Refractory anemia with ring sideroblasts and marked thrombocytosis cases harbor mutations in SF3B1 or other spliceosome genes accompanied by JAK2V617F and ASXL1 mutations | Haematologica

摘要

Refractory anemia with ring sideroblasts and marked thrombocytosis (RARS-T) is a rare entity with characteristics of both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN).1 Patients have been shown to be frequently JAK2V617F and SF3B1, and less commonly MPLW515, mutated (mut).1–4 Gene mutations that have been occasionally analyzed in limited cohorts include TET2, ASXL1 and DNMT3A.2,5,6 Recently, also CALRmut have been reported with varying frequencies of 0%–25%.7–10 However, a comprehensive mutational analysis of both MPN and MDS related markers has still not been made. To further characterize the genetic landscape of RARS-T, we analyzed 17 genes (ASXL1, CALR, CBL, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, MPL, NPM1, RUNX1, SF3B1, SRSF2, TET2, U2AF1, ZRSR2) in a large cohort of 92 RARS-T patients, and we were able to create a comprehensive mutational landscape in 75 patients.Patients were diagnosed with RARS-T if they strictly fulfilled the criteria according to the WHO classification 2008.1 Part of the data has been published previously in Flach et al. (18 of 92),5 Jeromin et al. (47 of 92),4 and Broseus et al. (54 of 92)3 and (54 of 95),9 respectively, but these have been included in our study with the purpose of analyzing features not examined in the aforementioned articles. Of 92 patients, data for 36 (39.1%) have not been published before. Patients gave their informed consent to laboratory analyses and scientific studies. The study design adhered to the tenets of the Declaration of Helsinki and was approved by our institutional review board.Male:female ratio was 1:1.4 and median age was 75 years (range: 44–89 years). Median white blood cell count (WBC) was 7.5 ×109/L (2.9–60.0 ×109/L), hemoglobin level 9.6 g/dL (6.9–13.2 g/dL), platelet count 659×109/L (454–1500 ×109/L), and percentage of ring sideroblasts (RS) 61% (18%–97%). Eighty-six patients were cytogenetically analyzed. Seventy-one patients (82.6%) had normal karyotype and 15 chromosomal aberrations (Online Supplementary Table S1). Screening for mutations in ASXL1, CALR, SF3B1, and SRSF2 was performed by direct Sanger sequencing. JAK2V617F, JAK2exon12 and MPLW515 were analyzed by melting curve analysis. All other genes were analyzed by a 454 deep-sequencing approach (NGS). For analysis of mutation burdens, cases were re-analyzed by NGS or a real-time quantitative approach for JAK2V617F (Online Supplementary Appendix).Ninety-two patients were analyzed for mutations in the above-mentioned 17 different genes. As material was limited in some cases, 7.7% of all intended analyses could not be performed (Online Supplementary Table S2). The five most frequently mutated genes were: SF3B1 (90.2%), JAK2V617F (58.7%), TET2 (23.3%), DNMT3A (16.7%), and ASXL1 (14.3%). JAK2exon12, CALR, IDH1 and NPM1 showed no mutation. The remaining gene mutations occurred in less than 10% of cases (Online Supplementary Table S2). Altogether, in 98.9% of the patients at least one mutation was found. Only one patient carried no mutation and had normal karyotype. We further analyzed this case for mutations in PRPF8 that have recently been shown to occur in myeloid malignancies and to associate with ring sideroblasts.11 In addition, a pan-myeloid NGS gene panel was applied providing higher sensitivity and data on 11 additional genes (Online Supplementary Appendix). However, also with these approaches no mutation was detectable.For the final comprehensive analysis we focused on 75 patients that had available mutation status for all 14 genes mutated in at least one of the cases (Figure 1A). JAK2V617F presented in combination with SF3B1mut in 49.3% (37 of 75). Interestingly, nearly all SF3B1 wild-type (wt) cases carried a JAK2V617F (85.7%; n.s.) and an ASXL1mut (85.7% vs. 7.4%; P<0.001). Furthermore, mutations in SRSF2 (2.9% vs. 42.9%; P=0.005) and U2AF1 (1.5% vs. 42.9%; P=0.002) were rare in SF3B1mut cases, but were associated with ASXL1mut (SRSF2mut: 36.4% vs