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

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.