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.

Characterization of MDS Harboring TET2 Mutations and/or TET2 Deletions

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4288-4288 ◽  
Author(s):  
Claudia Haferlach ◽  
Anna Stengel ◽  
Manja Meggendorfer ◽  
Wolfgang Kern ◽  
Torsten Haferlach
Keyword(s):  
Overall Survival ◽  
Relative Risk ◽  
Molecular Genetic ◽  
Normal Karyotype ◽  
Prognostic Impact ◽  
Complex Karyotype ◽  
Wild Type ◽  
Employment Equity ◽  
Total Cohort ◽  
Equity Ownership

Background: TET2 mutations and deletions have been reported in MDS. A detailed analysis of the prognostic impact of TET2 deletions and their association to TET2 mutations is lacking. Aim: To characterize MDS with TET2 mutations (mut) and/or TET2 deletions (del) with respect to accompanying cytogenetic and molecular genetic abnormalities and their impact on prognosis. Patients and Methods: First 788 unselected MDS cases (cohort A) were evaluated. As in this cohort only 8 cases with TET2 deletion were detected, further MDS were screened for TET2 deletions. In total 77 MDS harboring a TET2 deletion were identified and included in cohort B. Both cohorts were analyzed by chromosome banding analysis, FISH, genomic arrays and mutation analysis of TET2. Cases from cohort A were also analyzed for mutations in ASXL1, ATM, BCOR, BRCC3, CBL, CTCF, DNMT3A, ETV6, EZH2, FBXW7, IDH1, IDH2, JAK2, KRAS, LAMB4, MPL, NCOR1, NCR2, NF1, NRAS, PHF6, PRPF8, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2. Results: In cohort A 248 cases (31%) with TET2mut were identified. TET2del and a normal karyotype were more frequent in MDS with TET2mut as compared to those with TET2 wild-type (wt) (3% vs 1%, p=0.006; 89% vs 78%, p<0.001). SF3B1 and ASXL1 were frequently mutated in both TET2mut and TET2wt MDS (32% and 34%, 22% and 18%, respectively). In MDS with TET2mut compared to MDS with TET2wt the following genes were less frequently mutated: ATM (0.5% vs 3%, p=0.05), DNMT3A (9% vs 15%, p=0.02), ETV6 (0.5% vs 3%, p=0.03), IDH1 (0.5% vs 3%, p=0.02), IDH2 (1% vs 5%, p=0.002), TP53 (2% vs 7%, p=0.004), U2AF1 (4% vs 9%, p=0.04), while the following genes were more frequently mutated: CBL (6% vs 2%, p=0.01), EZH2 (8% vs 2%, p<0.001), SRSF2 (27% vs 12%, p<0.001), and ZRSR2 (15% vs 3%, p<0.001). Overall spliceosome genes were more frequently mutated in TET2mut than in TET2wt MDS (77% vs 56%, p<0.001). In the total cohort A neither TET2mut nor TET2del had an impact on overall survival (OS). In TET2mut MDS and TET2wt MDS SF3B1mut were associated with favorable outcome, while TP53mut were associated with shorter OS in both subsets (table 1). However in TET2mut MDS mutations in RUNX1 (p<0.0001), CBL (p=0.001), and U2AF1 (p=0.03) were independently associated with shorter OS, while in TET2wt MDS mutations in KRAS (p=0.03), EZH2 (p=0.02), NRAS (p=0.02), SRSF2 (p=0.007), IDH2 (p=0.05), and ASXL1 (p=0.01) were independently associated with shorter OS. In cohort B 40/77 (52%) MDS with TET2del also harbored a TET2mut. The 4q deletion encompassing the TET2 gene was < 10 MB in size and thus cytogenetically cryptic in 77% of cases with TET2mut, while the TET2 deletion was cryptic in only 24% of cases without TET2mut. A normal karyotype was present in 37 cases (48%), a complex karyotype in 29 (38%) and other abnormalities in 11 cases (14%). TET2mut were frequent in cases with a normal karyotype (68% vs aberrant karyotype: 32%, p<0.001) and were rare in cases with a complex karyotype (13%). Relating the mutation load of TET2mut to the proportion of cells with TET2del as determined by FISH revealed in 60% of cases that both TET2 alterations were present in the main clone, while in 23% of cases the TET2mut was present in a subclone only and in 17% the TET2del was observed in a subclone only. In the subset of patients with TET2del in a subclone only, 83% showed a normal karyotype and none a complex karyotype, while in the subset of cases with TET2mut in a subclone only, 43% showed a normal and 29% a complex karyotype. In the total cohort B the presence of a TET2mut in addition to the TET2del had no prognostic impact, while the presence of a complex karyotype was associated with shorter OS (RR: 8.0, p=0.004). Conclusions: 1) TET2 deletions are rare in TET2 mutated MDS (3%). 2) TET2 mutations are frequent in MDS with TET2 deletion (52%). 3) TET2 mutations are highly correlated to a normal karyotype and are rare in complex karyotype. 3) Neither TET2 mutations nor TET2 deletions have a prognostic impact in MDS. 4) In TET2 mutated MDS mutations in RUNX1, TP53, CBL, and U2AF1 have the strongest negative independent impact on OS, which in TET2 wild-type MDS is the case for mutations in TP53, KRAS, EZH2, NRAS, SRSF2, IDH2 and ASXL1. Table The relative risk of parameters significantly (p<0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Table. The relative risk of parameters significantly (p<0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Stengel:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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

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

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

Gene Amplifications In 84 Patients With Acute Myeloid Leukemia and 31 Patients With Myelodysplastic Syndrome Investigated By Array CGH

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3724-3724
Author(s):  
Andreas Roller ◽  
Simone Weber ◽  
Alexander Kohlmann ◽  
Melanie Zenger ◽  
Marita Staller ◽  
...  
Keyword(s):  
Array Cgh ◽  
Chromosomal Abnormalities ◽  
Ring Chromosome ◽  
Normal Karyotype ◽  
Structural Type ◽  
Single Copy ◽  
Chromosome 8 ◽  
Complex Karyotype ◽  
Employment Equity ◽  
Equity Ownership

Abstract Background Gains and losses of chromosomal material are frequent in AML and MDS and usually lead to loss or gain of a single copy of a whole chromosome, a chromosome arm or small stretches of the chromosome that may be microscopically invisible. More rarely, amplifications of chromosomal regions (defined as the presence of more than 6 copies of a region per cell) are observed. These supernumerary copies are located either extrachromosomally as small acentric chromosomal structures - so called double-minutes (dmin) - or intrachromosomally as large contiguous stretches of amplified DNA, so called homogeneously staining regions (HSR). Aims Characterize AML and MDS cases with gene amplifications with respect to size, affected genes and accompanying chromosomal abnormalities as well as TP53 status. Patients and Methods 84 AML and 31 MDS cases with cytogenetically visible amplifications were selected for this study. All cases were analyzed by array CGH, chromosome banding analysis, sequencing for TP53 mutations as well as FISH for TP53 deletions. Results The cohort comprised 55 (47.8%) males and 60 (52.2%) females with a median age of 72.0 years (range 38.0 - 90.3 years). A complex karyotype (≥4 aberrations) was present in 92/115 (80.0%) cases (AML=65/84 (77.4%); MDS=27/31 (87.1%)). In total, 385 amplified regions were identified by array CGH. In more detail: 3q26 (AML: n=6; MDS: n=3), 8q24 (AML: n=15; MDS: n=1), 11q21-25 (AML: n=42; MDS: n=13), 13q12 (AML: n=3; MDS: n=1), 13q31 (AML: n=3; MDS: n=2), 19p13 (AML: n=2; MDS: n=4), and 21q21-q22 (AML: n=24; MDS: n=5). The median number of amplified regions was 3 (range 1-18). In 14/115 (12.2%) cases, the amplification was located in dmins (AML: n=11; MDS: n=3) and in 101/115 (87.8%) patients in HSR (AML: n=73; MDS: n=28). In 40 of the latter 101 cases (39.6%) (AML: n=24; MDS: n=16) the amplification was located on a ring chromosome (rc). In patients with complex karyotypes we detected a significantly higher number of amplified regions as compared to non-complex karyotypes (3.5 vs. 2.8; p=0.015). No association between the complexity of the karyotype and the structural type of the amplification (dmin vs rc) was observed. Cases with non-complex karyotypes frequently harbored a 5q deletion (6/23; 26.1%) or chromosome 8 abnormalities (3/23; 13.0%). Within the subgroup of non-complex karyotypes del(5q) cases showed a tendency to a higher number of amplified regions (3.6 vs. 1.9; p=0.140). Further, amplifications of 11q genes were more frequent in complex karyotypes (54.4% vs. 21.7%; p=0.005), whereas 8q amplifications were more frequent in non-complex karyotypes (43.5% vs. 4.4%; p<0.001). We detected a large region on band 11q24, which was amplified in 41/53 (77.4%) cases. This commonly amplified region contains 1,575 genes including the MLL gene. Cases harboring dmins had shorter amplified regions compared to cases with rc (4,428,112.5 bp vs. 18,265,496.9 bp; p=0.028). Moreover, we detected a positive correlation of patients having a rc and gene amplification on chromosome 11q23-25 (p<0.05). On chromosome 3q, 8/9 (88.9%) cases shared a minimal amplified region covering the EVI1 gene. In comparison to samples obtained from healthy donors (n=47), the EVI1 expression was significantly higher in cases with EVI1 amplification (87.4 vs. 0.5; p=0.048). On chromosome 21q the regions of amplifications were heterogeneous. However, we detected a minimal region containing 11 genes including ERG which was amplified in 26/29 (89.7%) patients. ERG expression data was available in 8 cases and was significantly higher compared to a control cohort of AML with normal karyotype (n=331) (729.2 vs. 229.0; p=0.05). On chromosome 8 an amplified region was identified in 15/16 cases. In 14 of these cases (87.5%) the region included MYC. TP53mut were present in 93/115 (80.9%) patients, accompanied by a TP53del in 28/93 (30.1%) cases. Interestingly, cases harboring a TP53mut had more amplified regions compared to TP53wt (3.4 vs. 1.7; p<0.001). Conclusions 1. MLL is the most frequently amplified gene in AML and MDS. 2. Patients with complex karyotypes or TP53mut harbored more amplified regions compared to patients with non-complex karyotypes and TP53wt. 3. Amplifications on 11q were more frequent in complex karyotype whereas gene amplifications on 8q were predominantly observed in non-complex karyotypes. 4. EVI1 and ERG gene amplifications lead to a higher expression of the respective genes. Disclosures: Roller: MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller: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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals The Number of RUNX1 Mutations and the Presence of One Intact RUNX1 Allele Influence Cytogenetic Abnormalities, Additional Molecular Mutations and Prognosis in RUNX1 Mutated AML

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 284-284 ◽  
Author(s):  
Anna Stengel ◽  
Wolfgang Kern ◽  
Manja Meggendorfer ◽  
Karolina Perglerová ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Genetic Alterations ◽  
Adverse Impact ◽  
Missense Mutations ◽  
Employment Equity ◽  
Frameshift Mutations ◽  
Total Cohort ◽  
Equity Ownership ◽  
The Impact ◽  
Runx1 Mutation ◽  
Mutation Analyses

Abstract Background: According to the revised WHO classification of 2016, AML with mutated RUNX1 constitutes a new provisional entity. We previously reported that the subgroup of RUNX1-mutated AML with RUNX1 wild-type (WT) loss is associated with a distinct pattern of cytogenetic and molecular genetic abnormalities and with an adverse prognosis. However, the impact of multiple RUNX1 mutations is unclear yet. Aims: Evaluation of associated genetic alterations and prognosis in AML with >1 RUNX1 mutation as compared to those with (1) RUNX1 WT loss and (2) one RUNX1 mutation. Patient cohorts and methods: The total cohort comprised 467 AML cases with RUNX1 mutations (mut) (296 male, 171 female). Median age was 72 years (range: 18-91 years). All patients were investigated using chromosome banding analysis (CBA) and amplicon sequencing of RUNX1. The cohort was split into the subgroups with RUNX1 WT loss (UPD or RUNX1 deletion detected by genomic arrays) (n=50), cases with >1 RUNX1mut (n = 94) and cases with 1 RUNX1mut and conservation of the RUNX1 WT allele (n = 323). Of these, 50, 55 and 58 cases, respectively, were selected for further mutation analyses of ASXL1, BCOR, CBL, CEBPA, DNMT3A, ETV6, EZH2, FLT3-ITD, FLT3-TKD, GATA2, IDH1, IDH2, KIT, KRAS, MLL-PTD, NPM, NRAS, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and WT1. Variants of unknown significance were excluded from statistical analysis. Results: In the total cohort of 467 cases, CBA revealed a normal karyotype (NK) in 53% of patients, 18% harbored trisomies, 3% showed a complex karyotype (>3 abnormalities), 26% other aberrations. The proportion of cases with trisomies was largest in cases with RUNX1 WT loss (26%), followed by >1 RUNX1mut (19%) and 1 RUNX1mut (16%). In more detail, in the total cohort, 56% of cases with trisomies harbored +8, in 30% +13 was found. A similar pattern was observed for 1 RUNX1mut, whereas in cases with WT loss +13 was the most abundant trisomy (+8: 66% in 1 RUNX1mut vs. 31% in WT loss, p=0.022; +13: 15% vs. 62%, p<0.001). Cases with >1 RUNX1mut showed an intermediate distribution (+8: 44%, +13: 50%). Missense mutations were the most abundant mutation type (53%) inWT loss cases, followed by frameshift mutations (28%). By contrast, in cases with 1 RUNX1mut, frameshift mutations were found more frequent (45%, p=0.016), whereas missense mutations were detected at a frequency of 31% (p=0.006). In cases with >1 RUNX1mut, both were observed at similar frequencies (missense: 36%, frameshift: 38%). Mutation analyses of 163 selected cases revealed SRSF2 (39%), ASXL1 (36%), DNMT3A (19%), BCOR (18%), IDH2 (17%), SF3B1 (17%) and TET2 (17%) mutations as most frequently mutated genes in the total cohort. Cases with RUNX1 WT loss showed a higher frequency of ASXL1mut compared to the other cases (50% vs. 29%, p=0.013), while U2AF1mut were absent from this group (0% vs. 15%, p=0.019). Differences between cases with and without RUNX1 WT loss were also detected for DNMT3A, TET2, SF3B1 (more abundant in WT loss) and IDH2, WT1 (less abundant in WT loss), although these were not statistically significant. For many genes, the group of cases with >1 RUNX1mut showed an intermediate abundance pattern, or mutation frequencies similar to cases with 1 RUNX1mut were observed. Mutations in spliceosome genes (SF3B1, SRSF2, U2AF1, ZRSR2) were very abundant in all subgroups but less frequent (although not statistically significant) in cases with 1 RUNX1mut. Median overall survival (OS) in the total cohort was 14 months. WT loss (OS: 5 months) and >1 RUNX1mut (14 months) showed an adverse impact on prognosis compared to 1 RUNX1mut (22 months; p=0.002 and p=0.048, respectively). Mutations in ASXL1 and KRAS showed a negative impact on OS in the total cohort (10 vs. 18 months, p=0.028; 1 vs. 15 months, p<0.001), whereas DNMT3A mutations negatively affected OS in WT loss only (in WT loss: 1 vs. 7 months, p=0.005). Conclusion: 1) RUNX1 mutated AML cases with WT loss show a high frequency of +13, RUNX1 missense mutations and accompanying ASXL1 mutations. 2) They clearly separate from cases with 1 RUNX1mut, which depict a predominance of +8, RUNX1 frameshift mutations and mutations in IDH2 and WT1. 3) Patients with >1 RUNX1mut show a pattern intermediate between the former two subgroups. 4) Loss of RUNX1 WT and > 1 RUNX1mut showed an adverse impact on OS. Thus, not only the presence and number of RUNX1mut but also the conservation of an intact RUNX1 allele is biologically and clinically relevant. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

A NUP98-NSD1 Fusion Gene Can Be Detected in a Small Subset of Adult AML Patients with Normal Karyotype

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2531-2531
Author(s):  
Annette Fasan ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Normal Karyotype ◽  
Fusion Transcript ◽  
Chromosomal Band ◽  
Event Free Survival ◽  
Employment Equity ◽  
Aberrant Expression ◽  
Free Survival ◽  
Platelet Counts ◽  
Equity Ownership

Abstract Abstract 2531 Introduction: Fusion genes can be detected in approximately 30–35% of all AML cases and usually are the result of a cytogenetically detectable chromosomal rearrangement. Very recently, a novel fusion gene has been described in AML with normal karyotype (Hollink et al, Blood, 2011). This cryptic fusion involves nucleophosmin 98kD (NUP98) in chromosomal band 11p15 and the non homeobox gene NSD1 in chromosomal band 5q35. NUP98-NSD1 has been described in this single study with a frequency of 16.1% in pediatric and 2.3% in adult AML patients with distinct characteristics (e.g. mutual exclusivity with NPM1) and dismal prognosis. Aim: The aim of this study was to further evaluate NUP98-NSD1 rearrangements in adult AML with normal karyotype (NK) for frequency, association with other mutations and impact on outcome. Patients and Methods: Screening for NUP98-NSD1 fusion gene was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) in a total cohort of 148 de novo AML patients with NK and NPM1 wildtype status. The NUP98-NSD1 positive cases were verified by direct Sanger Sequencing of the PCR products. The cohort was composed of 84 males and 64 females. Median age was 55.4 years (range: 15.7 to 85.8 years). Further mutation analysis was available in subcohorts: FLT3-ITD (n=32 mut/117 screened), CEBPA (n=22 mut/124 screened), MLL-PTD (n=32 mut/117 screened) and RUNX1 (n=26 mut/83 screened). Results: In total, in 8/148 (5.4%) patients a NUP98-NDS1 fusion transcript was detected. NUP98-NDS1-positive cases had significantly higher platelet counts (median 221 vs 87 × 10e9/L; p=0.001). Patients with NUP98-NDS1 were younger than the NUP98-NDS1-negative patients (median: 43.5 years vs 55.4 years, p=0.067). Sex (5 male vs. 3 female), white blood cell count and hemoglobin levels at diagnosis were not different compared to NUP98-NDS1-negative cases. Cytomorphology revealed AML with minimal differential differentiation (n=4), with maturation (n=1), and myelomonocytic AML (n=3). In 3 NUP98-NDS1-positive cases immunophenotyping data was available and all 3 cases aberrantly expressed CD7. NUP98-NDS1-positive cases have a higher frequency of FLT3-ITD compared to NUP98-NDS1-negative cases (5/8, 62.5% vs. 27/140, 19.3%; p=0.015) and were mutually exclusive of CEBPA and RUNX1 mutations. With respect to survival the NUP98-NDS1-positive cases had a worse event free survival compared to NUP98-NDS1-negative cases (median 5.1 months vs. 25.2 months; p=0.054). Conclusions: A NUP98-NSD1 fusion transcript was detected in 5.4% of normal karyotype adult AML patients without NPM1 mutation. NUP98-NSD1-positive cases are characterized by younger age, high coincidence of FLT3-ITD, aberrant expression of CD7, relatively high platelet counts, and a short event free survival. Thus NUP98-NSD1 translocations seem to define a new subgroup of NK-AML. Importantly, in this prognostically adverse and so far cytogenetically undetectable group close and sensitive PCR based monitoring for minimal residual disease is available. Thus, this data suggests to perform PCR based screening for NUP98-NSD1 in AML with normal karyotype that lack NPM1 and CEBPA mutations. Disclosures: Fasan: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Comprehensive Molecular Profiling of T-Cell Acute Lymphoblastic Leukemia Identifies Mutations in 76 (97.4%) of 78 Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2548-2548
Author(s):  
Vera Grossmann ◽  
Alexander Kohlmann ◽  
Sandra Weissmann ◽  
Susanne Schnittger ◽  
Valentina Artusi ◽  
...  
Keyword(s):  
Promoter Methylation ◽  
Kinase Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Normal Karyotype ◽  
Genetic Alterations ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 2548 Introduction: At present, the diagnosis of T-ALL is based on immunophenotyping and specific chromosomal rearrangements. However, the knowledge about recurrent somatic mutations is limited. Patients and Methods: We studied a cohort of 78 adult T-ALL cases (n=33 early, n=33 cortical, n=2 mature T-ALL, n=10 subtype not available), including 57 males and 21 females. Age ranged from 18.8–87.7 yrs (median: 42 yrs). A deep-sequencing assay was used to investigate for specific molecular alterations in genes involved in transcriptional regulation: NOTCH1, FBXW7, CDKN2A, DNMT3A, FLT3-ITD, FLT3-TKD, NPM1, PTEN, and RUNX1. Further, chromosome banding analysis and FISH with probes for DNMT3A (2p23), SEC63 (6q21), MYB (6q23), CDKN2A (9p21), PTEN (10q23), CDKN1B (12p13) and TP53 (17p13), as well as CDKN2B promoter methylation analyses were performed. Results: Cytogenetic data was available in 68 patients: normal karyotype: n=22 (2 of these harbored a PICALM-MLLT10-rearrangement), SIL-TAL1-rearrangement: n=3, t(5;14)(q35;q32): n=2, t(10;14)(q24;q11)/t(7;10)(q34;q24): n=9, t(10;11)(p13;q21): n=3, other abnormalities n=29. Importantly, molecular mutations were detected in 67/78 patients (85.6%). In detail, NOTCH1 was the most frequently mutated gene (55/77 cases, 71.4%). Other alterations were detected in DNMT3A (16/78; 20.5%); RUNX1 (13/78; 16.6%); FBXW7 (11/75; 14.6%); PTEN (7/78; 10.0%); CDKN2A (3/58; 5.2%); FLT3-ITD (2/78; 2.5%); and FLT3-TKD (1/70; 1.4%). By FISH analyses, heterozygous deletions of the following loci were observed: DNMT3A (1/43; 2.3%), SEC63 (7/43; 16.3%), PTEN (1/32, 3.1%), CDKN1B (8/43; 18.6%) and TP53 (3/43; 7.0%). CDKN2A deletions were detected in 30/72 (41.6%) cases: n=14 heterozygous, n=15 homozygous, n=1 showed a clone with a heterozygous and a subclone with a homozygous deletion. Further, the CDKN2B promoter methylation status was analyzed. Here, 36/74 (48.6%) cases demonstrated hypermethylation. As such, when combining molecular mutations, CDKN2A deletions, and CDKN2B hypermethylation, in median 2 alterations per case were observed (range 1–5). Moreover, almost every patient (76/78) harbored at least one aberration resulting in a mutation rate of 97.4%. Interestingly, considering alterations in the group of cyclin-dependent kinase inhibitors (CDKN2A/1B deletions, CDKN2A mutations, and CDKN2B hypermethylation), 61/78 (78.2%) cases carried at least one such alteration. With respect to associations amongst molecular mutations, no specific pattern was observed except for a strong correlation between RUNX1 and DNMT3A mutations, i.e. 6/13 RUNX1 mutated cases concomitantly harbored DNMT3A mutations (p=0.021). Furthermore, we observed that DNMT3A and RUNX1 alterations were associated with higher age (DNMT3A: mean±SD 60.9±16 vs. 39.6±16 years; p<0.001; RUNX1: mean±SD 55.4±18 vs. 41.7±18 yrs; p=0.013) whereas PTENmut were associated with younger age (mean±SD 32.9±10 vs. 45.0±19 yrs; p=0.019). With regard to cytogenetics, DNMT3A was significantly correlated with normal karyotype (9/23, 39.1% vs. 6/45, 13.3%; p=0.028). Moreover, RUNX1mut were associated with lower WBC count (mean±SD 26.4±41 vs. 63.4±90 cell count G/L; p=0.025). With respect to immunophenotypes, cases with RUNX1mut showed a trend to be associated with early T-ALLs (9/23, 39.1% vs. 6/45, 13.3%; p=0.082). CDKN2B hypermethylation was significantly correlated with early T-ALLs (21/32, 65.6% vs. 10/31, 32.2%; p=0.012). In contrast, FBXW7mut were associated with the cortical subgroup (1/32, 3.1% vs. 9/32, 28.1%; p=0.013). With regard to clinical outcome, patients with RUNX1mut had a shorter overall survival (OS) compared to RUNX1wt patients (alive at 2 yrs: 44.4% mutated vs. 64.0% wild-type, p=0.011). Further, for NOTCH1mut cases (alive at 2 yrs: 67.4% mutated vs. 33.6% wild-type, p=0.060) a trend towards a better OS was detectable. Conclusions: 1. T-ALL is characterized by a high number of genetic alterations since 46/68 (67.6%) showed cytogenetic aberrations. In addition, at least one molecular alteration was observed in 76/78 (97.4%) patients. 2. The most frequent alterations observed were mutations in NOTCH1, DNMT3A, RUNX1 and FBXW7. 3. The cyclin-dependent kinase inhibitors were altered by deletion, mutation or hypermethylation in 78.2% of cases. 4. RUNX1 mutations are associated with shorter and NOTCH1 mutations with longer OS. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Artusi:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Stadler: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.

Prognostic Value of “Monosomal Karyotype” in Comparison to “Complex Aberrant Karyotype” in AML: A Study on 824 Cases with Aberrant Karyotype

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 418-418
Author(s):  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
Torsten Haferlach
Keyword(s):  
Cox Regression ◽  
Complex Karyotype ◽  
Employment Equity ◽  
Cytogenetic Abnormalities ◽  
Cox Regression Analysis ◽  
Poor Risk ◽  
Risk Patients ◽  
Equity Ownership ◽  
The Impact ◽  
Monosomal Karyotype

Abstract Abstract 418 Background: Several classifications based on cytogenetics have been proposed in AML. Typically 3 major categories for prognostication are defined: favorable, intermediate and unfavorable. The assignment to the unfavorable group shows minor differences between the different cytogenetic classifications currently used, however certain cytogenetic subgroups are assigned to the unfavorable subgroup concordantly: −5/5q−, 7q−/−7, −17/abn17p, inv(3)(q21q26)/t(3;3)(q21;q26) and complex karyotype (CK). With respect to CK 3 definitions are used: ≥3, ≥4 or ≥5 unrelated abnormalities. Recently, a so-called “monosomal karyotype” (MSK) defined as a karyotype showing “two or more distinct autosomal chromosome monosomies or one single autosomal monosomy in the presence of structural abnormalities” was introduced (Breems et al. JCO 2008). It was suggested that patients with MSK have a poor outcome being even inferior to CK. Aim: We here evaluated the prognostic power of differently defined cytogenetic subsets in order to identify the best definition for the prognostically most unfavorable subgroup. Patients: From our initial cohort of newly diagnosed AML (n=1,959) patients with t(15;17), t(8;21) or inv(16) (n=170) and AML with normal karyotype (n=965) were excluded. Thus, 824 patients with cytogenetic abnormalities remained for further investigation. Results: 428/824 (51.9%) patients showed an intermediate risk karyotype according to revised MRC criteria (MRC-I) (Grimwade et al. Blood 2010), while the remaining 396/824 (48.1%) cases belonged to the unfavorable MRC group (MRC-U). 162/824 cases (19.7%) fulfilled the criteria of MSK. According to MRC, 4 of these 162 cases with MSK were classified MRC-I while 158 were classified MRC-U. The overlap in classification between CK and MRC-U differed depending on the number of aberrations used to define CK. As such, the number of cases with CK was 272 (33.0%; MRC-I: 17, MRC-U: 255) using ≥3 clonal aberrations, and decreased to 222 (26.9%; all MRC-U) patients using ≥4 clonal aberrations or 196 (23.8%; all MRC-U) cases when applying the criterion of ≥5 clonal aberrations, respectively. Univariate Cox regression analysis revealed that unfavorable cytogenetics as defined by MRC-U, MSK, CK defined as ≥3, ≥4 or ≥5 unrelated abnormalities were all significantly associated with inferior OS as compared to the respective remaining intermediate group (for all p<0.001). Hazard ratios were 1.61, 1.93, 1.68, 1.94, and 1.92, respectively. Median OS in the respective categories was 8.5, 5.7, 6.3, 5.7, and 5.7 months, respectively. We then performed further analyses within the unfavorable risk group defined according to MRC and tested the impact of the 4 definitions for unfavorable subsets. In each comparison the median OS was significantly shorter for the subset with MSK, or CK defined as ≥3, '4 or ≥5 unrelated abnormalities as compared to the remaining MRC-U cases (5.7 vs 11.7 mo p=0.005; 6.3 vs 10.6 mo, p=0.031; 5.7 vs 11.0 mo, p=0.003; 5.7 vs 10.9 mo, p=0.006). Furthermore OS of patients within MRC-U excluding cases with MSK, or CK with ≥3, ≥4 or ≥5 unrelated abnormalities did not differ from patients with cytogenetic abnormalities assigned to MRC-I (median OS 11.7, 10.6, 11.0 and 10.9 mo, respectively vs 21.1 mo, p=0.072, p=0.16, p=0.28, and p=0.11, respectively). Within the MRC-U cohort only 124 cases fulfilled both criteria: MSK and CK≥4 (median OS 5.3 mo), 97 were CK≥4 only (median OS 6.3 mo) and 35 MSK only (median OS 6.7 mo). OS did not differ between these 3 subgroups but was significantly shorter for all comparisons to patients included in none of these subgroups (p<0.001, p=0.009, p=0.012, respectively). On the other hand OS of the 33 cases with 3 unrelated abnormalities did not differ from MRC-U cases with 1 or 2 abnormalities (18.9 vs 10.6, p=0.48). Conclusions: All definitions of very poor risk AML patients allow to identify a subset within MRC-U that shows significantly shorter OS than the remaining MRC-U cases. However, “complex karyotype defined as ≥4 unrelated abnormalities” is the best parameter as it identifies the largest proportion of very poor risk patients. Even more important, the application of the monosomal karyotype for prognostication and clinical guidance in AML misses 24.5% of the very poor risk patients identified based on CK ≥4. This may lead to suboptimal treatment decisions in this clinically proven very high risk patients. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Cytogenetic and Molecular Genetic Clonal Evolution in CLL Is Associated with an Unmutated IGHV Status and Frequently Leads to a Combination of Loss of 17p and TP53 mutation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3213-3213
Author(s):  
Claudia Haferlach ◽  
Sabine Jeromin ◽  
Niroshan Nadarajah ◽  
Melanie Zenger ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Median Time ◽  
Tp53 Mutation ◽  
Clonal Evolution ◽  
Molecular Genetic ◽  
Normal Karyotype ◽  
Employment Equity ◽  
Mutation Status ◽  
Equity Ownership ◽  
Mutation Analyses

Abstract Background: The clinical course in CLL is very heterogeneous ranging from stable disease to a rather rapid progression requiring treatment. The acquisition of genetic abnormalities termed clonal evolution (CE) is likely to correlate with clinical progression and might be used to guide treatment strategies. Aim: The aim of this study was to evaluate the frequency of CE on the cytogenetic (CCE) and molecular genetic (MCE) levels and its association with the IGHV mutation status and clinical outcome. Methods: 179 CLL cases were selected on the basis that chromosome banding analysis (CBA) and mutation analyses in TP53 and SF3B1 all having been performed at least at two time points. The median age at first evaluation was 72 years (range: 46-95). The first time point of analysis was at primary diagnosis (n=131) or during course of disease but prior to any treatment (n=48). In all patients interphase FISH was performed with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), and the centromeric region of chromosome 12 and the IGHV mutation status was evaluated. A total of 465 CBA, 417 TP53 and 424 SF3B1 mutation analyses were evaluated. The median number of samples per patient was 2 (range: 2-9). The time between samples ranged from 1 month to 9.8 years (median 21 months). For all patients clinical follow-up data was available with a median follow-up of 7.4 years and 5-year overall survival (OS) of 88%. Results: At first investigation CBA revealed a normal karyotype in 31 (17%) patients. In cases with an aberrant karyotype the pattern of abnormalities was typical for CLL: del(13q); 51% (homozygous: 15%), +12: 18%, del(11q): 16%, and del(17p): 5%. A complex karyotype (≥3 abnormalities) was present in 18%. The IGHV status was unmutated (IGHV-U) in 56% of cases and TP53 and SF3B1 mutations were detected in 10% and 15%, respectively. CCE was observed in 63/179 patients (35%). The median time to CCE was 46 months (range 3-111). The most frequent abnormalities gained during CCE were loss of 17p (14/63; 22%), 13q (11/63; 18%), and 11q (10/63; 16%). Acquired loss of 17p was more frequent in SF3B1mutated CLL (19% vs 6%, p=0.04). MCE was observed in 29/179 cases (16%). TP53 and SF3B1 mutations were acquired during the course of the disease in 23 (14%) and 7 (5%) cases, respectively. The median time to MCE was 61 months (range 1.5-109). Of note, in 2 cases with TP53 deletion a TP53 mutation was acquired and in 2 cases with TP53 mutation a TP53 deletion was acquired. In 12 CLL both a TP53 deletion and a TP53 mutation were acquired (table). CCE and MCE were significantly associated with IGHV-U (p=0.003; p<0.001) and with each other (p<0.001). In more detail, in 71% of cases with CCE and 90% of cases with MCE an IGHV-U was present. Thus, CCE and MCE were less frequent in IGHVmut CLL (23% and 4%). In 30% of CLL with CCE also MCE occurred. In addition CCE was associated with an aberrant karyotype at first investigation (p<0.001). CCE occurred in only 3% of CLL with a normal karyotype but in 42% of CLL with an aberrant karyotype. Time to treatment was significantly shorter in patients with CCE, MCE and both compared to the respective patients without (2.1 vs 5.5 yrs, p=0.004; 1.8 vs 4.8 yrs, p=0.07; 2.2 vs 5.3 yrs; p=0.04). While no impact of CCE on OS was observed in patients with a mutated IGHV status, in patients with an unmutated IGHV status a tendency to shorter OS was observed in cases with CCE compared to those without (7 year OS: 67% vs 83%; p=0.2). No impact on OS was observed for MCE. This may be due to rather short follow up after CE. However, if CCE and MCE resulted in CLL harboring both TP53 deletion and TP53 mutation 5 year OS was significantly shorter than in CLL with neither TP53 deletion nor TP53 mutation (75% vs 91%, p=0.03). Conclusions: 1) We observed CCE in 35% and MCE in 16% of CLL. 2) The pattern of cytogenetic abnormalities acquired during the course of the disease is similar to the pattern observed in CLL at diagnosis, however the frequency varies with del(17p) being the most frequently gained in CE. 3) CCE and MCE were highly correlated to IGHV-U. 4) In 25% of CLL with CCE and MCE CE resulted in the co-occurrence of TP53 deletion and TP53 mutation, which was associated with a significantly shorter OS emphasizing the necessity to reevaluate the TP53 status during the course of the disease to guide treatment. 5) The frequency and impact of CE needs to be further studied in unselected patient cohorts in which CBA and mutational analysis is performed on a regular basis. Table Table. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals A Comprehensive Panel of Molecular Mutations Notably Improves a Cytogenetic Prognostication System in Routine AML Diagnostics

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 286-286
Author(s):  
Claudia Haferlach ◽  
Manja Meggendorfer ◽  
Annette Fasan ◽  
Karolina Perglerová ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Molecular Genetics ◽  
Risk Group ◽  
Classification Systems ◽  
Prognostic Impact ◽  
Intermediate Risk ◽  
Complex Karyotype ◽  
Employment Equity ◽  
Mutation Status ◽  
Cox Regression Analysis ◽  
Equity Ownership

Abstract Background: Based on sequencing studies the molecular landscape of AML has been unraveled. Novel prognostic scores combining molecular mutations and karyotype have been proposed (Grimwade et al. Blood, 2016, Döhner et al. NEJM 2015). However, these proposed classification systems differ in several aspects and yet no consensus has been established which genetic information is required for prognostication in AML today. Aims: 1) Test the prognostic value of a panel of molecular markers in addition to a cytogenetic score in a large cohort of AML patients. 2) Determine the proportion of patients with a suitable molecular marker for disease monitoring (MRD) applying molecular genetics. Patients and Methods: 867 de novo AML cases younger than 60 years were investigated (median age: 48 years, median follow up of 41 months). All patients were evaluated for karyotype, KMT2A-PTD, FLT3-ITD and in addition for mutation status of ASXL1, CEBPA, DNMT3A, NPM1, RUNX1 and TP53 according to the proposal by Grimwade et al. Blood 2016. Results: First, AML were classified according to the refined MRC cytogenetic classification with AML with t(15;17)/PML-RARA regarded as a separate group (n=89 (10%), 90% overall survival (OS) at 5 years). 89 cases (10%) were assigned to the favourable risk group (t(8;21)/RUNX1-RUNX1T1: n=42; inv(16)/t(16;16)/CBFB-MYH11: n=47), 570 (68%) to the intermediate risk group and 119 (14%) to the adverse risk group. OS at 5 years was 66%, 53% and 28%, respectively, and differed significantly between all four subgroups (for all comparisons p<0.001). Next, the following subgroups were separated: CEBPA double mutated (dm) cases (n=44 (5%); OS at 5 years: 83%), NPM1mut/FLT3-ITD- AML (n=181 (21%); OS at 5 years: 62%), and NPM1mut/FLT3-ITD+ AML (n=137 (16%); OS at 5 years: 47%; for all comparisons between these 3 groups p<0.001). Thus, prognosis of CEBPAdm cases was comparable to PML-RARA+ AML. OS in NPM1mut/FLT3-ITD- AML is comparable to CBF-leukemias. In NPM1mut AML no prognostic impact of DNMT3Amut was found. In all these 3 groups defined on molecular genetics no prognostic impact of additional karyotype information on OS was observed. Next, in the remaining cases of the cytogenetic intermediate risk group (n=209) the prognostic impact of mutations in ASXL1, DNMT3A, RUNX1, TP53, KMT2A-PTD and FLT3-ITD was evaluated. In multivariate Cox regression analysis mutations in TP53 (relative risk (RR): 3.5; p=0.04), ASXL1 (RR: 2.2, p=0.004), and FLT3-ITD (RR: 1.8; p=0.04) were independently associated with shorter OS. OS at 5 years was 25% in cases carrying at least one of these mutations compared to 54% in cases with none of these mutations (p=0.001). Within the adverse cytogenetic risk group cases with either a complex karyotype (n=27) or a KMT2A (n=25) or MECOM rearrangement (n=14) had the worst outcome compared to the remaining cases (OS at 5 yrs: 19% vs 54%, p=0.02). In the remaining cases the presence of at least one mutation in either ASXL1, TP53 or FLT3-ITD was associated with worse outcome (OS at 5 yrs: 33% vs 74%, p=0.04). Thus, AML with complex karyotype, KMT2A or MECOM rearrangements had the worst prognosis, while cases with adverse cytogenetics and at least one mutation in either ASXL1, TP53 or FLT3-ITD have a slightly better outcome which is comparable to AML with intermediate risk cytogenetics harbouring one of these mutations. OS in AML with adverse cytogenetics without mutations in ASXL1, TP53 or FLT3-ITD is not worse than in AML with intermediate cytogenetics also lacking these mutations. A fusion gene or a molecular mutation as target for MRD monitoring was present in 791 patients (91%) when all genes analysed were taken into account. If only markers showing prognostic relevance, i.e. fusion genes, mutations in NPM1, CEBPA, FLT3-ITD, ASXL1 and TP53 were considered a MRD marker was still available in 726 cases (84%). Conclusions: 1) In AML a prognostication system is feasible based on the identification of t(15;17)/PML-RARA, t(8;21)/RUNX1-RUNX1T1, inv(16)/t(16;16)/CBFB-MYH11, 11q23/KMT2A rearrangements, 3q26/MECOM rearrangements, complex karyotype, and mutation status of NPM1, CEBPA, ASXL1, TP53, and FLT3-ITD (figure 1). 2) The analysis of these parameters allows to identify an MRD marker in 84% of patients. 3) The analysis of additional genes may be required in a comprehensive AML work-up as soon as novel targeted treatment strategies will become available. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Fasan: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.

TET2 Mutations Are Not Specific for Certain MPN Entities but More Likely Seem to Indicate Disease Progression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 438-438
Author(s):  
Susanne Schnittger ◽  
Claudia Tschulik ◽  
Nicole Wendland ◽  
Sonja Schindela ◽  
Frank Dicker ◽  
...  
Keyword(s):  
Genetic Markers ◽  
Myeloproliferative Neoplasms ◽  
Molecular Genetic ◽  
Normal Karyotype ◽  
Mutation Load ◽  
Coding Region ◽  
Total Cohort ◽  
Tet2 Mutation ◽  
Equity Ownership

Abstract Abstract 438 TET2 mutations have recently been described in various myeloid malignancies. To further evaluate the role of TET2 mutations in myeloproliferative neoplasms (MPN) we have analysed 96 MPN that have been well characterized by cytomorphology, cytogenetics and molecular genetics. The cohort consisted of 53 males and 43 females with a median age of 64.9 years (range: 16.6-86.3 years). Diagnosis was ET (n=22), HES (n=5), PMF (n=12), PV (n=32), MPN unclassifiable (MPN-u) (n=25). The ET, PMF and MPN-u were mainly selected for unmutated JAK2 status. Cytogenetics was availabel in 94/96 cases (98%). All ET and HES cases had a normal karyotype. In MPN-u 3 of 25 (12%), in OMF 3 of 12 (25%) and in PV 4 of 31(12.9%) revealed chromosomal aberrations. In all cases a BCR-ABL rearrangement was excluded. In addition in all cases mutation analysis for JAK2V617F, JAK2exon12, MPLW515 and CBL was performed. The total cohort was composed of 39 cases with JAK2V617F (3 × ET, 6 × PMF, 27 × PV, 3 × MPN-u), 5 cases with JAK2exon12 (all PV), 4 cases with MPLW515 (3 × ET, 1 × PMF), 2 cases with CBL mutation (both MPN-u). TET2 mutations were analyzed by amplification and sequencing of 21 PCR fragments covering the total coding region. Within the total cohort 20/96 cases (20.8%) revealed a TET2 mutation. Two different TET2 mutations in parallel were detected in three cases: one with MPN-u and two PV with homozygous JAK2V617F mutations. Throughout the gene the mutations were distributed as follows: exon4 (n=11), exon6 (n=4), exon7 (n=3), exon11 (n=5). 14 were missense, 3 nonsense and 6 were frameshift mutations. To analyze a further potential gene defect based on a TET2 deletion 15/20 cases from which methanol/acidic acid fixed cells were availabel were also analyzed by FISH (fluorescence in situ hybridization) for TET2 deletions. No deletion was detected in any of these cases. Thus with the exception of three cases with two different mutations all other mutated cases probably have retained one intact TET2 allele. With respect to diagnostic entities the TET2 mutations were distributed as follows, ET: 2/22 (9.1%), HES: 1/5 (20%), PMF: 4/12 (33.3%), PV: 9/31 (29%) and MPN-u: 4/27 (14.8%). With respect to other molecular genetic markers the TET2 mutations were distributed as follows: JAK2V617F: 10/20 (50%) (PV: n=8; PMF: n=2) from which 7/10 had JAK2V617F with a high mutation load (classified on the absence of a JAK2 wildtype allele) (PV: n=7; PMF: n=1), JAK2exon12: 1/5 (PV), MPLW515: 1/4 (PMF), CBL: 1/2 (MPN-u) , FIP1L1-PDGFRA: 1/5 case. Thus, 14/20 TET2 mutated cases (70%) revealed a detectable second mutation, 7 (50%) of which even with a high JAK2V617F mutation load. Taking also cytogenetics into account three further cases revealed aberrations resulting in a total of 17/20 TET2 mutated cases (85%) that had genetic markers in addition. 8/20 (40%) even had two or more genetic events in addition to the TET2 mutation. 2/3 cases with two TET2 mutation also had a very high JAK2V617F load. And five high load JAK2V617F cases had only a 50% TET2 mutation load, indicating that JAK2V617F was the first mutation in these cases followed by TET2 mutation as a second hit. There was no independent correlation of TET2 mutation with any of the analyzed MPN entities (p=0.359, n.s.). On the other hand TET2 mutations are more frequent in cases with further mutations compared to those without any other mutation irrespective of diagnosis (p=0.059). These data indicate that TET2 mutations 1) occur in all different subtypes of MPN and thus are no markers that indicate a specific entity, 2) are associated with other genetic markers that are more specific for certain MPN entities like FIP1L1-PDGFRA for HES, MPL for ET and PMF, JAK2exon12 for PV , 3) seem to be more likely associated with progression of MPN e.g. accumulation of mutations at least in MPN. Disclosures: Schnittger: MLL Munich Leukemia Lab: Equity Ownership. Tschulik:MLL Munich Leukemia Lab: Employment. Wendland:MLL Munich Leukemia Lab: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Lab: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Sign in / Sign up

Export Citation Format

Share Document