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

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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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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The Importance of Identification of M-BCRABL Oncogene and JAK2V617F Mutation in Myeloproliferative Neoplasms

Acta Medica Marisiensis ◽

10.2478/amma-2014-0010 ◽

2014 ◽

Vol 60 (2) ◽

pp. 44-48

Author(s):

Annamária Szántó ◽

Zsuzsanna Pap ◽

Z Pávai ◽

I Benedek ◽

Judit Beáta Köpeczi ◽

...

Keyword(s):

Molecular Markers ◽

Molecular Biology ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Gene Mutations ◽

Chronic Phase ◽

Final Diagnosis ◽

Jak2v617f Mutation ◽

Myeloproliferative Disease

Abstract Background: The elucidation of the genetic background of the myeloproliferative neoplasms completely changed the management of these disorders: the presence of the Philadelphia chromosome and/or the BCR-ABL oncogene is pathognomonic for chronic myeloid leukemia and identification of JAK2 gene mutations are useful in polycytemia vera (PV), essential thrombocytemia (ET) and myelofibrosis (PMF). The aim of this study was to investigate the role of molecular biology tests in the management of myeloproliferative neoplasms. Materials and methods: We tested the blood samples of 117 patients between April 2008 and February 2013 at the Molecular Biology of UMF Târgu Mureș using RQ-PCR (for M-BCR-ABL oncogene) and/or allele-specific PCR (for JAK2V617F mutation). Results: Thirty-two patients presented the M-BCR-ABL oncogene, 16 of them were regularly tested as a follow-up of the administered therapy: the majority of chronic phase patients presented decreasing or stable values, while in case of accelerated phase and blast phase the M-BCR-ABL values increased or remained at the same level. Twenty patients were identified with the JAK2V617F mutation: 8 patients with PV, 4 with ET, 3 with PMF, 4 with unclassifiable chronic myeloproliferative disease and 1 patient with chronic myelomonocytic leukemia. There was no case of concomitant occurance of both molecular markers. Conclusions: Molecular biology testing plays an important role in the management of myeloproliferative neoplasms: identification of the molecular markers confirms the final diagnosis, excluding secondary causes of abnormal blood count parameters. Regular monitoring of MBCR- ABL expression level is useful in the follow-up of therapeutic efficiency.

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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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Health Care Utilization and Associated Costs in Elderly Persons with Non-CML Myeloproliferative Neoplasms: Real-World Evidence From a United States Medicare Population

Blood ◽

10.1182/blood.v120.21.4273.4273 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4273-4273 ◽

Cited By ~ 1

Author(s):

Sudeep Karve ◽

Gregory L Price ◽

Keith L Davis ◽

Gerhardt M Pohl ◽

Richard A Walgren

Keyword(s):

Health Care ◽

Health Care Utilization ◽

Index Date ◽

Myeloproliferative Neoplasms ◽

Care Utilization ◽

Cell Transplant ◽

Elderly Persons ◽

Employment Equity ◽

Equity Ownership

Abstract Abstract 4273 Background: Non-CML myeloproliferative neoplasms (MPNs), which include essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF) and MPN not otherwise specified (MPN-NOS), are characterized by activation of JAK2 signaling and abnormal blood cell production. Median survival ranges from months to years for MF and up to a decade or more for PV and ET. Some symptomatic treatment options exist, but with the exception of hematopoietic stem cell transplant, none are curative. Although MPN incidence is highest in persons aged ≥65 years, little is known about overall health care utilization and costs in elderly persons with these diseases. MPNs are more prevalent in the elderly and therefore Medicare enrollees are a highly relevant source for US-based resource utilization and cost data for these diseases. Objective: To compare all-cause health care utilization and costs from four subtypes of elderly MPN patients (ET, PV, MF and MPN-NOS) with matched non-MPN/non-cancer controls. Methods: Retrospective data were taken from the Survey, Epidemiology, and End Results (SEER)-Medicare linked database in the US, which combines clinical information from the SEER cancer registry (MPN reporting has been required since 2001) with medical and pharmacy claims for Medicare enrollees. Patients with a new MPN diagnosis between Jan 1, 2001 and Dec 31, 2007 were selected and evaluated for all-cause health care utilization and costs from Jan 1, 2008 (index date) through Dec 31, 2008 (follow-up end date). Patients were classified by MPN subtype based on the most recent diagnosis information (ICD-O-3 from the SEER registry or ICD-9-CM from Medicare claims) before the index date. Patients who died before follow-up end, had HMO or discontinuous Medicare enrollment during the follow-up year, had enrollment based on end stage renal disease, or a diagnosis of a non-MPN malignancy before follow-up end were excluded from the study. Separate non-MPN/non-cancer control groups were selected for each MPN subtype and matched (5:1) on birth year, gender, ethnicity, geography, and reason for Medicare eligibility. Per patient health care utilization and costs during the follow-up year were aggregated and stratified by care setting. Costs were adjusted to 2010 US$ and represent amounts reimbursed by Medicare to providers. Costs were compared between MPN cases and controls using univariate t-tests. Results: A total of 1,355 MPN patients (n = 445 ET, 684 PV, 81 MF, 145 MPN-NOS) were identified for study inclusion and assigned matching controls. For ET, PV, MF and MPN-NOS cases, respectively, mean [SD] age at index was 75.5 [9.7], 70.8 [11.3], 70.8 [10.4] and 74.1 [8.9] years and % female was 69.0, 43.9, 54.3, and 55.2. Mean [SD] years between first MPN diagnosis and study index date was 3.1 [2.0], 3.4[1.9], 2.7 [2.0], and 3.1 [2.1] for ET, PV, MF and MPN-NOS cases, respectively. A significantly (p<0.05) higher proportion of MPN cases, regardless of subtype, had ≥1 hospitalization during follow-up vs. controls (ET vs. control: 22% vs. 16%, PV vs. control: 27% vs. 15%, MF vs. control: 31% vs. 12%, MPN-NOS vs. control: 36% vs. 17%). Mean [SD] total days of hospital care were similarly higher in MPN cases (ET vs. control: 2.7 [12.8] vs. 1.6 [6.6], PV vs. control: 2.6 [7.0] vs. 1.7 [9.5], MF vs. control: 2.5 [6.2] vs. 1.2 [5.9], MPN-NOS vs. control: 4.0 [10.0] vs. 2.1 [13.7]), although the PV vs. control difference was not statistically significant. The ER visit rate during follow-up was also significantly (p<0.05) higher in MPN cases (ET vs. control: 34% vs. 24%, PV vs. control: 38% vs. 25%, MF vs. control: 46% vs. 21%, MPN-NOS vs. control: 44% vs. 29%). All-cause costs for MPN cases vs. matched controls are presented in the figure. Mean total costs per patient, driven equally by inpatient and outpatient services, were significantly (p<0.001) higher in MPN cases (ET vs. control: $11,259 vs. $8,897, PV vs. control: $13,337 vs. $8,530, MF vs. control: $20,917 vs. $7,367, MPN-NOS vs. control: $20,174 vs. $9,800). Conclusions: Total health care costs during a given year for elderly patients with MPNs are 1.3 to 3 times higher (depending on subtype) than those of matched controls. These findings may help inform future cost effectiveness evaluations of novel MPN treatments, as well as decision making in the provision of optimal MPN care within a Medicare system in which resources are finite and must be allocated ethically and efficiently. Disclosures: Karve: RTI Health Solutions: Consultancy, Research Funding. Price:Eli Lilly and Company: Employment, Equity Ownership. Davis:Eli Lilly, Merck, GlaxoSmithKline, Bristol-Myers Squibb, Pfizer, Eisai, Sanof-Aventis, Gilead Sciences, MedImmune: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Pohl:Eli Lilly and Company: Employment, Equity Ownership. Walgren:Eli Lilly and Company: Employment, Equity Ownership.

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Comprehensive Molecular Analyses of BCR-ABL1 Negative MPN Show That PMF Is Genetically Much More Heterogeneous Than ET and PV

Blood ◽

10.1182/blood.v124.21.3224.3224 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3224-3224

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

Susanne Schnittger ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Normal Karyotype ◽

Great Majority ◽

Primary Myelofibrosis ◽

Gene Panel ◽

Prognostic Impact ◽

Prognostic Information ◽

Employment Equity ◽

Equity Ownership

Abstract Introduction: In the WHO classification (2008) JAK2 and MPL mutations are major criteria for the diagnosis of myeloproliferative neoplasms (MPN): polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocythemia (ET). Cytogenetic aberrations are rare in these entities. Although the prognostic impact of JAK2 mutations beside some other gene mutations has been shown in PMF patients, the driving events for establishing accelerated phase or blast crises are unknown. In recent years, novel molecular markers such as ASXL1, SRSF2, and CALR were identified and PMF was investigated in several studies. However, comprehensive mutational analyses of MPN entities in comparison to each other are still rare. Aim: To identify gene mutations beyond JAK2, CALR, and MPL using a 28 gene panel, and to compare mutational data with clinical data and prognostic information in order to identify a risk profile. Patients and Methods: We in the first step investigated 56 patients (19 ET, 18 PMF, and 19 PV; 21 females, 35 males) diagnosed by cytomorphology following WHO criteria and accompanied by genetic studies. All patients underwent mutation analyses by a 28 gene panel containing: ASXL1, BCOR, BRAF, CALR, CBL, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, NRAS, KRAS, MPL, NPM1, PHF6, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. The library was generated with the ThunderStorm (RainDance Technologies, Billerica, MA) and sequenced on MiSeq instruments (Illumina, San Diego, CA). BCR-ABL1 fusion transcripts were shown to be negative in all cases by PCR. Not yet described genetic variants (n=6) were excluded from statistical analyses. Cytogenetics was available in 55/56 cases and grouped in normal karyotype (n=45, 82%) or aberrant karyotype (n=10, 18%). Results: In the total cohort JAK2 (44/56, 79%) was the most frequently mutated gene, followed by TET2 (13/56, 23%), ASXL1 (11/56, 20%), SRSF2 (7/56, 13%), and CALR (6/56, 11%). All other analyzed genes showed mutation frequencies below 10% (10 genes) or even no mutation (13 genes). Analyzing the number of mutations per patient revealed that only 4 patients showed no mutation (4/56, 7%), the great majority had 1 mutation (19/56, 34%) and 2 mutations (23/56, 41%), while 5 patients showed 3 mutations (5/56, 9%), 4 patients had 4 (4/56, 7%) and 1 patient even 5 mutations (1/56, 2%). Accordingly, the mean number of mutations per patient was 1.9. Summing up the mutations in JAK2, CALR, and MPL resulted in 52/56 (93%) patients that had a mutation in at least 1 of these genes, indicating that most of the patients had just 1 or 2 additional gene mutations to one of the 3 known key player MPN genes (mean: 1.3 additional mutations). Cytogenetically there were no significant differences between the 3 entities in frequencies of normal (65-90%) and aberrant karyotypes (11-35%), although in the PMF cohort there were more aberrant karyotypes (6/17, 35%) in comparison to ET and PV (for each 2/19, 11%). Addressing the mutation patterns of these 3 MPN entities revealed similar frequencies of TET2 mutations. In contrast, as expected JAK2 was more often mutated in PV (18/19, 95%) compared to ET (12/19, 63%, p=0.042) and PMF (14/18, 78%) and CALR was more often mutated in ET (5/19, 26%) in comparison to PMF (1/18, 6%) and PV (0/19, 0%, p=0.046). In PMF ASXL1 (8/18, 44%) and SRSF2 (6/18, 33%) were more often mutated compared to ET (1/19, 5%, p=0.008; 1/19, 5%, p=0.042) and PV (2/19, 11%; p=0.029; 0/19, 0%; p=0.008), respectively. Investigating the numbers of mutated genes per patient resulted in a significantly different distribution within MPN entities: in the ET and PV cohorts patients carried mostly 1 or 2 mutations (36/38, 95%; mean: 1.5), while in PMF 9/18 (50%) patients carried >2 mutations (mean: 2.5; p=0.045). Looking at the affected genes besides JAK2 and CALR showed that in ET and PV 4 more genes were affected, while in PMF 11 different additional genes showed mutations, indicating that PMF is genetically much more heterogeneous than ET or PV. This nicely matches to the finding that PMF is also marked by the highest cytogenetic aberration rate of these 3 BCR-ABL1 negative MPN (24-42%). Conclusions: 1)JAK2 is the most and TET2 the second most frequently mutated gene in BCR-ABL1 negative MPN. 2) Most patients carry only 1 or 2 gene mutations. 3) However, PMF patients are genetically much more heterogeneous than ET and PV patients regarding both cytogenetic and molecular alterations. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment; Novartis: Research Funding. Alpermann: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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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DNMT3A is a Powerful Follow-up Marker in NPM1 mutated AML

Blood ◽

10.1182/blood.v124.21.122.122 ◽

2014 ◽

Vol 124 (21) ◽

pp. 122-122 ◽

Cited By ~ 2

Author(s):

Susanne Schnittger ◽

Claudia Haferlach ◽

Tamara Alpermann ◽

Niroshan Nadarajah ◽

Manja Meggendorfer ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Melting Curve ◽

Melting Curve Analysis ◽

Employment Equity ◽

Low Level ◽

Mutational Status ◽

Equity Ownership ◽

The Stability

Abstract Background: NPM1 mutated (mut) FLT3-ITD negative acute myeloid leukemia (AML) is a distinct prognostically favorable subtype of AML. Robust data is available demonstrating that monitoring therapy response using NPM1mut-specific real time PCR is an important tool to early detect relapses and provides important information to guide therapy. Since next generation sequencing techniques have become available further gene mutations were detected that accompany NPM1mut in AML. Of these DNMT3Amut were the most frequent and stable ones (Krönke et al., Blood, 2013). Aim: 1) Analyse the stability of DNMT3Amut in paired diagnostic and relapsed samples. 2) Evaluate whether monitoring of DNMT3Amut provides additional information to monitoring of NPM1mut. Patients and Methods: Samples were selected from a cohort of 359 NPM1mut de novo AML cases with an available DNMT3Amut status.First, toevaluate the stability of DNMT3Amut paired diagnostic and relapse samples of 103 patients were analyzed (44 males, 59 females; median age 60 years, range: 26-82 years). Median time to relapse was 11 months (range: 3-68 months). NPM1mut status was assessed at diagnosis with a LightCycler melting curve analysis assay. Non type A mutations were further characterized by Sanger sequencing. Second, all diagnostic and follow-up samples (n=1,813) were quantified by real time PCR specific for the individual NPM1mut. Analysis for DNMT3Amut was performed using either the 454 technology (454 Life Sciences, Branford, CT) or the MiSeq instrument (Illumina, San Diego, CA). Deep DNMT3A sequencing of remission samples was performed using the 454 technology. Results: Out of 103 paired samples 61 (59.2%) carried a DNMT3Amut at diagnosis. 57/61 (93.4%) patients stably retained the mutation at relapse, in 4 (6.6%) the DNMT3Amut was lost. On the other hand 2 of 42 (4.8%) cases with DNMT3A wildtype at diagnosis gained the mutation at relapse. Thus, DNMT3Amut status was shown to be relatively stable (97/103; 94.1%) and thus qualifies as a promising target for follow-up controls. For comparison of DNMT3Amut and NPM1mut status during follow-up 54 patients that were NPM1/DNMT3A double mutated at diagnosis were selected according to the availability of at least one sample in first remission with an NPM1mut level <0.01%. These samples were reanalyzed by deep sequencing for the respective DNMT3A amplicons that had identified mutations at diagnosis. Two of these 54 cases (3.7%) showed morphologic relapse but NPM1mut was negative at relapse (sensitivity of 10-7). In one of these two cases at diagnosis NPM1mut and TET2mut were observed while at relapse IDH1mut and RUNX1mut were present. However, at both time points the DNMT3Amut was identified. The second case lost NPM1mut and CEBPAmut and retained the DNMT3Amut and TET2mut. Thus in these 2 cases the DNMT3Amut can be regarded as the common ancestor. 1 case retained the NPM1mut at relapse but lost the DNMT3Amut. Of note, in 32/54 (59.3%) cases the DNMT3Amut persisted in the remission samples (NPM1mut low level <0.01% or negative) with high DNMT3Amut loads (median: 20%, range: 2-59%) that was only slightly below the load at diagnosis (median: 45%, range: 38-58%). To analyze the clinical importance of these persisting DNMT3Amut survival analysis was performed. Median overall survival for patients with persisting DNMT3Amut (n=32) was 69 vs 96 months in those who lost also the DNMT3Amut in remission (n=22, p=0.053). Median event free survival was 38 vs. 96 months (p=0.031). Thus the DNMT3A mutational status in remission of NPM1mut AML is a further important parameter for prognostication. The mechanisms underlying this observation are obscure. As NPM1mut disappeared in remission and DNMT3A was retained and with the exception of 2 cases all others (n=18) relapsed with an NPM1mut (even the same type as at diagnosis) two mechanisms may be discussed: 1) Persisting DNMT3Amut cells predispose by a yet unknown mechanisms to the development of a secondary NPM1mut or 2) a residual DNMT3Amut/NPM1mut very low level survivor is able to overgrow the DNMT3Amut sole mutated clone in remission and cause relapse. Conclusions: 1) DNMT3Amut persist in remission of NPM1mut AML in the majority of cases (59.2 %). 2) DNMT3Amut analysis in remission of NPM1mut AML is an important parameter for prognostication. 3) Clones with DNMT3Amut as the sole mutation may have a normal phenotype and thus DNMT3Amut may even be regarded as premalignant mutation. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Comorbidity Burden in Elderly Persons with Non-CML Myeloproliferative Neoplasms: Real-World Evidence From a United States Medicare Population

Blood ◽

10.1182/blood.v120.21.1734.1734 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1734-1734

Author(s):

Keith L Davis ◽

Gregory L Price ◽

Sudeep Karve ◽

Gerhardt M Pohl ◽

Richard A Walgren

Keyword(s):

Heart Failure ◽

Congestive Heart Failure ◽

Renal Disease ◽

Index Date ◽

Myeloproliferative Neoplasms ◽

Cost Of Care ◽

Employment Equity ◽

Comorbidity Burden ◽

Equity Ownership

Abstract Abstract 1734 Background: Non-CML myeloproliferative neoplasms (MPNs), which include essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF) and MPN not otherwise specified (MPN-NOS), are characterized by activation of JAK2 signaling and abnormal blood cell production. MPN patients, especially those aged ≥65 years (in whom MPN incidence is highest), are at increased risk for cardiovascular and other comorbidities (Vannucchi et al. Blood 2007;110:840-6, Marchioli et al. J Clin Oncol 2005;23:2224-32). However, comorbidity rates in elderly MPN patients as compared with non-MPN controls have not been described in previous literature. MPNs are more prevalent in the elderly and therefore Medicare enrollees are a highly relevant source for US-based epidemiological data for these diseases. Objective: To compare comorbidity rates from four subtypes of elderly MPN patients (ET, PV, MF and MPN-NOS) with matched non-MPN/non-cancer controls. Methods: Retrospective data were taken from the Survey, Epidemiology, and End Results (SEER)-Medicare linked database in the US, which combines clinical information from the SEER cancer registry (MPN reporting has been required since 2001) with medical and pharmacy claims for Medicare enrollees. Patients with a new MPN diagnosis between Jan 1, 2001 and Dec 31, 2007 were selected and evaluated for comorbidities from Jan 1, 2008 (index date) through Dec 31, 2008 (follow-up end date). Patients were classified by MPN subtype based on the most recent diagnosis information (ICD-O-3 from the SEER registry or ICD-9-CM from Medicare claims) before the index date. Patients who died before follow-up end, had HMO or discontinuous Medicare enrollment during the follow-up year, had enrollment based on end stage renal disease, or were diagnosed with a non-MPN malignancy before follow-up end were excluded. Comorbidities were defined by ICD-9-CM diagnosis codes comprising the Charlson Comorbidity Index (CCI) (Charlson et al. J Chron Dis 1987;40:373–83; Deyo et al. J Clin Epidemiol 1992;45:613-9) as well as other adverse conditions. Separate non-MPN/non-cancer control groups were selected for each MPN subtype and matched (5:1) on birth year, gender, ethnicity, geography, and reason for Medicare eligibility. The proportion of patients with individual comorbidities and mean CCI during the follow-up year were compared between MPN cases and controls using univariate chi-square tests and t-tests. Results: A total of 1,355 MPN patients (n = 445 ET, 684 PV, 81 MF, 145 MPN-NOS) were identified for inclusion and assigned matching controls. For ET, PV, MF and MPN-NOS cases, respectively, mean [SD] age at index was 75.5 [9.7], 70.8 [11.3], 70.8 [10.4] and 74.1 [8.9] years and % female was 69.0, 43.9, 54.3, and 55.2. Mean [SD] years between first MPN diagnosis and study index date was 3.1 [2.0], 3.4[1.9], 2.7 [2.0], and 3.1 [2.1] for ET, PV, MF and MPN-NOS cases, respectively. Comorbidity rates during the follow-up period for MPN cases and matched controls are shown in the figure. Compared with controls, ET, PV and MPN-NOS cases had significantly (p<0.05) higher rates of serious cardiovascular events and comorbidities during the follow-up year, including myocardial infarction (ET vs. control: 8.1% vs. 4.0%, PV vs. control: 8.6% vs. 4.3%, MPN-NOS vs. control: 9.7% vs. 5.0%), congestive heart failure (CHF) (ET vs. control: 16.4% vs. 12.7%, PV vs. control: 18.4% vs. 10.1%, MPN-NOS vs. control: 22.1% vs. 12.4%), peripheral vascular disease (PVD) (ET vs. control: 20.0% vs. 15.4%, PV vs. control: 19.4% vs. 13.6%, MPN-NOS vs. control: 27.6% vs. 15.7%), and stroke (ET vs. control: 17.8% vs. 13.4%, PV vs. control: 17.8% vs. 13.1%, MPN-NOS vs. control: 22.1% vs. 13.9%). MF cases also had higher rates of CHF, PVD and stroke, but due to small sample size, only congestive heart failure was significant. Other comorbidities were significantly higher in all MPN subtypes, notably thromboembolism, renal disease, moderate-to-severe liver disease, and infections. Conclusions: Medicare enrollees with MPNs generally experienced significantly higher comorbidity rates and overall comorbidity burden (based on mean CCI scores) than matched controls. These findings have implications for both the clinical management of MPN patients as well as for health economic assessments, since a substantial portion of the cost of care for MPNs may reside in treatment of comorbidities not directly coded to MPNs. Disclosures: Davis: Eli Lilly, Merck, GlaxoSmithKline, Bristol-Myers Squibb, Pfizer, Eisai, Sanof-Aventis, Gilead Sciences, MedImmune: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Price:Eli Lilly and Company: Employment, Equity Ownership. Karve:RTI Health Solutions: Consultancy, Research Funding. Pohl:Eli Lilly and Company: Employment, Equity Ownership. Walgren:Eli Lilly and Company: Employment, Equity Ownership.

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

Blood ◽

10.1182/blood.v122.21.105.105 ◽

2013 ◽

Vol 122 (21) ◽

pp. 105-105 ◽

Cited By ~ 1

Author(s):

Manja Meggendorfer ◽

Tamara Alpermann ◽

Torsten Haferlach ◽

Carina Schrauder ◽

Rabea Konietschke ◽

...

Keyword(s):

Bone Marrow ◽

Myeloproliferative Neoplasms ◽

Melting Curve ◽

Additional Patient ◽

Employment Equity ◽

Chronic Neutrophilic Leukemia ◽

Total Cohort ◽

Frame Shift ◽

Frame Shift Mutation ◽

Equity Ownership

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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SF3B1 Mutations in AML, MDS and MDS/MPN-RS-T Are Accompanied By Different Other Gene Mutations: Impact for Targeted Treatment Studies

Blood ◽

10.1182/blood.v128.22.1976.1976 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1976-1976

Author(s):

Manja Meggendorfer ◽

Sabine Jeromin ◽

Karolína Perglerová ◽

Claudia Haferlach ◽

Wolfgang Kern ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Specific Gene ◽

Prognostic Information ◽

Employment Equity ◽

Additional Mutation ◽

Potential Biomarker ◽

Leukemia Treatment ◽

Equity Ownership ◽

Relevant Gene

Abstract Introduction: Precision medicine aims at the molecular profiling of patients to specifically target gene mutations. Targeted therapies now enter leukemia treatment, e.g. by targeting FLT3-ITD or mutations in DNMT3A, TET2, IDH1/2 or JAK2. Recently, luspatercept, a fusion protein (ACE-536), was shown to inhibit different signaling cascades, resulting in differentiation and maturation of erythropoietic progenitors in anemic patients (Platzbecker et al., Haematologica 2015). Interestingly, only patients with myelodysplastic syndrome (MDS) and ring sideroblasts (RS) responded to luspatercept, suggesting SF3B1 to be a potential biomarker. Besides MDS, SF3B1 mutations occur also in acute myeloid leukemia (AML) and MDS/myeloproliferative neoplasms with RS and thrombocytosis (MDS/MPN-RS-T). However, concomitant gene mutations bearing prognostic information and/or also being therapeutic targets as well as the cytogenetic background may need to be addressed in addition before further investigation. Aim: To investigate the mutation pattern and cytogenetic background of patients with AML, MDS and MDS/MPN-RS-T carrying SF3B1 mutations. Patients and Methods: In a cohort of 365 patients - all showing SF3B1 mutations and the diagnosis of AML (n=51), MDS (n=263) or MDS/MPN-RS-T (n=51) - cytomorphology, cytogenetics and mutation status were available. The cohort comprised 145 females and 220 males, the median age was 75 yrs (range: 42-93 yrs). In all patients ASXL1, RUNX1, TP53 as important prognostic markers as well as DNMT3A, FLT3-TKD, IDH1/2, JAK2, K/NRAS and TET2 as optional targets were analysed for mutations. Furthermore, additional entity specific gene mutations were investigated in respective subcohorts (AML: CEBPA, FLT3-ITD, MLL-PTD, NPM1; MDS: ETV6, EZH2, SRSF2, U2AF1, ZRSR2; MDS/MPN-RS-T: MDS genes, CBL, MPL). Results: 73% of all patients (268/365) showed normal karyotypes. Addressing molecular genetics resulted in 370 mutations beside SF3B1 in 238 patients, leaving only 23% of patients (84/365) showing no other aberration than in SF3B1. The variant allele frequencies (VAF) of SF3B1 mutations were high in nearly all cases with only few (9/353) subclonal cases (VAF <10%). In AML the median VAF of SF3B1 was 45% (range: 5-70%) with 3 cases showing subclonal mutations, likewise in MDS with a VAF of 39% (range: 3-50%) and 6 subclonal cases, while in MDS/MPN-RS-T the VAF was also 39% (range: 15-50%) without any subclonal case. In detail, 63% of AML cases showed normal karyotypes. Looking at gene mutations revealed that 49/51 patients (96%) had additional gene mutations (median: 2, range: 0-4), while 28/51 cases (55%) showed mutations in at least one of the therapeutically relevant genes. Of note, 37/51 patients (73%) had a mutation known to be associated with adverse prognosis. Therefore, in AML just one patient had a sole SF3B1 mutation and only 3/51 cases (6%) showed only other targetable mutations beside SF3B1. In MDS 73% of patients showed normal karyotypes. MDS patients showed in median 1 additional mutation (range: 0-4), leaving 115/263 (44%) patients without additional mutations. Furthermore, 124/263 (47%) patients carried mutations in a therapeutically relevant gene, while only 32/263 cases (12%) had mutations worsening prognosis. This results in 74/263 MDS patients (28%) without any additional aberration and 85/263 patients (32%) showing only other targetable mutations beside SF3B1. Furthermore, 84% of MDS/MPN-RS-T showed normal karyotypes. In median 1 additional mutation (range: 0-7) was identified in MDS/MPN-RS-T patients, while 10/51 cases (20%) showed no additional mutation. Looking at therapeutically relevant gene mutations revealed in 39/51 patients a respective mutation, while 7/51 patients carried prognostically adverse mutations. Therefore, MDS/MPN-RS-T patients show also a high proportion of cases without additional aberration (9/51, 18%) and even 47% (24/51) of patients having only targetable gene mutations. Conclusion: 1) SF3B1 mutation is supposed to be in the main clone. 2) AML, MDS and MSD/MPN-RS-T differ in their respective patterns of molecular aberrations beside SF3B1 mutations. 3) MDS patients show most frequently SF3B1 mutations as sole abnormality and might therefore benefit best from SF3B1 targeting treatment. 4) Treatment decisions should in all cases consider additional targetable mutations but also those worsening prognosis. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Other: Part Owner MLL Munich Leukemia Laboratory.

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RARS-T Patients Harbor SF3B1 Mutations In 90.2% and Can Be Characterized By Mutations In ASXL1 and Other Spliceosome Genes In Most Of The Remaining Cases

Blood ◽

10.1182/blood.v122.21.2764.2764 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2764-2764

Author(s):

Sabine Jeromin ◽

Christiane Eder ◽

Sandra Weissmann ◽

Manja Meggendorfer ◽

Tamara Alpermann ◽

...

Keyword(s):

Who Classification ◽

Melting Curve ◽

Gene Mutations ◽

Curve Analysis ◽

Lower Percentage ◽

Refractory Anemia ◽

Melting Curve Analysis ◽

Employment Equity ◽

Ring Sideroblasts ◽

Equity Ownership

Abstract Introduction Refractory anemia with ring sideroblasts and marked thrombocytosis (RARS-T) is a rare entity with characteristics of both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms and is grouped as a provisional entity in the current WHO classification. RARS-T patients have been shown to be frequently JAK2V617F and less commonly MPLW515 mutated. Recently, SF3B1 mutations (mut) were described to occur at a high frequency of up to 85% and it seems that RARS-T is genetically best characterized by SF3B1 and JAK2V617F mutations. However, a comprehensive mutational landscape analysis is still missing und genetic events in the SF3B1wild-type (wt) cases remain to be clarified. Aim Comprehensively characterize a large cohort of RARS-T patients for gene mutations. Patients and Methods We investigated 92 cases that all strictly met the criteria for RARS-T according to the WHO classification 2008. JAK2V617F and MPLW515 were analyzed by melting curve analysis. Screenings for mutations in SF3B1, SRSF2 and ASXL1 were performed by direct Sanger sequencing. ZRSR2 and TET2 were analyzed by an amplicon next generation deep-sequencing approach (NGS). U2AF1 was either analyzed by melting curve analysis or NGS. The cohort comprised 54 females (58.7%) and 38 males (41.3%). Median platelet count was 659x109/L (range: 454 – 1,500x109/L) and median percentage of ring sideroblasts (RS) was 61% (range: 18 - 97%). Cytogenetic data was available in 86 patients: 71 patients (82.6%) had normal and 15 an aberrant karyotype. Results All patients were analyzed for mutations in SF3B1, JAK2V617F and MPLW515. SF3B1 was the most frequently mutated gene (83/92, 90.2%), followed by JAK2V617F (54/92, 58.7%). MPLW515mut occurred only rarely (2/92, 2.2%) and in both cases were accompanied by SF3B1mut. SF3B1mut cases occurred concomitantly with JAK2V617F (46/83, 55.4%). However, JAK2V617F showed a tendency to be more frequent in patients with SF3B1wt (8/9, 88.9% vs. 46/83, 55.4%, p=0.076). Additionally, a subset of the cases, especially those with SF3B1wt, was analyzed for other genes. Mutations occurred with following frequencies: TET2, 14/61, 23.0%; ASXL1, 11/85, 12.9%; SRSF2, 5/86, 5.8%; U2AF1, 4/88, 4.5%; ZRSR2, 2/83, 2.4%. In 98.9% (91/92) of all patients at least one mutation in the analyzed eight genes could be found. Only one patient carried no gene mutation in any of these genes and had normal karyotype. We further analyzed this case with a pan-myeloid genes NGS panel providing data on 19 additional genes. However, no mutation could be detected. Interestingly, nearly all SF3B1wt cases carried an ASXL1mut (7/9, 77.8% vs. 4/76, 5.3%, p<0.001). Accordingly, mutations in the spliceosome genes SRSF2 (2/78, 2.6% vs. 3/8, 37.5%, p=0.005) and U2AF1 (1/79, 1.3% vs. 3/9, 33.3%, p=0.003) were rare in SF3B1mut cases, but were associated with ASXL1mut (SRSF2mut: 3/11, 27.3% vs. 1/73, 1.4%, p=0.006; U2AF1mut: 3/11, 27.3% vs. 1/74, 1.4%, p=0.006). In contrast, the only two ZRSR2 mutated cases had concomitant SF3B1mut (n.s.). TET2mut showed no association with any of the other gene mutations. Analysis of patients with mutation status of all following genes: SF3B1, JAK2V617F, MPLW515, ASXL1, SRSF2, U2AF1, ZRSR2 (n=82), revealed that only SF3B1mut occurred as a sole alteration (31/82, 37.8%). In detail, SF3B1mut cases rarely showed more than 2 gene mutations, whereas nearly all SF3B1wt cases had 3 different gene mutations (5/75, 6.7% vs. 6/7, 85.7%, p<0.001). These 6 SF3B1wt cases all carried a JAK2V617F and ASXL1mut accompanied by either an SRSF2mut (n=3) or U2AF1mut (n=3). Furthermore, SF3B1mut were associated with higher percentage of RS (mean: 61% vs. 41%, p=0.006), whereas JAK2V617F had higher platelet counts (807 vs. 599 x109/L, p<0.001). ASXL1mut had lower percentage of RS (mean: 42% vs. 61%, p=0.007), so had U2AF1mut (mean: 36% vs. 60%, p=0.028), but not SRSF2mut. Conclusions 1. RARS-T patients are characterized by high occurrence of mutations in SF3B1 (90.2%), in 37.8% detected as sole mutation. 2. Most of the SF3B1wt cases show various gene mutations, harboring a JAK2V617F and ASXL1mut together with a third mutation in either SRSF2 or U2AF1. Disclosures: Jeromin: MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Alpermann: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. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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