scholarly journals Molecular Analyses of MDS/MPN Overlap Entities According to WHO Classification Reveal a Distinct Molecular Pattern for MDS/MPN, Unclassifiable

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4618-4618
Author(s):  
Manja Meggendorfer ◽  
Torsten Haferlach ◽  
Sabine Jeromin ◽  
Claudia Haferlach ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Gene Mutation ◽  
Who Classification ◽  
Gene Mutations ◽  
Normal Karyotype ◽  
Employment Equity ◽  
Ras Pathway ◽  
Molecular Abnormalities ◽  
Molecular Pattern ◽  
Equity Ownership ◽  
Stat Pathway

Abstract Introduction: The World Health Organization (WHO) classification defines myelodysplastic/myeloproliferative neoplasms (MDS/MPN) based on clinical, morphologic, and laboratory findings that show features of MDS and characteristics more consistent with MPN. This category includes atypical chronic myeloid leukemia (aCML), chronic myelomonocytic leukemia (CMML), MDS/MPN, unclassifiable (MDS/MPN, U), and refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). In recent years RARS-T, CMML, and also aCML were deciphered by several molecular studies, while MDS/MPN, U cases warrant closer investigations. Aim: To comprehensively investigate mutations in 17 genes known to be mutated in aCML, CMML, MDS/MPN, U, and RARS-T and to define entity specific mutation patterns in comparison to cytogenetic and clinical data. Patients and Methods: We investigated 179 patients diagnosed by cytomorphology, immunophenotyping and genetic studies following WHO criteria: 35 patients were diagnosed as aCML, 58 as CMML, 39 as MDS/MPN, U, and 47 as RARS-T. All patients underwent mutation analyses by a gene panel containing: ASXL1, TET2, DNMT3A, SRSF2, SF3B1, U2AF1, JAK2, CALR, MPL, NRAS, KRAS, CBL, BRAF, CSF3R, RUNX1, SETBP1, and NPM1. Gene mutations were analyzed by Sanger sequencing, next generation sequencing, melting curve analyses, or gene scan. Cytogenetics was available in 172/179 cases and was grouped as normal karyotype (n=128, 74%) or aberrant karyotype (n=44, 26%). Results: In the total cohort the most frequently mutated gene was ASXL1 (41%), followed by TET2 (40%), and the spliceosomal genes SF3B1 (31%) and SRSF2 (30%). Also frequently mutated were JAK2 (21%), NRAS (15%), RUNX1 (12%), and CBL (12%). All other investigated genes showed mutation frequencies below 10%. There were no significant differences between the 4 entities regarding frequencies of aberrant karyotypes (14-37%) and no correlation of the number of molecular mutations (0-6/patient) with any specific karyotype. Addressing the mutation patterns of these 4 entities showed that ASXL1 and TET2 are frequently mutated in all entities (19-60% and 26-53%, respectively), although significant differences between the entities exist (see figure): ASXL1 is less frequently mutated in RARS-T (19%) in comparison to aCML (60%; p<0.001) and CMML (52%; p=0.001), TET2 is more often mutated in CMML (53%) in comparison to MDS/MPN, U (26%; p=0.007) and RARS-T (32%; p=0.031). SRSF2 is more frequently mutated in CMML (53%) than in RARS-T (9%; p<0.001) and MDS/MPN, U (15%; p<0.001), SF3B1 is more often mutated in RARS-T (92%) than in all other entities (aCML: 11%, CMML: 5%, MDS/MPN, U: 13%; for all p<0.001). One important difference between aCML and CMML versus MDS/MPN, U and RARS-T was reflected by two different signaling pathways: i) JAK2/CALR/MPL (JAK/STAT pathway) were significantly more often affected in MDS/MPN, U (33%) and RARS-T (53%), (aCML: 9%, CMML: 7%; p<0.001). ii) NRAS/KRAS/CBL (RAS pathway) were more often mutated in aCML (37%) and CMML (52%), (MDS/MPN, U: 5%, RARS-T: 9%; p<0.001). The MDS/MPN, U cohort included most patients with no mutation in any analyzed gene (11/39, 28%) in contrast to aCML (2/23, 6%), CMML (5/58, 9%), and RARS-T (0/47, 0%). Furthermore all these MDS/MPN, U patients with no gene mutation had a normal karyotype. Looking at co-ocurrences of gene mutations in MDS/MPN, U revealed that SRSF2 and TET2 mutations occur together more frequently (4/10 vs. 2/29 in TET2wt; p=0.028). Of notice, in MDS/MPN, U U2AF1 (18%) was the most frequently mutated spliceosomal gene which was only rarely mutated in the other entities (5%, p=0.015). Conclusions: 1) ASXL1 and TET2 are the most frequently mutated genes found overall in MDS/MPN overlap. 2) SF3B1 mutations are specific for RARS-T. 3) SRSF2 is most frequently mutated in CMML, but also in aCML. 4) MDS/MPN, U is affected by mutations in all spliceosomal genes. 5) The JAK/STAT pathway is more often affected in MDS/MPN, U and RARS-T. 6) The RAS pathway is more often affected in aCML and CMML. 7) MDS/MPN, U shows a specific molecular pattern with characteristics reflecting a mixture of all other MDS/MPN entities. Red: gene mutation, orange: gene mutations combined, light grey: no mutation/normal karyotype, black: aberrant karyotype, white: not analyzed. Figure: Molecular abnormalities and cytogenetics in MDS/MPN entities. Figure:. Molecular abnormalities and cytogenetics in MDS/MPN entities. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin: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.

scholarly journals In CLL with Normal Karyotype By Conventional and Genomic Array Karyotyping the Prognostic Impact of an Unmutated IGHV Status Is Stronger Than the Impact of Gene Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4357-4357 ◽  
Author(s):  
Calogero Vetro ◽  
Torsten Haferlach ◽  
Manja Meggendorfer ◽  
Sabine Jeromin ◽  
Constance Regina Baer ◽  
...  
Keyword(s):  
Gene Mutation ◽  
Mutation Rate ◽  
Gene Mutations ◽  
Normal Karyotype ◽  
Prognostic Impact ◽  
Employment Equity ◽  
Mutation Status ◽  
Genomic Array ◽  
Equity Ownership ◽  
The Impact

Abstract Background: In 15-20% of CLL cases no aberrations are detected by chromosome banding analysis (CBA) and FISH due to limited resolution, lack of evaluable metaphases or presence of aberrations in loci not covered by standard-panel FISH probes. As reported in our previous study (Haferlach C. et al., ASH 2015, abs ID#79545), genomic arrays (GA) detected abnormalities in almost 20% of cases classified as normal by CBA and FISH and these showed an impact on time to first treatment (TTT) (Vetro C. et al., EHA 2016, abs ID# E1069). The CLL subgroup without abnormalities in CBA, FISH, and GA has not been characterised in detail, so far. Aims: 1) to describe CLL without abnormalities by CBA/FISH/GA by evaluating an extended gene panel, the IGHV mutation status and the B-cell receptor (BCR) stereotypy; 2) to determine prognostic impact of these factors. Patients and Methods: CLL diagnosis was based on cytomorphology and immunophenotyping according to standard guidelines. From a cohort of 1190 patients at diagnosis, 133 (11%) were selected based on normal karyotype by CBA, no abnormalities by interphase FISH with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), centromeric region of chromosome 12 and t(11;14)(q13;q32) (IGH-CCND1) and no abnormalities by GA (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany). IGHV mutation status and BCR stereotypy were determined according to Agathangelidis et al., Blood 2012, and DNA sequencing was performed for the following genes: ATM; SF3B1; TP53; KLHL6; KRAS; MYD88; NOTCH1; NRAS; POT1; FBXW7; HIST1H1E; XPO1; ITPKB; MAPK1; BIRC3; BRAF; DDX3X; EGR2; RIPK1; RPS15; CND2. Results: Median age was 66 years (range: 33-83). Median follow-up was 5.6 years, 33 patients (25%) received treatment since genetic analyses. 10-year overall survival (OS) was 76% and median TTT was 9.2 years. Mutations were observed in 53 patients (40%): SF3B1 (n=17; 13%); NOTCH1 (n=10; 8%); KLHL6 (n=6; 5%); TP53 (n=6; 5%); ATM (n=5; 4%); XPO1 (n=4; 3%); FBXW7 (n=3; 2%); MYD88 (n=3; 2%); DDX3X (n=2; 2%); POT1 (n=2; 1.5%); ITPKB (n=1; 1%); KRAS (n=1; 1%); NRAS (n=1; 1%); and no mutation in RPS15, CCND2, MAPK1, EGR2, BRAF, HIST1H1E, RIPK1, BIRC3. 6 patients had 2 simultaneous gene mutations and 1 patient had 3 (i.e. NOTCH1, ATM and TP53). A mutated IGHV status (IGHV-M) was present in 100 patients (75%) and an unmutated IGHV status (IGHV-U) in 33 patients (25%). IGHV-U was related to both the occurrence of any gene mutation (p<0.001) and the number of gene mutations (p=0.001). NOTCH1 was mutated in 7 out of the 33 IGHV-U patients (21%), but only in 3 out of 99 IGHV-M patients (3%) (p=0.001). XPO1 mutation occurred in 4 IGHV-U patients (12%) and none out of IGHV-M (p<0.001). Two IGHV-U patients showed POT1 mutation (6%), but no IGHV-M case (p=0.014). 9 patients out of 133 (7%) showed BCR-stereotypy. 2 were in cluster CLL#1 (both showing NOTCH1 mutation), 2 in cluster CLL#2 (both of them with SF3B1 mutation), 2 in CLL#4, 1 in CLL#8 (showing NOTCH1 and XPO1 mutations), 1 in CLL#201 (with KLHL6 mutation) and 1 in CLL#202 (with mutations in ATM, TP53 and NOTCH1 genes). In Kaplan-Meier analysis, IGHV-M patients did not reach a median TTT, while IGHV-U had a median of 5.1 years (p<0.001). Stereotypy rate was too low for reliable statistics. At univariate analysis, TTT was only influenced by: IGHV-U (relative risk (RR): 3.9, p<0.001), TP53 mutation (RR: 3.7, p=0.03), % CLL cells (RR: 1.2 per 10% increase, p=0.013), and number of mutations (RR: 1.8 per each mutation, p=0.031). Multivariate Cox regression analysis showed an independent role for IGHV-U status (RR: 3.3, p=0.002) and % CLL cells (RR: 1.2 per 10% increase, p=0.038) Only age showed an impact on OS (RR: 1.2 per decade, p<0.001). Conclusions: 1. The CLL subset without any genomic event by CBA/FISH/genomic array is characterized by very low frequency of IGHV-U status; 2. IGHV-U subgroup showed higher gene mutation rate compared to IGHV-M subgroup, in particular higher NOTCH1, XPO1 and POT1 mutation rate; 3. BCR stereotypy is less frequent than in CLL in general. 4. IGHV-U, as well as the higher disease burden (i.e. % CLL cells), has an independent negative impact on TTT. 5. Requirement for treatment is low and prognosis very favorable in CLL without any genomic event by CBA/FISH/genomic array and a mutated IGHV status. Disclosures Vetro: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Recently Suggested MDS/MPN Subgroup “Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis (RARS-T)” Demonstrates Unique Clinical, Cytogenetic, and Molecular Aspects: A Comprehensive Analysis of 57 Cases Strictly Defined According to WHO Criteria

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3081-3081
Author(s):  
Ulrike Bacher ◽  
Johanna Flach ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Who Classification ◽  
Normal Karyotype ◽  
Favorable Outcome ◽  
Refractory Anemia ◽  
Employment Equity ◽  
Who Criteria ◽  
Ring Sideroblasts ◽  
Equity Ownership ◽  
Wbc Count ◽  
Who 2008

Abstract Abstract 3081 Introduction: In the new WHO 2008 classification, “refractory anemia with ring sideroblasts associated with marked thrombocytosis” (RARS-T) represents a provisional entity defined by platelets ≥450 ×109/l (being lowered from 600 ×109/l; WHO 2001), proliferation of large megakaryocytes, bone marrow (BM) blasts <5%, and ring sideroblasts ≥15% of nucleated erythropoiesis. The separation of RARS-T from other myeloproliferative/myelodysplastic neoplasms is still under debate. Patients: To further characterize this subtype and to evaluate whether its separate position in the WHO classification is justified from biologic/genetic aspects, we analyzed 57 patients with a diagnosis of RARS-T (strictly defined according to WHO 2008 criteria) for peripheral blood parameters, BM morphology, cyto-/molecular genetics, and clinical profiles. The study cohort consisted of 34 females and 23 males (median age, 76 years, range, 51–92 yrs; 52 de novo; 5 therapy-associated). At the time of analysis, all pts were at diagnosis or therapy naïve. Patients with a sole del(5q) or >5% of blasts were excluded according to WHO criteria. Methods: All BM samples underwent May Giemsa Gruenwald and iron stainings. Chromosomal banding analysis (and FISH if needed) were performed in 56/57 cases. PCR was done for the following markers: JAK2V617F (investigated: n=47), MPLW515 (n=46), NRAS (n=24), TET2 mutations (TET2mut, n=14), MLL-PTD (n=13), FLT3-ITD (n=12), and CBL (n=16). Result: Median WBC count was 7.9 ×109/l (range, 3.1–60.0 ×109/l), median hemoglobin (Hb) level was 10 g/dl (range, 6–13 g/dl), and median platelet count was 572 ×109/l (range, 454-1, 737 ×109/l). The median ring sideroblast count was 60% (range, 18–92%). Karyotypes (KT) were as follows: normal KT: n=52 (52/56; 93%); +8: n=2; -Y: n=1. The most frequent mutation was the JAK2V617F (18/47; 38%); an MPLW515 mutation was detected in 3/46 (7%). From the 46 pts being analyzed both for the JAK2 as for the MPLmut, 21 (45.6%) were observed with one of both markers; there was no coincidence of the JAK2 and the MPL mutations. Furtheron, 5/14 (36%) had a TET2 mutation. Coincidences of molecular markers were observed in 3 pts who had a JAK2V617F and a TET2mut in parallel (TET2mut: 3/10; 30% in JAK2mut pts; vs. 2/4 in JAK2 wildtype pts; n.s.). No patient had a JAK2V617F and MPLW515 in parallel. There was no mutation of the NRAS, MLL-PTD, FLT3, or CBL genes in pts investigated for these markers. A positive JAK2V617F mutated status correlated significantly with higher platelets (p=0.038; T-test), whereas no significant correlations were observed for the respective medians taken as thresholds for leukocytes (≥7.9 ×109/l vs. <7.9 ×109/l vs.), Hb (≥10.0 g/dl vs. <10.0 g/dl), or ring sideroblast percentages (≥60% vs. <60%). All 3 pts with MPLW515mut had platelets ≥600×109/l. Cytogenetic aberrations were independent from the JAK2mut status (normal karyotype: 17/45 JAK2mut; 38%; vs. aberrant KT: 1/2 JAK2mut; n.s.) and the MPLmut status (normal KT: 3/44 MPLmut; 7%; vs. aberrant KT: 0/2; n.s.). Higher WBC count (≥7.9 ×109/l) was correlated to a higher Hb level (≥10 g/dl) (p=0.47) and to higher platelets (≥600 ×109/l) (p=0.011). The patients with RARS-T had a favorable outcome with 84.6% being alive at 2.5 years. Conclusion: Investigation of 57 patients strictly fulfilling the criteria of the WHO 2008 classification was able to confirm the unique profile of RARS-T in all aspects: patients with the RARS-T had a normal karyotype (>90% of all cases), had no prognostically adverse cytogenetic alterations, and frequently showed mutations of the JAK2 (V617F) or MPL (W515K/L) genes (45.6% in total). The molecular profile was even more homogeneous in RARS-T cases with ≥600 ×109/l platelets (the WHO threshold from 2001) due to significantly higher proportions of JAK2V617F positive cases when compared to cases with platelets between 450 and <600 ×109/l. However, from clinical aspects, patients with RARS-T had a favorable outcome in our study independent of the molecular state or the number of platelets. These data support to include RARS-T as definite subtype in the next edition of the WHO classification. The frequent occurrence of TET2 mutations in our cohort has to be noted for future diagnostic and classification approaches. Therefore, in cases suspicious for RARS-T but without evidence of a JAK2V617F, molecular screening should be performed including analysis for alterations of the TET2 and MPL genes. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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

Blood ◽  
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.

In CML Patients with Good Response to TKIs Other Gene Mutations Are Frequently (37%) Present in Addition to Philadelphia Negative, Cytogenetically Aberrant Clones but Are Rare (4%) in Cases with MMR and Normal Karyotype

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3126-3126 ◽  
Author(s):  
Susanne Schnittger ◽  
Manja Meggendorfer ◽  
Niroshan Nadarajah ◽  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Gene Mutations ◽  
Clinical Importance ◽  
Normal Karyotype ◽  
Imatinib Treatment ◽  
Mutation Load ◽  
Employment Equity ◽  
Cytogenetic Aberration ◽  
Equity Ownership ◽  
Over Time

Abstract Background: In chronic myeloid leukemia (CML) clonal chromosome aberrations in metaphases not carrying a t(9;22)(q34;q11) have been described during treatment with tyrosine kinase inhibitors (TKI), so-called Philadelphia-negative (Ph-) clones. Very rarely transformation to MDS was observed in patients carrying such Ph- clones but mainly restricted to patients harboring -7. Overall, the clinical significance of this phenomenon remains obscure. Aim: 1) Analyze in a large cohort of TKI-treated CML patients who developed Ph- clones the presence and occurrence of molecular mutations over time. 2) Evaluate whether molecular mutations are also present in CML patients who were at least in major molecular remission (MMR) and presented with a normal karyotype. Patients and Methods: First Cohort: 51 CML patients (pts, 23 males, 28 females; median age: 60 yrs, range: 37-84 yrs) with response to TKI (imatinib only: n=32, nilotinib only: n=2, imatinib and dasatinib or nilotinib: n=11, all three TKIs: n=6) who developed Ph- clones. Cytogenetics in these pts were as follows: +8 sole (n=24), -Y (n=8), -7 sole (n=4), +9 (n=2), other trisomies (n=4), 9 had other aberrations including some with combinations of two different clones (n=4). In median these abnormalities were present in 30% (range 8-100%) of analyzed metaphases. BCR-ABL1 levels at the time point of analysis were between 0 and 3.8 (median: 0.023) according to international scale. Second Cohort: 50 CML pts (24 males, 26 females; median age: 56 yrs, range: 21-83 yrs), who were at least in MMR and without development of any cytogenetic aberration after 3 years of imatinib treatment. Median time from start of therapy to analysis was 2.6 years (range 3 months to 14 yrs). All cases were analyzed with a pan-myeloid gene panel of 29 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, NOTCH1, NPM1, NRAS, PRPF40B, PTPN11, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2 and ZRSR2. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Billerica, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). In addition, RUNX1 was sequenced on the 454 NGS platform (454 Life Sciences, Branford, CT). Results: In the first cohort 28 mutations were found in 19 patients, as 5 patients had 2 and 2 patients even 3 mutations.Thus,in 19/51 pts (37.3%) ≥1 mutation was detected. Median mutation load was 11.5% (range: 2-56%). In detail, mutations in the following genes were detected: ASXL1 (n=9), DNMT3A (n=7), RUNX1 (n=3), NRAS (n=2), TET2 (n=2) and one each in CBL, EZH2, IHD1, PRPF40B, and TP53. Subsequently, these mutations were evaluated in samples from earlier or later time points (18 pts with a total of 235 samples, range: 3-20 samples/pt). In 12 cases a sample from diagnosis of CML was available. In 2 cases a CBL and an ASXL1 mutation were already detectable at low levels, 1.4% and 2%, respectively, at the time of diagnosis and later increased with decreasing BCR-ABL1 levels. In all other 10 cases the mutations were not detectable at diagnosis and were for the first time detectable during TKI treatment (in median after 24 months after diagnosis, range 2-73 months). In the remaining 6 cases date of occurrence could not be determined by backtracking as all earlier samples available were positive for the respective mutation. However, the over time mutation levels were inversely related to BCR-ABL1 expression indicating the presence in independent clones. Within the second cohort with cases in MMR that remained cytogenetically normal only in 2 of the 50 pts (4%) mutations were detected. In one patient a DNMT3A mutation was detected that could be monitored for 8 years with constant low mutation load (3-6%). This was not detectable at diagnosis and occurred after 6 months on imatinib. Very similarly, in the second case a TET2 mutation was first detected after 6 months on imatinib with a mutation load of 2% that very slowly increased to 7% within 8 years. Conclusions: 1) In CML patients that develop Ph- clones other mutations occur in 37.3%. 2) In contrast, in randomly selected CML pts with MMR that are cytogenetically normal, molecular mutations can be detected in only 4%. 3) The clinical importance of molecular mutations in CML in MMR remains unclear. 4) However, these results implicate that chromosomal aberrations are an indicator for genomic instability, also at the molecular level. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann: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.

Comprehensive Genetic Analyses Of 213 PMF Patients Revealed 13% Of SRSF2 Mutations Being Strongly Correlated To ASXL1 Mutations

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2828-2828
Author(s):  
Manja Meggendorfer ◽  
Tamara Alpermann ◽  
Christiane Eder ◽  
Claudia Haferlach ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Molecular Marker ◽  
Scoring System ◽  
Sanger Sequencing ◽  
Gene Mutations ◽  
Normal Karyotype ◽  
Prognostic Impact ◽  
Wild Type ◽  
Employment Equity ◽  
Cytogenetic Aberrations ◽  
Equity Ownership

Abstract Introduction Primary myelofibrosis (PMF) is a myeloproliferative neoplasm, characterized amongst others by stem-cell derived clonal myeloproliferation, bone marrow fibrosis, anemia, splenomegaly, constitutional symptoms and leukemic progression. Diagnosis is based in most cases on cytomorphology/histology demonstrating fibrosis as well as on mutations in JAK2 or MPL. The Dynamic Prognostic Scoring System (DIPSS)-plus is the current base for prognostication using different clinical parameters including karyotype. Furthermore, molecular genetic alterations are currently addressed to provide additional prognostic information. Recently, besides JAK2 and MPL further gene mutations have been described in a limited number of patients, including ASXL1 and SRSF2. Aim To analyze in a large cohort the frequency of SRSF2 and ASXL1 mutations in PMF patients, and to identify their prognostic impact in the context of other previously described gene mutations. Patients and Methods Diagnosis was done according to WHO classification. The cohort comprised 82 female and 131 male patients. In all cases a BCR-ABL rearrangement was excluded by RT-PCR or fluorescence in situ hybridization. JAK2V617F mutation was analyzed in all cases by melting curve analysis, MPLW515 mutation was subsequently analyzed in JAK2V617 wild type (wt) patients. In addition, we analyzed all patients for SRSF2 mutations by Sanger sequencing of the mutational hot spot region coding for amino acid Pro95. Cytogenetics was available in 139 patients. Patients were grouped in favorable (n=121) and unfavorable (n=18) karyotypes based on the DIPSS-plus-scoring system. Based on the previously described correlation of SRSF2mut with ASXL1mut and SETBP1mut in other myeloid entities, SRSF2 mutated cases were also analyzed for mutations in ASXL1 and SETBP1by Sanger sequencing. Follow-up data was available for 136 patients. Results 56% (120/213) of the patients showed JAK2V617F mutations and 18.0% (16/89) of JAK2wt patients carried a mutation in MPLW515 summing up to 65.1% of patients with an already established molecular marker. Of note, SRSF2 was mutated in 12.7% (27/213) of all PMF patients. Patients with SRSF2 mutation had higher white blood cell counts in comparison to SRSF2wt patients (20.00x109/L vs. 7.35x109/L; p=0.005), but there was no correlation to gender, age, hemoglobin level, platelet count or % of myeloblasts in the peripheral blood. In 17 SRSF2mut cases the karyotype was available, 12 were normal karyotype, while two cases showed an unfavorable karyotype according to DIPSS-plus with +8 and i(17)(q10), respectively. The remaining three aberrations belong to the favorable aberration group (del(20q), del(13q), and der(14)). There was no correlation of SRSF2 mutations to the cytogenetic subgroups normal karyotype (n=91) or DIPSS categories favorable and unfavorable aberrations. SRSF2 mutations were also equally distributed between both JAK2V617 or MPLW515 mutated and wild type cases. 18/27 SRSF2mut cases carried also either a JAK2 or MPL mutation, while 9 cases showed no additional JAK2 or MPL mutation. Therefore 30.6% patients remain that carry no mutation in at least one of the three genes investigated first. Interestingly, ASXL1 was frequently mutated in SRSF2 mutated patients (16/23 analyzed SRSF2mut patients) while none of the 24 analyzed SRSF2 mutated cases showed a mutation in SETBP1. To evaluate a potential influence of gene mutations on clinical outcome the overall survival (OS) was calculated. We could confirm that JAK2V617F had no prognostic impact. The same was true for MPLW515 mutations. In contrast to other studies we could not find any impact of SRSF2mutations on OS. Only cytogenetics, i.e. the normal karyotype showed a trend to a prognostic relevance: the median 3 year OS was 70.8% in patients with normal karyotype (n=56) but 58.8% in patients with cytogenetic aberrations (n=29; p=0.153). Conclusion 1) SRSF2 is mutated in 13% of PMF patients. 2) SRSF2 mutated patients show frequently an additional ASXL1 mutation but no coincidence with SETBP1. 3) The prognostic relevance of cytogenetic aberrations was confirmed, while the molecular marker SRSF2 shows no impact on prognosis. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Eder: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.

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 ◽  
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.

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

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

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

Deletion of the Tumor Suppressor Gene NF1 Is An Alternative Mechanism for Aberrant Activation of the RAS Pathway and Is Found in 11% of Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 401-401
Author(s):  
Claudia Haferlach ◽  
Alexander Kohlmann ◽  
Sonja Schindela ◽  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Normal Karyotype ◽  
Loss Of Function ◽  
Chi Square ◽  
Ras Pathway ◽  
Myeloid Malignancies ◽  
Expression Intensity ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Nf1 Gene ◽  
Equity Ownership

Abstract Abstract 401 The NF1 gene encodes neurofibromin 1, a Ras-specific guanosine triphosphatase-activating protein (GAP) that negatively regulates the p21ras (Ras) family of signaling proteins by accelerating GTP hydrolysis. Germline loss of function mutations of NF1 lead to Neurofibromatosis type I. Carriers of the mutations develop benign neurofibromas and are predisposed to neuronal tumors, but also to juvenile myelomonocytic leukemia (JMML), MDS, and AML. In addition, in children suffering from CMML without germline NF1 mutations, acquired loss of function mutations of NF1 have been reported. In contrast, the role of NF1 in adult myeloid malignancies has not been studied in detail. Therefore, we first evaluated NF1 gene expression in 272 AML cases which were analyzed by Affymetrix HG-U133 Plus 2.0 microarrays. These included cases with t(15;17)(q22;q12) (n=15), t(8;21)(q22;q22) (n=16), inv(16)(p13q22) (n=7), t(11q23)/MLL-rearrangement (n=10), complex aberrant karyotype (n=47), normal karyotype (n=97), and with various other genetic abnormalities (n=80). The median NF1 expression intensity was 131.6 (range 35.2 - 457.5). 68 cases showed an expression intensity of NF1 below 98.6 (first quartile). In this cohort cases with t(8;21) (n=10) or complex karyotype (n=18) were over-represented (Chi-square: p<0.0001 and p=0.021, respectively), while cases with normal karyotype (n=16) were under-represented (Chi-square: p=0.016). In 54/68 of the latter cases with low expression material was availabel for FISH analysis with a probe spanning the NF1 locus. Remarkably, in 11/54 of these cases (20.4%) a NF1 deletion was observed by interphase FISH (% of cells with NF1 deletion median 90% (range: 60-99%). The mean±SD NF1 expression intensity in cases with NF1 deletion was 60.4±17.7 as compared to 75.5±18.0 in cases with 2 NF1 copies (p=0.023). Chromosome banding analysis in these 11 cases revealed a complex karyotype (n=7), a normal karyotype (n=2), an inv(3)(q21q26), and a 5q-deletion accompanied by trisomy 21, respectively. To further investigate the incidence of NF1 deletion in myeloid malignancies 425 additional patients were analyzed by FISH for NF1 deletion using a 420 kb probe spanning the NF1 gene. A heterozygous NF1 deletion was observed in 30/425 (7.1%) patients: de novo AML: 13/142 (9.2%), s-AML: 5/39 (12.8%), t-AML: 4/13 (31%), CMML: 5/99 (5.6%), MDS: 0/122 (0%), MPN: 3/10 (30%). Chromosome banding analysis in the NF1-deleted cases revealed a normal karyotype (n=4), an inv(16)(p13q22) (n=6), an inv(3)(q21q26) (n=6), a complex aberrant karyotype (n=5) or other abnormalities (n=9). In conclusion, NF1 deletions occur in 11% of AML, 5% of CMML and 3/10 MPN and therefore are a frequent and important alternative genetic mechanism for activating the RAS pathway in adult myeloid malignancies. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

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

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

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

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 ◽  
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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