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Whole-Exome Sequencing of Polycythemia Vera Revealed Novel Driver Genes and Somatic Mutation Shared by T Cells and Granulocytes

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Whole-Exome Sequencing of Polycythemia Vera Revealed Novel Driver Genes and Somatic Mutation Shared by T Cells and Granulocytes Leukemia (2014) 28, 935–979 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu LETTERS TO THE EDITOR Whole-exome sequencing of polycythemia vera revealed novel driver genes and somatic mutation shared by T cells and granulocytes Leukemia (2014) 28, 935–938; doi:10.1038/leu.2014.7 in exon 12. This was a sixfold highermutationratethanpreviously reported9 and is similar to other myeloproliferative neoplasm. Somatic DNMT3A mutations were reported at low frequency in Polycythemia vera (PV) is a myeloproliferative neoplasm characte- PV (2.7%).10 The reported mutations were identified in the rized by an elevated red cell mass caused by excessive myelopoiesis terminal exon, at position M880 and R882. In this study, we with propensity to transformation to myelofibrosis and acute identified three somatic DNMT3A mutations (9.7%), one was leukemia.1 JAK2V617F mutation2 and acquired uniparental disomy on identified at the known hotspot, R882, and the other two were chromosome 9p (9p UPD)3,4 are the most frequent somatic novel frame-shift mutations at codon K456. SF3B1 encodes alterations. JAK2 is mutated in over 90% of PV patients;2 however, subunit 1 of the splicing factor 3b, which is important for in spite of its prevalence it is regarded as necessary but not anchoring the spliceosome to precursor mRNA. Mutation of SF3B1 sufficient to cause the disease.5 Yet, systematic mutation analysis of is frequent in most myeloproliferative neoplasm, having been exome coding sequence has to be described. We here analyzed 31 reported in myelodysplasia with ring sideroblasts (65%),9 JAK2V617F-positive patients by whole-exome sequencing and single myelodysplastic syndrome (20%),9 primary myelofibrosis (7%)11 nucleotide polymorphism array and further investigated the and essential thombocythemia (3%), but it has not been reported mutational evolution using longitudinal samples collected from in PV.9 In this study, we identified three SF3B1 mutations in two seven patients (Supplementary Methods). patients (9.7%), patient PV5 carried two mutations and both were Whole-exome sequencing targeting B43 Mb of coding sequence reported by COSMIC (http://cancer.sanger.ac.uk/cancergenome/ yielded an average of 125-fold coverage (Supplementary Figure 1), projects/cosmic/). Interestingly, phosphodiesterase 4C, PDE4C,is with 95% of targeted bases covered to a depth of  20 mutated in two patients. PDE4C hydrolyzes the second messenger (Supplementary Figure 2). In this study, we use the exomes of T cells cAMP and therefore mediates a variety of responses to as a reference genome and we defined sequence variants present in extracellular signals. Although mutation in this gene is rarely granulocytes but not in the T cells from the same patient as somatic observed in cancer, one of the mutations we discovered was mutations. DNA copy-number analysis revealed multiple recurrent reported in COSMIC, suggesting it may be functionally relevant. chromosomal arm-length changes including 9p UPD, trisomy of 9p The fraction of reads with a given mutation, the variant allele and 1q, LOH at 18p and single patients with 2p and 9q LOH, trisomy fraction, is proportional to the number of nuclei in the tumor 2q and UPD at 22q. Through serial sampling, we observed a focal sample harboring the mutation. As the granulocytes in PV patients hemizygous deletion in PV28 (Supplementary Figure 3) at 9q22.31-32. are clonal as determined by X-inactivation in females,12 the variant No known tumor suppressors or oncogenes were found in this region allele fraction of the mutations reported in Figure 1, should and its functional importance is unclear at this time. correspond to the order in which they appeared in the patient. We validated 87 somatic non-silent mutations and a subset of Three patients, PV5, PV8 and PV24, exhibited tumor variant allele important germline variants (Supplementary Table 1). The average fraction in the key epigenetic modifier genes, ASXL1, DNMT3A or non-silent somatic mutation rate was 0.08 per megabase, which SF3B1 that were higher than JAK2V617F (Figure 2a). Interestingly was much lower than other adult cancers.6 G–4T transversion mutation in a gene associated with immunosuppression in solid was the most frequent base change, followed by C–4T transition tumors, IDO1, is also found at higher variant allele burden than (Supplementary Figure 4). The recurrent G1849T mutation leading that of JAK2V617F. These results suggest that mutation of these to JAK2V617F accounted for the majority of G–4T transversions as genes might have preceded the acquisition of JAK2V617F. all patients harbored JAK2V617F. The most frequently mutated class In seven patients, we could determine the order of appearance of genes (Figure 1) was that involved in epigenetic modification. of mutations directly by longitudinal sampling (Figure 2b). Forty-two percent of patients had at least one somatic mutation in Patients PV1, PV3, PV8, PV10, PV23 and PV29 harbored only an epigenetic modifier, indicating that epigenetic regulation has a JAK2V617F mutation in 2011. Upon follow-up in 2013, four of them key role in PV pathogenesis, as it does in other myeloid had acquired additional mutations, particularly in key epigenetic neoplasms. Many other known cancer-related genes were modifier genes DNMT3A, SMARCA2 and TET2, but also in SF3B1, mutated in single patients as shown in Figure 1. STK11, CDH3 and TP53. One patient harbored IDH2 and JAK2in Recurrent somatic mutations were observed in: ASXL1, DNMT3A, 2011, but did not acquire any additional mutations. Results from a TET2, SF3B1 and PDE4C (Figure 1). ASXL1 belongs to the Enhancer of parallel study of these patients showed that 10% of them acquired trithorax and Polycomb gene families, which are responsible for 9p UPD prior to JAK2V617F mutations (Figure 1, Group III patients), maintaining activation and silencing of PcG and trxG proteins.7 whereas 42% had acquired JAK2V617F mutation first (Figure 1, Disruption of ASXL1, through inactivating mutations in exon 12 is Group II, AMBER13-LEU-1191). prevalent in post-PV myelofibrosis, but is identified only rarely in Novel sequence variants found in both granulocytes and T cells cases of essential thrombocytosis or PV.8 Stein and co-workers from the same patients are putative germline mutations. However, sequenced exon 12 of ASXL1 in 42 PV cases but found only one in seven patients, these putative germline variants were in genes patient (2%) with a nonsense mutation.8 We identified four that were somatically mutated in other patients in the cohort inactivating somatic mutations in ASXL1 (12.9%), two frame-shift (Figure 1, blue tiles). Moreover, a high proportion of these and two nonsense. All four loss-of-function mutations were identified so-called germline mutations were likely to be functionally Accepted article preview online 13 January 2014; advance online publication, 31 January 2014 Letters to the Editor 936 12 8 4 0 Gene Freq PV24 PV21 PV16 PV26 PV28 PV3 PV23 PV1 PV8 PV5 PV31 PV25 PV6 PV22 PV4 PV15 PV2 PV9 PV12 PV27 PV10 PV17 PV13 PV18 PV7 PV20 PV29 PV19 PV14 PV30 PV11 JAK/STAT JAK2 0.00.4 0.9 0.3 1.0 0.6 0.5 0.2 0.4 0.00.4 0.9 0.5 0.5 0.4 0.1 0.3 0.1 1.0 0.5 0.7 0.2 0.10.2 0.1 1.0 0.8 1.0 0.0 0.5 0.1 31 ASXL1 FC F NC NC C C 6 Histone modification KDM4D 1 SETD8 1 IDH2 C C 2 DNA TET2 FC C FC 4 methylation F DNMT3A FFC F 4 ARID4A S 1 Chromatin CHD6 1 remodeling CHD4 1 SMARCA2 1 NRAS C 1 BRAF 1 RTK/RAS RASGRF1 1 FGFR2 C 1 NF1 C CC F FC 5 GATA3 1 GATA2 C 1 Transcription NOTCH3 F 1 factors FOXP2 1 PPARGC1 1 STK11 1 Tumor TP53 C 1 suppressors PPARGC1A 1 PRF1 1 RNA SF3B1 C 2 processing NXF1 N 1 Protein CSNK2A1 N 1 Kinases MAST4 1 PDE4C C 2 DSG1 1 ASCC3 1 IDO1 S 1 C6 N 1 CCDC88A 1 HEATR8 1 HSD17B4 1 HUWE1 1 Others PARP3 1 ROBO2 F 1 MYPN C 1 PPHLN1 1 SHROOM2 1 TRPC4 1 USP5 1 HCN1 1 PTPRB 1 1q TT2 Copy number 18p LL2 alterations 9p U UUT UUT U UUUUUU UUU 17 9q L 1 2p L 1 2q T 1 22q U 1 Transformation MMMM 0 102030 Spleen size 0033330210013000000031000300001 Stage 2232332222232222222233222322232 Clinical data Response 111001 1121012201211111210111 Gender MF MF F F MMMMMMF MF MMMMMMF MMMMF MMMF Age 66 78 72 54 78 61 92 69 58 90 72 77 54 71 43 64 79 64 85 44 70 60 47 76 83 74 65 82 61 87 27 Group I III III II I I I III II I I II I III IIIII II II III I I I II II II I I I Granulocytes only F Frameshift N Nonsense Missense S Splicing C at COSMIC codon Granulocytes + T-cells N Nonsense F Frameshift C at COSMIC codon CNAs T Trisomy U UPD L LOH Figure 1. Summary of somatic mutations, germline variants and somatic DNA copy-number alterations identified in 31 PV cases. Somatic mutations were shown in orange and red based on their mutation type and germline variants were shown in blue. For germline variants, only those frame-shift variants, nonsense variants or those variants reported in COSMIC database (http://cancer.sanger.ac.uk/cancergenome/ projects/cosmic/) were shown and only those genes with somatic mutations identified in the cohort were listed. The total number of somatic mutations identified per patient and gene was showed at the top and right, respectively. The allelic fraction of JAK2V617F was shown at the top row and color scaled.
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