NRAS Mutations in AML: Biology, Cytogenetics, and Prognostic Impact in 2502 Patients.
Authors: Bacher Ulrike , Haferlach Torsten , Kern Wolfgang , Schoch Claudia , Schnittger Susanne 
Published: April 2020

Abstract
In AML NRAS mutations were reported in 11–30%. The prognostic impact is not yet clarified. We studied 2502 pts. between 18.3 and 91.8 years of age (median 63.4 years). 2037 pts. had de novo AML, 281 secondary AML following MDS, and 181 pts. suffered from therapy-related AML. All samples were evaluated by cytomorphology, cytochemistry, multiparameter flow cytometry, cytogenetics, FISH, and molecular genetics in parallel. The screening for NRAS mutations was performed around the mutational hotspots at codons 12, 13, and 61 using a melting curve based LightCycler assay. All cases tested positive for NRAS mutations were confirmed by sequence analysis. 257/2502 pts. (10.3%) showed NRAS mutations (NRASmut), whereas 2245 cases had wildtype NRAS (NRASwt). In 3 of the 257 mutated cases (1.2%) two different mutations were detected. None of these cases was homozygous for one mutation. The majority of mutations was found at codon 12 (n=112/257; 43.6%). Mutations at codon 13 were found in 64/257 pts. (24.9%). In both cohorts the changes were most frequently from glycin to asparagin (codon 12: n=76; 29.6%; codon 13: n=41; 16%). Mutations at codon 61 were detected in 54/257 pts. (21.0%), mostly from glycin to arginin. For 105 cases paired samples from diagnosis and relapse were analysed. The status was stable in 101 cases (96.2%). The history of AML did not differ significantly in association to NRAS mutations. The median leukocyte count was 28.0 G/l (range 0.4–514.0 G/l) in pts. with NRASmut and 63.4 G/l (range 18.3–91.8G/l) in the pts. with NRASwt (p&lt;0.0001). Cytogenetic analyses were available in all cases. Pts. were grouped as followed: normal karyotype, t(15;17), t(8;21), inv(3)/t(3;3), inv(16)/t(16;16), 11q23/MLL rearrangements, complex aberrant karyotype, +8, 5q- or loss of chromosome 5, 7q- or loss of chromosome 7, and other aberrations. In the subgroups with inv(16)/t(16;16) and with inv(3)/t(3;3) NRASmut showed a high frequency of 37.6% (50/133) (p&lt;0.0001), and 11/41 (26.8%) (p=0.0004), respectively. In contrast, NRASmut were significantly underrepresented in t(15;17) (2/102; 2.0%) (p=0.0048), in the subgroup with MLL/11q23 rearrangements (3/77; 3.9%) (p=0.0616), and in cases with complex aberrant karyotype (4/258; 1.6%)(p&lt;0.0001). With 80% and 55% mutations of codon 61 were significantly overrepresented in pts. with inv(16) and with inv(3) compared to the total group where mutations at codon 12/13 were the most common (both p&lt;0.0001). We did not find a significant prognostic impact of NRASmut for OS, EFS and DFS. This was also true in detail for subgroups with inv(16), inv(3), t(8;21), normal karyotype and other aberrations. In AML with normal karyotype we further took the FLT3 mutation status into account. For pts. without FLT3 mutations the EFS was slightly better if NRAS mutations were detected (426+/−73 days) in comparison to pts. negative for NRAS mutations (378+/−21 days) (p=0.0603). In conclusion, we found a clear pattern of NRAS mutations in defined subgroups of AML supporting the hypothesis of a two hit mutation model in AML. However, a prognostic impact was not found.