Analyses of 164 RUNX1 mutations (RUNX1mut) in 147 of 449 patients (32.7%) with normal karyotype or noncomplex chromosomal imbalances were performed. RUNX1mut were most frequent in acute myeloid leukemia French-AmericanBritish classification M0 (65.2%) followed by M2 (32.4%) and M1 (30.2%). Considering cytogenetics, RUNX1mut were most frequent in cases with ؉13 (27 of 30, 90%), whereas frequencies were similar in other cytogenetic groups (26%-36%). The molecular genetic markers most frequently associated with RUNX1mut were partial tandem duplication in the MLL gene (19.7%), internal tandem duplication in the FLT3 gene (FLT3-ITD; 16.3%), and NRAS mutations (9.5%). Patients with RUNX1mut had shorter overall and event-free survival compared with RUNX1 wild-type cases (median, 378 days vs not reached, P ؍ .003; and median, 285 vs 450 days, P ؍ .003, respectively). In addition, it was shown that the adverse effect of RUNX1 was independent of the adverse effect of FLT3-ITD as well as of the high frequency of prognostically favorable NPM1mut and CEBPAmut in the RUNX1wt group. No effect of the type or localization of the individual RUNX1 mutations was observed. Multivariate analysis showed independent prognostic relevance for overall survival for RUNX1mut (P ؍ .029), FLT3-ITD (P ؍ .003), age (P < .001), and white blood cell count (P < .002). (Blood. 2011;117(8):2348-2357)
999 Poster Board I-21 NPM1 mutations are frequently reported to be typical for de novo AML and are regarded as prognostically favorable if not associated with FLT3-ITD. These mutations have rarely been reported in secondary AML after myelodysplastic syndrome (MDS) or after myeloproliferative neoplasms (MPN). We have detected NPM1 mutations in 37/283 patients with AML after a previous MDS (s-AML) (13.1%) and in 6/67 after a previous MPN (9%). Here we describe the characteristics of these 43 NPM1 mutated s-AML cases to show the involvement of NPM1 mutations in development of secondary AML. The total cohort of 43 cases was composed of 22 males and 21 females with a median age of 71.3 years (range: 29.3-87.7 years). Cytogenetics was available in 40 of the 43 cases (93%). 27 of these had a normal karyotpye whereas 13 revealed one of these aberrations: +4 (n=3), t(1;14)(p34;q32) (n=1); -7 (n=1), del(9q) (n=2), +13 (n=1); +21 (n=1), -Y (n=1); i(X)(p10) (n=1), [+1,der(1;13)(q10;q10),+i(5)(p10),+8] (n=1) and a t(5;12)(q33;p13) (n=1). All 43 samples were analysed for MLL-PTD, FLT3-ITD, FLT3-TKD, NRAS, CEBPA, RUNX1 mutations as well as for KITD816 and JAK2V617F mutations. The incidence of additional cooperating mutations was similar to de novo AML. FLT3-ITD was detected in 14/37 AML after MDS (37.8%) and only once (1/6) after MPN. FLT3-TKD was observed in 3/37 case after MDS (8.1%) and never after MPN. In addition there was one case with RUNX1 and 4 cases (10.8%) with NRAS mutation after MDS. In none of the cases a CEBPA mutation or MLL-PTD was observed. Thus a total of 18/37 cases (48.8%) after MDS revealed a further molecular mutation in addition to NPM1. Of those without additional molecular mutations (only NPM1) 4 cases revealed cytogenetic aberrations resulting in 22/37 cases (59.5%) with additional cytogenetic or molecular mutations. Also in the 6 cases with NPM1 after MPN we detected a high proportion of additional mutations. Two of these 6 cases defined to be after MPN had a history of KITD816V mutated mastocytosis. Two further cases had preceding JAK2V617F mutated MPN and one additional carried an ETV6-PDGFRB rearrangement. In all these 5 transformed MPN cases the initial typical MPN mutation was retained in AML (blast crisis) whereas the NPM1 mutation was acquired and may have served as a second hit in the development to AML. One of the two JAK2+/NPM1+ cases in addition also acquired an FLT3-ITD. From 11 of the s-AML cases a paired sample from the timepoint of MDS was available. Retrospectively the NPM1 mutations was retraced by mutation specific realtime PCR and also all other markers were analysed. Three different patterns were observed: 1) in two cases the NPM1 mutation was not detectable in MDS (analysed 35 and 11 months before diagnosis of s-AML). In one case an NPM1/ABL1 level of 1.6% was detectable 6 months after diagnosis of MDS and a level of 2129% eleven months after diagnosis of MDS. 2) In six cases the NPM1 mutation was not detectable with standard methods in MDS, but with sensitive Real time PCR a ratio of 1-4 log below the s-AML level was already detectable 6-17 months before onset of s-AML. 3) In three further cases a high NPM1 level comparable to that in s-AML was already detectable in MDS 2-12 months before s-AML evolved. These three cases gained an FLT3-ITD at the time point of transformation from MDS to AML. These pattern show that NPM1 can be an early or a late event in transformation to s-AML and although the acquisition of mutations seems to be important in the transformation to AML the sequence of the single events seem to be secondary. As NPM1 have a favourable prognosis in de novo AML if not associated with FLT3-ITD we did a respective analysis for overall survival (OS) and (EFS) for our cohort of s-AML after MDS. For this analysis 278 s-AML patients were available: NPM1-/FLT3- (n=223); NPM1+/FLT3- (n=20), NPM1-/FLT3+ (n=20) and NPM1+/FLT3+ (n=12). The total cohort revealed a bad outcome (median OS: 56.6 days and median EFS: 43.5 days; range 2-1049 days for both). The median time for MDS until transformation to AML was 316 days (range: 15-6310 days). No difference with respect to outcome was detected between the four different molecular genetic subgroups. In conclusion, these data 1) show that NPM1 mutations play a major role in the evolution of AML following MDS or MPN. 2) NPM1 mutations can be the first as well as the second hit during transformation. 3) Support the theory of a multistep genetic principle in development of secondary AML. 4) s-AML with a NPM1+/FLT3-ITD- status can not be regarded as prognostically favorable. Disclosures: Schnittger: MLL Munich Leukemia Lab: Equity Ownership. Weiss:MLL Munich Leukemia Lab: Employment. Dicker:MLL Munich Leukemia Lab: Employment. Sundermann:MLL Munich Leukemia Lab: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Lab: Equity Ownership. Haferlach:MLL Munich Leukemia Lab: Equity Ownership.
3067 Poster Board III-4 During the last years it has been shown that PCR based detection of minimal residual disease (MRD) has high relevance for early detection of relapse and overall prognostication. This has been proven for several fusion transcripts but also for NPM1 as a target in normal karyotype AML (NK-AML). Other mutations frequently found in NK-AML are RUNX1 mutations (8-10%) and CEBPA mutations (10-15%). However, these mutations are distributed throughout the entire coding reading frames of CEBPA and RUNX1 making mutation analyses more laborious compared to analysis of genes with mutational hotspots like NPM1. In addition, it is nearly impossible to establish high sensitive real time PCR assays for every patient specific mutation. In contrast, DHPLC (denaturing high performance liquid chromatography) is a method that effectively can detect unknown mutations and for known mutations has a sensitivity of up to 1%. Therefore we analyzed the impact of DHPLC analysis for the applicability and value as predictive MRD analysis. At diagnosis mutation screening by DHPLC was performed first. Both genes were amplified each by four different PCR reactions that were subsequently analysed on a WAVE system (Transgenomic, Inc., Omaha, USA). Positive reactions were further characterized by sequencing. The respective fragment or fragments containing the mutations was/were subsequently also analyzed in the follow up samples. The sensitivity was dependent on the kind of the mutation and its position within the PCR fragment and was between 1 and 10% as estimated by limited dilution experiments. Paired diagnostic and relapse samples were available in 15 cases (12 RUNX1 and 3 CEPBA). The respective mutations were retained at relapse in all cases indicating the stability of both markers, rendering them eligible for follow up evaluation. Next, we analysed 30 patients with CEBPA mutation detected at diagnosis and further investigated 91 samples during follow up. 12 of these cases had two different mutations that were localized on two different DHPLC fragments and thus could be analysed in parallel. For RUNX1 we analysed 144 follow up samples of 60 patients that revealed one (n=51) or two (n=9) RUNX1 mutations at diagnosis. Six of the CEBPA mutated and 13 of the RUNX1 mutated cases had an FLT3-ITD in addition. The median follow up sample number per patient was 3 (range 2-13) and the median follow up time was 339 days (range: 57-3001 days). In the subsequent analysis both cohorts were combined. The follow up samples were simply rated as negative or positive. According to previous studies in fusion gene positive patients and NPM1 mutated patients the impact of the DHPLC results on survival was analysed for defined time intervals after start of treatment: interval 1 (days 18-60), interval 2 (days 61-120), interval 3 (day 121-365), interval 4 (days >365). DHPLC results within these intervals were as follows: interval 1 (positive: n=16; negative n=17), interval 2 (positive: n=14; negative n=19) interval 3 (positive: n=38; negative n=65), interval 4 (positive: n=22; negative n=38). Whenever in the follow up samples two different mutations were available (n=99), the results were shown to be concordant. The impact of the results was analysed by Kaplan Meier analysis. For overall survival a trend for significance was found for interval 1 (medians not reached; p=0.157) and interval 2 (medians not reached; p=0.090) and a significant impact for interval 3 (median: not reached vs. 981 days; p=0.015) and interval 4 (median not reached: vs. 285 days; p=0.048), demonstrating that negative DHPLC results correlate with longer OS. This effect could even more clearly be shown for event free survival with respective results for interval 1 (median: 463 vs 731; p=0.048), interval 2 (median: 499 vs 731 days; p=0.109) and interval 3 (p<0.001) (too few samples for interval 4). As neither age, WBC or pretreatment FLT3 status were significantly associated with outcome in this cohort a multivariate analysis could not be performed. These data clearly show that in the absence of sensitive markers for RQ-PCR low sensitive PCR can be very useful for follow up controls at least in RUNX1 and CEBPA mutated AML. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Lab: Employment. Sundermann:MLL Munich Leukemia Lab: Employment. Spiel:MLL Munich Leukemia Lab: Employment. Wendland:MLL Munich Leukemia Lab: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Lab: Equity Ownership. Kern:MLL Munich Leukemia Lab: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
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