RUNX1 Mutations Can Be Found in 34% of De Novo AML with Normal Karyotype or Single Chromosomal Imbalances and Are Associated with Good Prognosis but Turn to Unfavourable Outcome If Further Cytogenetic or Molecular Mutations Are Acquired

Mutations in the DNA-binding domain, the Runt homology domain of the AML1/RUNX1 gene have been described mostly in therapy-related myelodysplastic syndrome, in therapy-related AML as well as in AML after MDS (s-AML). Recently we have shown that RUNX1 mutations also can be found in de novo AML with normal karyotype and single or simple chromosomal imbalances. To further address the importance of RUNX1 in these kind of AML we analyzed the RUNX1 mutational status in a selected cohort of 389 de novo AML with: normal karyotype (NK): n=221, +8 (n=40), +11 (n=9), +13 (n=26), +21 (n=14), rare trisomies (n=11), −7/7q- (n=22), 5q- (n=3), 9q- (n=6), 20q- (n=4), or any combinations of these (n=33). Median age was 67.5 years (range: 20.4–88.2), male:female ratio was 216:173. In this selected cohort 134/389 mutations (34.4%) were detected, showing that RUNX1 is one of the most frequently mutated genes in certain de novo AML. The mutations were not randomly distributed according to FAB subtypes: AML M0 (58.3%, n=36), M1 (32.1%, n=84), M2 (34%, n=126), M4 (20.5%, n=39), but only 10.5% in M6 (n=19) and never detected in M5 (n=12). Also within the single different cytogenetic groups the RUNX1 mutations (RUNX1mut) revealed different frequencies: NK: 28.5%; +8: 35%, +11: 44 %, +13: 96%, +21: 56%, −7/7q-: 27%, 20q-: 75% and 23.5% in the combination group. The patients (pts) were also analyzed for CEBPA, FLT3ITD, FLT3TKD, JAK2, MLLPTD, NPM1 and NRAS. 49/134 RUNX1mut cases (36.6%) revealed at least one of these additional mutations. CEBPA and JAK2 mutations were never detected in combination with RUNX1. An NPM1 mutation was observed in one RUNX1mut pts with +21. The mutation found most frequently together with RUNX1 was MLLPTD that was detected in 28/134 pts (20.9%) followed by FLT3-ITD that was detected in 24/134 cases (17.9%). The distribution of additional mutations in the different cytogenetic groups was heterogeneous. Of the 63 pts with RUNX1mut NK 20 had MLLPTD (31.7%) and 13 had FLT3ITD (20.6%). In 14 pts with +8 and RUNX1mut no MLLPTD but 3 (21.4%) FLT3ITD were detected, in addition to one case with NRAS. Two of the 4 RUNX1mut +11 pts had MLL-PTD. The 8 cases with RUNX1 mut and +21 had a very high additional mutational rate with 2 MLLPTD and 6 FLT3ITD and 1 FLT3TKD, 1 NPM1 and 1 NRAS (3 double mutated cases). In contrast only 3 of 25 (12%) RUNX1mut pts with +13 had additional mutations (2 MLLPTD and 1 FLT3ITD). Similarily the −7/7q- group with RUNX1mut (n=6) revealed no additional markers. This finding suggest that some chromosomal aberrations are biologically “ more potent” than others and require less additional molecular events to cause overt leukemia. Clinical follow up data were available for 213 pts (74 RUNX1mut and 139 RUNXunmut). A direct comparison of these two groups showed a trend to shorter overall survival (OS) in the RUNX1mut pts (p=0.094) and a significantly shorter event free survival (EFS) (p=0.008). A subanalysis for RUNX1 and MLLPTD showed worse EFS for all three groups (RUNX1+/MLLPTD+ (n=21), RUNX1+/MLLPTD− (n=53), RUNX1−/MLLPTD+ (n=18) compared to the unmutated group (n=121) (p=0.064, 0.009, 0.081). Similar results were obtained for the combination of RUNX1/FLT3ITD with (RUNX1+/FLT3ITD+ (n=15), RUNX1+/FLT3ITD− (n=59), RUNX1−/FLT3ITD+ (n=24)) compared to the unmutated group (n=114) (p=0.006, 0.033, 0.223). Subsequently an analysis in the NK group was performed (25 RUNX1mut, 82 RUNX1wt). Surprisingly, RUNX1mut AML were not worse than the RUNX1unmut AML. In a subanalysis taking MLL-PTD and FLT3-ITD into account there were only 12 sole RUNX1mut pts and all revealed no event for OS and EFS (median follow up time: 196 days). In a further analysis taking cytogenetic as well as molecular aberrations into account it could be shown that RUNX1mut without any of these further aberrations have no event and are favourable compared to pts with single aberrations (p=0.063) and even more favourable compared to those with two or more cytogenetic or molecular aberrations (p=0.031). A multivariate analysis including age, cytogenetics, molecular genetics, and additional further genetic events shows that only age (p=0.027) and additional genetic events (p=0.005) are independent prognostic markers in this analysis. In conclusion, RUNX1 mutations

1. are frequent in AML with NK or single chromosomal imbalaces,

2. tend to go together with additional mutations which differ dependent on underlying cytogenetics.

3. cooperate most frequently with MLL-PTD 4) per se are prognostically favourable as single genetic aberration but deteriorate by acquisition of other cytogenetic and/or molecular aberrations.