ASXL1 exon 12 Mutations Are Frequent in AML with Intermediate Risk Karyotype and Are Independently Associated with An Extremely Poor Outcome

Abstract Abstract 416 Introduction: ASXL1 mutations have recently been described in a number of different myeloid malignancies. Data on frequency, association with other markers and outcome in AML are rare. Aim: The aim of this study was to evaluate ASXL1 mutations (ASXL1mut) in AML with intermediate risk karyotype for frequency, association with other mutations and impact on outcome. Methods: We analyzed 476 cases with intermediate risk de novo AML for ASXL1 mutations by direct Sanger sequencing of exon 12. Other mutations were analyzed as described previously and were available in part of the patients (NPM1: n=474, FLT3-ITD: n=473, FLT3-TKD: n=407, MLL-PTD: n=474, CEBPA: n=447, RUNX1: n=150, WT1: n=384, IDH1: n=464 and IDH2: n=444, TET2: n=109, NRAS: n=191; KRAS: n=110, DNMT3A: n=83). 397 cases had a normal karyotype (NK) and 79 had intermediate risk aberrant cytogenetics (according to MRC). Female/male ratio was 221/255 and age ranged from 18.5–100.4 y (median: 66.4). Results: Overall, in 70/476 patients (14.7%) ASXL1mut were detected. In detail, the most frequent mutation was p.G646WfsX12 (n=36) followed by p.E635RfsX15 (n=9), and p.Y591X (n=2). The remaining 21 mutations were non-recurrent consisting of 2 frameshift, 13 nonsense and 6 missense mutations. All mutations were detected with a mutation/wildtype load of 40–50% and none of the cases had more than one ASXL1mut. ASXL1mut were more frequent in males than in females (56/255, 22.0% vs 14/221, 6.3%, p=0.001) and were associated with higher median age (72.4 yrs vs 64.1 yrs, p<0.001). In detail, in the cohort > 65 yrs 21.7% (n=55/254) and in those <65 yrs only 6.8% (n=15/222) were ASXL1mut (p<0.001). With respect to morphology ASXL1mut were more frequent in AML without maturation than in all others (37.5% vs 14.3%, p=0.022). In 242 cases immunophenotyping data was available and cases with ASXL1mut (n=34) had a higher expression of CD13 (mean±SD, 55±23% vs. 43±25%, p=0.012), CD34 (46±32% vs. 24±26%, p<0.001), CD133 (29±27% vs. 16±23%, p=0.006) and HLA-DR (42±25% vs. 30±24%, p=0.009) as well as a lower expression of CD33 (66±21% vs. 77±21%, p=0.005) and thus had a more immature immunophenotype as compared to ASXL1wt. There was no association with leukocyte or platelet counts. With regard to cytogenetics ASXL1mut were more frequent in those with aberrant karyotype than in NK (20/79, 25.3% vs 50/397, 12.6%, p=0.008). Generally, ASXL1mut were observed together with all other molecular mutations but there was a strong correlation to RUNX1mut (n=18/43, 41.9% vs 19/107, 17.8% in RUNX1wt, p=0.003) and a negative correlation with NPM1mut (n=9/274; 3.3% vs. n=61/200, 30.5% in NPM1wt, p<0.001) and DNMT3Amut (1/26, 3.8% vs. 19/55 in DNMT3A, 34.5%, p=0.002). Patients with ASXL1mut had a shorter overall survival (OS) (median: 11.2 vs 38.8 months, p<0.001) and event free survival (EFS) (median: 9.0 vs 23.9 months, p<0.001). In detail, this adverse impact could be shown for both NK (OS: median: 10.9 vs 38.3 months, p<0.001; EFS: 9.8 vs. 26.5 months, p<0.001) and intermediate risk aberrant cytogenetics (OS: median: 8.6 vs 38.8 months, p<0.001; EFS: 5.3 vs 21.5 months, p=0.011), separately. Although the ASXL1mut were much more frequent in the elderly and compared to the ASXL1wt had a shorter OS (median: 7.0 vs 16.3 months, p=0.002) an adverse effect on survival could also be shown in the cohort <65 yrs (median OS: 11.6 vs 47.3 months, p<0.001 and median EFS: 9.3 vs 34.5 months, p<0.001). Because of the high coincidence of the two mutations the impact of ASXL1mut in dependence of RUNX1 status was analyzed. In the RUNX1mut (n=43) the ASXL1mut (n=18) still had an adverse impact on EFS (median: 5.3 vs 15.6 months, p=0.010) and a trend for shorter OS (10.7 vs. 20.5 months, p=0.079). In a multivariable analysis ASXL1 is an unfavourable factor for OS independent of age and RUNX1 mutational status (p=0.026, RR: 2.0). Conclusions:ASXL1 mutations belong to the most frequent mutations in intermediate risk AML. There is a strong association with male sex, high age, immature phenotype and RUNX1mut. Still, ASXL1mut retained its independent very poor prognostic impact. Although the number of known molecular markers in AML is continuously increasing and selection of the most import markers for diagnostic work-up seems challenging this data indicates that ASXL1 is one of the most prominent candidates. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Characterization and Prognostic Impact of Multiple Productive IGHV Rearrangements in CLL

Blood ◽

10.1182/blood.v128.22.3207.3207 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3207-3207

Author(s):

Sabine Jeromin ◽

Claudia Haferlach ◽

Frank Dicker ◽

Manja Meggendorfer ◽

Torsten Haferlach ◽

...

Keyword(s):

Prognostic Factors ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Employment Equity ◽

Mutational Status ◽

Treatment Naïve ◽

Trisomy 12 ◽

Equity Ownership ◽

The Impact

Abstract Background: In chronic lymphocytic leukemia (CLL) one of the strongest prognostic factors is IGHV mutational status. Infrequently, patients present not only with a single IGHV rearrangement but with multiple productive rearrangements. In about 2% of all CLL patients analyzed on cDNA level multiple rearrangements display the same mutational status and are categorized accordingly following ERIC recommendations. In another 1% rearrangements with discordant IGHV mutational status are detected and preclude a definite risk assignment. Only limited data exist on these rare subgroups. Aim: To characterize treatment-naive CLL patients with multiple productive IGHV rearrangements and determine the impact on prognosis. Patients and Methods: Out of 8,016 treatment-naive CLL patients between 2005 and 2015 and with data on IGHV mutational status we identified 204 (3%) with multiple productive rearrangements. IGHV mutational status was analyzed on cDNA and in all cases according to ERIC recommendations. IGHV mutated status (M) was defined by sequence identity <98% and unmutated status (U) by ≥98%. Chromosome banding analysis was available in 102 cases and interphase FISH with probes for 17p13, 13q14, 11q22 and centromeric region of chromosome 12 in 191. Male:female ratio was 3:1 and median age 68 years (range: 38-89). Additionally, data on SF3B1 and TP53 mutations was present in all cases. Follow-up data on time to first treatment (TTT) and overall survival (OS) was available in 105 cases with a median follow-up of 4 years. For statistical comparison we used a cohort of 1,262 untreated CLL patients with single IGHV rearrangement (median age: 67 years; range: 30-91, median follow-up: 6 years). Results: Out of 204 patients with multiple, productive rearrangements 199 (98%) presented with two and 5 patients (2%) with three IGHV rearrangements. Concordant IGHV mutated status (MM) was present in 120 cases (59%), whereas concordant unmutated status (UU) was seen in 34 patients (17%). In 50 cases (25%) a mixed IGHV status (UM) was detected. We analyzed frequencies of complex karyotype by CBA, biclonality according to immunophenotype (concurrent kappa restricted and lambda restricted subpopulations) and/or CBA, TP53 disruption (TP53mut and/or del(17p)), SF3B1mut, del(11q), trisomy 12, and del(13q). Overall, a higher frequency of biclonality was detected in patients with multiple vs. single IGHV rearrangements (16% vs. 1%, p<0.001). However, association to neither MM, UU nor UM existed. MM presented with molecular and cytogenetic characteristics similar to M. Correspondingly, UU showed similar frequencies of mutations and aberrations to U, except for higher frequency of trisomy 12 in UU vs. U (42% vs. 19%, p=0.003). Interestingly, UM presented with characteristics similar to U and UU. UM was associated with TP53 disruption vs. M (16% vs. 5%, p=0.003) and vs. MM (5%, p=0.035) as well as with SF3B1mut vs. M (16% vs. 5%, p=0.008). Furthermore, UM cases showed high frequency of del(11q) vs. M (29% vs. 3%, p<0.001) and vs. MM (1%, p<0.001) and less frequently del(13q) sole vs. M (41% vs. 60%, p=0.011) and MM (41% vs. 69%, p=0.001). No significantly differences in TTT were observed between MM and M (median: 13 vs. 14 years) and between UU and U (6 vs. 4 years), respectively. However, the difference between MM vs. UU (p=0.022) and M vs. U (p<0.001) was significant. The UM subgroup presented with a TTT (median: 4 years) similar to U and UU, whereas it was significantly shorter vs. M (p=0.003) and MM (p=0.006), respectively. A similar picture emerged for survival. 5-year OS of MM was not different vs. M (94% vs. 90%) but vs. U (78%, p=0.001). The statistical analysis of OS in UU was hampered by low case numbers. UM presented again with similar 5-year OS vs. U (81% vs. 78%, n.s.) and significantly worse OS vs. M (90%, p=0.049) and vs. MM (94%, p=0.014). Conclusions: (1) Patients with multiple productive IGHV rearrangements and concordant IGHV status show similar prognosis and characteristics to patients with single rearrangement with the respective IGHV status. (2) Cases with mixed IGHV status show similar prognosis to patients with IGHV unmutated status and accordingly are characterized by high frequencies of adverse prognostic factors like TP53 disruption, SF3B1mut, and del(11q), whereas del(13q) sole is less frequent. Disclosures Jeromin: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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WT1 Mutations Are Secondary Events In AML and Show Varying Frequencies Within Genetic Subgroups and Different Impact On Prognosis

Blood ◽

10.1182/blood.v122.21.2542.2542 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2542-2542

Author(s):

Susanne Schnittger ◽

Christiane Eder ◽

Tamara Alpermann ◽

Frank Dicker ◽

Madlen Ulke ◽

...

Keyword(s):

Multivariate Analysis ◽

Median Time ◽

Univariate Analysis ◽

Normal Karyotype ◽

Adverse Impact ◽

Prognostic Impact ◽

Employment Equity ◽

Genetic Aberrations ◽

Equity Ownership ◽

The Impact

Abstract Background Mutations (mut) in the WT1 gene belong to the first genetic aberrations described in AML. In contrast to recurrent fusion genes or NPM1mut WT1mut do not seem to be disease defining. Also in contrast to other mutations in AML, for most of which a certain prognostic value has been established, the impact of WT1mut still is discussed controversially. Aim Analyze the frequency and prognostic impact of WT1 mutations in comparison to other genetic aberrations. Patients and Methods 3,157 unselected AML patients (pts) were analyzed (de novo: n=2,699, s-AML: n=234, t-AML: n=224). 1,708 pts were male and 1,449 female. Median age was 67.1 years (y) (range: 17.8-100.4 y) with 1,108 pts <60 y and 2,049 ≥60 y. The mutational hot spot regions of WT1 (exons 7 and 9) were analyzed by direct Sanger sequencing with a sensitivity of ∼10%. Karyotype and WT1 mutation status was available in all cases. Other mutations were assessed in subsets: ASXL1 (n=1,951), CEBPA (n=2,670), DNMT3A (n=1,293), FLT3-ITD (n=3,149), FLT3-TKD (n=3,004), IDH1R132 (n=2,431), IDH2R140 (n=2,380), IDH2R172 (n=2,412), KRAS (n=1,409), NRAS (n=1,780), NPM1 (n=3,003), MLL-PTD (n=2,961), RUNX1 (n=2,390), TET2 (n=1,016) and TP53 (n=1,215). Results A total of 189 WT1 mutations were detected (exon 7: n=151, exon 9: n=38). The total frequency of WT1mut pts was 175/3,157 (5.5%). 11 pts were double to quadruple mutated. The frequency was heterogeneous with respect to AML subtypes. Compared to all others, significantly higher frequencies were detected in biallelic CEBPAmut (15/110; 13.6%; p=0.001), followed by t(15;17)/PML-RARA (18/164; 11.0%, p=0.004), and FLT3-ITD (58/682; 8.5%, p<0.001). Lower frequencies were observed in DNMT3Amut (18/412; 4.3%, p=0.014, ASXL1mut (6/355; 1.7%, p<0.001), IDH2R140 (5/286; 1.7%, p=0.001), and IDH1R132 (2/222; 0.9%, p<0.001). WT1mut were never detected in pts with complex karyotypes (0/175; p=0.047) or those with IDH2R172 (0/68; p=0.020). Further, WT1mut were more frequent in females (95/1,449, 6.6%) than in males (80/1,708, 4.7%) (p=0.014) and in younger pts (<60 y: 102/1,108, 9.2% vs ≥ 60 y: 73/2,049, 3.6%; p<0.001). Median age of pts with WT1mut was 55.5 y compared to 63.6 in WT1wt (p<0.001). Further, WT1mut were associated with lower platelet count (58.4 vs 84.7 x109/L; p<0.001) and lower hemoglobin level (8.8 vs 9.3 g/dL, p=0.001). There was no association to the history of the disease or white blood cell count. Stability of WT1mut was analyzed in 35 paired diagnostic and relapse samples (median time of relapse after diagnoses: 11.1 months (m); range: 2.6-60.6 m). In 23 cases (65.7%) the WT1mut was retained at relapse and in 12 cases (34.3%) it was lost. In 5 cases a sample at 2nd relapse was available (median time from 1st relapse: 8.5 m, range: 6.0-18.0 m). 3 of these cases retained and 2 lost the WT1mut. Analysis of prognostic impact was restricted to intensively treated pts (n=1,936, WT1mut: n=132, 6.8%). In the total cohort, there was no impact of WT1mut on prognosis. In pts ≥60 y there was a trend to shorter event free survival (EFS) for WT1mut (9.3 vs 12.3 m, p=0.052). In the two prognostically favorable groups with high WT1mut incidences (biallelic CEBPAmut and PML-RARA) no effect on outcome was seen. When restricting the analysis to normal karyotype AML (WT1mut: n=85, WT1wt: n=1,093) WT1mut pts had shorter EFS (10.8 vs 17.9 m, p=0.008). This was true for the younger (12.2 vs 29.0 m, p=0.007) as well as for the older pts (9.3 vs 13.9 m, p=0.016). In a multivariate analysis all parameters with significant impact on EFS in univariate analysis were included: age (p<0.001, HR: 1.24), ASXL1mut (p<0.001, HR: 1.36), FLT3-ITD (p<0.001, HR: 1.55), NPM1mut/FLT3-ITD wild-type (p<0.001, HR:1.55), RUNX1 (p=0.019, HR: 1.23, and WT1mut (p=0.009, HR: 1.64). In multivariate analysis WT1mut was found to have independent adverse impact on EFS (p=0.002, HR: 1.64) besides FLT3-ITD status (p<0.001, HR: 1.71) and age (p<0.001, HR: 1.28). Conclusions WT1 mutations are 1) more frequent in females and younger AML, 2) more frequent in t(15;17)/PML-RARA, biallelic CEBPAmut, FLT3-ITD mutated AML, and nearly mutually exclusive of ASXL1, IDH1, IDH2 and complex karyotype. 3) The distribution pattern in different genetic subtypes and the instability during follow-up as shown by paired sample analyses clearly emphasize a secondary character of this mutation. 4) For AML with normal karyotype an independent adverse impact of WT1mut on EFS was shown. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Ulke:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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

Blood ◽

10.1182/blood.v116.21.102.102 ◽

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.

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

Blood ◽

10.1182/blood.v124.21.123.123 ◽

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.

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Prognostic Impact of the Interaction between DNMT3A mutation and Internal Tandem Duplication of the FLT3 Gene (FLT3-ITD) in Patients with De Novo Mutated NPM1 (NPM1mut) acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-115337 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1492-1492

Author(s):

Guadalupe Oñate ◽

Ana Garrido ◽

Jordi Esteve ◽

Rosa Coll ◽

Montserrat Arnan Sangerman ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukemia ◽

Complete Remission ◽

Myeloid Leukemia ◽

De Novo ◽

Prognostic Impact ◽

Intermediate Risk ◽

Mutational Status ◽

De Novo Aml ◽

Acute Myeloid

Abstract Introduction The association of NPM1mut and FLT3-ITD in de novo acute myeloid leukemia (AML) with intermediate-risk cytogenetics has different prognostic impact depending on the FLT3 allelic burden. Previous studies published by our cooperative group showed that patients with de novo AML of intermediate-risk cytogenetics with NPM1mut and FLT3-ITD low ratio (<0.5, FLT3low) at diagnosis presented an overall survival and relapse rate similar to those with NPM1mut and FLT3wt. Therefore, in the CETLAM-2012 protocol, patients with FLT3low NPM1mut AML are not considered for allogenic hematopoietic stem cell transplant (allo-HSCT) in first complete remission (CR1). Recent studies suggest that the co-occurrence of DNMT3A mutation in FLT3-ITD NPM1mut AML patients confers a worse prognosis regardless of FLT3-ITD ratio. We analysed our data to determine whether these findings were confirmed in our cohort, specifically in the low FLT3-ITD ratio patients, since this could have therapeutic implications. Methods and patients A total of 163 patients with de novo AML, intermediate-risk cytogenetics and NPM1mut were analysed (median age 53 years (18-72); male:female 72:91 (0.79)). Eighty patients (49%) harboured an FLT3-ITD, with a high allelic ratio in 42 of 76 patients with available ITD/wt ratio (55%). They were included in the AML-2003 (n=49) and AML-2012 (n=114) CETLAM protocols. Proportion of patients undergoing alloHSCT in CR1 is detailed in table 1. Bone marrow samples from diagnosis were studied for DNMT3A mutations as previously described. The definition of complete remission (CR), overall survival (OS), leukemia-free survival (LFS) and risk of relapse (RR) followed recommended ELN criteria. The Kaplan-Meier method was used to estimate the distribution of LFS and OS, for RR cumulative incidence was used. Results Out of the 163 patients with AML of intermediate risk cytogenetics and NPM1mut, 78 presented DNMT3A mutations (48%). Of these, 62 (79%) presented mutations in codon R882 or corresponded to DNA insertions/deletions while 16 (21%) harboured missense mutations. Presence of DNMT3A mutation did not associate with FLT3-ITD (ITD/85 DNMT3Awt vs ITD/78 DNMT3Amut, p=0.394). In the entire cohort, 5-year OS, LFS and RR were 58±4.5%, 59±4.6% and 27±13.9%. FLT3-ITD ratio confirmed its prognostic impact when analysing FLT3wt (n=83) vs FLT3low (n=34) vs FLT3high (n=42) patients (5-year OS of 68±6% vs 62±8.7% vs 37±8.6%; p=0.002; and 5-year RR of 18±9.4% vs 27±16.1% vs 41±23.2%; p=0.023). On the contrary, DNMT3Amut did not exert any effect on overall outcome (5-yr OS DNMT3Awt vs DNMT3Amut 61±6.2% vs 55±6.2%; p=0.234) When DNTM3A mutational status was considered, the impact of FLT3-ITD on outcome was mitigated in wild-type DNMT3A population. Thus, we found that DNMT3Awt patients presented no statistical differences in OS according to FLT3 mutational status or ratio: FLT3wt (n=46) vs FLT3-ITD (n=39) was 67±8.5% vs 57±8.2%; p=0.122, whereas FLT3wt (n=46) vs FLT3low (n=18) vs. FLT3high (n=19) was 67±8.5% vs. 66±11.5% vs 46±11.8%; p=0.088 (image 1A).This was also seen in relation to LFS and RR according to FLT3 ratio: 5-yr LFS of FLT3wt vs FLT3low vs FLT3high was 72±7.9% vs 61±12.6% vs 51±13.4%; p=0.244 and 5-year RR of the same groups: 19±8.8% vs 26±12.5% vs 27±21.9%; p=0.724 (image 2A). In the DNMT3Amut group, patients with FLT3-ITD (n=41) presented shorter OS than those with FLT3wt (n=37) with an OS of 37±10.7% vs 69±7.8%; p=0.028. When FLT3 ratio was considered, FLT3wt (n=37) vs FLT3low (n=16) vs FLT3high (n=23) showed an OS of 69±7.8% vs. 58±13.2% vs 27±13.1%; p=0.038 (image 1B). Similar results were seen in LFS according to FLT3 ratio (FLT3wt (n=29) vs FLT3low (n=16) vs FLT3high (n=20) 71±8.6% vs 53±12.9% vs 18±13.8%; p=0.012). Finally, we observed significant differences in the 5-year RR when considering DNMT3Amut patients in relation to FLT3 ratio (FLT3wt vs FLT3low vs FLT3high 18±10.6% vs 27±20% vs 54±28.8%; p=0.021)(image 2B). Conclusions In this study, patients with NPM1mut and FLT3-ITDlow presented a similar outcome to patients with NPM1mut and FLT3wt regardless of DNMT3A mutational status. These results support the modification of alloHCST policy in CR1 in CETLAM-2012, which do not consider alloHSCT for patients with FLT3low. On the other hand, concurrence of DNMT3A mutation may have an added negative effect in patients with NPM1mut and FLT3-ITDhigh, which should be further confirmed in larger studies. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v128.22.284.284 ◽

2016 ◽

Vol 128 (22) ◽

pp. 284-284 ◽

Cited By ~ 1

Author(s):

Anna Stengel ◽

Wolfgang Kern ◽

Manja Meggendorfer ◽

Karolina Perglerová ◽

Torsten Haferlach ◽

...

Keyword(s):

Genetic Alterations ◽

Adverse Impact ◽

Missense Mutations ◽

Employment Equity ◽

Frameshift Mutations ◽

Total Cohort ◽

Equity Ownership ◽

The Impact ◽

Runx1 Mutation ◽

Mutation Analyses

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

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

Blood ◽

10.1182/blood.v128.22.4357.4357 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4357-4357 ◽

Cited By ~ 1

Author(s):

Calogero Vetro ◽

Torsten Haferlach ◽

Manja Meggendorfer ◽

Sabine Jeromin ◽

Constance Regina Baer ◽

...

Keyword(s):

Gene Mutation ◽

Mutation Rate ◽

Gene Mutations ◽

Normal Karyotype ◽

Prognostic Impact ◽

Employment Equity ◽

Mutation Status ◽

Genomic Array ◽

Equity Ownership ◽

The Impact

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

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Prognostic Impact of Genetic Characterization in the GIMEMA LAM99P Study for Newly Diagnosed Adult AML. Relevance of Combined Analysis of Conventional Karyotyping, FLT3 and NPM Mutational Status.

Blood ◽

10.1182/blood.v106.11.226.226 ◽

2005 ◽

Vol 106 (11) ◽

pp. 226-226

Author(s):

G. Saglio ◽

Francesco Lo Coco ◽

A. Cuneo ◽

F. Pane ◽

G. Rege Cambrin ◽

...

Keyword(s):

Multivariate Analysis ◽

High Risk ◽

Genetic Characterization ◽

High Risk Group ◽

Low Risk ◽

Prognostic Impact ◽

Intermediate Risk ◽

Mutational Status ◽

Molecular Studies ◽

The Impact

Abstract Between 1998 and 2002, 509 patients with AML (median age 46 yrs, range 15–60) were enrolled in the multicenter LAM99P study of the Italian GIMEMA group. To better evaluate the clinical impact of genetic characterization, all patients received a uniform protocol and diagnostic samples were centralised for cytogenetic and molecular studies. Therapy consisted of HU pre-treatment (2g/m2 for 5 days) followed by induction with DNR (50 mg/m2 d 1, 3, 5), cytarabine (100 mg/m2 d 1–10) and etoposide (100 mg/m2 d 1–5) and consolidation with cytarabine (500 mg/m2/q12 hrs d 1–6) and DNR (50 mg/m2 d 4–6). After consolidation, eligible patients with an identical HLA donor were to receive allogeneic SCT and the remaining peripheral blood autologous SCT. Cytogenetic and molecular genetic characterization (including analysis of major fusion genes, FLT3 and NPM status) was available in 397 (78%) patients. Compared to previous GIMEMA studies, the possibility to collect samples during the 5d of HU pretreatment considerably improved genetic characterization and in particular centralised karyotyping by overcoming the problem of sampling and shipment over the w-end. After induction, 269/397 (68%) patients achieved CR. For induction response, conventional K identified 3 distinct risk groups as follows: low risk (inv. 16 and t8;21), intermediate (normal K and other anomalies not comprised in the high risk group) and high risk (t3;3, inv.3, t9;22, 11q23, 5/7 abnormalities complex K,) with CR rates of 92%, 67% and 39%, respectively (P<.0001). NPM mutations were significantly associated with older age, higher WBC, normal K and FLT3-ITD. CR rates in NPM+ (mutated) vs. NPM- (wildtype) groups were 76% vs. 60% for the whole population and 81% vs, 61% for patients in the normal K group (P<.001 for both comparisons). Multivariate analysis for CR indicated that low risk K and NPM+ were independent factors favorably affecting CR achievement while FLT3 status had no significant impact on CR. The analysis of prognostic factors for DFS and OS was carried out in 269 patients in CR (median follow-up of 39 mos.) and multivariate analysis performed after adjusting for unfavorable factors (WBC count). Multivariate analysis of variables influencing OS showed the following: low vs intermediate K, P=.0005; high vs intermediate K, P<.0001; FLT3+ vs. FLT3−, P=.06. Multivariate analysis for DFS showed: low risk vs. intermediate risk K, p=.01; high risk vs. intermediate risk K, p= .03; FLT3+ vs. FLT3-, p=.0002. NPM status did not significantly influence DFS in either the whole population or in the normal K group. In particular, there was no difference in the DFS rates among patients NPM+ and NPM- in the normal K/FLT3- group while in the normal K/FLT3+ group there was a trend (p=.06) for lower relapse rate for NPM+ patients as compared to NPM- ones. These results highlight the relevance of combining cytogenetic and molecular studies in the diagnostic work up of AML and confirm the impact of karyotype on all outcome estimates as well as of FLT3 status on DFS. As to NPM mutations, these appear a favorable predictor of CR achievement. Further investigations in large clinical trials are needed to assess the prognostic value of NPM mutations on outcome in AML with normal karyotype.

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Impact of FLT3 Mutation Status and Other Genetic Parameters In Acute Promyelocytic Leukemia (APL) with t(15;17)(q22;q12)/PML-RARA.

Blood ◽

10.1182/blood.v116.21.1685.1685 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1685-1685

Author(s):

Susanne Schnittger ◽

Claudia Haferlach ◽

Tamara Alpermann ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Platelet Count ◽

Acute Promyelocytic Leukemia ◽

Univariate Analysis ◽

Promyelocytic Leukemia ◽

Prognostic Impact ◽

Employment Equity ◽

Transcript Levels ◽

Cytogenetic Aberrations ◽

Mutational Status ◽

Equity Ownership

Abstract Abstract 1685 Acute promyelocytic leukemia (APL) with t(15;17)(q22;q12)/PML-RARA displays the most favourable entity among the different subtypes of acute myeloid leukemia with a five year overall survival (OS) of more than 80%. However, around 10–20% still experience relapse. In order to find pretreatment parameters that may be predictive for outcome in APL, we have comprehensively analyzed 147 PML-RARA-positive patients (pts) at diagnosis. All pts were treated with ATRA in addition to standard chemotherapy. Patients were selected according to availability of cytogenetics, FLT3-ITD mutational status and FLT3-TKD mutational status. The cohort was comprized of 85 males and 62 females. Median white blood cell count (WBC) was 1,8×109/L (range: 0.2–183.4 × 109/L), median platelet count was 30.0 (range: 1.0–228.0 × 109/L) and median hemoglobin level (Hb) was 9.7 (range: 3.6–16.4 g/dL). In 115 pts bone marrow smears were available: 68 were classified as M3 and 47 as M3v (FAB criteria). According to PML-RARA fusion type 89 pts had a bcr1 (long variant), 52 a bcr3 (short variant) and 6 a bcr2 (exon6) breakpoint. Diagnostic %PML-RARA/ABL1 transcript levels were heterogeneously distributed ranging from 0.6 to 96.7 (median: 18.5). In 57/147 pts (38.8%) additional cytogenetic aberrations (ACA) were detected (+8/+8q: n=26, i(17q): n=11, 9q-: n=3, complex: n=2, all others: n=15). A FLT3-ITD was detected in 47 pts (32.0%) and a FLT3-TKD mutation in 19 pts (12.9%). Thus, a total of 65 pts (44.2%) had a mutated FLT3 status (one case revealed both ITD and TKD mutations). FLT3-ITD was highly associated with bcr3 breakpoints (32 FLT3-ITD vs. 20 FLT3wt compared to 14 FLT3-ITD vs 75 FLT3wt in bcr1 and 1 FLT3-ITD vs. 5 FLT3wt in bcr2; p<0.001). Furthermore, FLT3-ITD was associated with higher WBC (mean: 33,153 compared to 5,170 × 109/L in FLT3wt pts, p<0.001) and a lower platelet count (mean: 30,351 vs. 63,324 × 109/L in FLT3wt pts, p=0.001). All parameters mentioned above were analyzed for a possible impact on OS and EFS. Median follow up time of this cohort was 16 months. OS was significantly better in males (2 year OS: 94.2% vs. 78.5% in females; p=0.038). Age as a continuous variable was found significantly related to both OS and EFS (p=0.002, each). Overall, the presence of ACAs had no impact on OS or EFS. In a next step the different ACAs as defined above were evaluated separately. The only group with significantly shorter OS and EFS was the one including the non recurrent “other” ACAs (2 years OS/EFS: 63.6% each, compared to 87.5%/81.4% in the remaining 5 cytogenetic groups, p=0.014/p=0.040). Importantly, these differences exclusively are due to four early deaths in this “other non recurrent” group. No significant effect on OS or EFS was found for WBC, Hb, platelet count, M3/M3v, PML-RARA breakpoint, diagnostic %PML-RARA/ABL1 transcript levels as well as FLT3-ITD, FLT3-TKD or combined FLT3-ITD/TKD status. However, when the FLT3-ITD/wildtype ratio was taken into account a significantly worse EFS was found for those with a FLT3-ITD/wildtype ratio >0.5 (n=21; 2 years EFS: 61.2% vs. 83.5% in the combined group with FLT3wt or FLT3-ITD/wildtype ratio < 0.5, p=0.009). Parameters with significant impact in univariate analysis were included into the multivariate analyses. For OS this was performed for gender, age and “other non recurrent ACA”. All three parameters were proven independent prognostic factors (p=0.026, RR: 0.24; p=0.004, RR: 1.44/decade; and p=0.013, RR: 4.32, respectively). For EFS age, FLT3-ITD/wildtype ratio >0.5, and “other non recurrent ACA” were analyzed (p=0.003, RR: 1.41/decade; p=0.077, RR: 2.73, and p=0.049, RR: 2.46, respectively). In conclusion, specific treatment in APL is extremely efficient what results in minor prognostic impact of otherwise established pretreatment parameters like WBC count, additional cytogenetic aberrations and mutated FLT3 status. Age is the strongest prognostic factor for OS and EFS. Non-recurrent ACAs are associated with an inferior OS. Most importantly, FLT3-ITD mutations with high allelic burden of more than 0.5 are associated with a shorter EFS. This data should be confirmed in controlled prospective studies to draw final conclusions for clinical decision making. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Analysis of the IDH1G105 (SNPrs11554137) Polymorphism in 961 AML Patients and in a Large Cohort of 475 Healthy Controls

Blood ◽

10.1182/blood.v118.21.2518.2518 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2518-2518

Author(s):

Susanne Schnittger ◽

Christiane Eder ◽

Tamara Alpermann ◽

Thomas Illig ◽

Norman Klopp ◽

...

Keyword(s):

Minor Allele ◽

Adverse Impact ◽

Favourable Effect ◽

Intermediate Risk ◽

Control Cohort ◽

Employment Equity ◽

Male Ratio ◽

Healthy Control ◽

Major Allele ◽

Equity Ownership

Abstract Abstract 2518 Introduction: Mutations in IDH1 and IHD2 at arginines 132 and 140 or 172, respectively, have recently been shown to play an important role in AML. Also the IDH1105GGT minor allele of the IDH1G105 (SNPrs11554137) polymorphism that is localized in the same exon as the IDH1R132 mutation has recently been reported to be an adverse prognostic factor in AML (JCO: 14, 2356–2364, 2010). Aim: We aimed at further delineating the frequency and impact of the IDH1105GGT minor allele in AML and also analyzed a healthy control cohort. Methods:IDH1G105 (SNPrs11554137) was analyzed in 961 AML patients by a LightCycler-based melting curve assay. Female/male ratio was 433/528 and age ranged from 13.1–100.4 years (median, 66.7 years). The results were compared to a healthy control cohort from the KORA (Cooperative Health Research in the Region of Augsburg) survey S4, which consists of 475 cases who where matched to the leukemia samples with respect to sex (193f/282m) and age (median: 67, range 32–81 years). Informed consent for participation in anonymized genetic studies was obtained from all individuals. IDH1G105 in the healthy cohort was analyzed by Sanger sequencing. Further mutation analyses were available in subsets of the AML patients, respectively, as follows: (IDH1R132 n=625, IDH2R140 n=587, IDH2R172 n=590, FLT3-ITD n=629, FLT3-TKD n=503, NPM1 n=628, CEBPA n=587, RUNX1 n=231, MLL-PTD n=629, NRAS n=273, KRAS n=133, ASXL1 n=470) and were analyzed as described previously. A subcohort of 634 AML with intermediate risk karyotype was analyzed for survival. Female/male ratio of this subcohort was 280/354 and age ranged from 15.7–86.6 years (median, 66.9 years). The adverse impact of IDH1R132, IDH2R140 and IDH2R172 on the NPM1+/FLT3-ITD- group has been shown previously for this group (Blood 2010 116: Abstract 102). Results: The IDH1105GGT minor allele was detected in 11.2% (108/961) in AML and in 8.8% (42/475) of the KORA control. This slight difference does not reach statistical significance (p=0.17) and thus there is no indication that this variance is a predisposing factor for leukemia. Also the frequency of a homozygous IDH1105GGT minor allele was not different between the AML cohort (3/108, 2.8%) and the KORA cohort (2/42, 4.8%, n.s.). In the AML cohort there was no association of the IDH1105GGT minor allele with age, WBC, platelet count or any of the above mentioned molecular mutations. In contrast, some differences in survival were observed: patients with the IDH1105GGT minor allele had a longer event free survival (EFS) than those with the IDH1105GGC major allele (median: 30.1 vs. 21.6 months, p=0.052) in the intermediate risk cohort. This prognostically favourable effect of the IDH1105GGT minor allele was most prominent in the NPM1+/FLT3- group with a median EFS of 45.1 vs 23.5 months as compared to those with the IDH1105GGC major allele (p=0.015). Conclusions: 1) The polymorphic IDH1105GGT minor allele was not found to be a marker predisposing for AML. 2) No association of the IDH1105GGT minor allele to any mutation or other biological parameters was detected. 3) We were not able to reproduce the previously published adverse impact of the IDH1105GGT minor allele on survival in AML. In contrast, in our cohort of 475 patients with intermediate risk AML the EFS was even better in patients carrying the IDH1105GGT minor allele, especially in the subcohort with NPM1+/FLT3−ITD−. Thus, we would suppose that the IDH1105GGT minor allele is a favorable molecular marker in intermediate risk AML. However, as these findings are in contrast to previously published data, further confirmation in additional studies is necessary to draw firm conclusions on the utility of the IDH1105 polymorphism as a marker for diagnostics and prognosis in AML. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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