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.

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.

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.

RUNX1 Deletions Are a Novel Mechanism of Loss of Function in AML and Are Associated with Adverse Cytogenetics.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2517-2517
Author(s):  
Claudia Haferlach ◽  
Vera Grossmann ◽  
Melanie Zenger ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Copy Number ◽  
De Novo ◽  
Chromosome 21 ◽  
Prognostic Impact ◽  
Complex Karyotype ◽  
Loss Of Function ◽  
Array Data ◽  
Runx1 Gene ◽  
Equity Ownership ◽  
Wbc Count

Abstract Abstract 2517 Background: RUNX1 is a crucial hematopoietic transcription factor located on the long arm of chromosome 21 (21q22). Three types of acquired alterations of the RUNX1 gene have been described in AML so far: 1. translocations leading to fusion genes such as t(8;21)(q22;q22)/RUNX1-RUNX1T1 2. molecular mutations, which usually lead to loss of normal RUNX1 function, and 3. amplifications, which are predominately found in AML with complex karyotype. Another possible mechanism that causes loss of function is a partial or complete deletion of the gene. Aim: To clarify whether RUNX1 is also affected by partial or complete deletions in AML. Patients and Methods: We screened 623 AML patients (pts) for RUNX1 deletions (del) by interphase fluorescence in situ hybridization using probes spanning the complete RUNX1 gene (MetaSystems, Altlussheim, Germany). The cohort comprised 472 de novo, 85 secondary (s-AML), and 66 therapy-related AML (t-AML). Median age was 67.3 yrs (range: 18 to 91.5 yrs). For all pts cytogenetics was available and categorized according to refined MRC criteria (Grimwade et al. Blood 2010). Cytogenetics were favorable in 164, intermediate in 210 and adverse in 249 pts, respectively. RUNX1 mutation analysis was performed in 252 cases. In addition, 19 pts with RUNX1 del were analyzed by genomic arrays (Human CGH 12×270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI (n=12); SNP 6.0 arrays, Affymetrix, Santa Clara, CA (n=7)). Results: Frequency and clinical characteristics: In 57/623 (9.1%) cases deletions affecting RUNX1 were identified. The frequency of RUNX1 del did not vary significantly between de novo (8.9%), s-AML (11.8%) and t-AML (7.6%). However, RUNX1 del were more frequent in pts with adverse cytogenetics (18.1%) compared to intermediate (5.7%) or favorable cytogenetics (0%, p<0.0001). In contrast, RUNX1 mutations (mut) were more frequent in intermediate (26.1%) as compared to favorable (0%) and unfavorable cytogenetics (10.5%, p=0.003). Pts harboring RUNX1 del were significantly older than those with 2 RUNX1 copies (mean: 70.0 vs 61.6 yrs, p<0.0001) and showed lower WBC count (mean: 13,225 vs 21,419/μl, p=0.010), while no difference was observed with respect to hemoglobin level and platelet count. Cytogenetics: 38/57 (66.7%) pts with RUNX1 del harbored a complex karyotype, one case showed a normal karyotype while a variety of different abnormalities were found in the remaining 18 pts. Type of cytogenetic alteration: In 5 cases one RUNX1 allele was lost due to monosomy 21. In two cases a deletion of the long arm of chromosome 21 was cytogenetically visible. 29 pts showed derivative chromosomes 21 resulting from unbalanced translocations or duplications of the long arm of chromosome 21. Ring chromosomes 21 were found in 3 cases. In 7 pts a translocation involving 21q22 was identified. 11 cases with a RUNX1 del showed cytogenetically normal chromosomes 21. Remarkably, 6 of these 11 pts showed a trisomy 21. RUNX1 mutations: 36 pts with RUNX1 del were also evaluated for RUNX1 mut. In 7 pts (19.4%) a RUNX1 mut in the remaining allele was detected. Thus, the mutation frequency did not differ from pts without RUNX1 del (40/216, 18.5%). Genomic array data: The size of the deletion on chromosome 21 varied between 258 kb and 11,792 kb (median: 1,987 kb). In one case a homozygous RUNX1 deletion was detected. Based on array data pts with RUNX1 del were subdivided into two groups: The first group comprised 7 pts with a non-complex karyotype and an interstitial deletion on chromosome 21 encompassing RUNX1. Secondly, in 12 pts with RUNX1 del and a complex karyotype array data revealed between 2–16 changes in copy number state on chromosome 21. Interestingly, in 4/12 (30%) cases the ERG gene, located 2.6 Mb telomeric to RUNX1, was amplified. Conclusions: 1. Partial or complete deletions of RUNX1 are frequent recurrent genetic events in AML. 2. They are associated with adverse cytogenetics, lower WBC count and are more frequent in elderly patients. 3. Loss of one RUNX1 copy results either from clear-cut interstitial deletions on chromosome 21 or occurs based on highly rearranged chromosomes 21 showing several changes in copy number states accompanied by gains and losses of several regions on chromosome 21. Whether the pathogenetic impact of RUNX1 del is comparable to RUNX1 mut and if they have prognostic impact independent of cytogenetics remains to be studied. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: 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.

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.

Analysis of ASXL1, IDH1, IDH2, c-CBL, and WT1 Mutations in De Novo Acute Myeloid Leukaemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1450-1450
Author(s):  
Mariam Ibañez ◽  
Esperanza Such ◽  
Jose Cervera ◽  
Irene Luna ◽  
Sandra Dolz ◽  
...  
Keyword(s):  
De Novo ◽  
Prognostic Impact ◽  
Significant Implication ◽  
Wild Type ◽  
Idh Mutations ◽  
Flt3 Mutations ◽  
Exon 4 ◽  
De Novo Aml ◽  
Cebpa Mutations ◽  
Acute Myeloid

Abstract Abstract 1450 The clinical relevance and prognostic implications of some recently identified mutations in acute myeloid leukemia (AML) is not yet well established. Among them, we have selected to be analyzed those affecting the following genes: Additional Sex Combs-Like 1 (ASXL1), Isocitrate Dehydrogenase (IDH1 and IDH2), Casitas B-lineage Lymphoma (c-CBL), and Wilms Tumor 1 (WT1). They have been previously reported with a variable incidence: ASXL1 mutations in 10.8% patients with normal karyotype (NK), IDH1 and IDH2 mutations in 8 – 33% of de novo AML, c-CBL mutations in 2% of de novo AML, and WT1 mutations in 5–12% of de novo AML patients. In order to know the incidence and prognostic impact of these mutations and their possible cooperative role in leukemogenesis, we have screened for ASXL1, IDH1, IDH2, c-CBL, WT1, FLT3, NPM1 and CEBPa, mutations in a cohort of de novo AML patients from a single centre. We studied 174 de novo AML patients [98M/76F; median age: 62 yr. (range: 16 – 88); favourable (n= 13), intermediate (n= 86) and high (n= 51) cytogenetic risk classification by the MRC group]. DNA was isolated from bone marrow samples obtained at diagnosis. In order to determine cooperating mutations, we developed a new combination of high-resolution melting (HRM) assays on a LightCycler® 480 and lastly direct sequencing, to detect somatic mutations for ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), WT1 (exons 7, 8 and 9) and c-CBL (exons 8 and 9). All mutations reported in this study were confirmed al least twice. FLT3 (ITD and D835Y), NPM1 (exon 12) and CEBPa were performed as described previously by standard methods. Sequence analysis was checked by its corresponding GeneBank Accession Number. The number of patients found to carry mutations in our series was: 16 patients with ASXL1 mutations (9.2%), 16 patients with IDH mutations (2.9% had a IDH1R132, 12.6% the SNP rs11554137 and 6.3% IDH2R140), 5 patients with WT1 mutations (2.9%), 37 patients with FLT3 mutations (21.3%), 44 patients with NPM1 mutations (25,3%) and 8 patients with CEBPa mutations (4.6%). No mutations where found in c-CBL. We could not found a pattern of cooperating mutations in the studied group of genes. WT1, FLT3 and NPM1 were associated with leukocyte count >30 × 109/L at diagnosis (80% vs. 31% for WT1, P =0,022; 68% vs. 22% for FLT3, P= 0.001; and 50% vs. 24% for NPM1, P= 0.002; in mutated vs. wild-type patients, respectively). WT1 was also associated with a platelet count > 50 × 109/L at diagnosis (100% vs. 57% in mutated vs. wild-type patients, respectively; P =0,048). Besides, FLT3 and NPM1 mutations were more frequent in the intermediate cytogenetic risk group (82% and 74%; P =0.004 and P =0.047; respectively). ASXL1 and IDH mutations were not correlated with any of the clinical and biological features studied. In univariate analysis, only age and cytogenetics had an impact on overall survival (OS, median of 12mo vs. 3mo, for patients < and ≥65 yr., P <0.001 and 24mo, 11mo and 3mo for favourable, intermediate and high risk, P =0.005). Mutational status of ASXL1, IDH1, IDH2, WT1, FLT3, NPM1 and CEBPa did not impact on outcome in the whole series. However, when the analysis was restricted to patients with intermediate cytogenetic risk, patients with FLT3 mutations had a shorter OS (19mo vs. 8mo, wild-type vs. mutated patients; P =0.047) and those with WT1 mutations showed a trend towards an inferior OS (11mo vs. 1mo, wild-type vs. mutated patients; P = 0.066). In multivariate analysis in patients with intermediate cytogenetic risk, the age [HR (95% CI) = 3.3 (1.9 − 5.9) P <0.001], and FLT3 status [HR (95% CI) = 2.2 (1.2–3.9) P =0.008] retained an independent adverse significance for OS. In terms of relapse free survival any of the variables showed a significant implication. To sum up, the incidence found for the studied genes was lower than the previously reported: ASXL1, 9.2%; IDH1R132, 2.9%; IDH2R140, 6.3%; WT1, 2.9%; and c-CBL, 0%. We were unable to find a pattern of cooperating mutations in the studied group of genes or any impact of these mutations on the outcome. Disclosures: No relevant conflicts of interest to declare.

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.

Modulation of the Clonal Composition in Relapsed CLL: A Study Based on Targeted Deep-Sequencing of ATM, BIRC3, NOTCH1, POT1, SF3B1, SAMHD1 and TP53

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1721-1721
Author(s):  
Sabine Jeromin ◽  
Wolfgang Kern ◽  
Richard Schabath ◽  
Tamara Alpermann ◽  
Niroshan Nadarajah ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Clonal Evolution ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutation Load ◽  
Employment Equity ◽  
Time Points ◽  
Mutational Status ◽  
Equity Ownership ◽  
Time Point

Abstract Background: Relapse or refractory disease is a challenging clinical problem in the majority of chronic lymphocytic leukemia (CLL) patients. Treatment influences the clonal composition by selection and eventually induction of additional genetic abnormalities. Aim: To characterize the clonal evolution in relapsed CLL patients by deep-sequencing analysis of mutations in ATM, BIRC3, NOTCH1, POT1, SF3B1, SAMHD1 and TP53. Patients and Methods: Sequential samples of 20 relapsed CLL patients at three time-points were evaluated: A: at diagnosis (n=16) or in untreated state (n=4), B: at first relapse (n=20) and C: at second relapse (n=2). Patients were treated with diverse treatment schemes and had temporarily achieved either complete or partial remission during the course of the disease. The median time from diagnosis to first-line treatment was 13 months (1 - 169 months). All geneswere sequenced by a deep sequencing approach (Illumina, San Diego, CA). IGHV mutational status was determined by direct Sanger sequencing at time-point A. Chromosome banding analysis (CBA) and FISH data on del(17p), del(11q), trisomy 12 (+12), and del(13q) were available in 33/42 and 36/42 samples, respectively. Results: Initially, samples at first relapse were sequenced. Mutations in SF3B1 (6/20, 30%), TP53 (5/20, 25%), ATM (5/20, 25%), NOTCH1 (4/20, 20%), and SAMHD1 (3/20, 15%) were detected at high frequencies. No mutations were detected in BIRC3 and POT1. In total, 75% of cases presented with at least one mutation (Figure 1): 8 (40%) cases had one, 6 (30%) cases had two and one patient had three genes concomitantly mutated (mut). Patients were also analyzed for IGHV mutational status at diagnosis and presented with unmutated status at a frequency of 85% (17/20). Subsequently, samples from cases with mutations were analyzed at time-point A. In 12/15 (80%) cases the mutations at relapse were already detectable at time-point A with a similar load indicating presence of the main clone before and after chemotherapy. However, in 7/15 (47%) patients new gene mutations were acquired either additionally to existing mutations (n=4) or in previously wild-type cases (n=3). In 5/7 (71%) cases mutations were located in TP53. TP53 mut were the only mutations that were not detected in samples before treatment (sensitivity of 3%). Thus, TP53 mutations might have been initiated by chemotherapy or exist in a minor subclone subsequently selected by chemotherapy. Furthermore, only 4 cases had low-level mutations (3-6% mutation load) at diagnosis in either SAMHD1 or SF3B1 that eventually increased in their burden during disease course. Of note, in two patients a multibranching clonal evolution could be identified (#2, #9). For patient #2 three time-points were analyzed. At diagnosis 2 ATM mutations were detected with mutation loads of about 20%, each. In the course of the disease these mutations were lost, whereas SF3B1 mut showed a stable mutation load in all three time-points of about 40%. In contrast, mutation load of SAMHD1 increased over time from 4% to 87%. CBA was performed at diagnosis and detected independent clones with del(11q) and del(13q). Accordingly, del(11q) detected by FISH at diagnosis was lost and the percentage of cells with del(13q) increased from diagnosis to time-point C. Therefore, patient #2 shows different genetic subclones in parallel that were eradicated or selected by chemotherapy. In patient #9 two SF3B1 mutations were initially detected with the same mutation load of 10%. After treatment one mutation was lost, whereas the load of the second mutation increased indicating at least two different subclones with only one of them being sensitive to chemotherapy. This might be due to different additional aberrations. Indeed, CBA identified two clones: one with +12 alone and one in combination with del(13q). FISH revealed unchanged percentage of +12 at time-point B, whereas del(13q) positive cells were diminished. Conclusions: In 75% of relapsed CLL cases mutations in SF3B1, TP53, ATM, NOTCH1, and SAMHD1 are present at high frequencies. 80% of these mutations are already detectable before treatment initiation representing the main clone. Remarkably, TP53 mutations were the only mutations that were not detected before but only after chemotherapy. Figure 1. Distribution of gene mutations in 15 CLL cases with mutations at diagnosis or before treatment (D) and at relapse (R). Red = mutated, grey = wild-type, white = not analyzed. Figure 1. Distribution of gene mutations in 15 CLL cases with mutations at diagnosis or before treatment (D) and at relapse (R). Red = mutated, grey = wild-type, white = not analyzed. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schabath:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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