Comprehensive Analysis Of U2AF1 Mutations In 843 Patients With Myeloid Neoplasms With Respect To Other Genetic Alterations

Genes affecting the splicing machinery have been found to be frequently mutated in MDS patients. U2AF1 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 (7-11%), but was so far investigated only in small subsets of AML and MPN and was found rarely mutated.

To determine the frequency of U2AF1 mutations (U2AF1mut) in different myeloid entities and to evaluate the correlation of U2AF1mut with other gene mutations, cytogenetics and clinical features.

The total cohort consisted of 843 patients, whereof 74 were diagnosed as AML, 201 as MDS, 243 as MPN, and 325 as MDS/MPN overlap. 331 patients were female, 512 male. Cytogenetics was available in 830 patients and these were grouped by the following karyotypes: normal karyotype (n=561), +8 (n=39), -7 (n=15), del(20q) (n=95), -Y (n=29), other aberrations (n=59), and complex karyotype (n=32). Based on the previously described association of U2AF1mut with del(20q) there was an intended selection bias to this abnormality. Mutational analyses for U2AF1 were performed by either melting curve analyses or next generation sequencing. In subcohorts we investigated mutations in ASXL1 (n=505), CBL (n=647), CEBPA (n=68), CSF3R (n=213), DNMT3A (n=260), ETV6 (n=129), EZH2 (n=355), FLT3-ITD (n=352), FLT3-TKD (n=239), IDH1/2 (n=367 and 286, respectively), JAK2 (n=681), KITD816 (n=244), KRAS (n=393), MLL-PTD (n=384), MPLW515 (n=612), NPM1 (n=477), NRAS (n=509), RUNX1 (n=516), SETBP1 (n=336), SF3B1 (n=839), SRSF2 (n=784), TET2 (n=428), and TP53 (n=239) by Sanger sequencing, next generation sequencing, gene scan, or melting curve analysis.

In the total cohort we detected U2AF1 mutations in 55/843 (6.5%) patients, the two mutational hot spots were equally affected with 29 p.Ser34 and 26 p.Gln157 mutations, respectively. Mutation frequencies were 10.9% in MDS, 9.5% in AML, 7.1% in MDS/MPN overlap and 1.2% in MPN. U2AF1mut patients were older (median: 72.6 vs. 71.8 years; p=0.012), the mutation was more frequent in males (42/512 (8.2%) vs. 13/331 (3.9%) in females; p=0.015) and associated with lower hemoglobin levels (median: 9.5 vs. 11.0g/dL; p<0.001), and platelet counts (median: 78x10⁹/L vs. 179x10⁹/L; p=0.002). Regarding cytogenetics we found a high association of U2AF1mut to del(20q): in 18 of 95 cases (18.9%) with del(20q) a U2AF1 mutation was detected compared to 37 U2AF1mut in 735 cases (5.0%) with any other karyotype (p<0.001). This was true for AML (5/16 vs. 2/56; p=0.005), MDS (11/49 vs. 11/150; p=0.007) and MDS/MPN overlap cases (1/8 vs. 21/309; p=0.441). In contrast in MPN none of the 21 del(20q) patients showed a U2AF1 mutation compared to 18/74 in all other entities (p=0.01). Mutations in the two other genes of the splicing machinery, SF3B1 and SRSF2, occurred in 122/839 (14.5%) and 198/784 (25.3%) cases and were mutually exclusive with U2AF1mut. Only one case each showed an U2AF1mut and a SF3B1 (p=0.002) or SRSF2 (p<0.001) mutation. We furthermore analyzed a number of other gene mutations frequently mutated in myeloid entities and their association to U2AF1mut. There was no correlation to mutations in NPM1, FLT3-ITD and FLT3-TKD, MLL-PTD, and CEBPA in AML patients. In MDS patients there was also no correlation to mutations in ASXL1,ETV6, EZH2, TP53, RUNX1, NRAS, and KRAS. This was also true for JAK2, MPL, CBL, and TET2 mutations in MPN. However in MDS/MPN overlap patients U2AF1mut were more frequently found in cases with ASXL1mut (14/115 (12.2%) in ASXL1mut vs. 7/179 (3.9%) in ASXL1wt; p=0.01) and together with KITD816mut (3/10 (30%) in KITD816mut vs. 15/212 (7%) in KITD816wt; p=0.038).

1) U2AF1 is most frequently mutated in MDS, followed by AML and MDS/MPN overlap and in contrast rarely mutated in MPN. 2) U2AF1mut highly correlates with del(20q) in MDS, AML and MDS/MPN overlap but not in MPN cases. 3) In MDS/MPN overlap U2AF1mut associates significantly with ASXL1mut and KITD816mut.

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