Evaluation of the New Genetic Risk Classification of the European LeukemiaNet Recommendations in 1,110 Patients with De Novo AML and Proposal of a Refined Version

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 413-413
Author(s):  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
Torsten Haferlach
Keyword(s):  
Molecular Markers ◽  
De Novo ◽  
Prognostic Impact ◽  
Employment Equity ◽  
Female Ratio ◽  
Mutation Status ◽  
Equity Ownership ◽  
De Novo Aml ◽  
Favorable Group ◽  
Refined Version

Abstract Abstract 413 Background: The recently published recommendations for prognostication in AML (Döhner et al. for ELN, Blood 2010;115,453–474) were based on a review of the literature and included cytogenetics as well as NPM1, CEBPA and FLT3-ITD mutation status for risk stratification. We here aimed to evaluate the prognostic impact of this approach in an independent cohort. Patients: We started with a cohort of 1,428 adults with newly diagnosed AML, which were investigated by cytomorphology, immunophenotyping, cytogenetics, and molecular genetics. We first excluded patients with t(15;17) (n=59), therapy-associated AML (n=111) and secondary AML (n=148). Thus, 1,110 patients with de novo AML and cytogenetics available in all cases were further evaluated according to ELN criteria. The following molecular markers were investigated: NPM1 (1,064/1,110), FLT3-ITD (1,066/1,110), CEBPA (880/1,110), MLL-PTD (1,064/1,110) and RUNX1 (454/1,110). Results: Male/female ratio was 1.2 (598/512), median age was 66.6 years (range 18.3 – 100.4). According to the ELN proposal 297 (26.8%) pts were assigned to the favorable group (CBF leukemias, NPM1mut/without FLT3-ITD in normal karyotype (NK), or CEBPAmut in NK), 363 (32.7%) pts were classified as intermediate I (NPM1mut/FLT3-ITD+, or NPM1wt/FLT3-ITD+, or NPM1wt without FLT3-ITD; all NK), 249 (22.4%) as intermediate II (t(9;11) or cytogenetic abnormalities not classified as favorable or adverse), and 201 (18.1%) as adverse (inv(3)/t(3;3); t(6;9); t(v;11)(v;q23); −5 or del(5q); −7; abn(17p); complex karyotype, i.e. ≥ 3 chromosome abnormalities)). Evaluation according to these criteria revealed significant differences in overall survival between the favorable subgroup and all other subgroups (inter I p<0.001; inter II 0.008, adv <0.001). Also adverse vs all other subgroups (all p<0.001) differed significantly with respect to OS. However, no significant differences were observed between both large cohorts of inter I and inter II (together 55.1% of all pts). We therefore intended to revise the ELN criteria for better discrimination of the intermediate groups. In addition to ELN recommendations we considered a threshold of 0.5 for the FLT3-ITD ratio (mut/wt) which had been suggested more valid for prognostication than the mutation status per se. For the revised classification molecular markers were mandatory for all cases with intermediate risk cytogenetics. Therefore, 100 cases had to be excluded due to missing data. Thus, 1,010 pts were reclassified into our new subgroups defined as: favorable I: CBF leukemias; favorable II:NPM1mut or biallelic CEBPAmut (without any other molecular marker and no fav or adv cytogenetics); intermediate I:FLT3-ITD ratio <0.5 (without RUNX1 or MLL-PTD and no fav or adv cytogenetics); intermediate II:FLT3-ITD ratio ≥0.5 and/or RUNX1mut and/or MLL-PTD+ (and no fav or adv cytogenetics); adverse: as defined by ELN. Patients were distributed as follows: fav I: 68 (6.7%), fav II: 286 (28.3%), inter I: 157 (15.5%), inter II: 298 (29.5%), adv: 201 (19.9%). Fav I and fav II had no significant differences in OS (median n.r. vs 62.2 mo, n.s.) and therefore were grouped together as “favorable”. This finally leads to four different prognostic subgroups: favorable: CBF leukemias; NPM1mut or biallelic CEBPAmut, intermediate I:FLT3-ITD ratio <0.5, intermediate II:FLT3-ITD ratio ≥0.5 and/or RUNX1mut and/or MLL-PTD+, adverse. Patients were distributed as follows: fav: 354 (35.0%), inter I 157 (15.5%), inter II: 298 (29.5%), adv: 201 (19.9%). Median OS differed between all subgroups: fav 62.2, inter I 24.3, inter II 12.4, adv 8.7 mo. (fav vs inter I p=0.058, vs inter II <0.001, vs adv <0.001; inter I vs inter II 0.004, vs adv <0.001; inter II vs adv 0.039). Conclusion: The new ELN proposal for prognostication in de novo AML is based on cytogenetic and molecular genetic data. Based on this proposal we further improved prognostication in a series of 1,010 pts by integrating the following molecular markers besides cytogenetics: NPM1mut, biallelic CEBPAmut and FLT3-ITD ratio <0.5 for the favorable group and FLT3-ITD ratio ≥0.5, other CEBPAmut, MLL-PTD+, or RUNX1mut for the intermediate group, and adverse based on cytogenetics only. This refined version may contribute to a better risk assessment in de novo AML patients allowing to separate 4 subgroups with striking differences in OS. Disclosures: Alpermann: 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.

AML with RUNX1 Mutations and Loss of RUNX1 Wild-Type - a Distinct Subset?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2578-2578
Author(s):  
Claudia Haferlach ◽  
Niroshan Nadarajah ◽  
Annette Fasan ◽  
Karolína Perglerová ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
De Novo ◽  
Chromosome 21 ◽  
Prognostic Impact ◽  
Wild Type ◽  
Mutation Load ◽  
Employment Equity ◽  
Runx1 Gene ◽  
Equity Ownership ◽  
De Novo Aml ◽  
Runx1 Mutation

Abstract Background: Mutations in RUNX1 have been reported in 5 to 20% of AML. RUNX1 mutated AML is associated with a myeloid rather than monocytic differentiation, shows a typical pattern of cytogenetic abnormalities with a high frequency of trisomy 8 or 13, has a typical pattern of additional molecular mutations with a high frequency of accompanying ASXL1 and SF3B1 mutations and is nearly mutually exclusive of NPM1 and CEBPA double mutations and other entity-defining genetic abnormalities. In a subset of patients with RUNX1 mutations loss of the wild-type allele can be assumed due to a high mutation load. The aim of this study was the detailed analysis of a subset of RUNX1 mutated AML with RUNX1 wild-type loss with respect to accompanying cytogenetic and molecular genetic abnormalities and prognostic impact. Patients and Methods: A cohort of 467 AML with RUNX1 mutations (mut) at diagnosis identified during diagnostic work-up in our laboratory were the basis of this study. Median age was 72 years (yrs) (range 18-91 yrs), and male:female ratio 296: 171. 366 patients had de novo AML, 77 s-AML following MDS, 24 t-AML. For all patients (pts) cytogenetics and for 341 data on FAB subtype was available. Mutation data was available for NPM1 (n=456), MLL-PTD (n=453), CEBPA (n=449), FLT3-ITD (n=457), FLT3-TKD (n=457), WT1 (n=398), ASXL1 (n=313), TP53 (n=231), DNMT3A (n=177), TET2 (n=174), NRAS (n=305), KRAS (n=213) and SF3B1 (n=119). 64 patients with a mutation load of RUNX1 mutation >70% evaluated by sequencing analysis were selected for further analysis. All 64 cases were analysed by genomic arrays (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany) to determine the copy number state and copy neutral loss of heterozygosity (CN-LOH). Median age was 73 yrs (range 24-87 yrs), and male:female ratio was 27: 37. 50 patients had de novo AML, 11 s-AML following MDS, 3 t-AML. Results: Array CGH revealed a cytogenetically cryptic deletion on the long arm of chromosome 21 encompassing the RUNX1 gene in 5/64 (8%) patients while a CN-LOH on 21q including the RUNX1 gene was observed in 45 cases (70%). Thus in 50 cases (78%) with a high RUNX1 mutation load a RUNX1 wild-type loss (wt-loss) was detected by array CGH. In 43% (6/14) of the remaining cases the high RUNX1 mutation load was caused by amplification of the long arm of chromosome 21 either due to gain of whole chromosomes 21 or to an isochromosome 21q. First we focused on the characterization of RUNX1 mutated cases with RUNX1 wt-loss. In 22/50 cases (44%) an aberrant karyotype was observed with a distinct aberration pattern. 11 cases harbored +13, 5 had +8 and 6 cases a loss of 7q. No other recurrent abnormalities were observed. With respect to concurrent mutations the following frequencies were found: ASXL1 (42%), FLT3 -ITD (34%), TET2 (21%), KRAS (11%), MLL-PTD (8%), NRAS (7%), and FLT3-TKD (6%). No NPM1 mutation or CEBPA double mutations were identified. Comparison of those cases with RUNX1 wt-loss to all other RUNX1 mutated AML (n=417) revealed a significantly higher frequency of +13 (22% vs 9%, p=0.01) and FLT3 -ITD (34% vs 19%, p=0.015). FAB subtypes M0 and M1 were more frequent (46% vs 12%, p<0.001; 35% vs 22%, n.s.) and M2 and M4 less frequent (14% vs 46%, p<0.0001; 5% vs 17%, n.s.). Survival analyses were restricted to 212 de novo AML pts with RUNX1 mut who received intensive chemotherapy (median overall survival (OS): 20 months (mo), median event-free survival (EFS): 12 mo). Median OS and EFS was shorter in patients with RUNX1 wt-loss compared to those without (15 vs 20 mo, n.s., 10 vs 12 mo, p=0.04). In univariate Cox regression analysis a negative impact on OS was observed for RAS mut (relative risk (RR): 2.2, p=0.005), male gender (RR: 1.6, p=0.02), and age (RR: 1.3 per decade, p<0.001). Shorter EFS was associated with RUNX1 wt-loss (RR: 1.7, p=0.04), RAS mut (RR: 1.9, p=0.02) and age (RR: 1.2 per decade, p<0.001). In multivariate analysis RAS mut (OS: RR: 2.4, p=0.002; EFS: RR: 2.0, p=0.008) and age (OS: RR: 1.3 per decade, p<0.001; EFS: RR: 1.2 per decade, p<0.001) had independent prognostic impact. Conclusions: RUNX1 mutated AML with wild-type loss is a distinct AML subset that does not overlap with any of the genetically defined WHO categories and is characterized by an immature phenotype (81% FAB Subtype M0 and M1) and a higher frequency of +13 and FLT3-ITD as compared to RUNX1 mutated AML without wild-type loss. Wild-type loss and RAS mutations are associated with inferior outcome in RUNX1 mutated AML. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah: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. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

IDH Mutations Can Be Detected In 28.7% of All Normal Karyotype AML and Have Unfavourable Impact on the NPM1+/FLT3-ITD- Genotype

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 102-102
Author(s):  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
Torsten Haferlach
Keyword(s):  
De Novo ◽  
Hot Spot ◽  
Normal Karyotype ◽  
Prognostic Impact ◽  
Employment Equity ◽  
Therapeutic Decisions ◽  
Idh Mutations ◽  
Equity Ownership ◽  
De Novo Aml ◽  
The Impact

Abstract Abstract 102 Introduction: Mutations in IDH1 and IHD2 have recently been shown to play an important role in AML. As they code for enzymes from the citric acid cycle mutations within these genes from the mechanistical point of view are a totally new kind of mutation associated with AML. In IDH1 one mutational hot spot (amino acid R132) and in IDH2 two hotspots (R140 and R172) have been reported. We aimed at further delineating the impact of IDH1 and IDH2 mutations in AML and analyzed the interaction with other mutations in normal karyotype (NK) AML. Methods: 526 AML patients were selected according to normal karyotype and availability of mutational status for FLT3-ITD, NPM1 and MLL-PTD. Further mutation analyses were available in subgroups of the cohort (FLT3-TKD: n=318, CEBPA: n=369, RUNX1: n=174, NRAS: n=220). Female/male ratio was 283/243 and age ranged from 20.0–90.1 years (median, 66.9 years). 435 had de novo AML (82.6%), 71 AML following MDS (s-AML,13.5%) and 20 AML after previous treatment of other malignancies (t-AML, 3.8%). The respective base exchanges in R132, R140, and R172 were analysed by a melting curve assay with subsequent sequencing of the positive samples. Results: Overall, in 151 pts (28.7%) IDH mutations (IDHmut) were detected. In detail, 68 mutations (12.9% of all cases) were detected in IDH1 (R131C: n=35, R131L: n=17, R131H: n=7, R131G: n=6, R131S: n=3) and 83 mutations (15.8%) in IDH2 (R140Q: n=72, R140L: n=2, R140W: n=1, N141G: n=1, R174K: n=7). IDH1mut and IDH2mut were mutually exclusive in this cohort. IDH1mut were more frequent in females (18.2% vs 8.6 % in males, p=0.001), whereas there was no sex difference for IDH2. According to history IDH1 was equally distributed in de novo AML, s-AML and t-AML whereas IDH2 was more frequent in de novo compared to s- and t-AML (19.6% vs. 7.6 vs 11.8%, p=0.048). According to FAB the most prevalent subtype was FAB M1 with IDHmut in 23.2% compared to 9.8% in all other FAB (in detail: IDH1: 44.8% vs. 23.9%, IDH2: 27.0% vs. 15.1%; p<0.001, for both). IDH1 was underrepresented in M4 (4.9% vs. 15.0 % in all other subtypes, p=0.004), whereas the distribution of IHD2 was not different in M4 vs. all others. The immunophenotype (n= 297) of IDHmut cases tended to be more immature and featured a lower expression of monocytic markers. The analyzed 78 IDHmut cases, as compared to 219 IDHwt cases, showed a significantly higher expression of MPO and CD117 while CD116, CD11b, CD14, CD15, CD36. CD56, CD64, CD65 and CD7 were lower expressed. Age, WBC count, and platelet count were not different between IDH1, IDH2 and IDHwt cases. IDH mutations are not mutually exclusive of other mutations. However, the frequency of CEBPAmut in IDHmut compared to IDHwt was decreased (7.7% vs. 13.7, p=0.001) (IDH1: 0% vs 11.7%, p=0.022 and IDH2: 7.7% vs 13.4%, p=0.053). MLL-PTD was more frequent in IDHmut vs. IDHwt (44.7 vs. 5.8%, p=0.039), however, this is restricted to IDH1mut vs. IDH1wt (26.3 vs. 6.3%, p=0.018). RUNX1mut are distributed equally in IDH2mut and IDH2wt (20.0% vs 27.3%) but are underrepresented in IDH1mut compared to IDH1wt (2.2% vs. 28.7%, p=0.068). FLT3-ITDs are equally distributed between IDHmut and IDHwt, however, those IDH1mut with FLT3-ITD have lower FLT3-ITD/FLT3wt ratios compared to FLT3-ITD+ IDH1wt cases (mean: 0.16 vs. 0.72; p=0.005). All other mutations were distributed equally in IDHmut compared to IDHwt. For survival analysis only cases with de novo AML <65 years were included (n=164, IDHmut: n=37, n=, IDHwt: 127). In the total analysis there was no effect on overall survival or event free survival (EFS). However there was a trend for shorter EFS of the IDHmut vs. IDHwt (median: 439 days vs. not reached, p=0.080) in cases with NPM1+/FLT3-ITD- genotype. For IDH2 there was a significant adverse effect in the NPM1+/FLT3-ITD- group (median EFS: 397 vs. 679 days, p=0.045). Summary: IDH mutations belong to the most frequent mutations in NK AML and can occur together with all other known mutations. There is a high preponderance for the FAB M1 subtype and a more immature immunophenotype for both IDH mutations and a strong female preponderance for IDH1. In addition, an adverse prognostic impact of IDH mutations was shown for the NPM1+/FLT3-ITD- genotype. Further analyses should focus on the definition of the role and place of IDH mutations for therapeutic decisions in patients with AML. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. 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.

Further Insights Into The Molecular Landscape Of De Novo Acute Myeloid Leukemia (AML) Investigating 1,291 Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 607-607
Author(s):  
Torsten Haferlach ◽  
Ulrike Bacher ◽  
Tamara Alpermann ◽  
Wolfgang Kern ◽  
Alexander Kohlmann ◽  
...  
Keyword(s):  
De Novo ◽  
Median Number ◽  
Genetic Alterations ◽  
The Cancer Genome Atlas ◽  
Research Network ◽  
Prognostic Impact ◽  
Coding Region ◽  
Employment Equity ◽  
Equity Ownership ◽  
De Novo Aml

Abstract Background The Cancer Genome Atlas Research Network (TCGA) published a hallmark sequencing study on molecular mutations in 200 fully characterized adult de novo AML (NEJM 2013). According to their data AML harbor in average 13 mutations in the coding region of the genome of which 5 are in genes known to be recurrently mutated in AML. Further, detailed data on co-occurrence and mutual exclusiveness of molecular mutations was presented. However, given the heterogeneity of AML a cohort of 200 AML might not be fully representative. Aims 1. Compare the published mutation frequency to our cohort 2. Evaluate, whether the mutation frequencies vary with age. 3. Determine the number of additional mutations in genetically defined AML subgroups 4. Analyze the co-occurrence of molecular mutations. Patients and Methods 1,291 adult de novo AML (700 m/591 f; median: 68 yrs; 18-100 yrs) were analyzed for mutations by different PCR assays and next-generation sequencing including the 11 most frequently mutated genes reported by TCGA (FLT3-ITD/-TKD, NPM1, DNMT3A, IDH1, IDH2, TET2, RUNX1, TP53, NRAS, CEPBA, WT1) and also ASXL1, KRAS, MLL-PTD (that had been found at lower frequencies by TCGA), and CBL. Cytogenetics was performed in all cases. Results Mutations were found in NPM1: n=410/1,189 (34.5%), DNMT3A: n=105/340 (30.9%), TET2: n=104/349 (29.8%), FLT3-ITD: n=305/1,231 (24.8%), RUNX1: n=201/1,045 (19.2%), IDH2: n=154/938 (16.4%), ASXL1: n=157/1,000 (15.7%), TP53: n=97/743 (13.1%), NRAS: n=101/842 (12.0%), IDH1: n=93/1,053 (8.8%), FLT3-TKD: n=94/1,132 (8.3%), MLL-PTD: 98/1,181 (8.3%), CEPBA: n=84/1,105 (7.6%) (double-mut: n=38; single-mut: n=46), KRAS: n=38/552 (6.9%), WT1: n=58/918 (6.3%), and CBL: 8/352 (2.3%). These mutation frequencies are comparable to those reported by TCGA. Only ASXL1 mutations were less frequently observed by TCGA (2.5%). The following mutations were more frequent in pts <60 yrs: FLT3-ITD (P=0.003), NPM1mut and WT1mut (P<0.001 for both). In contrast, ASXL1, RUNX1 (P<0.001, each) and TET2mut (P=0.005) were more frequent in pts ≥60yrs. A total of 802 pts were investigated for at least 9 markers (ASXL1, FLT3-ITD, FLT3-TKD, CEBPA, MLL-PTD, IDH1, IDH2, NPM1, RUNX1): The median number of molecular mutations was 2 (range, 0-5; mean±SD, 1.6±0.9). The lowest number of additional mutations was observed in pts with RUNX1-RUNX1T1 (0.3±0.6) and reciprocal MLL rearrangements (mean±SD, 0.4±0.6) followed by CBFB-MYH11 (0.6±0.8), NPM1 (0.9±0.7), CEPBAmut (0.9±1.0), and MLL-PTD (1.2±0.7). In concordance with TCGA results, a significant coincidence of ASXL1mut with IDH2mut and RUNX1mut was found. A total of 335 pts was screened for FLT3-ITD, DNMT3Amut, and NPM1mut in parallel and there was a high coincidence: 27/335 (8.1%) with all 3 mutations and further 63 (18.8%) with 2 out of 3; all combinations P<0.001, each). Beyond the observations within the TCGA study, we found additional positive correlations such as IDH1mut to DNMT3A (P=0.004) and as well to NPM1mut (P=<0.001), and FLT3-ITD to MLL-PTD (P=0.010) as well as to WT1mut (p=0.001). Furthermore, according to the TCGA data, the following mutations were mutually exclusive: TP53mut to NPM1mut and to FLT3-ITD (P<0.001, each), and in addition RUNX1mut to NPM1mut (P<0.001). However, we could not confirm the mutual exclusiveness of RUNX1mut and FLT3-ITD as 21.0% of RUNX1mut AML also showed FLT3-ITD. Beyond the TCGA data, we found the following mutations to show significant negative correlations: MLL translocations were significantly negatively correlated with FLT3-ITD, NPM1, DNMT3A, IDH2, and RUNX1mut, as well were RUNX1-RUNX1T1 rearrangements with FLT3-ITD, NPM1, and IDH2mut, and CBFB-MYH11 rearrangements with FLT3-ITD and NPM1mut. Conclusions 1) Investigation of a large cohort of de novo AML largely confirmed the mutation frequencies of the TCGA data, but revealed a higher frequency of ASXL1mut. 2) In addition, we depicted new patterns of positive and negative correlations of genetic alterations. 3) This further emphasizes the variety of pathways of leukemogenesis in de novo AML requiring additional analyses to delineate the prognostic impact of different marker combinations and their impact on treatment decisions. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bacher:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Mutations In SETBP1 Occur In 3.1% Of De Novo AML and Show a Distinct Genetic Pattern That Highly Resembles Atypical CML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2560-2560
Author(s):  
Manja Meggendorfer ◽  
Tamara Alpermann ◽  
Elisabeth Sirch ◽  
Claudia Haferlach ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
De Novo ◽  
Complex Karyotype ◽  
Employment Equity ◽  
Cytogenetic Abnormalities ◽  
Myeloid Malignancies ◽  
Genetic Pattern ◽  
Equity Ownership ◽  
De Novo Aml ◽  
Atypical Cml

Abstract Introduction Recently, mutations in SETBP1 (SETBP1mut) have been identified in different myeloid malignancies. We previously determined mutation frequencies in the range of 5-10% in MPN and MDS/MPN overlap, while we found SETBP1 more frequently mutated in atypical CML (32%). SETBP1mut has been shown to associate with CBL and ASXL1 mutations, as well as the cytogenetic abnormalities -7 and i(17)(q10). While SETBP1 mutations have been detected in 3% of s-AML cases, so far no mutations of SETBP1 in de novo AML have been described. Aim To analyze the mutation frequency of SETBP1 mutations in de novo AML with corresponding cytogenetic abnormalities and their respective correlation to clinical data and other gene mutations. Patients and Methods We investigated 422 adult de novo AML patients, diagnosed by cytomorphology, immunophenotyping and genetic studies following WHO classification. SETBP1 was analyzed by Sanger sequencing of the coding region for amino acids 800 to 935. The cohort comprised 229 males and 193 females, the median age was 65.8 years (range: 19.3 – 89.0). Cytogenetics was available in all 422 cases. Based on the previously described association of SETBP1mut with -7 and i(17)(q10) in other myeloid malignancies there was a selection bias to these karyotypes. Cases were grouped according to cytogenetic abnormalities: normal karyotype (n=88) and aberrant karyotype (n=334), i.e. i(17)(q10) (n=15), +14 (n=20), -7 (n=100), other abnormalities (n=129), and complex karyotype (n=114; 44 contained i(17)(q10), +14 or -7). Within the SETBP1mut cases the following molecular markers were analyzed: ASXL1, CBL, CEBPA, FLT3-ITD, FLT3-TKD, IDH1/2, KRAS, NRAS, NPM1, MLL-PTD, RUNX1, SRSF2, TP53 and WT1 by Sanger sequencing, next generation sequencing, gene scan or melting curve analyses. These data were also available in sub-cohorts of SETBP1 negative cases. Results In the total cohort mutations in SETBP1 were detected in 3.1% (13/422) of all cases. SETBP1mut patients were older (median age: 73.5 vs. 65.7 years; p=0.05) and showed a slightly higher white blood cell count (14.5 vs. 13.8x109/L; p<0.001). There was no correlation to gender, hemoglobin level and platelet count. However, analyzing the cytogenetic groups SETBP1mut showed, like in other myeloid malignancies, a strong co-occurrence with -7 and i(17)(q10), since 4/13 SETBP1 positive cases carried a monosomy 7, and 7/13 an i(17)(q10). The two remaining cases showed a trisomy 14 or a complex karyotype that also contained a i(17)(q10). No SETBP1mut was found in any other cytogenetic subgroup. Therefore, SETBP1mut correlated significantly with i(17)(q10) (8/15 i(17)(q10) were SETBP1mut vs. 5/407 in all other karyotypes; p<0.001). Further, we analyzed the association of SETBP1 mutations with other molecular markers. SETBP1mut correlated with ASXL1mut, 9/33 (27%) ASXL1mut patients showed a mutation in SETBP1, while only 2 (1%) showed a SETBP1 mutation in 229 ASXL1 wild type (wt) patients (p<0.001). This was also true for CBLmut, where 4/8 (50%) CBLmut cases were SETBP1mut, while only 8/158 (5%) were SETBP1mut in the group of CBLwt (p=0.001). This was even more prominent in SRSF2mut patients, where all 9 SRSF2mut were also SETBP1mut, while only 4/8 (50%) patients carried a SETBP1 mutation within the SRSF2wt group (p=0.029). In contrast, SETBP1mut were mutually exclusive of mutations in TP53 (0/67 in TP53mut vs. 12/194 in TP53wt; p=0.04), possibly reflecting the exclusiveness of TP53mut in i(17)(q10) patients. There was no correlation to any other analyzed gene mutation. Remarkably, while there was a high coincidence of SETBP1mut, SRSF2mut (9/13) and ASXL1mut (9/11), none of these patients showed mutations in the typical AML markers NPM1, FLT3-ITD, CEBPA, MLL-PTD, or WT1. Comparing the mutational loads of SETBP1, ASXL1 and SRSF2 resulted in SRSF2 having in most cases the highest mutational loads (range: 30-70%) while ASXL1 and SETBP1 showed equal or lower mutational loads (15-50% and 10-50%, respectively), possibly indicating that SRSF2 mutation is a former event followed by ASXL1 and SETBP1 mutation. Conclusions 1) For the first time we describe, that SETBP1 mutations occur in de novo AML. 2) SETBP1 mutations are correlated with a distinct genetic pattern with high association to i(17)(q10), ASXL1 and SRSF2 mutations and are mutually exclusive of TP53mut. 3) Thus, the genetic pattern of SETBP1 mutated AML highly resembles that of atypical CML. Disclosures: Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Sirch:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Assessment Of Human Equilibrative Nucleoside Transporter 1 (hENT1) – Expression By Flow Cytometry In Acute Myeloid Leukemia and Myelodysplastic Syndromes and Correlation To Clinical Outcome In Acute Myeloid Leukemia Patients Treated With Intensive Chemotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2582-2582 ◽  
Author(s):  
Frauke Bellos ◽  
Bruce H. Davis ◽  
Naomi B. Culp ◽  
Birgitte Booij ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Molecular Genetic ◽  
Newly Diagnosed ◽  
Employment Equity ◽  
Equity Ownership ◽  
De Novo Aml ◽  
Very High ◽  
Acute Myeloid

Abstract Background Nucleoside analogs depend on cellular hENT1 expression for entry into cells and cytotoxic activity. Studies suggest low cellular hENT1 levels correlate with poor response to such chemotherapies in solid tumors, data on AML and MDS is scarce. Aim To examine hENT1 expression by multiparameter flow cytometry (MFC) in newly diagnosed AML and MDS and correlate results to morphologic, cytogenetic (CG) and molecular genetic (MG) findings. To examine hENT1 expression with respect to clinical outcome in AML patients (pts) treated with intensive cytarabine-based chemotherapy (CHT). Methods We studied pts with newly diagnosed AML (n=145) and MDS (n=96), 133/108 male/female, median age 67.3 (AML) and 73.3 years (MDS). CG was done in 130 AML and 86 MDS. Pts included 107 de novo AML, 9 t-AML, 29 s-AML; FAB: 9 M0, 27 M1, 50 M2, 9 M3, 21 M4, 8 M4eo, 7 M5, 14 not classified; by CG (MRC): 21 favorable, 75 intermediate, 34 adverse. 91 were de novo MDS, 5 t-MDS; 1 RARS, 17 RCMD-RS, 37 RCMD, 3 5q- syndrome, 3 RAEB-1, 5 RAEB-2, 1 CMML, 24 not classified; 2 IPSS-R very low, 55 IPSS-R low, 8 IPSS-R intermediate, 8 IPSS-R high, 13 IPSS-R very high. hENT1 expression was quantified by a novel four color intracellular staining assay using monoclonal antibodies against hENT1, CD45, CD64 and myeloperoxidase. Median fluorescence intensities (MFI) of hENT1 were determined in myeloid progenitors (MP), granulocytes (G) and monocytic cells (Mo) and correlated to hENT1 MFI in lymphocytes to derive hENT1 index (index). Results No correlation of index to age, gender, hemoglobin level or counts for blasts, WBC or platelets was detected. In AML, we generally saw higher index by trend in the more favorable prognostic subgroups. M3/t(15;17)/PML-RARA+ displayed higher index in MP than non-M3 AML (4.24 vs 2.56, p<0.001). G index was lower in M0 (3.01) vs M1, M2, M4 and M4eo (5.66, 4.34, 5.35, 4.77; p=0.01, 0.028, 0.004, 0.043, respectively) and in M2 compared to M1 and M4 (4.34. vs 5.66 and 5.35, p=0.01 and 0.033, respectively). M2 showed lower MP index than M5 (2.42 vs 2.99, p=0.016). Considering CG, index in MP was higher in favorable vs intermediate and adverse pts (3.05 vs 2.58 and 2.53, p=0.034 and 0.023, respectively), Mo index was higher ín favorable vs adverse pts (3.17 vs 2.71, p=0.044). By MG, higher index in Mo and G was observed in RUNX1-RUNX1T1+ AML (4/83, 4.32 vs 3.04, p=0.01; 8.16 vs 4.60, p=0.002, respectively). Higher index for MP was found in FLT3-ITD mutated (mut) (18/111; 3.19 vs 2.62, p=0.012), CEPBA mut (4/26, 3.15 vs 2.35, p=0.004) and for Mo in NPM1 mut AML (23/104; 3.72 vs 2.84, p=0.02), whereas lower index for MP was found in RUNX1mut pts (13/65; 2.17 vs 2.59, p=0.031). De novo AML displayed higher MP index than s-AML (2.7 vs 2.28, p=0.008). Using lowest quartile of index for MP (2.1185) as cut-off, AML pts in the MRC intermediate group treated with CHT (n=38) had inferior OS if MP index was below vs above this cut-off (OS at 6 months 63% vs. 95%, p=0.017, median follow up 4.6 months). MDS showed lower Mo and MP index than AML (2.68 vs 2.96, p=0.021, 1.84 vs 2.65, p<0.001, respectively). By IPSS-R, significance was reached for higher index in Mo and MP in very low risk compared to low risk pts (3.39 vs 2.54, p=0.013 and 4.07 vs 1.78, p<0.001, respectively), MP in very low compared to intermediate and high risk pts (4.07 vs 1.95, p=0.004; 4.07 vs 1.76, p=0.002), and MP and G in very low vs very high risk pts (4.07 vs 1.71, p=0.005; 5.86 vs 3.85, p=0.001, respectively). IPSS-R intermediate vs poor and very poor showed lower G index (5.47 vs 3.59, p=0.018 and vs 3.85, p=0.034 respectively). Conclusion AML with genetic and molecular genetic good risk profile had higher hENT1 expression in MP, G and Mo, suggesting a causal mechanism for better response to CHT and better outcome. Consequently, AML with poor risk molecular genetics (RUNX1 mut) showed lower levels of hENT1 in MP. The detection of higher levels in FLT3-ITD mut pts is in line with reportedly good response to CHT, overall worse outcome being mostly due to early relapses. Strikingly, we saw differences in outcome in pts treated with CHT according to hENT1 expression with shorter OS in pts with low index for MP. Higher index in de novo AML than s-AML and MDS may be causal for better response to nucleoside-based CHT in de novo AML. Data for MDS may be interpreted accordingly, lower risk cases showing higher index in MP, G and Mo. Further analyses are needed to explore hENT1 expression in AML and MDS more comprehensively. Disclosures: Bellos: MLL Munich Leukemia Laboratory: Employment. Davis:Trillium Diagnostics, LLC: Equity Ownership. Culp:Trillium Diagnostics, LLC: 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. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Myeloid Malignancies with Isolated 7q Deletion Can be Further Characterized By Their Accompanying Molecular Mutations

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3811-3811
Author(s):  
Claudia Haferlach ◽  
Annette Fasan ◽  
Manja Meggendorfer ◽  
Melanie Zenger ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Array Cgh ◽  
De Novo ◽  
Jak2 V617f ◽  
Terminal Deletion ◽  
Employment Equity ◽  
Myeloid Malignancies ◽  
Additional Chromosome ◽  
Equity Ownership ◽  
De Novo Aml ◽  
7Q Deletion

Abstract Background: 7q deletions (del(7q)) are recurrent cytogenetic abnormalities. They occur either as the sole abnormality or accompanied by additional chromosome aberrations in AML, MDS, MDS/MPN and MPN. Cases with del(7q) as the sole abnormality are rare and poorly characterized. Aim: In patients with myeloid malignancies and del(7q) as the sole abnormality we determined 1. Type and size of the del(7q) 2. Spectrum of accompanying molecular mutations and their impact on the phenotype. Patients and Methods: 81 cases with myeloid malignancies and del(7q) as the sole abnormality were included in this study. Of these 38 had AML (27 de novo, 7 secondary, 4 therapy-related), 17 MDS (14 de novo, 3 therapy-related), 10 MDS/MPN (9 CMML, 1 MDS/MPN unclassifiable) and 16 MPN. The median age was 72 years (range: 29-89 years). All cases were investigated by array CGH (Agilent, Waldbronn, Germany) and for mutations in ASXL1, CALR, CBL, DNMT3A, ETV6, EZH2, JAK2, KRAS, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TET2, and TP53. Results: Array CGH revealed an interstitial del(7q) in 67 cases, while 14 cases showed terminal del(7q). Further characterization of these deletions using 24 color FISH revealed unbalanced translocations in 10 of the 14 cases with terminal deletion. Partner chromosomes were X, 8, 9, 12, 13, 17 (n=2), 19 (n=2), and 22. The breakpoints on chromosome 7 were diverse ranging from 7q11 to 7q32. In two cases the breakpoint was within the CDK6 gene. In two cases with terminal del(7q) the complete loss of 7q was due to an idic(7)(q11.21). In the remaining two cases the terminal deletion could not be further resolved. In the 67 cases with interstitial del(7q) the size of the del(7q) varied between 1.8 and 158.9 Mb (median: 52.6 Mb). No commonly deleted region could be identified for all cases. However, in 57 cases the deleted region encompassed genomic position 101,912.442 (7q22.1) to 119,608.824 (7q31.31) including 111 genes. The size of the 7q deletion was smaller in cases with interstitial deletion as compared to terminal deletion (57.7 MB vs 70.9 MB, p=0.04) and in MPN as compared to all other entities (48.7 MB vs 62.8 MB, p<0.001). The mutation analyses revealed mutations in TET2 37% (25/67), ASXL1 35% (27/78), RUNX1 26% (18/69), DNMT3A 21% (14/68), SRSF2 18% (13/73), JAK2 V617F 14% (11/79), CBL 9% (7/75), NRAS 9% (7/77), MLL -PTD 5% (4/80), KRAS 5% (3/66), EZH2 4% (3/72), TP53 4% (3/74), SF3B1 4% (3/75), ETV6 3% (2/73), NPM1 3% (2/77), CALR 1% (1/77), MPL 1% (1/76). ASXL1 and TET2 were frequently co-mutated as 56% of ASXL1 mutated cases also harbored a TET2 mutation (p=0.02). 39 cases were analysed for all 16 molecular mutations. The majority of patients (n=27, 69%) had more than one mutation (range: 2-4), 9 patients (23%) had one mutation and in 3 patients (8%) no mutation was detected. The number of mutations per patient was lower in patients <70 years as compared to patients ≥70 years (0, 1,2,3,4 mutations detected in: 23%, 15%, 15%, 46%, and 0% vs 0%, 27%, 27%, 31%, and 15%, p=0.05). CBL mutations were most frequent in CMML (44%) but rare in all other subtypes (5%, p=0.003), while RUNX1 mutations were most frequent in AML (43% vs 9%; p=0.002) and JAK2 V617F mutations most frequent in MPN (50% vs 5%, p<0.001). DNMT3A mutations and MLL -PTD were significantly more frequent in de novo AML than in all other entities (43% vs 11%, p=0.007; 15% vs 0%, p=0.009), while no significant differences in frequency were observed between the different entities for any of the other mutations or the number of mutations per case. In CMML CBL mutations were associated with del(7q) (44%) as CBL mutations were present in only 17% of non del(7q) CMML (n=101, p=0.07). The frequency of RUNX1 mutations was significantly higher in AML with del(7q) as the sole abnormality (43%) as compared to all other AML (n=2273, 21%; p=0.001). Median overall survival (OS) for the total cohort was 25 months and did not differ significantly between AML, MDS, MDS/MPN and MPN (26, 27, not reached, 15 months, respectively). Conclusions: 1. Sizes and localisations of the del(7q) largely overlapped between AML, MDS, MDS/MPN and MPN. 2. 92% of all patients with 7q deletion harbored at least 1 molecular mutation. 3. TET2 and ASXL1 were the most frequently mutated genes and were present at comparable frequencies in all subtypes. 4. AML with del(7q) is closely associated with RUNX1 mutations while CMML with del(7q) frequently harbored CBL mutations suggesting a cooperative leukemogenic potential in these entities. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Fasan:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Zenger: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.

scholarly journals The RUNX1 Gene Is Altered in 26% of AML Patients Either By Translocation, Mutation, Gain or Deletion

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 123-123
Author(s):  
Claudia Haferlach ◽  
Niroshan Nadarajah ◽  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
De Novo ◽  
Intermediate Risk ◽  
Missense Mutations ◽  
Employment Equity ◽  
Runx1 Gene ◽  
Genetic Abnormalities ◽  
Partner Gene ◽  
Different Types ◽  
Equity Ownership ◽  
De Novo Aml

Abstract Background: In AML four types of acquired alterations of the RUNX1 gene have been described: 1. translocations involving RUNX1 leading to fusion genes such as RUNX1-RUNX1T1, 2. molecular mutations, 3. amplifications of RUNX1, 4. Partial or complete deletions of the RUNX1 gene. Aim: To determine the frequency of different RUNX1 alterations and to characterize the spectrum of accompanying genetic abnormalities. Patients and Methods: We screened 726 de novo AML patients (pts) for RUNX1 deletions (del) and translocations using a dual color break-apart probe covering the 5' and 3' part of RUNX1 (MetaSystems, Altlussheim, Germany) and in addition evaluated RUNX1 mutations (mut) by Sanger or next-generation amplicon deep-sequencing. Median age was 67 yrs (range: 18 to 100 yrs). For all patients cytogenetics was available and categorized according to MRC criteria (Grimwade et al. Blood 2010). Partial deletions of RUNX1 as detected by FISH were confirmed by array CGH (Agilent Technologies, Santa Clara, CA). Results: In 89/726 pts (12.3%) abnormalities of the RUNX1 gene were detected by FISH: 10 pts (1.4%) showed a deletion encompassing the whole RUNX1 gene while additional 9 pts (1.2%) showed a partial loss of one RUNX1 copy. A gain of one RUNX1 copy was present in 45/726 (6.2%) pts. In 3 pts a gain of the 5' part of RUNX1 was accompanied by a loss of the 3' part while in 2 pts one copy of the 3' part was gained accompanied by a loss of the 5' part. A translocation affecting the RUNX1 gene was detected in 31 pts (4.3%). The partner gene was RUNX1T1 in 29 pts and located on 16q13 and 18p11 in one pt each. One pt with a RUNX1 translocation also showed a 5' RUNX1 deletion. In 110/726 pts (15.2%) a RUNX1mut was detected. Of these, 16 pts showed two and 5 pts three mutations in RUNX1. Thus, in total 136 mutations were detected in 110 pts: 58 (42.6%) were frameshift, 42 (30.9%) missense, 21 (15.4%) nonsense, 9 (6.6%) splice-site and 6 (4.4%) in-frame insertions/deletions. The RUNX1mut was homozygous in 15 pts, these were predominantly missense mutations (9/15; 60%). Within the subset of pts with RUNX1mut 2 harbored an additional RUNX1del and 9 pts a gain of a RUNX1 copy, while no RUNX1 translocation was present. In AML FAB type M0 both RUNX1mut and RUNX1del showed the highest frequencies (33.3% and 14.8%). 48.4% and 45.2% of cases with RUNX1 translocations were FAB type M1 and M2. While RUNX1mut were most frequent in the cytogenetic intermediate risk group (19.1%; favorable: 2.2%, adverse: 9.7%), RUNX1del were most frequent in pts with adverse risk cytogenetics (9.7%; favorable: 1.1%, intermediate: 0.8%). A comparable distribution was observed for a gain of RUNX1 copies (adverse: 19.4%, favorable: 4.5%, intermediate: 2.6%). With respect to additional molecular mutations all types of RUNX1 alterations were mutually exclusive of NPM1mut. Further, the frequency of DNMT3Amut and CEBPAmut was significantly lower in pts with RUNX1 alterations as compared to those without (14.3% vs. 34.3%; p<0.0001 and 6.4% vs. 13.4%; p=0.012). However, some striking differences between the different types of RUNX1 alterations were detected: ASXL1mut were significantly more frequent in pts with RUNX1mut (36.7%) but rather infrequent in pts with RUNX1del, gain and translocation (12.5%, 6.1%, and 6.7%). A comparable association was noticed for SF3B1mut which were frequent in RUNX1mut pts (23.8%) and rather infrequent in pts with RUNX1del, gain and translocations (0%, 10.5%, and 0%). In contrast, pts with either RUNX1del or RUNX1 gains showed a significantly higher TP53mut frequency (66.7% and 35.3%) as compared to RUNX1mut or RUNX1 translocated pts (7.1% and 4.8%). In the total cohort median overall survival (OS) was 18.7 months and differed significantly between the different types of RUNX1 alterations: for RUNX1 translocations, mutations, gains and deletions it was 35.5, 14.1, 12.4 and 4.3 months. Conclusions: 1. The RUNX1 gene is altered in 26% of AML. 2. All types of RUNX1 alterations predominantly occur in AML M0, M1 and M2 and are rare in the remainder AML. 3. They are mutually exclusive of NPM1 mutations and show a negative association with DNMT3A mutations. 4. While RUNX1 mutations were most frequent in patients with intermediate risk cytogenetics, RUNX1 deletions and gains were most frequent in patients with adverse cytogenetics. 5. Outcome differs significantly and is best in patients with RUNX1 translocations and worst in cases with RUNX1 deletions. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah: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.

scholarly journals A 17-Gene Leukemia Stem Cell (LSC) Score in Adult Patients (Pts) with Acute Myeloid Leukemia (AML) Reveals a Distinct Mutational Landscape and Refines Current European Leukemianet (ELN) Genetic Risk Stratification

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 289-289 ◽  
Author(s):  
Marius Bill ◽  
Deedra Nicolet ◽  
Ann-Kathrin Eisfeld ◽  
Krzysztof Mrózek ◽  
Christopher J. Walker ◽  
...  
Keyword(s):  
De Novo ◽  
Gene Mutations ◽  
Disease Free Survival ◽  
Prognostic Impact ◽  
Expression Data ◽  
Age Related ◽  
Mutational Status ◽  
Multivariable Analyses ◽  
De Novo Aml ◽  
Favorable Group

Abstract Introduction: Prognosis of AML pts is still poor mainly because of refractoriness to or relapse after intensive chemotherapy. High rates of relapse are also attributed to LSCs, which are a small subset of cells with acquired abnormal self-renewal capacity and increased resistance to chemotherapy. A better understanding of LSCs is critical to improve outcomes of pts with AML. Ng et al. (Nature 2016;540:433) defined a 17 stemness-associated gene score that was highly prognostic. Aims: The aim of this study was to validate the prognostic relevance of the 17-gene LSC score and explore its utility in the context of the ELN classification. We also examined gene mutations associated with the 17-gene LSC score. Methods: We analyzed a total of 934 pts [729 aged <60 years (y) and 205 aged ≥60 y] with de novo AML. We used whole transcriptome expression data (RNAseq) to calculate the aforementioned 17-gene LSC score for each pt in our cohort. Similar to Ng et al., we used the median of the whole cohort to discriminate between pts with LSChigh and LSClow scores. The mutational status of 80 cancer- and leukemia-associated genes (Eisfeld et al. Leukemia 2017;31:2211) were determined using a targeted next-generation sequencing panel, CEBPA mutations using Sanger sequencing, and an internal tandem duplication (ITD) of the FLT3 gene using fragment analysis in pretreatment bone marrow or blood samples. All pts were treated on frontline Cancer and Leukemia Group B/Alliance protocols. Results: A comparison of pretreatment clinical and genetic features revealed that LSChigh pts were older (P<.001; median age, 53 vs 46 y) and had higher platelet counts (P<.001; median, 63 vs 50x109/L) than LSClow pts. Pts with a LSChigh score more frequently had FLT3-ITD (P<.001) and mutations in the ASXL1 (P=.001), DNMT3A (P<.001), RUNX1 (P=.002), SRSF2 (P=.02), STAG2 (P=.009), TET2 (P=.008) and TP53 (P<.001) genes. Conversely, these pts had a lower frequency of biallelic CEBPA (P<.001), GATA2 (P=.008) and KIT (P<.001) mutations. Because of differences in treatment intensity, we analyzed outcomes of younger and older pts separately. Younger pts with a LSChigh score had a lower complete remission (CR) rate (P<.001; 63% vs 87%), shorter disease-free survival (DFS; P<.001; 3-y rates, 26% vs 48%; Figure 1A) and overall survival (OS; P<.001; 3-y rates, 27% vs 59%; Figure 1B) compared to those of LSClow pts. In multivariable analyses including clinical and genetic factors that impact on outcome, a LSChigh score associated with lower remission rates (P<.001; HR: 0.36), shorter DFS (P<.001; HR: 1.67) and OS (P<.001; HR: 1.88) after adjusting for other co-variates. We also analyzed the prognostic impact of the LSC score with respect to the 2017 ELN classification. We found that LSC score associated with different ELN groups (P<.001), with LSChigh pts being more often classified in the Adverse or Intermediate group and less often in the Favorable group. Within the ELN Favorable and Adverse groups, LSChigh score retained its prognostic impact and identified pts with a lower CR rate and shorter DFS and OS (Table1). In older pts, a LSChigh score also associated with lower CR rate (P=.004; 50% vs 72%), shorter DFS (P=.04; 3-y rates, 6% vs 17%; Figure 1C) and OS (P<.001; 3-y rates, 9% vs 27%; Figure 1D). In multivariable analyses, LSC score remained significant only for OS (P<.003; HR: 1.70) after adjusting for other co-variates. Regarding the ELN classification, pts with LSChigh score in the Favorable group had shorter OS (P=.05; 3-y rates, 17% vs 50%) and, by trend, shorter DFS (P=.09; 3-y rates, 17% vs 39%); no significant differences were found in Intermediate or Adverse groups. Conclusions: We used RNAseq expression data and applied the previously established 17-gene LSC signature to score 934 de novo AML pts. We detected distinct mutational differences between LSChigh and LSClow pts, with LSChigh pts more often carrying gene mutations associated with age-related clonal hematopoiesis (i.e., ASXL1, DNMT3A, TET2, SRSF2 and TP53 mutations). Moreover, this score, derived from the expression of stemness-associated genes, has not only a prognostic impact on its own but also in the context of the current 2017 ELN classification. Disclosures Kolitz: Magellan Health: Consultancy, Honoraria. Powell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

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

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

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

ETV6 Rearrangements Are Recurrent Genetic Events In Myeloid Malignancies and In AML Are Associated with NPM1- and RUNX1-Mutations and An Intermediate Prognosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2758-2758
Author(s):  
Claudia Haferlach ◽  
Susanne Schnittger ◽  
Wolfgang Kern ◽  
Torsten Haferlach
Keyword(s):  
De Novo ◽  
Normal Karyotype ◽  
Employment Equity ◽  
Childhood All ◽  
Myeloid Malignancies ◽  
Equity Ownership ◽  
De Novo Aml ◽  
Diagnostic Work ◽  
Work Up

Abstract Abstract 2758 Introduction: The ETV6 gene (formerly TEL) is located in the chromosomal band 12p13 and is a frequent target of deletions and chromosomal translocations in both myeloid and lymphoid leukemias. In ALL the most frequent partner gene of ETV6 is RUNX1. ALL with ETV6-RUNX1 fusions are observed in 20% of childhood ALL and are associated with favorable outcome. In contrast ETV6 rearrangements are less frequent and not well described in myeloid malignancies. Therefore, the aim of this study was to analyze ETV6 rearrangements in myeloid malignancies with respect to frequency, partner genes and impact on prognosis. Patients/Methods: 55 cases with ETV6 rearrangements were identified in a total cohort of 9,550 cases (0.5%) with myeloid malignancies (de novo AML: n=3,090, s-AML: 486, t-AML: 222, MDS: n=3,375, MDS/MPN overlap: n=210, CMML: n=447, MPN: n=1,720) which had been sent to our laboratory between 08/2005 and 07/2010 for diagnostic work-up. In all cases chromosome banding analysis was performed and in cases with abnormalities involving 12p13 FISH was carried out in addition to verify the ETV6 rearrangement. Results: ETV6 rearrangements were observed in 31 patients with de novo AML (1.0% of investigated cases), 8 with s-AML (1.7%), 5 with t-AML (2.3%), 6 with MDS (0.2%) and 5 with MPN (0.3%). No ETV6 rearrangements were detected in the cohorts of MDS/MPN or CMML. ETV6 rearrangements were significantly more frequent in s-AML and t-AML as compared to de novo AML (p<0.001). Median age in AML was 59.9 years. In 15 cases with de novo AML FAB-subtypes were available: M0: n=8, M1: n=4, M2: n=1, M4: n=1, and M7: n=1. Thus, ETV6 rearrangements are closely related to immature AML subtypes. In 25/55 cases (45.5%) the ETV6 rearrangement was the sole abnormality. Recurrent additional abnormalities were 7q-/-7 in 10 cases and del(5q) in 8 cases. 36 different partners of ETV6 were observed, recurrent partners were located on 3q26 (EVI1, n=11), 5q33 (PDGFRB, n=4), 22q12 (n=3), 2q31 (n=2), 5q31 (ACSL6, n=2), 12p12 (n=2), 17q11 (n=2). Molecular analysis was performed in addition in AML with ETV6 rearrangements for mutations in NPM1 (n=26 investigated), FLT3-ITD (n=33), FLT3-TKD (n=11), MLL-PTD (n=25) and RUNX1 (n=7). NPM1-mutations were observed in 5 cases (19.2%), FLT3-ITD in 3 cases (9.1%), FLT3-TKD in 2 cases (18.2%), MLL-PTD in 1 case (4%) and RUNX1 mutations in 4 cases (57.1%), respectively. Clinical follow-up data was available of 47 cases. No differences in overall survival (OS) and event-free survival (EFS) were observed in cases with ETV6 rearrangement whether or not additional cytogenetic abnormalities or 7q-/-7 or del(5q) were present. Next 30 de novo AML with ETV6 rearrangement were compared to 819 AML without ETV6 rearrangement. Based on cytogenetics cases were assigned into 9 subgroups: 1) t(15;17)(q22;q21), n=48; 2) t(8;21)(q22;q22), n=29; 3) inv(16)(p13q22)/t(16;16)(p13;q22), n=19; 4) 11q23/MLL abnormalities, n=28; 5) inv(3)(q21q26)/t(3;3)(q21;q26), n=6; 6) normal karyotype, n=424; 7) complex karyotype, n=71; 8) other abnormalities, n=194 and 9) ETV6 rearrangements, n=30. Median OS was not reached for groups 1, 2, 3, 4, and 6 and was 10.6 mo, 11.8 mo, 32.2 and 26.3 mo for groups 5, 7, 8, and 9 respectively. OS at 2 yrs was 95.6%, 96.3%, 76.6%, 64.9%, 26.7%, 63.3%, 23.9%, 58.5% and 60.1% for groups 1–9, respectively. The respective data for median EFS were: not reached for groups 1 and 2 and 15.9 mo, 13.5 mo, 5.1 mo, 16.6 mo, 7.5 mo, 12.5 mo and 14.0 mo for groups 3–9, respectively. Conclusions: ETV6 rearrangements are rare in myeloid malignancies. ETV6 is rearranged with a large variety of partner genes. The highest frequency of ETV6 rearrangements was observed in s-AML and t-AML. OS and EFS of AML with ETV6 rearrangements are comparable to AML with normal karyotype. Thus, the detection of ETV6 rearrangements is associated with in intermediate prognosis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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