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.

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.

CBL Mutations Are Correlated with CMML, Frequently Associated with RUNX1 but Mutually Exclusive of JAK2V617F Mutations.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 962-962
Author(s):  
Susanne Schnittger ◽  
Andreas Reiter ◽  
Madlen Ulke ◽  
Alexandra Spiel ◽  
Frank Dicker ◽  
...  
Keyword(s):  
Chromosomal Aberrations ◽  
Myeloproliferative Neoplasms ◽  
Molecular Genetic ◽  
Normal Karyotype ◽  
Allelic Loss ◽  
Missense Mutations ◽  
Equity Ownership ◽  
Highly Correlated ◽  
Atypical Cml ◽  
Allelic Burden

Abstract Abstract 962 Mutations in exons 8 and 9 of the CBL gene containing the RING and linker domains were recently described in 10 % of CMML , 8% of atypical CML and single cases of PMF or HES (Grand et al., Blood 2009). However, the number of cases is still limited and further biological characterization scarce. We therefore performed CBL mutation analysis in a large cohort of well characterized myeloproliferative neoplasms (MPN). This cohort was selected according to the availability of cytomorphology, cytogenetics and further molecular genetic characterization. In total, 418 MPN patients were analysed. The cohort was composed of 182 males and 236 females with a median age of 68.3 years (range 16.3-93.3). Diagnoses according to cytomorphology were as follows: ET (n=88), PMF (n=33), PV (n=59), HES (n=14), CMML-1 (n=73), CMML-2 (n=26), MDS/MPN overlap (n=8), RARS-T (n=18), MPN unclassifiable (MPNu) (n=99). Cytogenetics was available in 382/418 cases (91.4%) and revealed a normal karyotype in 312 cases (81.7%) whereas 70 (18.3%) had chromosomal aberrations. A BCR-ABL rearrangement was excluded in all cases. Further molecular characterization revealed a JAK2V617F-mutation in 120/391 (30.7%), diverse JAK2exon12 mutations in 7/37 (18.9%) (all these cases were JAK2V617F unmutated PV), a MPLW515 mutation in 12/160 (7.5%), and TET2 mutations in 26/110 (23.6%) cases. Within the total cohort, CBL mutations were detected in 26/418 cases (6.2%). As recently described, all mutations were missense mutations and were clustered in a region spanning 50 amino acids around the RING and linker domains. Two of 28 mutations were detected in two cases each (C419Y and A420G), all others were single and as follows (L380P, L381G, L381P, I383M, C384T, I384T, D388G, D390V, C396Y, G397V, H398A, C404Y, W408C, C416R, G415S, C416S, C416Y, P417H, R420L, R421L, I423N, I429F, I429N, V430M) (according to ENST00000264033). In 12 /26 cases the detection of the CBL wildtype (wt) only indicated allelic loss, and two patients had two different mutations. The mutations were most frequently found in CMML-1 (14/73; 19.2%), CMML-2 (2/26; 7.9%), PMF (4/32; 12.5%) and MPN unclassifiable (7/99; 7.1%). In contrast, CBL mutations were not detected in PV, ET, HES, RARS-T and MDS/MPN overlap. The presence of CBL mutations was not correlated to age, gender, chromosomal aberrations or WBC in comparison to CBL unmutated cases within the respective subcohorts. In addition, all 26 CBL mutated cases were analysed for JAK2V617F, JAK2exon12, MPLW515, FLT3-ITD, MLL-PTD, NPM1, NRAS and RUNX1 mutations. TET2 sequence analysis was available in two cases. The CBL mutations were exclusive of all these mutations with the exception of RUNX1 and TET2. Four of 16 CMML cases (25%) with CBL mutations had a RUNX1 mutation and one MPNu case revealed a TET2 mutation. In CMML-1 11/14 (78.6%) cases showed loss of the wt allele or two different mutations and thus lack a functionally intact CBL allele. In contrast, nearly all CBL mutations in MPNu and all PMF had a low allelic burden. From four of the CBL mutated cases also a sample at an earlier time point (3 to 14 months) was available. In three cases the mutation was already present two to three months before when MPN was suspected but not diagnosed. One case clearly gained a D388G within 14 months between first appearance of leukocytosis and diagnosis of MPN which indicates that CBL mutation is a later event in this case. In conclusion, these data show that CBL mutations i) are highly correlated with CMML, ii) in CMML are associated with high allelic burden or two CBL mutations, iii) in CMML are frequently associated with RUNX1 mutations, iv) are rarely found in PMF and MPNu and are rare or absent in all other MPN, v) seem to be mutually exclusive of other mutations typical for MPN such as JAK2 and MPLW515 mutations. Thus patients with suspected MPN who lack other MPN typical mutations should be routinely screened for CBL mutations. Disclosures: Schnittger: MLL Munich Leukemia Lab: Equity Ownership. Ulke:MLL Munich Leukemia Lab: Employment. Spiel:MLL Munich Leukemia Lab: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Lab: Equity Ownership.

Development of An Oligonucleotide Resequencing Array for Rapid Mutation Analysis in Acute Myeloid Leukemia with Normal Karyotype.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 705-705 ◽  
Author(s):  
Susanne Schnittger ◽  
Michael Bonin ◽  
Christopher Schroeder ◽  
Christiane Eder ◽  
Frank Dicker ◽  
...  
Keyword(s):  
De Novo ◽  
Molecular Genetic ◽  
Normal Karyotype ◽  
High Sensitivity ◽  
Turnaround Time ◽  
Coding Region ◽  
Prognostic Relevance ◽  
Standard Design ◽  
Equity Ownership ◽  
New Mutations

Abstract Abstract 705 During the last years AML with normal karyotype (NK-AML) has been gradually elucidated by molecular genetic mutations. The constellation of specific mutation patterns was shown to have prognostic relevance (e.g. NPM1 mutated/FLT3 unmutated correlate with good prognosis). The growing prognostic relevance and the development of specific drugs forces the need for tools that costsaving analyze a large number of genes within a short turn around time. Oligonucleotide resequencing arrays can explore base exchanges by standard design or known mutations by specific probe designs representing the exact sequence of the mutation. For analysis of NK-AML we have developed a customized oligonucleotide resequencing array (Affymetrix, Santa Clara, USA) covering 13 AML relevant genes (CEBPA, FLT3, JAK2, KIT, KRAS, MLL, MPL, NPM1, NRAS, PTPN11, RUNX1, TP53 and WT1). Special probe designs were used for the most frequent mutations like NPM1_A, NPM1_B, NPM1_D, JAK2V617F, KITD816V, FLT3D836, NRAScodon12, 13, and 61 mutations. 2 μg of DNA for each patient were required and the workflow comprises 128 PCR reactions, pooling, fragmentation, hybidization, staining, washing, scanning, and evaluation. All steps were optimized to allow completion within 3 days. For each patient the whole coding region of all genes (34 kb) was amplified in 128 PCR reactions. In total 64 NK-AML patients were analyzed that were precharacterized by standard methods for CEBPA, FLT3-ITD, FLT3-TKD, JAK2, KITD816, MLL-PTD, NPM1, NRAS, RUNX1 and WT1exon 7 and 9.The patients were selected based on the detection of at least 2 different mutations and these included CEBPA: n=16, FLT3-ITD: n=29, FLT3-TKD: n=11, JAK2: n=2, KITD816: n=4, MLL-PTD: n=11, NPM1type A: n=15, NPM1 type B: n=3, NPM1 type D: n=2, rare NPM1 types: n=8, NRAS: n=8, and RUNX1: n=12. Using this newly designed resequencing array and after evaluation with the JSI software (Medical System Kippenheim, Germany) identification of the previously known base exchanges was achieved in 11/11 in FLT3-TKD, 2/2 JAK2V617F, 8/8 NRAS, 3/4 KITD816V, 2/2 CEBPA and 2/2 in RUNX1 resulting in a sensitivity of 27/28 (96.4%). One KITD816 mutation was not detected as it was present in less than 10% of cells and thus was below the specifications of the array. In addition, by use of the mutation specific designs all NPM1 type A (n=15), B (n=3) and D (n=2) mutations and 7/8 of the rare NPM1 mutations were detectable, resulting in an NPM1 total detection rate of 27/28 (96.4%). As expected, with currently available software tools it was not possible to detect mutations that cause dose effects like FLT3-ITD or MLL-PTD or insertion/deletion mutations. In contrast, new mutations were detected in genes and regions that were not covered by standard techniques. These latter mutations were subsequently verified by Sanger sequencing: five so far undetected mutations in FLT3 were V491L; G549A, G481E, V194M, and N676T. Although the clinical relevance of most of these mutations is unclear N676T has been implicated in PKC412 resistance. Furthermore, four previously not described mutations were detected in JAK2 (V446G, G571S, E846D, R1063H).Three new mutations were found in MPL (W416X, W632C, L594W). Similar MPL mutations have been described in familial thrombocythemia and although these patients have a high risk for development of AML these mutations have so far not been implicated in de novo AML. Three new mutations were detected in WT1 (G44V, E47V, H137R), 3 in KRAS (Q61L (n=2) and V114L), one in PTPN11 (E76K) and four mutations of unknown significance in MLL (G276G, E502K, A822V, S901R). Thus, in total 24 new mutations were detected in 64 patients (38%) in addition to the respective two mutations detected by conventional techniques indicating that a significant subset of AML carry more mutations than reflected by standard approaches. Although the clinical significance of most of these mutations is unclear this may have implications in new drug development and study design. In conclusion, using this newly designed customized AML resequencing array 34 kb per patient can be analysed with a turnaround time of approximately 3 days, including analysis. Beside the high sensitivity of 96.4% to detect common missense or known mutations this methods has a high potential to detect new mutations that can not be covered by present routine techniques. Disclosures: Schnittger: MLL Munich Leukemia Lab: Equity Ownership. Bonin:Microarray Facility Tübingen: Employment. Schroeder:Microarray Facility Tübingen: Employment. Eder:MLL Munich Leukemia Lab: 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.

MDS and AML With ≥15% Ring Sideroblasts Share Overlapping Features In Cytogenetics But Demonstrate Different Patterns and Incidences Of SF3B1 mutations

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2776-2776
Author(s):  
Tamara Alpermann ◽  
Sabine Jeromin ◽  
Claudia Haferlach ◽  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
Normal Karyotype ◽  
Amino Acid Position ◽  
Prognostic Impact ◽  
Mutation Load ◽  
Employment Equity ◽  
Total Cohort ◽  
Ring Sideroblasts ◽  
Equity Ownership

Abstract Background SF3B1 mutations (SF3B1mut) correlate with the presence of ring sideroblasts (RS) and can be found in MDS and in AML. Aim To evaluate the incidence of SF3B1mut in a large cohort of MDS and AML patients (pts) with ≥15% RS, and furthermore correlate to percentage of blasts, mutation load, concomitant genetic markers and to define their prognostic impact. Patients and Methods We investigated bone marrow (BM) in 1,238 newly diagnosed pts with MDS (n=770) and AML (n=468). In all cases MGG, MPO, NSE and iron staining was performed according to WHO criteria. 717 pts showed ≥15% RS and thus were included in this study. In all pts SF3B1mut and cytogenetic analysis was available. Results 579/717 (80.8%) pts were diagnosed with MDS (93.3% de novo; 6.7% therapy-related), and 138/717 (19.2%) with AML (61.6% de novo, 33.3% secondary, and 5.1% therapy-related). MDS subtypes were distributed as follows: 329 (56.8%) RCMD, 126 (21.8%) RARS, 63 (10.9%) RAEB-1, 55 (9.5%) RAEB-2, and 6 (1.0%) MDS with isolated del(5q). AML FAB subtypes were as follows: 11 (8.0%) M0; 10 (7.2%) M1; 70 (50.7%) M2, 14 (10.1%) M4, and 33 (23.9%) M6. Mean percentage of RS was 50.4% and differed between MDS and AML (52.9% vs 40.1%; p<0.001). Within the MDS cohort mean RS differed between the MDS WHO categories following an ascending order from MDS with isolated del(5q) (36.8%), RAEB-2 (40.0%), RAEB-1 (45.0%), RCMD (55.3%), to RARS (57.0%). In contrast, no differences were seen within the different AML FAB subtypes (mean RS M0: 40.3%, M1: 37.0%, M2: 40.1%, M6: 44.2%). Per definition, mean BM blasts differed between MDS and AML (3.6% vs 32.6%; p<0.001). Of note, percentages of RS and BM blasts were negatively correlated in the total cohort (p<0.001; r: -0.253) as well as for the cohort of MDS (p<0.001; r: -0.238) and showed a respective trend within the cohort of AML (p=0.072; r: -0.154). Within the cohort of MDS percentages of RS were higher in SF3B1mut vs wild-type (wt) pts (59.1% vs 42.3%; p<0.001) and mutation load of SF3B1mut (median 37.5%; range 10%-60%) correlated to the amount of RS (p<0.001, r: 0.258). No respective difference or correlation was seen within the AML cohort. Regarding cytogenetics SF3B1mut were more frequent in pts with normal karyotype than in pts with aberrant karyotype in the MDS cohort (76.1% vs 43.7%; p<0.001) as well as in the AML cohort (48.7% vs 18.2%; p=0.001). Further in the total cohort SF3B1mut were less frequent in ASXL1mut than in ASXL1wt (24.0% vs 48.5%; p=0.041), and within the AML cohort SF3B1mut showed a positive correlation to MLL-PTD (71.4% vs 25.7%; p=0.019). Additionally, we analyzed the position of the SF3B1mut. Within the total cohort 21 different amino acid positions were affected. We focused on the most frequent positions: 700 (55.9%), 666 (16.2%), 662 (8.0%), 625 (7.5%), 622 (4.0%), and 663 (1.7%). Mutations at position 666 were less frequent within MDS than in AML pts (14.3% vs 35.1%; p=0.003) and mutations at amino acid position 662 indicated a trend to be prevalent in MDS only (8.8% vs 0.0%; p=0.059). In addition, an analysis was performed for the contiguous subcohorts of 69 MDS with BM blasts between 10-19% and 44 AML with 20-29% (formerly RAEB-t). Neither differences in mean percentage of RS (38.7% vs 39.3%; n.s.), frequencies of SF3B1mut (17.4% vs 22.7%; n.s.), nor differences within the position of the mutation were identified. Follow-up data was available in 304 patients. Within the cohort of MDS SF3B1mut pts had better overall survival (OS) than SF3B1wt pts (5-year-survival rate 72.7% vs 35.2%; p<0.001). This holds true within the subcohort of normal karyotype (75.0% vs 35.6%; p=0.004) and within aberrant karyotype (67.6% vs 39.7%; p=0.012). No respective effect on OS was seen within the AML cohort. Also within the subgroup of early MDS (RCMD, RARS, and MDS with isolated del(5q); n=222) SF3B1mut pts had better OS than SF3B1wt pts (74.9% vs 48.2%; p<0.001), this holds true in patients with normal as well as in patients with aberrant karyotype (77.4% vs 56.1%; p=0.095 and 68.5% vs 44.6%; p=0.042, respectively). In contrast SF3B1 mutation status had no impact on OS within the cohort of MDS with excess of blasts (RAEB-1 and RAEB-2 together n=45). Conclusions 1) Percentages of RS are decreasing with increasing BM blasts percentages. 2) Different mutations within the SF3B1 gene are correlated to either MDS or AML. 3) The prognostic impact of SF3B1mut was only observed in patients with early MDS, but not in RAEB-1/2 or AML. Disclosures: Alpermann: MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 999-999
Author(s):  
Susanne Schnittger ◽  
Tamara Weiss ◽  
Frank Dicker ◽  
Jana Sundermann ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
De Novo ◽  
Myeloproliferative Neoplasms ◽  
Blast Crisis ◽  
Nras Mutation ◽  
Npm1 Mutation ◽  
Secondary Aml ◽  
Second Hit ◽  
Total Cohort ◽  
Equity Ownership ◽  
De Novo Aml

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

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.

Recurrent ATM and BIRC3 Mutations in Patients with Chronic Lymphocytic Leukemia (CLL) and Deletion 11q22-q23

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1771-1771
Author(s):  
Vera Grossmann ◽  
Alexander Kohlmann ◽  
Susanne Schnittger ◽  
Sandra Weissmann ◽  
Sabine Jeromin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Deep Sequencing ◽  
Chromosome Band ◽  
Prognostic Impact ◽  
Coding Region ◽  
Chromosome 11 ◽  
Mutation Status ◽  
Frame Shift ◽  
Equity Ownership

Abstract Abstract 1771 Introduction: Deletion of the long arm of chromosome 11 (11q22-q23) is one of the most common chromosome aberrations (∼12%) in CLL and is associated with rapid disease progression and shorter overall survival (OS). The commonly deleted region contains the genes ATM and BIRC3. ATM (chromosome 11q22.3) plays a central role in activating TP53, DNA recombination processes, and cell cycle control. ATMmut were described to occur in ∼35% of CLL patients with 11q deletions. The apoptosis inhibitor BIRC3 is located on chromosome band 11q22.2, 6.0 Mb proximal of ATM. BIRC3mut have recently been described with a mutation frequency of 4% in CLL (Rossi D, Blood 2012). Aims: 1. Determine the frequency of deletions and mutations of ATM and BIRC3 in CLL with 11q deletion. 2. Analyze the association of ATM and BIRC3 mutations with ATM and BIRC3 deletions. 3. Characterization of the landscape and prognostic impact of ATM and BIRC3 mutations. Patient and Methods: The investigated cohort comprised 60 de novo CLL cases harboring del(11q) identified by chromosome banding analysis. The cohort comprised 47 males and 13 females, median age was 68.9 years. Survival data was available in 38 cases. For sensitive mutation detection of the complete coding region of ATM (3056 amino acids) and BIRC3 (604 amino acids) an amplicon deep-sequencing approach (454 Roche Life Sciences, Branford, CT) was developed. Patients were further characterized for mutation status of TP53 (n=60), SF3B1 (n=59), and IGHV (n=60). Additionally, patients were investigated by interphase FISH for ATM (n=60; MetaSystems, Altlussheim, Germany) and BIRC3 (n=39; BAC RP11–177O8, BlueGnome, Cambridge, UK) deletions. Results: Based on FISH results, all 60 cases showed an entire gene deletion of ATM. Of those cases investigated for BIRC3del, 34/39 (87.2%) cases showed a deletion, thus 5/39 (12.8%) carried an ATMdel only. Mutational screening of the ATM and BIRC3 genes identified 24 ATMmut in 19/60 (31.7%) patients and 3 BIRC3mut in 3/60 (5.0%) cases. Additional mutations were detected in TP53 (3/58; 5.2%), and the spliceosome machinery gene SF3B1 (10/59, 16.9%). 49/60 (81.7%) cases were IGHV unmutated, whereas 11/60 (18.3%) cases were IGHV mutated. The majority of ATMmut were found to be missense mutations (n=15) followed by nonsense (n=4), frame-shift (n=4), and in-frame (n=1) variants. In more detail, only one mutation occurred in two distinct patients (p.Ile2888Thr). Most cases showed only one mutation (n=15, 78.9%), whereas 3 cases (15.8%) showed two and one case (5.3%) three mutations. Mutations were spread across the entire coding region (exon 3–59). The median mutational burden as assessed by deep-sequencing read counts was 38.5%, ranging from 4.0–89.5%. For BIRC3 3 deletions (1–4 bp) were observed resulting in 2 frame-shift and 1 splice-site mutation. The median mutation load was 32.5% (range: 20.0%-92.0%). In addition, two cases with BIRC3mut showed also a BIRC3del (n=1 data not available). Interestingly, all of these 3 cases with BIRC3mut were wild-type in the ATM gene. To further address the role of ATMmut association analyses with respect to age, gender, white blood cell count, haemoglobin level, and platelet count were performed, however, no correlations were observed. Further, ATMmut were not correlated with IGHV (3/11 vs 16/49, P=1.00), TP53 (0/3 vs 19/55, P=0.544) or SF3B1 (3/10 vs 15/49, P=1.00) mutation status. However, the presence of ATMmut in CLL with del(11q) was associated with shorter OS (76.2% vs 95.0% at 3 years), although this difference was not significant. However, when comparing OS of ATMmut cases to cases without del(11q) (n=1,245) we detected a trend towards a shorter OS (76.2% vs 94.2% at 3 years, P=0.149) in contrast to cases with del(11q), but without ATMmut in comparison to cases with no del(11q) (95.0% vs 94.2%, P=0.731). Conclusions: 1. BIRC3 was mutated in only 5.0%, whereas ATM was mutated in 31.7% of patients with del(11q). 2. No mutation hotspot regions were observed, thus analyses of the whole genes are mandatory. 3. ATMmut were associated with a trend to shorter OS in CLL with del(11q). In comparison to cases without del(11q), cases with ATMmut showed shorter OS, indicating a more relevant role of ATMmut on prognosis versus cases with a del(11q) only. 4. In 38 cases (63.3%) with del(11q) neither ATM nor BIRC3 mutations were detected suggesting that further genomic alterations which are not yet identified play a role in CLL with del(11q). Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Weissmann:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Kienast:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Homozygosity of Mutated Calreticulin in Myeloproliferative Neoplasms By an Acquired Chromosome 19p UPD Is Associated with Deletions in the Long Arm of Chromosome 5 and SF3B1 Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3145-3145
Author(s):  
Anna Stengel ◽  
Manja Meggendorfer ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
Chromosomal Aberrations ◽  
Tp53 Mutation ◽  
Progressive Disease ◽  
Array Cgh ◽  
Mutation Load ◽  
Employment Equity ◽  
Tet2 Mutation ◽  
Equity Ownership ◽  
Calr Mutation

Abstract Background: CALR is mutated in about 70% of patients with essential thrombocythemia and primary myelofibrosis lacking mutations in JAK2 and MPL. High mutational loads (≥ 60%) are seen less frequently in CALR mutated cases than in JAK2 or MPL mutated cases. Especially for JAK2 uniparental disomy (UPD) of 9p24 is commonly detected leading to homozygosity of JAK2V617F and progressive disease. Until now, in CALR mutated patients UPD has only rarely been detected and data on these cases are scant. Aim: (1) Detection of cases with CALR UPD in a cohort of MPN cases with high CALR mutation loads and/or progressive disease, (2) cytogenetic and molecular genetic characterization of these cases in comparison to CALR mutated patients without UPD. Patients and Methods: Overall 50 cases with a CALR mutation were analyzed by genomic arrays (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany). Caseswere selected as follows: (1) mutation load ≥ 60% determined by gene scan analysis (n=26), (2) progressive disease (accelerated phase: n=7, blast crisis: n=5) according to cytomorphology and/or (3) displaying mutations in ASXL1, SRSF2, EZH2 and/or IDH1/2 as markers for progression (n=12). Additionally, amplicon next-generation sequencing was performed to detect mutations in ASXL1, CBL, DNMT3A, EZH2, IDH1/2, KRAS, NRAS, RUNX1, SF3B1, SRSF2, TET2, TP53 and U2AF1. Variants of unknown significance were excluded from statistical analysis. Results: 11/50 cases (22%) with UPD were identified by array CGH. While the type 1 (c.1099_1150del52) CALR mutation was the most frequent mutation in cases without UPD (25/39, 64%), type 1 mutation was rare in CALR UPD cases (1/11, 9%; p=0.009). Thus, in cases with UPD, mainly the type 2 (c.1154_1155insTTGTC) mutation (5/11, 45%) and rare mutation types were detected (5/11, 45%). Of note, the only case with CALR UPD showing the type 1 mutation was found to harbor a TET2 mutation and UPD encompassing 4q23q28, which includes the TET2 gene. As the CALR mutation load was clearly lower compared to the TET2 mutation load (50% vs. 100%), the CALR mutation might only belong to a subclone in this case. Cases with CALR UPD showed a higher mutation load than cases without UPD (mean: 77% vs. 48%, p<0.001) and presented more frequently with MPN in acceleration (5/11, 46% vs. 6/39, 15%, p=0.048). Almost all of the patients (4/5, 80%) in accelerated phase with UPD showed a mutation load ≥60%.The remaining case was the patient with concomitant TET2 and CALR UPD. Regarding chromosomal aberrations, del(13q) (12/50, 24%), del(5q) (9/50, 18%), del(20q) (6/50, 12%) and gain of 1q (5/50, 10%), have been detected as recurrent abnormalities by array CGH analyses in the total cohort. Del(5q) showed a trend to be more frequent in cases with UPD (4/11, 36% vs. 5/39, 13%; p=0.093). Corresponding to the advanced disease state of the selected cohort, mutation analyses revealed a high frequency of ASXL1 mutations (44%), followed by mutations in TET2 (19%), EZH2 (13%), TP53 (13%), U2AF1 (9%), NRAS (9%) and SF3B1 (7%). Interestingly, SF3B1 mutations were exclusively detected in cases with CALR UPD (3/11, 27% vs. 0/34, 0%; p=0.012), whereas ASXL1 mutations tended to be more frequent in cases without UPD (19/37, 51% vs. 2/11, 18%, p=0.083). TP53 mutations were also detected more often in the subgroup of cases with CALR UPD, although this was not statistically significant (3/11, 27% vs. 3/35, 9%). Additionally, TP53 mutations showed a significant correlation to del(5q) (TP53 mutation in cases with del(5q): 5/9, 56% vs. TP53 mutation in cases without del(5q): 1/37, 3%, p=0.001). Comparison of the CALR mutation loads with mutation loads of accompanying mutations revealed that in most cases, the CALR mutation is found in the main clone. Conclusions: In cases with high CALR mutation loads and/or progressive disease UPD of 19p13 is rather common (22%). These cases show a distinct pattern of chromosomal aberrations and additional molecular mutations, as they are associated with del(5q) and mutations in SF3B1 and TP53, whereas ASXL1 mutations are less frequent. Due to the limited number of cases, these results have to be verified in future analyses. Disclosures 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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment.

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.

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