RARS-T Patients Harbor SF3B1 Mutations In 90.2% and Can Be Characterized By Mutations In ASXL1 and Other Spliceosome Genes In Most Of The Remaining Cases

Refractory anemia with ring sideroblasts and marked thrombocytosis (RARS-T) is a rare entity with characteristics of both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms and is grouped as a provisional entity in the current WHO classification. RARS-T patients have been shown to be frequently JAK2V617F and less commonly MPLW515 mutated. Recently, SF3B1 mutations (mut) were described to occur at a high frequency of up to 85% and it seems that RARS-T is genetically best characterized by SF3B1 and JAK2V617F mutations. However, a comprehensive mutational landscape analysis is still missing und genetic events in the SF3B1wild-type (wt) cases remain to be clarified.

Comprehensively characterize a large cohort of RARS-T patients for gene mutations.

We investigated 92 cases that all strictly met the criteria for RARS-T according to the WHO classification 2008. JAK2V617F and MPLW515 were analyzed by melting curve analysis. Screenings for mutations in SF3B1, SRSF2 and ASXL1 were performed by direct Sanger sequencing. ZRSR2 and TET2 were analyzed by an amplicon next generation deep-sequencing approach (NGS). U2AF1 was either analyzed by melting curve analysis or NGS. The cohort comprised 54 females (58.7%) and 38 males (41.3%). Median platelet count was 659x10⁹/L (range: 454 – 1,500x10⁹/L) and median percentage of ring sideroblasts (RS) was 61% (range: 18 - 97%). Cytogenetic data was available in 86 patients: 71 patients (82.6%) had normal and 15 an aberrant karyotype.

All patients were analyzed for mutations in SF3B1, JAK2V617F and MPLW515. SF3B1 was the most frequently mutated gene (83/92, 90.2%), followed by JAK2V617F (54/92, 58.7%). MPLW515mut occurred only rarely (2/92, 2.2%) and in both cases were accompanied by SF3B1mut. SF3B1mut cases occurred concomitantly with JAK2V617F (46/83, 55.4%). However, JAK2V617F showed a tendency to be more frequent in patients with SF3B1wt (8/9, 88.9% vs. 46/83, 55.4%, p=0.076). Additionally, a subset of the cases, especially those with SF3B1wt, was analyzed for other genes. Mutations occurred with following frequencies: TET2, 14/61, 23.0%; ASXL1, 11/85, 12.9%; SRSF2, 5/86, 5.8%; U2AF1, 4/88, 4.5%; ZRSR2, 2/83, 2.4%. In 98.9% (91/92) of all patients at least one mutation in the analyzed eight genes could be found. Only one patient carried no gene mutation in any of these genes and had normal karyotype. We further analyzed this case with a pan-myeloid genes NGS panel providing data on 19 additional genes. However, no mutation could be detected. Interestingly, nearly all SF3B1wt cases carried an ASXL1mut (7/9, 77.8% vs. 4/76, 5.3%, p<0.001). Accordingly, mutations in the spliceosome genes SRSF2 (2/78, 2.6% vs. 3/8, 37.5%, p=0.005) and U2AF1 (1/79, 1.3% vs. 3/9, 33.3%, p=0.003) were rare in SF3B1mut cases, but were associated with ASXL1mut (SRSF2mut: 3/11, 27.3% vs. 1/73, 1.4%, p=0.006; U2AF1mut: 3/11, 27.3% vs. 1/74, 1.4%, p=0.006). In contrast, the only two ZRSR2 mutated cases had concomitant SF3B1mut (n.s.). TET2mut showed no association with any of the other gene mutations. Analysis of patients with mutation status of all following genes: SF3B1, JAK2V617F, MPLW515, ASXL1, SRSF2, U2AF1, ZRSR2 (n=82), revealed that only SF3B1mut occurred as a sole alteration (31/82, 37.8%). In detail, SF3B1mut cases rarely showed more than 2 gene mutations, whereas nearly all SF3B1wt cases had 3 different gene mutations (5/75, 6.7% vs. 6/7, 85.7%, p<0.001). These 6 SF3B1wt cases all carried a JAK2V617F and ASXL1mut accompanied by either an SRSF2mut (n=3) or U2AF1mut (n=3). Furthermore, SF3B1mut were associated with higher percentage of RS (mean: 61% vs. 41%, p=0.006), whereas JAK2V617F had higher platelet counts (807 vs. 599 x10⁹/L, p<0.001). ASXL1mut had lower percentage of RS (mean: 42% vs. 61%, p=0.007), so had U2AF1mut (mean: 36% vs. 60%, p=0.028), but not SRSF2mut.

1. RARS-T patients are characterized by high occurrence of mutations in SF3B1 (90.2%), in 37.8% detected as sole mutation. 2. Most of the SF3B1wt cases show various gene mutations, harboring a JAK2V617F and ASXL1mut together with a third mutation in either SRSF2 or U2AF1.

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