Analysis of SF3B1 in a Large Cohort of Refractory Anemia with Ring Sideroblasts with/without Marked Thrombocytosis (RARS-T/RARS) in an European Multicentre Study Reveals a High Incidence (86.3%) of Mutations and an Independent Favorable Association with Outcome

SF3B1 mutations (SF3B1mut) have recently been shown to be highly correlated with ring sideroblasts in MDS and a favorable prognosis (Papaemmanuil et al., NEJM, 2011). However, MDS with ring sideroblasts mainly comprise RARS and this per se is a favorable entity. A subsequent study showed that SF3B1mut retained their favorable impact in MDS with low or intermediate-1 IPSS (Malcovati et al., Blood, 2011) but these results remain controversial (Patnaik and Tefferi, Blood, 2012). The impact of SF3B1mut RARS-T still remains to be investigated.

To analyse the frequencies and clinical impact of SF3B1 in RARS-T especially in comparison to RARS and also to take the JAK2V617F and MPLW515 status into account.

To obtain a comprehensive data set of this rare entity samples from five European centres were collected. The cohort comprised of 146 cases (RARS-T: n=111) and RARS (n=35). Median age was 73.5 years (range: 44.4–96.1 years). 74 cases were male and 72 female. Karyotype was available in 130 cases: normal (n=110, 84.6%), aberrant (n=20, 15.6%).

SF3B1 was analyzed with next-generation amplicon deep-sequencing (454 Life Sciences, Branford, CT) with a at least 200-fold coverage. This approach is able to detect mutations with a sensitivity of below 2%. JAK2V617F was analyzed by allele specific real time PCR to estimate allele burden. MPLW515 was analyzed by melting curve analyses.

In the total cohort 126 of 146 cases (86.3%) revealed a SF3B1mut. With the exception of one p.Arg549Cys in exon 12 all other mutations were located in exon 14 and 15. With the exception of one del/ins mutation all were missense mutations. In detail, the most frequent mutation was p.Lys700Glu (68/126, 54.0%), followed by p.Lys666Arg/Asn/Glu/Thr mutations (n=18, 14.3%) and p.His662Asp/Gln (n=13, 10.3%). Other mutations including one p.Met784_Lys785delinsIle were detected rarely or in single cases only. Three patients harboured 2 different mutations. Median mutation/wildtype load was 40% (range: 15–78%). Small subclones with SF3B1mut were not detected. Frequencies of SF3B1mut were similar in RARS-T (96/111, 86.5%) compared to RARS (30/35, 85.7%). In contrast, both entities differed by the presence of JAK2V617F mutations that were detected in 55/146 (37.7%) in the total cohort but in 55/111 (49.5%) in RARS-T compared to none in RARS (p<0.001). JAK2V617F allele burden was very heterogenous with a median of 49% (range: 0.9–100 %). None of the cases had a JAK2exon12 mutation (122 tested) and 1/111 RARS-T had an MPLW515L mutation. The role of SF3B1mut was analyzed with respect to age, sex, white blood cell count (WBC), hemoglobin levels, platelet counts, blast counts, percentage of ring sideroblasts, karyotype and JAK2V617F allele burden. In RARS-T SF3B1mut were more frequent in females (56/59, 94.9%) compared to males (40/52, 76.9%) (p=0.01), and ring sideroblast counts were higher in SF3B1mut than SF3B1wt (55% vs 38%) (p=0.007). No further correlations were detected for these parameters. Overall survival (OS) was analyzed in 114 cases in which survival data was available (100 SF3B1mut and 14 SF3B1wt). Using Kaplan Meyer analysis, in the total cohort those with SF3B1mut had longer survival than SF3B1wt (median: not reached vs. 8.0 years (y)) (p=0.009). When restricted to RARS-T (74 SF3B1mut and 10 SF3B1wt) OS was 6.2 y in SF3B1mut vs 1.7 y in SF3B1wt (p=0.018). In RARS OS was 10.8 y in the SF3B1mut and not reached in SF3B1wt (n.s. as only 4 cases are SF3B1wt). In RARS-T, also JAK2V617F was associated with more favorable outcome (median OS: 8.1 vs 4.9 y in the JAK2V617wt, p=0.038). In RARS-T with Cox regression analysis the following parameters were relevant for survival: age (p=0.037), WBC (p=0.018), SF3B1mut (p=0.026), and JAK2V617F (p=0.046), and were included in multivariable analysis. All parameters retained an independent significant impact on survival: age (p=0.041), WBC (p=0.001), SF3B1mut (p=0.049), and JAK2V617F (p=0.002).

SF3B1mut is observed in a high percentage of RARS-T (86.5%) as well as in RARS (85.7%) However, both entities on the molecular level can be subdivided by the absence of JAK2V617F mutations in RARS compared to high frequency in RARS-T. In RARS-T SF3B1mut are associated with female sex, higher WBC, ring sideroblast counts and a longer OS. This data strengthen the relevance of molecular genetic markers in the diagnosis and prognostication also in RARS-T.

Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.