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

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.

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RUNX1 Mutated AML Are Associated with a Distinct Pattern of Cytogenetic and Additional Molecular Genetic Abnormalities

Blood ◽

10.1182/blood.v124.21.124.124 ◽

2014 ◽

Vol 124 (21) ◽

pp. 124-124

Author(s):

Niroshan Nadarajah ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach ◽

Claudia Haferlach

Keyword(s):

High Frequency ◽

De Novo ◽

Male Gender ◽

The Other ◽

Typical Pattern ◽

Employment Equity ◽

Cytogenetic Abnormalities ◽

Genetic Abnormalities ◽

Equity Ownership ◽

De Novo Aml

Abstract Background: Mutations in RUNX1 have been reported in 5 to 20% of AML. The aim of this study was to analyse a large cohort of RUNX1 mutated AML in detail with respect to accompanying cytogenetic and moleculargenetic abnormalities, mutation type and mutation load. Patients and Methods: We investigated 468 AML with RUNX1 mutations (mut) all identified during diagnostic work-up in our laboratory. Sequencing was performed by either Sanger or next-generation sequencing. Median age was 71.9 yrs (range 18-91 yrs), male : female ratio 297 : 171. 369 patients had de novo AML, 75 s-AML following MDS, 24 t-AML. For all patients (pts) cytogenetics was available and categorized according to MRC (Grimwade et al. Blood 2010). Mutation data was available for NPM1 (n=455), MLL-PTD (n=454), CEBPA (n=449), FLT3-TKD (n=422), WT1 (n=394), FLT3-ITD (n=362), ASXL1 (n=293), TP53 (n=204), DNMT3A (n=143), TET2 (n=143), and SF3B1 (n=99). Data on FAB subtype was available in 342 cases (73.1%). Results: The most frequent FAB subtype in RUNX1mut AML was AML M2 (145/342; 42.4%), followed by M1 (23.1%), M4 (15.8%), M0 (15.5%), other subtypes were rare (<2%). Cytogenetics were favorable in 2 (0.4%), intermediate in 397 (84.8%) and adverse in 69 (14.7%) pts. Most frequent cytogenetic abnormalities were +8 (76; 34.7%), +13 (46; 21.0%), +21 (15; 6.8%), +11 (12; 5.5%), -7 (22; 10%), del(7q) (18; 8.2%), del(5q) (12; 5.5%). Only 12 pts (5.5%) showed a complex karyotype (> 3 aberrations). The frequency of all other abnormalities was <5%. Two pts showed a t(15;17)(q24;q21) and 3 MLL rearrangements (partner genes on 7q32, 17q21, 19p13). None of the other recurrent cytogenetic abnormalities according to WHO classification was present. 249 (53.2%) pts had a normal karyotype. ASXL1mut were the most frequent accompanying mutations (36.5%). Mutation frequencies for the other genes were: SF3B1 (27.3%), TET2 (26.6%), FLT3-ITD (17.7%), DNMT3A (16.1%), MLL-PTD (10.8%), CEBPA (6.9%; single mutated (sm): 5.3%, double mutated (dm): 1.6%), WT1 (5.8%), FLT3-TKD (5.5%), TP53 (4.4%), and NPM1 (1.1%). While mutations in ASXL1 and TET2 frequently occurred concomitantly (37.5% of ASXL1mut cases also harboured a TET2mut, p=0.032), ASXL1mut and SF3B1mut rarely co-occurred (only 2 ASXL1mut were SF3B1mut, p=0.001). There were no differences in mutation frequencies and cytogenetic abnormalities observed between de novo AML, s-AML and t-AML. In 368 cases (78.6%) one RUNX1 mutation was detected, 81 pts (17.3%) showed two, 16 cases (3.4%) three, and 3 cases (0.6%) four. The difference in mutation loads between the 2 mutations with the highest load was ≤10% in 55/100 pts, suggesting that both mutations were present in the same clone. In total 592 RUNX1 mutations were detected, 242 (40.9%) were frameshift, 206 (34.8%) missense, 83 (14.0%) nonsense, 37 (6.3%) in frame insertion/deletions, 23 (3.9%) splice-site and 1 no stop change. No association between the types of 1st and 2nd mutations was observed. The mutations were homozygous in 65 pts (13.9%), these were predominantly missense mutations (38; 58.5%). A significantly higher frequency of homozygous mutations was observed in pts with +13 (28.3%, p=0.006), +21 (46.7%; p=0.002), AML M0 (35.8%; p<0.001) and M1 (22.8%; p=0.014) while they were less frequent in M2 (4.8%; p<0.001) and M4 (3.7%, p=0.017). Survival analyses were restricted to 203 de novo AML pts who were treated with intensive chemotherapy (median overall survival (OS) 20.4 months (mo)). Median OS was significantly longer in female than in male pts (45.6 vs 16.3 mo; p=0.003) and in pts ≤ 60 vs >60 yrs (44.4 vs 16.1 mo; p<0.001). Shorter OS was observed for pts with del(5q) (1.8 vs 21.1 mo; p=0.002) and adverse cytogenetics (12.4 vs 23.3 mo, p=0.016). Including these 4 parameters into a multivariate Cox regression analysis revealed that age, male gender and del(5q) were independently associated with shorter OS (relative risk: 1.8, 1.6, 3.4; p: 0.01, 0.038, 0.028) Conclusions: RUNX1 mutated AML: 1. is associated with a myeloid rather than monocytic differentiation, 2. shows a typical pattern of cytogenetic abnormalities with a high frequency of +8 and +13, 3. has a typical pattern of additional molecular mutations with a high frequency of accompanying ASXL1 und SF3B1 mutations, 4. is nearly mutually exclusive of NPM1 and CEBPAdm mutations and other entity defining genetic abnormalities, 5. Male gender, age >60 yrs and del(5q) were negative prognostic factors. Disclosures Nadarajah: 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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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

Blood ◽

10.1182/blood.v126.23.2578.2578 ◽

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.

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

Blood ◽

10.1182/blood.v122.21.2560.2560 ◽

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.

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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 ◽

10.1182/blood.v122.21.2582.2582 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2582-2582 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v126.23.3811.3811 ◽

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.

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AML with Inv(3)(q21q26) or t(3;3)(q21;q26) Are Frequently Accompanied by Mutations in RUNX1 and NRAS and Show a High Incidence of NF1 Deletions: a Study On 40 Cases.

Blood ◽

10.1182/blood.v114.22.273.273 ◽

2009 ◽

Vol 114 (22) ◽

pp. 273-273

Author(s):

Claudia Haferlach, ◽

Torsten Haferlach ◽

Frank Dicker ◽

Tamara Weiss ◽

Wolfgang Kern ◽

...

Keyword(s):

Cell Count ◽

Chromosome Aberrations ◽

De Novo ◽

Blast Cell ◽

Splice Mutation ◽

Acute Leukemias ◽

Genetic Abnormalities ◽

Additional Chromosome ◽

Equity Ownership ◽

De Novo Aml

Abstract Abstract 273 AML with inv(3)(q21q26) or t(3;3)(q21;q26) have been defined as a distinct entity in the WHO classification in the category of “acute myeloid leukemia with recurrent genetic abnormalities”. Whereas cases with t(8;21)(q22;q22), inv(16)(p13q22/t(16;16)(p13;q22) or t(15;17)(q22;q12) are considered as acute leukemias regardless of the percentage of blasts in the bone marrow, it is not clear whether cases with inv(3)(q21q26)/t(3;3)(q21;q26) should be categorized as such if blast cell count is <20%. To analyze the spectrum of myeloid neoplasms in which inv(3)/t(3;3) occurs, all cases with these abnormalities as diagnosed since 2005 in our laboratory were identified. CML cases showing a t(9;22) and inv(3)/t(3;3) were not included as here CML blast crisis was evident. To further decipher accompanying genetic lesions in AML with inv(3)(q21q26)/t(3;3)(q21;q26) we performed in addition to chromosome banding analyses, FISH for the detection of NF1 deletions and mutation screening of NPM1, MLL (PTD), FLT3 (ITD, TKD), RUNX1, KIT (D816), NRAS (codon12/13/61), CBL (exon8/9 splice mutation), MPL, and JAK2 (V617F, exon12). The study cohort included 20 males and 20 females. Median age was 64.8 years (range: 36.3–91.3), median WBC was 3.9G/l (range: 1.1–75.0G/l) and median platelet count 133G/l (range: 5–799G/l). Based on cytomorphology 23 cases were classified as AML (de novo: 18, t-AML: 2, s-AML: 3 (2 after MPN, 1 after MDS), 15 as MDS and 2 as MPN. 27 showed an inv(3)(q21q26) and 13 a t(3;3)(q21;q26). Additional chromosome aberrations were observed in 23/40 (57.5%) cases (AML: 16/23, 69.6%; MDS 7/17, 41.2%): one, two or more than two additional aberrations in 13, 6 and 4 cases, respectively. Recurrent abnormalities were −7 (n=17), del(5q) (n=4). The following molecular mutations were detected: FLT3-TKD (1/32, 3.1%), NRAS (7/29, 24%), RUNX1 (6/29, 20.7%), CBL exon8/9 splice mutation (n=2/24, 8.3%) and JAK2V617F (2/27, 7.4%). One case each with JAK2V617F was diagnosed with MPN and s-AML after MPN, respectively. No mutations were detected for: NPM1 (0/20), MLL-PTD (0/22), FLT3-ITD (0/35), KIT (0/25), JAK2exon12 (0/13) and MPL (0/13). One copy of the NF1 (neurofibromin 1) gene, which negatively regulates the RAS pathway, was found to be deleted in 4/26 (15.4%) cases using FISH (NF1/MPO probe from Kreatech, Amsterdam, The Netherlands). Overall, 22 molecular alterations were observed in the analyzed genes (15 in AML cases, 7 in MDS/MPN cases). Taking also additional cytogenetic aberrations into account 31/40 patients (20/23 AML, 11/17 MDS/MPN) showed further genetic abnormalities in addition to inv(3)/t(3;3). Survival data were available in 28 cases. No significant differences were observed with respect to overall survival (OS) and event-free survival (EFS) between cases diagnosed as MDS or MPN vs AML. Whereas no impact of additional chromosome aberrations or presence of molecular mutations on OS was observed, a trend for a shorter OS in cases with RUNX1 mutation was found (2 months vs 21.8 months, p=0.07). In addition 17 cases with inv(3)(q21q26)/t(3;3)(q21;q26) and de novo AML were compared to 814 de novo AML without inv(3)(q21q26)/t(3;3)(q21;q26). The median OS for the total cohort was 48.2 months (mo), the median EFS 16.3 mo. Based on cytogenetics cases were assigned into 8 subgroups: 1. t(15;17)(q22;q21), 2. t(8;21)(q22;q22), 3. inv(16)(p13q22)/t(16;16)(p13;q22), 4. 11q23/MLL abnormalities, 5. inv(3)(q21q26)/t(3;3)(q21;q26), 6. normal karyotype, 7. complex karyotype, 8. other abnormalities. Median OS was not reached for groups 1, 2, 3, 4, and 6 and was 23.4 mo, 11.8 mo and 32.2 mo for groups 5, 7, and 8, respectively. OS at 2 yrs was 95.6%, 96.3%, 76.6%, 64.9%, 47.5%, 63.5%, 23.9% and 58.5% for groups 1–8, respectively. The respective data for median EFS were: not reached for groups 1 and 2 and 15.9 mo, 13.5 mo, 6.3 mo, 16.9 mo, 7.5 mo and 12.5 mo for groups 3–8, respectively. In summary, inv(3)/t(3;3) is observed in MDS, MPN, de novo AML, s-AML and t-AML and frequently accompanied by additional cytogenetic or molecular genetic abnormalities. Especially frequent were mutations in RUNX1, NRAS and deletions of NF1. Prognosis of patients with inv(3)/t(3;3) is unfavourable irrespective of the cytomorphological diagnosis. These data suggest to consider cases with inv(3)/t(3;3) as one entity regardless of blast cell count. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

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

Blood ◽

10.1182/blood.v116.21.2758.2758 ◽

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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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 ◽

10.1182/blood.v118.21.413.413 ◽

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.

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Final Safety and Efficacy Results from the CPX-351 Early Access Program (EAP) for Older Patients with High-Risk/Secondary Acute Myeloid Leukemia (sAML)

Blood ◽

10.1182/blood-2018-99-111537 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1434-1434

Author(s):

Gail J. Roboz ◽

Melissa L. Larson ◽

S. Eric Rubenstein ◽

Scott R Solomon ◽

Gary J. Schiller ◽

...

Keyword(s):

High Risk ◽

Older Patients ◽

Research Funding ◽

De Novo ◽

Newly Diagnosed ◽

Open Label ◽

Employment Equity ◽

Phase 3 ◽

Equity Ownership ◽

De Novo Aml

Abstract Background: CPX-351 (Vyxeos®) is a dual-drug liposomal encapsulation of cytarabine and daunorubicin at a synergistic ratio. In a large randomized, open-label, multicenter, phase 3 study of CPX-351 versus conventional cytarabine/daunorubicin chemotherapy (7+3 regimen) in adults aged 60-75 years with newly diagnosed high-risk/sAML, patients treated with CPX-351 had significantly longer survival times and higher remission rates (Lancet JE, et al. J Clin Oncol. 2018). Based on these results, CPX-351 was approved by the US FDA in 2017 for the treatment of adults with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes (AML-MRC). This abstract reports the results of an EAP study that provided expanded access to CPX-351 for older patients who met the eligibility criteria for the phase 3 study and collected additional data on safety and efficacy. Methods: In this phase 4, single-arm, open-label EAP, patients were between 60-75 years of age and had confirmed high-risk/sAML (therapy-related AML [tAML], AML with a history of myelodysplasia [MDS] or chronic myelomonocytic leukemia [CMML], or de novo AML with MDS karyotype). Patients could receive up to 2 cycles of induction with CPX-351 100 units/m2 (cytarabine 100 mg/m2 + daunorubicin 44 mg/m2) on Days 1, 3, and 5 (2nd induction: Days 1 and 3). Patients with complete remission (CR) or CR with incomplete platelet or neutrophil recovery (CRi) could receive up to 4 cycles of consolidation with CPX-351 65 units/m2 (cytarabine 65 mg/m2 + daunorubicin 28.6 mg/m2) on Days 1 and 3. The primary endpoint was safety, and the secondary endpoint was the rate of CR+CRi. Results: Overall, 52 patients received ≥1 dose of CPX-351 and were included in the safety analysis. Among these patients, the median age was 70 years (range: 55-75) and 23% had an Eastern Cooperative Oncology Group score of 2. Median time since diagnosis was 0.30 months (range: 0.03-36.14) months. Patients with AML-MRC accounted for 77% of the safety analysis population, including those with antecedent MDS with prior hypomethylating agent (HMA) treatment (25%), antecedent MDS without prior HMA treatment (21%), antecedent CMML (8%), and de novo AML with MDS karyotype (23%); 23% of patients had tAML. All patients received 1 induction cycle and 25% received 2 induction cycles; 17%, 8%, 4%, and 2% of patients received 1, 2, 3, and 4 consolidation cycles, respectively. CR+CRi was achieved in 23 patients (44% [95% CI: 31%, 59%]), including 15 with CR (29% [95% CI: 17%, 43%]) and 8 with CRi (15% [95% CI: 7%, 28%]; Table). The median time to remission was 37 days (range: 15-72). At the end of the study, 47 (90%) of patients were still alive and 11 (21%) patients received transplant. All patients were alive at Day 30, and the mortality rate at Day 60 was 6%. The safety profile observed in this EAP study was consistent with that of the phase 3, randomized study (Table). Treatment-emergent adverse events (TEAEs) of any grade occurred in 96% of patients, including 44% of patients with an TEAE deemed related to treatment; the only treatment-related AE that occurred in >10% of patients was febrile neutropenia (31%). Only 2 patients (4%) discontinued treatment due to an AE (ejection fraction decrease and intercranial hemorrhage [n = 1 each]). Five patients (10%) had grade 5 AEs during the study, including disease progression, multiple organ dysfunction syndrome, acute respiratory distress syndrome, aspiration, and intracranial hemorrhage (n = 1 each). Conclusions: The data from this EAP study were consistent with results from the randomized, phase 3 study. The safety profile in the EAP study was similar to that observed in the phase 3 study and there was a similar CR+CRi rate (44% vs 48%, respectively) in this high-risk/sAML population. Disclosures Roboz: Sandoz: Consultancy; Cellectis: Research Funding; Argenx: Consultancy; Bayer: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Consultancy; Aphivena Therapeutics: Consultancy; Sandoz: Consultancy; AbbVie: Consultancy; Pfizer: Consultancy; Roche/Genentech: Consultancy; Novartis: Consultancy; Celgene Corporation: Consultancy; Celltrion: Consultancy; Jazz Pharmaceuticals: Consultancy; Roche/Genentech: Consultancy; Eisai: Consultancy; Otsuka: Consultancy; Astex Pharmaceuticals: Consultancy; Celgene Corporation: Consultancy; Janssen Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy; Eisai: Consultancy; Otsuka: Consultancy; Orsenix: Consultancy; Celltrion: Consultancy; AbbVie: Consultancy; Daiichi Sankyo: Consultancy; Aphivena Therapeutics: Consultancy; Bayer: Consultancy; Argenx: Consultancy; Jazz Pharmaceuticals: Consultancy; Astex Pharmaceuticals: Consultancy; Cellectis: Research Funding; Orsenix: Consultancy; Pfizer: Consultancy. Rubenstein:Alexion: Consultancy, Honoraria, Speakers Bureau; Cyclacel: Other: Travel support; Astex: Other: Travel support. Schiller:Celator/Jazz Pharmaceuticals: Research Funding; Pharmacyclics: Research Funding. An:Jazz Pharmaceuticals: Employment. Mancino:Jazz Pharmaceuticals: Employment. Chiarella:Celator/Jazz Pharmaceuticals: Employment, Equity Ownership. Louie:Celator/Jazz Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Lin:Jazz Pharmaceuticals: Honoraria.

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IDH1 Mutations Are Detected in 9.3% of All AML and Are Strongly Associated with Intermediate Risk Karyotype and Unfavourable Prognosis: a Study of 999 Patients

Blood ◽

10.1182/blood.v114.22.lba-3.lba-3 ◽

2009 ◽

Vol 114 (22) ◽

pp. LBA-3-LBA-3 ◽

Cited By ~ 2

Author(s):

Susanne Schnittger ◽

Claudia Haferlach ◽

Madlen Ulke ◽

Leyla Kaya ◽

Tamara Weiss ◽

...

Keyword(s):

De Novo ◽

Normal Karyotype ◽

Independent Effect ◽

Classical Type ◽

Intermediate Risk ◽

Base Exchange ◽

Equity Ownership ◽

Idh1 Mutations ◽

De Novo Aml ◽

Cebpa Mutations

Abstract Abstract LBA-3 Introduction: IDH1 is the gene coding for the soluble isocitrate dehydrogenease 1 (NADP+), which catalyzes the oxidative decaroxylation of isocitrate to 2-oxoglutarate. The gene has been shown to be frequently mutated in high-grade gliomas at residue p.R132, which is located in the substrate binding site of IDH1. So far, several other tumors have been analyzed without detection of the respective mutation (Bleeker et al., Human Mutation 2009). However, recently a next generation sequencing project found IDH1 mutated in 8.5% of AML with normal karyotype (Mardis et al., NEJM, 2009). Aim: To further evaluate the importance of IDH1R132 (IDH1mut) in AML we have analyzed a cohort of 999 comprehensively characterized AML cases. Methods: The respective base exchange was analysed by a LightCycler based melting curve assay with subsequent sequencing of the positive samples. Results: The cohort was comprised of 536 male and 463 female patients (median age: 65.9 years; range: 17.1- 93.3 years). 833 had de novo AML (83.4%), 122 AML following MDS (s-AML,12.1%) and 44 AML after previous treatment of different malignancies (t-AML, 4.4%). Karyotype was available in all cases: 681 had a normal karyotype (NK) AML, and 319 had chromosomal aberrations (t(15;17): n=29; inv(16): n=12, t(8;21): n=23, t(11q23): n=10, t(6;9): n=4, inv(3): n=3; -7: n=27, +8: n=29, +13: n=11, -Y: n=4; complex aberrant: n=60, others: n=106). Overall, in 93 pts IDH1 p.R132 mutations were detected (9.3%). Five different amino acid exchanges were observed: R132C (n=49), R132L (n=22), R132 H and G (n=7, each), and R132S (n=5). With respect to history of the patient IDH1mut were found in 80/833 of de novo AML (9.6%), 11/122 (9.0%) of s-AML, and 2/44 (4.5%) of t-AML, respectively (n.s.). More females (57/463, 12.3%) than males (36/536; 6.7%) had IDH1mut (p=0.003). Age was slightly higher in the mutated cases (63.9 vs. 61.9 years, n.s.). No differences were found for WBC count. IDH1mut were distributed differently between karyotypes: in NK 69/681 (10.1%) and in aberrant karyotypes 24/318 (7.5%). However, IDH1 was never mutated in inv(16), t(8;21), t(6;9), t(11q23), inv(3), or in complex aberrant karyotypes (n=112). In 2 of 27 cases (7.4%) with t(15;17) an IDH1 mutation was detected. Thus, the IDH1 mututations clustered in the intermediate risk karyotype group in comparison to the good or poor risk groups (91/771; 11.8% vs 2/134 (1.5%), p<0.001). The cohort was also characterized for several other molecular mutations. FLT3-ITD was present in 22% (212/954), FLT3-TKD in 6.7% (33/496), NPM1 in 35.4% (329/929), NRAS in 14.6% (48/328), MLL-PTD in 6.9% (64/932), CEBPA mutations in 7.4% (48/645) and RUNX1 mutations in 33.0% (99/299) of analysed cases, respectively. IHD1 mutations were found to be more frequent in NPM1 mutated than in NPM1wt cases (41/329; 12.4% vs 48/598; 8.0%, p= 0.019) and in those with MLL-PTD (11/64; 17.2% vs 77/867; 8.9%, p= 0.031). With lower frequencies IDH1mut were also detected together with RUNX1 mutations (n=8/99), CEBPA mutations (n=2/48), NRAS mutations (n=7/48), and FLT3-TKD (n=1/33). IDH1 was similarly distributed between FLT3-ITD mutated and unmutated cases (18/212; 8.5% vs. 72/744; 9.7%). In 22 (23.7%) of all IDH1mut AML no additional mutation was detected, whereas in 48 (51.6%) one additional, in 22 (23.7%) two additional and in one case three additional mutations were found. An unfavourable effect of IDH1mut on event free survival (EFS) was observed in the total group (median: 272 vs. 456 days; p=0.007) as well as in those with intermediate risk karyotype (median: 272 vs. 449 days; p=0.008). A shorter EFS of the IDH1mut was particularly seen in the NPM1wt cohort (median: 244 vs. 375 days; p=0.038) with a strong trend for an independent effect in a multivariate analysis (p=0.089). Conclusions: IDH1 mutations are frequent in AML and are prognostically unfavourable especially in the NPM1wt cohort. IDH1 mutations seem to be a new class of mutation probably complementing with the classical type 1 and type 2 mutations. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Ulke:MLL Munich Leukemia Lab: Employment. Kaya:MLL Munich Leukemia Lab: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

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