response to the treatment was observed in the bone marrow of 6 of the 7 newly-diagnosed patients for whom bone marrow analysis was available, 3 of whom had a continuous complete remission (CR) for 4 (ongoing), 8, and 10 months post-treatment, and 3 had a continuous partial remission (PR) for 3,7, and 7 (ongoing) months. The median overall survival (OS) of the patients with CR/PR is 7 months to date (table 1). No significant response was observed in the relapsed/refractory patients, with a median OS of 2.5 months. Twelve patients died from disease progression. Conclusions: Astarabine, a new composition of leukemia-targeted cytarabine, is safe and very well tolerated, even in patients over 80 years of age, resulting in response in 6 of 7 newly diagnosed patients with acute leukemia. To the best of our knowledge, this is the first report permitting high-dose of cytarabine, considered a cornerstone of leukemia therapy, to be given to a population of patients that heretofore did not have this option. Further dose escalation studies are currently ongoing at a cytarabine-equivalent dose of 4.5 and 6 g/m2/day. A phase II study is planned to confirm these encouraging results and define the use of Astarabine for patients otherwise unable to receive high doses of cytarabine. Disclosures Zuckerman: BioSight Ltd: Consultancy, Research Funding. Gengrinovitch:BioSight Ltd: Employment, Equity Ownership, Patents & Royalties: Inventor all of the patents. Ben-Yakar:BioSight Ltd: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor of all patents.

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EZH2 Mutations and Impact on Clinical Outcome Analyzed in 1604 Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114421 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1528-1528

Author(s):

Sebastian Stasik ◽

Jan Moritz Middeke ◽

Michael Kramer ◽

Christoph Rollig ◽

Alwin Krämer ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Employment Equity ◽

Advisory Committees ◽

Mutational Status ◽

Equity Ownership ◽

Acute Myeloid

Abstract Purpose: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and key epigenetic regulator involved in transcriptional repression and embryonic development. Loss of EZH2 activity by inactivating mutations is associated with poor prognosis in myeloid malignancies such as MDS. More recently, EZH2 inactivation was shown to induce chemoresistance in acute myeloid leukemia (AML) (Göllner et al., 2017). Data on the frequency and prognostic role of EZH2-mutations in AML are rare and mostly confined to smaller cohorts. To investigate the prevalence and prognostic impact of this alteration in more detail, we analyzed a large cohort of AML patients (n = 1604) for EZH2 mutations. Patients and Methods: All patients analyzed had newly diagnosed AML, were registered in clinical protocols of the Study Alliance Leukemia (SAL) (AML96, AML2003 or AML60+, SORAML) and had available material at diagnosis. Screening for EZH2 mutations and associated alterations was done using Next-Generation Sequencing (NGS) (TruSight Myeloid Sequencing Panel, Illumina) on an Illumina MiSeq-system using bone marrow or peripheral blood. Detection was conducted with a defined cut-off of 5% variant allele frequency (VAF). All samples below the predefined threshold were classified as EZH2 wild type (wt). Patient clinical characteristics and co-mutations were analyzed according to the mutational status. Furthermore, multivariate analysis was used to identify the impact of EZH2 mutations on outcome. Results: EZH2-mutations were found in 63 of 1604 (4%) patients, with a median VAF of 44% (range 6-97%; median coverage 3077x). Mutations were detected within several exons (2-6; 8-12; 14-20) with highest frequencies in exons 17 and 18 (29%). The majority of detected mutations (71% missense and 29% nonsense/frameshift) were single nucleotide variants (SNVs) (87%), followed by small indel mutations. Descriptive statistics of clinical parameters and associated co-mutations revealed significant differences between EZH2-mut and -wt patients. At diagnosis, patients with EZH2 mutations were significantly older (median age 59 yrs) than EZH2-wt patients (median 56 yrs; p=0.044). In addition, significantly fewer EZH2-mut patients (71%) were diagnosed with de novo AML compared to EZH2-wt patients (84%; p=0.036). Accordingly, EZH2-mut patients had a higher rate of secondary acute myeloid leukemia (sAML) (21%), evolving from prior MDS or after prior chemotherapy (tAML) (8%; p=0.036). Also, bone marrow (and blood) blast counts differed between the two groups (EZH2-mut patients had significantly lower BM and PB blast counts; p=0.013). In contrast, no differences were observed for WBC counts, karyotype, ECOG performance status and ELN-2017 risk category compared to EZH2-wt patients. Based on cytogenetics according to the 2017 ELN criteria, 35% of EZH2-mut patients were categorized with favorable risk, 28% had intermediate and 37% adverse risk. No association was seen with -7/7q-. In the group of EZH2-mut AML patients, significantly higher rates of co-mutations were detected in RUNX1 (25%), ASXL1 (22%) and NRAS (25%) compared to EZH2-wt patients (with 10%; 8% and 15%, respectively). Vice versa, concomitant mutations in NPM1 were (non-significantly) more common in EZH2-wt patients (33%) vs EZH2-mut patients (21%). For other frequently mutated genes in AML there was no major difference between EZH2-mut and -wt patients, e.g. FLT3ITD (13%), FLT3TKD (10%) and CEBPA (24%), as well as genes encoding epigenetic modifiers, namely, DNMT3A (21%), IDH1/2 (11/14%), and TET2 (21%). The correlation of EZH2 mutational status with clinical outcomes showed no effect of EZH2 mutations on the rate of complete remission (CR), relapse free survival (RFS) and overall survival (OS) (with a median OS of 18.4 and 17.1 months for EZH2-mut and -wt patients, respectively) in the univariate analyses. Likewise, the multivariate analysis with clinical variable such as age, cytogenetics and WBC using Cox proportional hazard regression, revealed that EZH2 mutations were not an independent risk factor for OS or RFS. Conclusion EZH mutations are recurrent alterations in patients with AML. The association with certain clinical factors and typical mutations such as RUNX1 and ASXL1 points to the fact that these mutations are associated with secondary AML. Our data do not indicate that EZH2 mutations represent an independent prognostic factor. Disclosures Middeke: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Scholl:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Abbivie: Other: Travel support; Alexion: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding. Brümmendorf:Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy, Research Funding; Merck: Consultancy; Pfizer: Consultancy, Research Funding. Burchert:AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Roche: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding; Roche: Research Funding; Johnson & Johnson: Research Funding; Affimed: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

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Randomized Phase II Study Evaluating the Efficacy and Safety of Romiplostim Treatment of Patients with Low or Intermediate Risk Myelodysplastic Syndrome (MDS) Receiving Lenalidomide.

Blood ◽

10.1182/blood.v114.22.1770.1770 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1770-1770 ◽

Cited By ~ 6

Author(s):

Roger M Lyons ◽

Richard A. Larson ◽

Michael A. Kosmo ◽

Sunil Gandhi ◽

Delong Liu ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Intermediate Risk ◽

Employment Equity ◽

Treatment Groups ◽

Platelet Counts ◽

Equity Ownership ◽

Clinically Significant ◽

Dose Reductions ◽

Platelet Transfusions

Abstract Abstract 1770 Poster Board I-796 Introduction Romiplostim is a peptibody protein designed to increase platelet production by binding to and activating the thrombopoietin receptor. Low platelet counts in patients with myelodysplastic syndromes (MDS) may be due to the underlying disease or to treatment with disease-modifying agents, and platelet transfusions are often the only treatment for clinically significant thrombocytopenia (CST) or bleeding. This was a phase 2 multi-center, randomized, double-blind, placebo-controlled, dose-finding study that evaluated the effect of romiplostim on the incidence of clinically significant thrombocytopenic events (grade 3 or 4 thrombocytopenia and/or receipt of platelet transfusions) and the safety of romiplostim in patients with low or intermediate risk MDS receiving lenalidomide. Patients and Methods Patients who were ≥18 years old, had MDS by bone marrow exam and WHO criteria, had low or Intermediate-1 risk category MDS using the IPSS, and were planning to receive lenalidomide were eligible. Patients were randomized 1:1:1 into treatment groups receiving placebo, 500 μg romiplostim, or 750 μg romiplostim by weekly subcutaneous injections in combination with lenalidomide (one 10 mg capsule by mouth daily for each 28-day cycle). Treatments continued for a total of four cycles. Results The median age of the 39 randomized patients was 74 years (range, 39 to 90); 15 (39%) had platelet counts <50 × 109/L, and 7 (18%) had del(5q). We report trends due to baseline imbalances between treatment groups, likely due to the limited sample size. The overall incidence rates of CST appeared to be greater in the placebo group than either romiplostim group (Table). In contrast to the placebo patients, median platelet counts remained above 50 × 109/L in both the 500 μg and 750 μg romiplostim groups for the treatment period. The incidence of platelet transfusions appeared to be lower in the 500 μg romiplostim group, and the incidence of adverse events was comparable between all of the groups. No deaths were reported during the treatment period. Twelve patients (31%) discontinued the study. Disease progression to AML was reported in 1 patient in the romiplostim 500 μg group. The patient withdrew consent and discontinued the study. No bone marrow was available to confirm AML by protocol-defined criteria. Fewer lenalidomide dose reductions and delays due to thrombocytopenia were seen in both of the romiplostim treated groups. The proportion of patients who achieved an MDS treatment response was 8%, 36% and 15% for the placebo, 500 μg romiplostim, and 750 μg romiplostim groups, respectively. MDS response rates appeared higher in the romiplostim group, regardless of baseline del(5q) status. Baseline imbalance between groups due to the small sample size limited our interpretation of the data. Conclusions Romiplostim appeared to be well-tolerated in low and intermediate risk MDS patients receiving lenalidomide. This preliminary information suggests that romiplostim may reduce the rate of clinically significant thrombocytopenic events in these patients while increasing platelet counts and decreasing the incidence of lenalidomide dose reductions and delays due to thrombocytopenia Disclosures Lyons: GlaxoSmithKline: Consultancy, Speakers Bureau; Johnson&Johnson: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy; Amgen Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau. Off Label Use: Use of romiplostim to treat Thrombocytopenia in MDS. Larson:Amgen Inc.: Equity Ownership, Research Funding. Liu:Amgen Inc.: Honoraria, Research Funding. Hu:Amgen Inc.: Employment, Equity Ownership. Franklin:Amgen Inc.: Employment, Equity Ownership. Berger:Amgen Inc.: Employment, Equity Ownership.

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Use of a New Rotary Powered Device for Bone Marrow Aspiration and Biopsy Yields Excellent Specimens Quickly and Efficiently.

Blood ◽

10.1182/blood.v114.22.4544.4544 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4544-4544

Author(s):

Ronan T. Swords ◽

Kevin R. Kelly ◽

Devalingam Mahalingam ◽

Stephen C. Cohen ◽

Larry J. Miller ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Biopsy ◽

Clinical Evaluation ◽

Research Funding ◽

Bone Marrow Aspiration ◽

Swine Model ◽

Employment Equity ◽

Equity Ownership ◽

Specimen Quality ◽

The Mean

Abstract Abstract 4544 Background The importance of bone marrow aspiration and biopsy in the evaluation of hematopoietic and non-hematopoietic disorders is well established. Recently, a new FDA-cleared battery powered bone marrow biopsy system was developed to allow operators access to the bone marrow space quickly and efficiently. Aims The first aim of this study was to evaluate the quality of core specimens using the new powered device compared to specimens obtained using the traditional manual technique in a swine model. The second aim was to evaluate the safety and efficacy of the device in patients presenting for outpatient hematology clinic visits. Materials and Methods For the pre-clinical evaluation of the device, three anesthetized pigs were used for the study. The powered device (OnControl, Vidacare Corporation, San Antonio, TX, USA) was comprised of a battery powered driver and needle set. The manual device used was a T-Handle Jamshidi bone marrow biopsy needle (Cardinal Health, Dublin, OH, USA). Core biopsy samples obtained were assessed for length and sample quality and then submitted for analysis to a pathologist blinded to the device used. The clinical evaluation of the device was conducted in accordance with practice guidelines and directions for use. Data collection included insertion success, time from insertion to removal, specimen quality, operator satisfaction with control/function of the device and overall operator satisfaction based on a scoring system (0-5; 0=totally unacceptable, 5=outstanding). Results Twenty six samples were collected from the swine model (19 samples using the powered device and 9 using the manual technique). No cellular artifact or thermal damage was reported in any of the samples obtained. The mean lengths for samples obtained using the powered and manual techniques were respectively 19.4mm±1.6mm and 18.6mm±5.3mm. For the clinical evaluation of the device, 16 patients were recruited from 2 centers. Mean insertion time was 11.25±3.39 seconds and mean time from needle contact with skin to needle removal was 38.5±13.94 seconds. No complications were reported. Five operators rated the overall use of the device as outstanding in 75% of cases. Conclusions In this study, the manual and powered samples were equivalent in specimen quality. The powered device however, captured longer biopsies when compared to the manual technique. In the patients evaluated, the device was easy to use as well as being safe and effective. The mean procedural time was significantly faster than previously reported with a manual technique. A randomized study of the powered device compared to the manual technique is underway. Disclosures: Swords: Vidacare Corporation: Research Funding. Kelly:Vidacare Corporation: Research Funding. Mahalingam:Vidacare Corporation: Research Funding. Cohen:Vidacare Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Miller:Vidacare Corporation: Employment, Equity Ownership. Philbeck:Vidacare Corporation: Employment, Equity Ownership. Brenner:Vidacare Corporation: Consultancy, Research Funding. Giles:Vidacare Corporation: Research Funding.

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NPM1 Mutations Have a High Impact On the Development of Secondary AML.

Blood ◽

10.1182/blood.v114.22.999.999 ◽

2009 ◽

Vol 114 (22) ◽

pp. 999-999

Author(s):

Susanne Schnittger ◽

Tamara Weiss ◽

Frank Dicker ◽

Jana Sundermann ◽

Wolfgang Kern ◽

...

Keyword(s):

De Novo ◽

Myeloproliferative Neoplasms ◽

Blast Crisis ◽

Nras Mutation ◽

Npm1 Mutation ◽

Secondary Aml ◽

Second Hit ◽

Total Cohort ◽

Equity Ownership ◽

De Novo Aml

Abstract Abstract 999 Poster Board I-21 NPM1 mutations are frequently reported to be typical for de novo AML and are regarded as prognostically favorable if not associated with FLT3-ITD. These mutations have rarely been reported in secondary AML after myelodysplastic syndrome (MDS) or after myeloproliferative neoplasms (MPN). We have detected NPM1 mutations in 37/283 patients with AML after a previous MDS (s-AML) (13.1%) and in 6/67 after a previous MPN (9%). Here we describe the characteristics of these 43 NPM1 mutated s-AML cases to show the involvement of NPM1 mutations in development of secondary AML. The total cohort of 43 cases was composed of 22 males and 21 females with a median age of 71.3 years (range: 29.3-87.7 years). Cytogenetics was available in 40 of the 43 cases (93%). 27 of these had a normal karyotpye whereas 13 revealed one of these aberrations: +4 (n=3), t(1;14)(p34;q32) (n=1); -7 (n=1), del(9q) (n=2), +13 (n=1); +21 (n=1), -Y (n=1); i(X)(p10) (n=1), [+1,der(1;13)(q10;q10),+i(5)(p10),+8] (n=1) and a t(5;12)(q33;p13) (n=1). All 43 samples were analysed for MLL-PTD, FLT3-ITD, FLT3-TKD, NRAS, CEBPA, RUNX1 mutations as well as for KITD816 and JAK2V617F mutations. The incidence of additional cooperating mutations was similar to de novo AML. FLT3-ITD was detected in 14/37 AML after MDS (37.8%) and only once (1/6) after MPN. FLT3-TKD was observed in 3/37 case after MDS (8.1%) and never after MPN. In addition there was one case with RUNX1 and 4 cases (10.8%) with NRAS mutation after MDS. In none of the cases a CEBPA mutation or MLL-PTD was observed. Thus a total of 18/37 cases (48.8%) after MDS revealed a further molecular mutation in addition to NPM1. Of those without additional molecular mutations (only NPM1) 4 cases revealed cytogenetic aberrations resulting in 22/37 cases (59.5%) with additional cytogenetic or molecular mutations. Also in the 6 cases with NPM1 after MPN we detected a high proportion of additional mutations. Two of these 6 cases defined to be after MPN had a history of KITD816V mutated mastocytosis. Two further cases had preceding JAK2V617F mutated MPN and one additional carried an ETV6-PDGFRB rearrangement. In all these 5 transformed MPN cases the initial typical MPN mutation was retained in AML (blast crisis) whereas the NPM1 mutation was acquired and may have served as a second hit in the development to AML. One of the two JAK2+/NPM1+ cases in addition also acquired an FLT3-ITD. From 11 of the s-AML cases a paired sample from the timepoint of MDS was available. Retrospectively the NPM1 mutations was retraced by mutation specific realtime PCR and also all other markers were analysed. Three different patterns were observed: 1) in two cases the NPM1 mutation was not detectable in MDS (analysed 35 and 11 months before diagnosis of s-AML). In one case an NPM1/ABL1 level of 1.6% was detectable 6 months after diagnosis of MDS and a level of 2129% eleven months after diagnosis of MDS. 2) In six cases the NPM1 mutation was not detectable with standard methods in MDS, but with sensitive Real time PCR a ratio of 1-4 log below the s-AML level was already detectable 6-17 months before onset of s-AML. 3) In three further cases a high NPM1 level comparable to that in s-AML was already detectable in MDS 2-12 months before s-AML evolved. These three cases gained an FLT3-ITD at the time point of transformation from MDS to AML. These pattern show that NPM1 can be an early or a late event in transformation to s-AML and although the acquisition of mutations seems to be important in the transformation to AML the sequence of the single events seem to be secondary. As NPM1 have a favourable prognosis in de novo AML if not associated with FLT3-ITD we did a respective analysis for overall survival (OS) and (EFS) for our cohort of s-AML after MDS. For this analysis 278 s-AML patients were available: NPM1-/FLT3- (n=223); NPM1+/FLT3- (n=20), NPM1-/FLT3+ (n=20) and NPM1+/FLT3+ (n=12). The total cohort revealed a bad outcome (median OS: 56.6 days and median EFS: 43.5 days; range 2-1049 days for both). The median time for MDS until transformation to AML was 316 days (range: 15-6310 days). No difference with respect to outcome was detected between the four different molecular genetic subgroups. In conclusion, these data 1) show that NPM1 mutations play a major role in the evolution of AML following MDS or MPN. 2) NPM1 mutations can be the first as well as the second hit during transformation. 3) Support the theory of a multistep genetic principle in development of secondary AML. 4) s-AML with a NPM1+/FLT3-ITD- status can not be regarded as prognostically favorable. Disclosures: Schnittger: MLL Munich Leukemia Lab: Equity Ownership. Weiss:MLL Munich Leukemia Lab: Employment. Dicker:MLL Munich Leukemia Lab: Employment. Sundermann:MLL Munich Leukemia Lab: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Lab: Equity Ownership. Haferlach:MLL Munich Leukemia Lab: Equity Ownership.

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Outcome of Refractory Anemia with Ringed Sideroblasts Associated with Marked Thrombocytosis (RARS-T) In a Large Cohort of Patients

Blood ◽

10.1182/blood.v116.21.4113.4113 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4113-4113 ◽

Cited By ~ 1

Author(s):

Francois Girodon ◽

Julien Broseus ◽

Lourdes Florensa ◽

Esther Zipperer ◽

Susanne Schnittger ◽

...

Keyword(s):

Platelet Count ◽

Myeloproliferative Neoplasms ◽

Hemoglobin Concentration ◽

Large Cohort ◽

Refractory Anemia ◽

White Blood Count ◽

Employment Equity ◽

Disease Evolution ◽

Ringed Sideroblasts ◽

Equity Ownership

Abstract Abstract 4113 Introduction: Most of the data related to RARS-T, a rare disorder, involve small cohorts of patients. We aimed to analyze more patients also considering a variety of myelodysplastic or myeloproliferative disorders. Objective: To compare a large cohort of patients with RARS-T to refractory anemia with ringed sideroblasts (RARS), refractory anemia with ringed sideroblasts and multilineage dysplasia (RARS-MD) or essential thrombocythemia (ET) at the time of diagnosis and during disease evolution, in terms of survival and complications. Materials: Data of a European multi-center study was used including 199 cases of RARS-T 173 cases of RARS, 102 cases of RARS-MD and 431 cases of ET. Results: At baseline, compared to RARS and RARS-MD patients, RARS-T patients had similar hemoglobin concentration, but a higher white blood count. The JAK2V617F mutation was observed in 43%, 12% and 5% in RARS-T, RARS and RARS-MD patients, respectively. When separated in 2 groups (450,000<platelet count <600,000 and platelet count >600,000 × 109/l), RARS-T patients were comparable for sex, age, hemoglobin level and survival. However, patients with platelet count > 600,000 × 109/l had higher WBC (11 ×109/l versus 7.5 ×109/l, p<0.001). Similarly, no difference was noted in the survival in the JAK2 positive and negative RARS-T patients. The age and sex standardised overall survival of RARS-T patients was similar to RARS and RARS-MD patients, but lower than ET patients (p<0.001). This was despite a higher risk of transformation in acute leukemia, relative to RARS-T afflicted individuals, of 2.4 and 3.5 in RARS-MD and RARS patients, respectively. Conclusion: According to our results, the outcome in RARS-T more closely mimics myelodysplastic syndromes rather than myeloproliferative neoplasms. Our results agree with the WHO 2008 classification that considers RARS-T as a separate disorder. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Gattermann:Novartis: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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In BCR-ABL1 Negative Myeloproliferative Neoplasms the Detection of JAK2exon12, MPLW515, CBL, KITD816V, FIP1L1-PDGFRA Mutations Are Closely Linked to Specific Entities, Whereas the JAK2V617F, TET2, or EZH2 Mutations Demonstrate a Broader Diversity: Patterns From Diagnostic Reports of 18,547 Patients Analyzed

Blood ◽

10.1182/blood.v116.21.458.458 ◽

2010 ◽

Vol 116 (21) ◽

pp. 458-458

Author(s):

Susanne Schnittger, ◽

Christiane Eder ◽

Frank Dicker ◽

Vera Grossmann ◽

Alexander Kohlmann ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Hematologic Malignancy ◽

Rare Event ◽

Jak2v617f Mutation ◽

Patient Specific ◽

Second Step ◽

Double Positive ◽

Employment Equity ◽

Equity Ownership ◽

Diagnostic Work

Abstract Abstract 458 The first mutation detected in BCR-ABL1 negative myeloproliferative neoplasms (MPN) was JAK2V617F that revolutionized diagnostics of MPN during the last five years. However, although this genetic marker is useful to discriminate MPN from reactive disorders, it is not specific for one entity. In addition, approximately 5% of all polycythemia vera (PV) and 50% of essential thrombocytosis (ET) and primary myelofibrosis (PMF) are not JAK2V617F mutated. In these entities other activating mutations, e.g. MPLW515 mutations or JAK2exon12 mutations, cover additional small proportions of patients without JAK2V617F mutation. To further improve the molecular genetic characterization of MPN research focuses on the identification of novel mutations and, recently, CBL, TET2, and EZH2 genes were identified to be mutated in MPN. We here report on our single centre experience in applying these markers in a daily diagnostic work flow comprizing a total cohort of 18,547 cases with suspected MPN that were investigated between 8/2005 und 8/2010 with individual patient specific combinations of these markers as soon as published. Thus, the most frequently tested marker was JAK2V617F that was applied in 17,027 pts. In 6,622/17,027 (38.9%) a definite diagnosis of MPN could be made or confirmed on the basis of the detection of JAK2V617F mutation. More detailed, the percentage of JAK2V617F positive cases varied depending on the suspected diagnoses: In patients with cytomorphologically confirmed or suspected ET 581/891 (65.2%) were JAK2V617F positive, in PMF: 168/290 (57.9%), in PV: 800/942 (84.9%), in MPN-U: 51/212 (24.0%), in CMML: 38/383 (9.9%), in “MPN” not further specified by the referring physician: 4741/11249 (42.1%), and in those with unexplained leukocytosis/thrombocytosis/splenomegaly or suspected hematologic malignancy: 139/2492 (5.6%). Many of the before mentioned cases were suspected MPN and therefore analyzed for both JAK2V617F and BCR-ABL1. Thus, in 9,924 pts BCR-ABL1 and JAK2V617F testing were performed in parallel. As such, in 541/9,924 (5.5%) analyses BCR-ABL1 positive CML was identified and 3,558 cases were JAK2V617F mutated (35.9%). Only 8 pts were BCR-ABL1/JAK2V617F double positive (0.08%), thus this is a very rare event. In cases with JAK2V617F negative PV in a second step JAK2exon12 mutation was analyzed and 27/147 (18.3%) were tested positive. JAK2V617F negative ET or PMF were analyzed in a second step for MPLW515 mutations. In ET 24/258 (9.3%) and in PMF 14/164 (8.5%) cases were tested positive. JAK2exon12 or MPLW515 were never concomitantly detected with JAK2V617 in our cohort (parallel assessments: n=3,769). PCR for detection of FIP1L1-PDGFRA was performed in 1,086 cases with suspected HES/CEL or unclear eosinophilia but only 26 (2.4%) were tested positive and a CEL could be diagnosed. However, in 36/130 (27.7%) FIP1L1-PDGFRA negative cases a KITD816V mutation was detected and thus a diagnosis of mastocytosis could be established. In addition, confirmation of mastocytosis was achieved in further 326/731 (44.6%) pts with suspected mastocytosis, three of these pts had a JAK2V617F mutation in addition. Further analyses were recently done on selected well characterized cohorts of MPN: CBL mutations were analyzed in 623 cases and tested positive in 54 (8.7%): 26/199 CMML (13.0%), 1/25 PMF, 27/293 MPN-U (9.2%), but never were detected in ET (n=61) or PV (n=45). TET2 sequencing detected mutations in 56/191 (29.3%) of pts analyzed: ET: 6/28 (21.4%), PMF: 4/12 (33.3%), PV: 10/31 (32.3%), CMML: 17/22 (77.3%) cases, MPN-U: 17/86: (19.8%), HES: 1/9 cases, Mastocytosis: 1/3 cases. Thus, TET2 mutations are widely spread in different entities and were frequently associated with other mutations: JAK2V617F: n=16, JAK2exon12: n=1, MPLW515: n=2, CBL: n=5, FIP1L1-PDGFRA: n=1, KITD816V: n=1, and EZH2: n=2. Finally, EZH2 sequence analysis detected mutations in 4/68 (5.9%) cases (1/16 PV, 2/11 PMF, 1/17 MPN-U, 0/20 ET, 0/4 CEL). In conclusion, these data show that the analysis of molecular mutations greatly improved the diagnostic work up of MPN in the last 5 years. The detection of some mutations (JAK2exon12, MPLW515, CBL) are useful to further subclassify MPNs. Others (JAK2V617F, TET2, EZH2) are widely distributed and are helpful for classification and also to discriminate MPN from reactive disorders. The individual power of each marker for prognostication in MPN remains to be defined in future studies. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann: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.

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Impact of Expression of BAALC, CDKN1B, ERG, EVI1, and MN1 on Prognosis and Their Association with Karyotype, FLT3-ITD, NPM1 and MLL-PTD Status In Adult AML: A Comprehensive Study on 286 Cases

Blood ◽

10.1182/blood.v116.21.953.953 ◽

2010 ◽

Vol 116 (21) ◽

pp. 953-953

Author(s):

Claudia Haferlach ◽

Alexander Kohlmann ◽

Sonja Schindela ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

...

Keyword(s):

Molecular Genetic ◽

Normal Karyotype ◽

Genetic Alterations ◽

Complex Karyotype ◽

Employment Equity ◽

Aberrant Expression ◽

Cox Regression Analysis ◽

Total Cohort ◽

Equity Ownership ◽

Low Expression

Abstract Abstract 953 Introduction: The WHO classification in 2008 listed for the first time aberrant expression of genes as molecular genetic alterations affecting outcome in AML. High expression of BAALC, ERG and MN1 were shown thus far to be associated with unfavorable outcome in normal karyotype AML (AML-NK). In addition high EVI1 expression was suggested to predict poor outcome. Recently, our group identified low expression of CDKN1B as a favorable prognostic marker. The aim of this study was to evaluate the expression of BAALC, CDKN1B, ERG, EVI1 and MN1 in AML comprising all cytogenetic risk groups with respect to their association with distinct cytogenetic and known molecular genetic subgroups and their impact on prognosis. Patients/Methods:: Expression levels of BAALC, CDKN1B, ERG, EVI1 and MN1 were determined by oligonucleotide microarrays (HG-U133 Plus 2.0, Affymetrix) in 286 AML (t(15;17) n=15; t(8;21) n=16; inv(16) n=7; normal karyotype n=99; 11q23/MLL-rearrangements n=10; complex karyotype n=51; other abnormalities n=88). Patients were further analyzed for mutations in NPM1, FLT3-ITD, CEPBA and MLL-PTD. Results: Expression of BAALC, CDKN1B, ERG, EVI1 and MN1 varied significantly between genetic subgroups: While t(15;17), t(8;21) and 11q23/MLL-rearrangements were associated with low CDKN1B expression, AML-NK and NPM+ cases showed a higher CDKN1B expression. Lower BAALC expression was observed in AML with t(15;17), 11q23/MLL-rearrangement and AML-NK as well as in FLT3-ITD+ AML and in NPM1+ AML, while in AML with other abnormalities a higher BAALC expression was observed. ERG expression was lower in AML with 11q23/MLL-rearrangement and normal karyotype, while it was higher in AML with complex karyotype. Low EVI1 expression was observed in AML with t(15;17), t(8;21), inv(16) and AML-NK, while it was higher in AML with 11q23/MLL-rearrangements. Low MN1 expression was associated with t(15;17), t(8;21) and AML-NK, while it was increased in cases with inv(16) or other abnormalities. Next, Cox regression analysis was performed with respect to overall survival (OS) and event free survival (EFS). In the total cohort high BAALC and ERG expression as continuous variables were associated with shorter OS and EFS while CDKN1B, EVI1 and MN1 had no impact. Furthermore the cohort was subdivided into quartiles of expression for each gene. After inspection of the survival curves the cut-off for high vs low expression was set as follows: BAALC: 75th percentile, CDKN1B: 25th percentile, ERG and MN1: 50th percentile. For EVI1 expression pts were separated into expressers (n=44) and non-expressers (n=242). Low CDKN1B expression was associated with longer OS and EFS in the total cohort (p=0.005, not reached (n.r.) vs 14.9 months (mo); p=0.013, 31 vs 9.7 mo). High BAALC expression had no impact on OS, but was associated with shorter EFS in the total cohort as well as in AML with intermediate cytogenetics and AML with other abnormalities (p=0.032, 6.2 vs 13.0 mo; p=0.027, 5.1 vs 11.3 mo; p=0.006, 2.3 vs 14.8 mo). High ERG expression was significantly associated with shorter OS and EFS in the total cohort (p=0.002, 12.5 mo vs n.r.; p=0.001, 8.1 vs 15.7 mo) as well as in AML-NK (p=0.001, 11.3 mo vs n.r.; p=0.010, 7.2 vs 22.1 mo). OS was also shorter in AML with unfavorable karyotype (p=0.048, median OS 9.3 mo vs n. r.). With respect to MN1 high expressers had a significantly shorter OS and EFS in the total cohort (p=0.004, 12.3 mo vs. n.r.; p=0.001, 8.1 vs 16.7 mo) as well as in AML-NK (p=0.001, 9.7 mo vs n.r.; p=0.001, 5.1 vs 22.1 mo). In a multivariate analysis including CDKN1B, ERG and MN1 all parameters retained their impact on OS as well as on EFS, while BAALC lost its impact on EFS. Adding MLL-PTD, NPM1+/FLT3-ITD-, favorable and unfavorable karyotype into the model demonstrated an independent significant adverse impact on OS for MLL-PTD (p=0.027, relative risk (RR): 2.38) and ERG expression (p=0.044, RR: 1.59) only. In the respective analysis for EFS only favorable karyotype showed an independent association (p=0.002, RR: 0.261). Conclusion: 1) Expression of BAALC, CDKN1B, ERG, EVI1 and MN1 varies significantly between cytogenetic subgroups. 2) BAALC as a continuous variable and CDKN1B, ERG and MN1 as dichotomized variables are independently predictive for OS and EFS in AML. 3) ERG expression even retains its independent prediction of shorter OS if cytogenetic and other molecular genetic markers are taken into account. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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