Somatic PHF6 Mutations in 1760 Cases with Various Myeloid Neoplasms

Letters to the Editor 2270 REFERENCES 9 Ota A, Tagawa H, Karnan S, Tsuzuki S, Karpas A, Kira S et al. Identification 1 Liu P, Cheng H, Roberts TM, Zhao JJ. Targeting the phosphoinositide 3-kinase and characterization of a novel gene, C13orf25, as a target for 13q31-q32 fi 64 – pathway in cancer. Nat Rev Drug Discov 2009; 8:627–644. ampli cation in malignant lymphoma. Cancer Res 2004; :3087 3095. 2 Staudt LM. Oncogenic activation of NF-kappaB. Cold Spring Harb Perspect Biol 10 He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S et al. 2010; 2: a000109. A microRNA polycistron as a potential human oncogene. Nature 2005; 435:828–833. 3 Kloo B, Nagel D, Pfeifer M, Grau M, Duwel M, Vincendeau M et al. Critical role 11 Hayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, Tomida S et al. of PI3K signaling for NF-kappaB-dependent survival in a subset of activated A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung B-cell-like diffuse large B-cell lymphoma cells. Proc Natl Acad Sci USA 2011; cancers and enhances cell proliferation. Cancer Res 2005; 65: 9628–9632. 108:272–277. 12 Jin HY, Oda H, Lai M, Skalsky RL, Bethel K, Shepherd J et al. MicroRNA-17 ~ 92 4 Qiao Q, Jiang Y, Li G. Inhibition of the PI3K/AKT-NF-kappaB pathway with cur- plays a causative role in lymphomagenesis by coordinating multiple oncogenic cumin enhanced radiation-induced apoptosis in human Burkitt's lymphoma. pathways. EMBO J 2013; 32: 2377–2391. 121 – J Pharmacol Sci 2013; :247 256. 13 Jin HY, Lai MY, Xiao CC. microRNA-17 ~ 92 is a powerful cancer driver and a 5 Hussain AR, Ahmed SO, Ahmed M, Khan OS, Al Abdulmohsen S, Platanias LC et al. therapeutic target. Cell Cycle 2014; 13:495–496. Cross-talk between NFkB and the PI3-kinase/AKT pathway can be targeted in primary effusion lymphoma (PEL) cell lines for efficient apoptosis. PLoS One 2012; 7: e39945. This work is licensed under a Creative Commons Attribution- 6 Sasaki Y, Derudder E, Hobeika E, Pelanda R, Reth M, Rajewsky K et al. Canonical NonCommercial-NoDerivs 4.0 International License. The images or NF-kappaB activity, dispensable for B cell development, replaces BAFF-receptor other third party material in this article are included in the article’s Creative Commons signals and promotes B cell proliferation upon activation. Immunity 2006; license, unless indicated otherwise in the credit line; if the material is not included under 24:729–739. the Creative Commons license, users will need to obtain permission from the license 7 Suzuki A, Kaisho T, Ohishi M, Tsukio-Yamaguchi M, Tsubata T, Koni PA et al. holder to reproduce the material. To view a copy of this license, visit http:// Critical roles of Pten in B cell homeostasis and immunoglobulin class switch creativecommons.org/licenses/by-nc-nd/4.0/ recombination. J Exp Med 2003; 197: 657–667. 8 Srinivasan L, Sasaki Y, Calado DP, Zhang B, Paik JH, DePinho RA et al. PI3 kinase signals BCR-dependent mature B cell survival. Cell 2009; 139:573–586. © The Author(s) 2016

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Somatic PHF6 mutations in 1760 cases with various myeloid neoplasms

Leukemia (2016) 30, 2270–2273; doi:10.1038/leu.2016.212 Written consent forms were obtained from all the patients. Genetic analysis was approved by the ethical review board in each institution. Somatic mutations were confirmed by paired DNA from Next-generation sequencing has enabled us to detect driver tumor and germline samples (buccal smear or CD3-positive cells). mutations in a sensitive manner. By whole-exome sequencing, we In case of non-paired DNA, the nonsense and frameshift mutations previously identified a somatic mutation of the plant home- were classified to be somatic, and the missense mutations odomain finger 6 (PHF6) gene (p.G291X) in 1 out of 29 cases with were classified as somatic if they were already reported as somatic myelodysplastic syndromes (MDS).1 Initially, germline mutations of in the Catalogue of Somatic Mutations in Cancer database PHF6, located at Xq26.2, are reported to cause congenital (http://www.sanger.ac.uk/genetics/CGP/cosmic/). Börjeson–Forssman–Lehmann syndrome (BFLS) with X-linked In total, we identified 62 somatic mutations of PHF6 in 54 cases recessive inheritance.2 BFLS is characterized by mental deficiency, (Table 1). By copy number analysis,1,8 deletions affecting the PHF6 epilepsy, hypogonadism, obesity and dysmorphic features.3 locus were identified in five cases, while no focal amplifications of Recently, it was found that germline PHF6 mutations are also PHF6 locus were identified. Among somatic mutations, 17 were responsible for the female cases with a congenital disorder similar missense, 16 frameshift, 23 nonsense and 6 affecting splice sites. to Coffin–Siris syndrome.4 Moreover, somatic PHF6 mutations Therefore, mutations leading to truncated transcripts were were reported in hematological neoplasms, including T-acute dominant (63%, 39/62). While PHF6 mutations were distributed lymphocytic leukemia (38% of cases were positive for mutations)5 to the whole coding region, 14 out of 17 (82%) missense and acute myeloid leukemia (AML) (3%).6 According to recent mutations were located at the PHD2 domain and 8 (47%) were studies, somatic PHF6 mutations were identified in 3% of cases recurrent (p.R274Q) (Figure 1a). The PHD2 domain of PHF6 is rich with de novo AML7 and in ~ 3% of those with MDS.8,9 Never- in positively charged amino acids including arginine and lysine, theless, pathophysiology due to PHF6 defects in myeloid which were confirmed to be essential for the DNA-binding neoplasms remains to be fully elucidated. capacity of PHF6 as recently reported.12 Consequently, missense In this study, we clarified the implications of somatic PHF6 mutations affecting these amino acids in the PHD2 domain mutations in the cases with various myeloid neoplasms (N = 1760), (p.R274Q and p.K235E) (Figure 1a) might result in loss of PHF6 including the cohort of MDS and AML that we previously function. Together with highly frequent truncating mutations and reported.8,10,11 To identify somatic mutations, we applied whole- dominant deletions, most of the PHF6 mutations (87%; 53/61) might exome sequencing to 49 cases. Subsequently, targeted sequencing be pathogenic in myeloid neoplasms due to loss of function. (SureSelect, Agilent, Santa Clara, CA, USA) and PCR-based pool Clinically, PHF6 mutations were detected in the cases with AML sequencing were performed in 1428 and 356 cases, respectively, 73 of with myelodysplasia-related changes (AML/MRC) (4/26, 15.4%), which were subjected to both methods (Supplementary Table 1). de novo AML (11/340, 3.2%), chronic myelomonocytic leukemia Detailed methods of the sequencing were previously reported.1,8 (CMML) (4/86, 4.7%), MDS (34/1139, 3.0%) and chronic

Accepted article preview online 1 August 2016; advance online publication, 2 September 2016

Leukemia (2016) 2232 – 2279 © 2016 Macmillan Publishers Limited, part of Springer Nature. Letters to the Editor 2271 blast ratio but not with platelet counts (Supplementary Table 3b). Table 1. Proportion of the cases with PHF6 mutations in each disease These findings were compatible to the high prevalence of PHF6 subtype mutations in advanced categories of myeloid neoplasms. In addition, Diagnosis All cases Cases with PHF6 mutations we assessed a relationship between PHF6 mutations and cytogenetics (in 944 patients with MDS and 156 with de novo AML whose information on karyotyping was available), which revealed that PHF6 N % mutations were not significantly associated with abnormal karyotype fi MDS 1139 34 3.0 (Supplementary Table 4). Subgroup analyses on the speci c Low risk 667 9 1.3 chromosomal abnormalities most frequently identified in cases with RA 49 3 5.4 PHF6 mutations (+8, t(8;21), and complex karyotype) showed that RCMD 228 3 1.3 neither of them was significantly associated with PHF6 mutations. 5q − 44 0 0 These results suggest that cytogenetic abnormalities are not likely RARS 344 3 0.9 related to PHF6-defective myeloid neoplasm. MDS-U 2 0 0 Therefore, we further investigated concomitant mutations of 20 High risk commonly evaluated genes in patients with PHF6 mutations, RAEB 466 25 5.3 Not informative 6 0 0 in whom several driver genes were frequently mutated (Supplementary Table 5). In our whole cohort, PHF6 mutations MDS/MPN 114 4 3.5 were significantly associated (false discovery rate o0.1) with CMML 86 4 4.7 those of RUNX1, U2AF1, SMC1A, ZRSR2, EZH2, and ASXL1.In MDS/MPN-U 28 0 0 contrast, SF3B1 mutations were exclusive with PHF6 mutations, MPN 141 1 0.7 which is compatible with the fact that PHF6 mutations were very CML 64 1 1.6 rarely detected in cases with refractory anemia with ringed PV 28 0 0 sideroblasts (0.9%). For subtypes of AML, RUNX1 mutations PMF 25 0 0 significantly co-occurred with PHF6 mutations in AML/MRC cases ET 23 0 0 (P = 0.009), and EZH2 (P = 0.008), SMC1A (P = 0.007) and RUNX1 Not informative 1 0 0 (P=0.04) mutations were also significantly frequent in de novo AML 366 15 4.1 AML cases with PHF6 mutations. In MDS cohort, PHF6 mutations de novo AML 340 11 3.2 were significantly coincident with U2AF1 (P = 0.0005), RUNX1 AML/MRC 26 4 15.4 (P = 0.0009), IDH1/2 (P = 0.01), and ASXL1 (P = 0.01) mutations, Total 1760 54 3.1 while SF3B1 mutations were exclusively identified (P = 0.002). Out fi − of these, RUNX1 mutations were signi cantly frequent in multiple Abbreviations: 5q , MDS with isolated del(5q); AML, acute myeloid disease subtypes with PHF6 mutations, suggesting this gene leukemia; AML/MRC, AML with myelodysplasia-related changes; CML, might have a pathogenic role in PHF6-related myeloid neoplasm. chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia; fi ET, essential thrombocythemia; MDS, myelodysplastic syndromes; MDS/ With regard to copy number alterations, two out of ve deletions MPN, myelodysplastic/myeloproliferative neoplasms; MDS/MPN-U, MDS/ at PHF6 locus were focal lesions, two others represented whole- MPN unclassifable; MDS-U, MDS unclassifable; MPN, myeloproliferative chromosomal losses and the fifth was an i(Xq). These two focal neoplasms; PV, polycythemia vera; PMF, primary myelofibrosis; RA, deletions of PHF6 also included the loci of STAG2 and SMC1A. refractory anemia; RAEB, refractory anemia with excess blasts; RARS, These findings suggest that the deletions and mutations of these refractory anemia with ringed sideroblasts; RCMD, refractory cytopenia genes with PHF6 on the same chromosome might have a with multilineage dysplasia. synergetic effect on leukemic evolution in myeloid neoplasms. To further clarify the PHF6 pathophysiology in MDS, we focused our study on the well-annotated MDS cohort (N = 944).8 First, the myelogenous leukemia (CML) (1/64, 1.6%) (Table 1). In MDS, PHF6 mutational number of the genes except for PHF6 on average per fi mutations were significantly more frequent in the cases with case was signi cantly higher in the cohort with PHF6 mutations refractory anemia with excess blasts (RAEB) (5.3%) than those with (3.8) compared to that with wild-type PHF6 (2.8) (P = 0.018). To the other phenotypes (1.3%, Po0.001). Such predominant further elucidate an impact of PHF6 mutations on the increased mutations in the cases with AML/MRC and RAEB suggest that number of other mutations, a two-way factorial analysis of PHF6 mutations might have an important role in leukemic variables including disease risks was performed. The number of fi evolution. To the contrary, PHF6 mutations were rare in the cases mutations was signi cantly associated with high-risk MDS o with myeloproliferative neoplasms (0.7%), including CML, which is (P 0.001), but not with PHF6 mutations. Accordingly, the similar to the findings reported in a recent paper from association between PHF6 mutations and a high number of other International Cancer Genome Consortium.13 mutations is indirect, and most likely the reason why cases with Although frequent PHF6 mutations in male cases with AML PHF6 mutations are associated with a high number of mutations is were reported,6 the unbiased comparisons of subgroups with that PHF6 mutations are more frequent in high-risk MDS harboring fi more mutations (Supplementary Figure 1). Second, the ratio of the matched clinical backgrounds did not con rm the male fi dominance (Supplementary Table 2). While another paper showed cases with intra-tumor heterogeneity was signi cantly higher in the cohort with PHF6 mutations (P = 0.006), which was evaluated shorter overall survival in de novo AML cases with PHF6 7 fi by comparing variant allele frequencies (VAFs) between PHF6 and mutations, PHF6 mutations were not signi cantly associated with the other genes as previously described.8 VAFs of PHF6 mutations poor outcomes in any phenotype of our data set (data not shown). tended to be low, and more than half of them (54%. 13/24) were Next, we investigated an impact of PHF6 mutations on clinical acquired in subclonal populations (Figure 1b). VAFs of mutations in parameters. By univariate analyses on several variables including the genes associated with RNA splicing and DNA methylation were bone marrow blast ratio (%), peripheral blood neutrophil counts, higher than those of PHF6 mutations, suggesting that PHF6 hemoglobin levels, and platelet counts, PHF6 mutations were mutations might be acquired in the later phase of the clinical course. significantly associated with higher bone marrow blast ratio In 7 out of 53 cases with PHF6 mutations, multiple PHF6 (P = 0.008) and lower platelet counts (P = 0.027) (Supplementary mutations were identified. Among these, five mutations and a Table 3a). A following multivariate analysis of these significant factors deletion of PHF6 were detected in an index female patient. By revealed that PHF6 mutations were significantly associated with high targeted sequencing, p.C297S and p.R319X mutations were

Frameshift Nonsense Missense p.R274

PHF6 PHD1 PHD2

PHF6 mutations Other mutations Average of VAFs 50

10 Variant allele frequency (VAF) (%) 0 12 3456789101112131415161718192021222324 Cases with PHF6 mutations Figure 1. Somatic PHF6 mutations identified by next generation sequencing. (a) In 54 out of 1760 sequenced patients with myeloid neoplasms, 62 somatic mutations of PHF6 were identified, and distributed over the whole coding region. Missense mutations were frequently located within the PHD2 domain. As indicated by an arrow, p.R274 was recurrently affected. (b) In 24 presented cases (x axis) out of 54 cases with PHF6 mutations, intra-tumor heterogeneity was identified by chi-square test as previously described.8 By the comparison of variant allele frequencies (y axis) between PHF6 mutations and the other mutations in each case, PHF6 mutations were supposed to be subclonal in 13 out of 24 (54%) cases (green arrows). An average of VAFs was calculated from the data of all mutations detected in each case.

observed on the independent sequence reads, suggesting that AUTHOR CONTRIBUTIONS these two mutations might be acquired in the distinct clones TM, YN, HidM, MS, SO and KY performed study design and interpretation of the concomitant with the deletion of the wild-type PHF6 allele data. TM, YN, MS, YusS, AK, TY, ASO, KK and RK performed molecular experiments (parallel mutations) (Supplementary Figure 2). and data analysis. YuiS, KC, HT and SaM performed bioinformatics. KI, ShM, HirM, PHF6 encodes a transcription factor with two PHD-type TsN, RK, HK, HPK, LYS, ToN, CH, WK and TH collected samples and clinical data, zinc-finger domains. Wang et al.14 showed that PHF6 in the contributed to the interpretation of the data and critically reviewed the draft. nucleolus regulates the cell cycle by controlling synthesis of ribosomal RNA, and that the loss of PHF6 increases DNA damage. T Mori1, Y Nagata1, H Makishima1, M Sanada1, Y Shiozawa1, A Kon1, This suggests the tumor suppressor potential of PHF6, which T Yoshizato1, A Sato-Otsubo1, K Kataoka1, Y Shiraishi2, K Chiba2, dovetails with our results that most of the PHF6 defects probably H Tanaka3, K Ishiyama4, S Miyawaki4, H Mori5, T Nakamaki6, resulted in the loss of function of the gene. Furthermore, PHF6 R Kihara7, H Kiyoi7, HP Koeffler8,9, L-Y Shih10, S Miyano2,3, mutations were often identified in the subclone and sometimes in T Naoe7,11, C Haferlach12, W Kern12, T Haferlach12, 1 1 a parallel manner, which suggests that these mutations were S Ogawa and K Yoshida 1 acquired during disease progression. PHF6 mutations were Department of Pathology and Tumor Biology, Graduate School of frequent in the cases with AML/MRC and significantly associated Medicine, Kyoto University, Kyoto, Japan; 2 with RUNX1 mutations. This result recapitulates the previous Laboratory of DNA Information Analysis, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; report showing RUNX1 mutations are frequent in the cases with 3 AML/MRC,15 suggesting a synergistic effect of these mutations on Laboratory of Sequence Data Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; leukemic evolution. 4 In summary, PHF6 defects most likely result in their loss of Division of Hematology, Tokyo Metropolitan Ohtsuka Hospital, Tokyo, Japan; function and have a substantial effect on the evolution into the 5Division of Hematology, Internal Medicine, Showa University aggressive types of myeloid neoplasms, associated with other Fujigaoka Hospital, Kanagawa, Japan; concomitant genetic defects including RUNX1 mutations. 6Division of Hematology, Department of Medicine, Showa University, Kanagawa, Japan; 7 CONFLICT OF INTEREST Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan; TH, CH and WK declare equity ownership of MLL Munich Leukemia Laboratory GmbH. 8Hematology/Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA; ACKNOWLEDGEMENTS 9National University of Singapore, Cancer Science Institute of Singapore, Singapore, Singapore; This work was supported by Grant-in-Aids from the Ministry of Health, Labor and 10 Welfare of Japan and KAKENHI (23249052, 22134006, 21790907, 15km0305018h0101, Division of Hematology-Oncology, Department of Internal 16H05338, 26115009, 26890016 and 15H05668) (Kyoto; SO, HM, MS and KY), project Medicine, Chang Gung Memorial Hospital, Chang Gung University, for development of innovative research on cancer therapies (p-direct) (Kyoto, SO), Taipei, Taiwan; 11 The Japan Society for the Promotion of Science (JSPS) through the ‘Funding Program National Hospital Organization Nagoya Medical Center, Nagoya, for World-Leading Innovative R&D on Science and Technology’, initiated by the Japan and 12 Council for Science and Technology Policy (CSTP) (Kyoto, SO) and NHRI-EX100- MLL Munich Leukemia Laboratory, Munich, Germany 10003NI Taiwan (Taipei LYS). E-mail: [email protected]

Leukemia (2016) 2232 – 2279 © 2016 Macmillan Publishers Limited, part of Springer Nature. Letters to the Editor 2273 REFERENCES 8 Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G et al. Landscape 1 Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R et al. Frequent of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia 28 – pathway mutations of splicing machinery in myelodysplasia. Nature 2011; 478:64–69. 2014; :241 247. 2 Lower KM, Turner G, Kerr BA, Mathews KD, Shaw MA, Gedeon AK et al. Mutations 9 Papaemmanuil E, Gerstung M, Malcovati L, Tauro S, Gundem G, Van Loo P et al. in PHF6 are associated with Borjeson-Forssman-Lehmann syndrome. Nat Genet Clinical and biological implications of driver mutations in myelodysplastic 122 – 2002; 32: 661–665. syndromes. Blood 2013; : 3616 3627. 10 Kihara R, Nagata Y, Kiyoi H, Kato T, Yamamoto E, Suzuki K et al. Comprehensive 3 Borjeson M, Forssman H, Lehmann O. An X-linked, recessively inherited syndrome analysis of genetic alterations and their prognostic impacts in adult acute myeloid characterized by grave mental deficiency, epilepsy, and endocrine disorder. Acta leukemia patients. Leukemia 2014; 28: 1586–1595. Med Scand 1962; 171:13–21. 11 Kon A, Shih LY, Minamino M, Sanada M, Shiraishi Y, Nagata Y et al. Recurrent 4 Wieczorek D, Bogershausen N, Beleggia F, Steiner-Haldenstatt S, Pohl E, Li Y et al. mutations in multiple components of the cohesin complex in myeloid neoplasms. A comprehensive molecular study on Coffin-Siris and Nicolaides-Baraitser Nat Genet 2013; 45: 1232–1237. syndromes identifies a broad molecular and clinical spectrum converging on 12 Liu Z, Li F, Ruan K, Zhang J, Mei Y, Wu J et al. Structural and functional insights 22 – altered chromatin remodeling. Hum Mol Genet 2013; : 5121 5135. into the human Borjeson-Forssman-Lehmann syndrome-associated protein PHF6. 5 Van Vlierberghe P, Palomero T, Khiabanian H, Van der Meulen J, Castillo M, Van J Biol Chem 2014; 289: 10069–10083. Roy N et al. PHF6 mutations in T-cell acute lymphoblastic leukemia. Nat Genet 13 Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC et al. Somatic 42 – 2010; : 338 342. CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J 6 Van Vlierberghe P, Patel J, Abdel-Wahab O, Lobry C, Hedvat CV, Balbin M et al. Med 2013; 369: 2391–2405. PHF6 mutations in adult acute myeloid leukemia. Leukemia 2011; 25: 14 Wang J, Leung JW, Gong Z, Feng L, Shi X, Chen J. PHF6 regulates cell cycle progression 130–134. by suppressing ribosomal RNA synthesis. JBiolChem2013; 288: 3174–3183. 7 Patel JP, Gonen M, Figueroa ME, Fernandez H, Sun Z, Racevskis J et al. Prognostic 15 Harada H, Harada Y, Niimi H, Kyo T, Kimura A, Inaba T. High incidence of somatic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med mutations in the AML1/RUNX1 gene in myelodysplastic syndrome and low blast 2012; 366: 1079–1089. percentage myeloid leukemia with myelodysplasia. Blood 2004; 103: 2316–2324.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Vascular events and risk factors for thrombosis in refractory anemia with ring sideroblasts and thrombocytosis

Leukemia (2016) 30, 2273–2275; doi:10.1038/leu.2016.216 review of the medical record. OS was calculated from time of initial diagnosis to the time of last follow-up or death. Thrombosis- free survival (TFS) was calculated from the time of diagnosis Refractory anemia with ring sideroblasts and thrombocytosis of RARS-T to development of thrombosis in uncensored patients (RARS-T) is a provisional entity in the World Health Organization and date of last follow-up or death in patients censored (WHO) classification of myelodysplastic/myeloproliferative overlap for thrombosis. Conventional statistics were utilized for all syndromes, with diagnostic features of RARS, along with a platelet analyses. count 4450 × 109/l and large atypical bone marrow (BM) Among the 82 study patients, 46 (56%) were males and median megakaryocytes.1,2 Patients with RARS-T have a unique mutational age was 72 years (range, 48–93). At a median follow-up of landscape, with frequent mutations involving; SF3B1 (~85%), JAK2 27 months (range, 0–163), 48 (59%) deaths, 16 (20%) thrombotic (~50%), ASXL1 (~30%), TET2 (~25%), SETBP1 (~15%) and DNMT3A events and 2 (2%) leukemic transformations were documented. (~15%).3,4 The frequency of thrombotic events in RARS-T is The median OS was 44 months.4 There were no women in the thought to be similar to that of essential thrombocytosis (ET) (3.6 reproductive age group (15–45 years) and no women in the study vs 3.9/100 patient-years), but more frequent than RARS (3.6 vs had prior adverse obstetric events. CV risk factors were present in 0.9/100 patient-years).5 However, unlike in ET, it remains unclear as to whether thrombotic events in RARS-T impact overall survival (OS).6 In a prior study (n = 15), 6 of 10 (60%) patients with SF3B1mut RARS-T had thrombotic events; whereas there were no thrombotic events in SF3B1wt patients.7 We carried out this study to (i) describe vascular events, (ii) identify risk factors for thrombosis and (iii) determine the impact of thrombotic events on OS in patients with RARS-T. Eighty two patients with WHO-defined RARS-T were included in the study.4 All patients had BM aspirates and core biopsies, iron stains for detection of BM ring sideroblasts (RS) and cytogenetics performed at diagnosis. A 27-gene panel-targeted capture assay was carried out on BM DNA specimens from 48 (59%) RARS-T patients obtained at diagnosis for the following genes: TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF and SETBP1, by previously described methods.4 Details of thrombotic events and cardiovascular (CV) risk factors, including hypertension, Figure 1. Thrombosis-free survival in 48 patients with RARS-T, diabetes, smoking and dyslipidemia, were obtained by careful stratified by their SF3B1 mutation status.

Accepted article preview online 1 August 2016; advance online publication, 2 September 2016