The Two Mutation Hot Spots Ser34 and Gln157 in U2AF1 Show Different Occurrence, Correlation and Clinical Features in Myeloid Malignancies: An Analysis of 785 Cases

Recently, genes affecting the splicing machinery have been found to be frequently mutated in MDS patients. The U2AF1 gene codes for one of these splicing components, showing two distinct mutational hot spots at amino acids Ser34 and Gln157. Mutations in U2AF1 induce global abnormalities in RNA splicing, producing intron containing unspliced RNAs. U2AF1 has been shown to be most frequently mutated in MDS cases, while rarely mutated in AML and MPN patients.

1. Determine the frequency of U2AF1 mutations in different myeloid malignancies. 2. Evaluate the correlation of U2AF1mut with mutations in other genes coding for splicing components, cytogenetics and clinical features.

The total cohort of 785 patients comprised of 323 MPN (MPN-u: n=153, MPN in AP: n=19, MPN in BC: n=5; PMF: n=24, PV: n=46, P, ET: n=58, HES/CEL: n=18), 316 MDS/MPN overlap (MDS/MPN-u: n=38; aCML: n=5, CMML: n=273), 89 MDS (MDS: n=11, RAEB: n=15, RARS: n=5, RARS-T: n=13, RCMD: n=16, RCMD-RS: n=25, t-MDS: n=4), and 57 AML (de novo AML: n=44, s-AML: n=13). U2AF1 mutations at Ser34 and Gln157 were analyzed by melting curve analysis. Additionally, mutational analyses of SF3B1 (n=698) and SRSF2 (n=381) were performed by Sanger sequencing. Cytogenetics was available in 770/785 cases.

Overall, U2AF1 was mutated in 45/785 cases (6%). Regarding the type of mutation 29/45 cases (64.4%) showed a missense mutation in Ser34 and 15/45 cases (33.3%) a missense mutation in Gln157, whereas one case had a 6 base pair insertion at Gln157 (c.470_475dup; p.158_159dup).

The two mutation hot spots were affected with different frequencies within the myeloid entities. While U2AF1Ser34 was mutated most frequently in MDS (13/89; 14.6%) and AML (7/57; 12.3%) and rarely in MDS/MPN overlap (7/316; 2.2%) and MPN (2/323; 0.6%), U2AF1Gln157 was never mutated in AML (0/57; 0%), rarely in MDS (1/88; 1.1%) and MPN (2/323, 0.6%), but more frequently in MDS/MPN overlap (11/315, 3.5%).

Also the gender distribution varied between the two mutational hot spots. U2AF1Ser34 mutations were more frequent in males than in females (23/481 (4.8%) vs. 6/304 (2.0%); p=0.051). No differences in gender distribution were observed for U2AF1Gln157 mutations (male: 12/479 (2.5%) vs. female: 4/303 (1.3%); p=0.308).

In MDS/MPN overlap patients with a U2AF1Gln157 mutation were older (mean: 79.0 vs. 72.4 years; p=0.024) in contrast to patients with a mutation in Ser34 (mean: 72.3 vs. 72.6 years; p=0.927). Furthermore, in this entity patients with a U2AF1Ser34 mutation had a lower hemoglobin level (mean: 8.9 vs. 11.0 g/dL; p<0.001) what was not true for cases with a mutation in Gln157 (mean:10.2 vs. 11.0 g/dL; p=0.317). In contrast, the platelet count was lower in MDS patients with a Ser34 mutation (mean: 98,778 vs. 304,992/μl; p<0.001), while there was no difference in patients with a Gln157 mutation (mean: 108,333 vs. 143,425/μl; p=0.534).

At next a comparison to other genes from the splicing machinery (SF3B1 and SRSF2) was performed. SF3B1 mutations were detected in 56/698 (8.0%) and SRSF2 in 141/381 (37.0%) cases of the subcohorts. U2AF1 mutations were nearly mutually exclusive of these two mutations. There was no U2AF1 mutated case with an additional SF3B1 mutation (0/56 vs. 37/642 in the SF3B1wt group; p=0.64). Only 1/141 case showed a mutation in U2AF1 and SRSF2, while 24/240 were U2AF1 mutated within the SRSF2wt group (p<0.001).

Analysis in comparison with cytogenetics revealed a high frequency of U2AF1 mutations in patients with del(20q) compared to in all other cytogenetic subgroups (21/126 (16.7%) vs. 23/644 (3.6%); p<0.001). In all patients with del(20q) a U2AF1Ser34 and no U2AF1Gln157 mutation was detected. U2AF1 was most frequently mutated in del(20q) cases in MDS (13/45; 28.9%) and AML (6/29; 20.7%), less frequently in MDS/MPN overlap (2/15; 13.3%) and never in MPN (0/37; 0%; overall, p=0.005).

1. Mutations in U2AF1 occur in 15.7% of MDS and 12.3% of AML, but rarely in MDS/MPN overlap (5.9%) and MPN (1.9%) patients. 2. The highest U2AF1 frequency was observed in MDS patients with del(20q) (28.9%). 3. The two mutation hot spot regions U2AF1Ser34 and U2AF1Gln157 are affected at different frequencies depending on gender and disease entity.

Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.