Exome Sequencing Reveals Novel and Recurrent Mutations with Clinical Impact in Blastic Plasmacytoid Dendritic Cell Neoplasm

ORIGINAL ARTICLE Exome sequencing reveals novel and recurrent mutations with clinical impact in blastic plasmacytoid dendritic cell neoplasm

J Menezes1, F Acquadro1,2, M Wiseman2,GGo´ mez-Lo´ pez3, RN Salgado1, JG Talavera-Casanãs4, I Bunõ5, JV Cervera6, S Montes-Moreno7, JM Hernańdez-Rivas8, R Ayala9, MJ Calasanz10, MJ Larrayoz10, LF Brichs11, M Gonzalez-Vicent12, DG Pisano3, MA Piris7,SA´ lvarez1 and JC Cigudosa1

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a very rare disease that currently lacks genomic and genetic biomarkers to assist in its clinical management. We performed whole-exome sequencing (WES) of three BPDCN cases. Based on these data, we designed a resequencing approach to identify mutations in 38 selected genes in 25 BPDCN samples. WES revealed 37–99 deleterious gene mutations per exome with no common affected genes between patients, but with clear overlap in terms of molecular and disease pathways (hematological and dermatological disease). We identified for the first time deleterious mutations in IKZF3, HOXB9, UBE2G2 and ZEB2 in human leukemia. Target sequencing identified 29 recurring genes, ranging in prevalence from 36% for previously known genes, such as TET2, to 12–16% for newly identified genes, such as IKZF3 or ZEB2. Half of the tumors had mutations affecting either the DNA methylation or chromatin remodeling pathways. The clinical analysis revealed that patients with mutations in DNA methylation pathway had a significantly reduced overall survival (P ¼ 0.047). We provide the first mutational profiling of BPDCN. The data support the current WHO classification of the disease as a myeloid disorder and provide a biological rationale for the incorporation of epigenetic therapies for its treatment.

Leukemia (2014) 28, 823–829; doi:10.1038/leu.2013.283 Keywords: BPDCN; blastic NK-cell lymphoma; TET2; ASXL1; IKAROS family

INTRODUCTION The overall prognosis for BPDCN is remarkably poor. Lymphoid- Blastic plasmacytoid dendritic cell neoplasm (BPDCN), previously like chemotherapy is the preferred treatment, however, most referred as ‘blastic NK-cell lymphoma/leukemia’ or ‘agranular patient relapse, resulting in a median overall survival (OS) of 12–14 16,17 CD4 þ /CD56 þ hematodermic neoplasm’, is a rare and aggressive months. Only high-dose therapy followed by allogeneic stem hematologic disease derived from precursors of a specialized subset cell transplantation can provide durable control in this otherwise 18,19 of dendritic cells, and hence, after some discussion, is considered a fatal condition. myeloid-related disease according to the 2008 WHO classification of We designed a study to discover BPDCN-associated mutations myeloid neoplasms.1–7 These tumor cells infiltrate skin, bone marrow, based on sequencing the complete exome of three tumors, peripheral blood and lymph nodes8–11 and mainly affect elderly matched with normal samples. The discovered mutations detected patients with the maximum incidence peaking at B65 years of age. were then further studied in an expanded cohort. These efforts led Diagnosis is based on the immunophenotype of the blast cells to the identification of several important disease-associated that are characterized by the expression of CD4, CD43, CD56, CD123, mutations, the reconstruction of the genetic pathways underlying BDCA-2/CD303, TCL1 and CTLA.12 In addition, immunohistochemical the BPDCN pathogenesis and revealed associations between genetic markers, such as SPIB, BDCA-4, IRF-8, BCL11A and CD2AP, have events and clinically important features of the disease. been recently reported as tools for BPDCN diagnosis.12,13 There are no established genetic biomarkers that assist in the clinical management of BPDCN. Preliminary results from molecular PATIENTS AND METHODS studies suggest that abnormalities of genes known to have a Patient samples putative role in the prognosis on other hematological malignancies, DNA samples for whole-exome sequencing (WES) were obtained from such as CDKN2A/CDKN2B, TET2 and TP53, are frequently seen in normal tissue (oral mucosa) and tumor bone marrow in three patients with BPDCN.14,15 BPDCN. Thirty-nine additional tumors were further included for the

1Molecular Cytogenetics Group, Human Cancer Genetics Program, Spanish National Cancer Research Centre—CNIO, Madrid, Spain; 2NIMGenetics, R&D Department, Tres Cantos, Madrid, Spain; 3Bioinformatic Unit, Structural Biology and Biocomputing Program, Spanish National Cancer Research Centre—CNIO, Madrid, Spain; 4Servicio de Hematologıáy Hemoterapia, Complejo Hospitalario Ntra. Sra. de Candelaria, Santa Cruz de Tenerife, Spain; 5Laboratorio de Gene´tica Hematolo´ gica, Servicio de Hematologıá, Hospital General Universitario Gregorio Maranõń, Instituto de Investigacio´ n Sanitaria Gregorio Maran˜ oń, Madrid, Spain; 6Hematology Department, Hospital Universitario La Fe, Valencia, Spain; 7Pathology Department, Hospital Universitario Marque´s de Valdecilla, Fundacioń IFIMAV, Santander, Spain; 8IBSAL, IBMCC, Centro de Investigacioń del Cańcer, Universidad de Salamanca-CSIC, Servicio de Hematologıá, Hospital Universitario de Salamanca, Salamanca, Spain; 9Hematology Department, Hospital Universitario 12 de octubre, Madrid, Spain; 10Departamento de Gene´tica, Universidad de Navarra, Pamplona, Spain; 11Laboratori de Citologia Hematolo`gica, Laboratori de Citogene`tica Molecular, Servei de Patologia, Hospital del Mar, GRETNHE, IMIM (Hospital del Mar Research Institute), Barcelona, Spain and 12Unidad de Trasplante Hematopoye´tico, Hospital Ninõ Jesus, Madrid, Spain. Correspondence: Dr JC Cigudosa, Molecular Cytogenetics Group, Human Cancer Genetics Program, Spanish National Cancer Research Centre—CNIO, Melchor Fernańdez Almagro, 3 28029, Madrid, Spain. E-mail: [email protected]. Received 27 June 2013; revised 26 August 2013; accepted 17 September 2013; accepted article preview online 27 September 2013; advance online publication, 18 October 2013 BPDCN coding genome J Menezes et al 824 targeted resequencing analysis: 11 tumor samples came from bone City, CA, USA). Data were analyzed with BioEdit Sequence Alignment marrow cells, and 28 samples from paraffin-embedded tissues. All samples Editor 7.0.5.3. were obtained at BPDCN diagnosis. The study was approved by the ethical research and animal care committee from the Institute of Health Carlos III Statistical analysis (CEI PI 32_2009). Clinical characteristics of the patients are summarized in Supplementary Table 1. DNA was extracted by the phenol–chloroform For survival analysis, only the 17 patients with complete follow-up clinical method and the quality and quantity of purified DNA was assessed by data were included. OS was calculated taking into account the first date of fluorometry (Qubit, Invitrogen, Life Technologies, Carlsbad, CA, USA) and diagnosis of BPDCN to death of disease or to last follow-up. Kaplan–Meier gel electrophoresis. method was used to estimate the distribution of OS and differences in survival between-groups were assessed using the log rank test with the SPSS v.19 software (SPSS, Chicago, IL, USA). A P-value of p0.05 was WES and bioinformatic analysis considered statistically significant. The preparation of shotgun libraries from the leukemic and non-leukemic genomic DNA obtained from the three patients followed the ‘SureSelect Human All Exon Kit’ protocol (Agilent Technologies, Santa Clara, CA, USA), RESULTS covering 50 Mb of coding exons (B1.60% of the genome). One to three Mutations in coding sequencing by WES micrograms of high molecular weight genomic DNA from each sample was To improve our understanding of the genes involved in the fragmented by acoustic shearing on a Covaris S2 instrument (Life pathogenesis and clinical evolution of BPDCN, WES was Technologies, Carlsbad, CA, USA). Fractions of 150–300 bp were ligated performed on matched tumor and normal samples from three to Illumina adapters and PCR-amplified for six cycles. For exon enrichment, 300 ng of library was hybridized to SureSelect Human All Exon Capture kit individuals with this disease. WES and bioinformatic analysis were for 24 h at 65 1C. Biotinylated hybrids were captured, and the enriched carried out according to the workflow in Figure 1a. In total, we libraries were completed by amplifying with 12 cycles of PCR. The resulting found 509, 590 and 1505 somatic alterations in case 1, case 2 and purified DNA library was applied to an Illumina flow cell for cluster case 3, respectively. Among them, 347 were intronic in case 1, 457 generation and sequenced using the Illumina Genome Analyzer IIx in case 2 and 961 in case 3, and therefore excluded from further (San Diego, CA, USA) generating 2 Â 76 bp, paired-end reads by following analysis. To gain insight into the genes affected by the the manufacturer protocols. transformation process in BPDCN, we focused our research on Exome variant analysis and biological impact predictions were 20 the mutations that passed our quality control and were predicted performed by RUbioSeq software using default parameters for somatic to result in protein-coding changes, identifying a total of 85 variation analysis. In detail, sequencing data were first checked by FastQC for quality control checks on raw sequence data and then aligned to the mutations in 81 affected genes: 28 mutations in case 1, 8 in case 2, human reference genome (GRCh37) using Burrows-Wheeler alignment.21 and 49 in case 3 (Table 1 and Supplementary Table 2). The That reads unmapped by Burrows-Wheeler alignment were realigned distribution of numbers and categories of somatically acquired using BFAST.22 Somatic variants were identified using the Unified exonic mutations and indels is represented in Figure 1b. Genotyper v2 available at the GATK.23 For variant calling, we used GATK Unfortunately, when we crossed these 81 affected genes, we did Unified Genotyper v2 applying the ‘Discovery’ genotyping mode and not identify a common mutated gene between patients default parameters for filtering. Thus, SNPs available at dbSNP132 (hg19) (Figure 1c). Of interest, however, we identified, for the first and those reported by the 1000 Genomes Project were filtered out from time, deleterious mutations in IKZF3 (IKAROS family zinc-finger VCF output files. The GATK QUAL field was employed for ranking selected 3—Aiolos), HOXB9 (homeobox B9), UBE2G2 (ubiquitin-conjugating somatic variants. The percentage of reads supporting the mutation of the total number of reads at a given position was taken as 20% in the tumor enzyme E2G 2) and ZEB2 (zinc-finger E-box binding homeobox 2) DNA, and 0.5% in normal DNA.24 Biological impact predictions for detected in human leukemia. variants were obtained from Ensembl Variant Effect Predictor.25 In order to A functional clustering analysis of the mutated genes, using IPA estimate the damaging character of the missense mutations, we used two software, showed a substantial enrichment of pathways involved different algorithms: SIFT and PolyPhen-2. in dermatological diseases and conditions, genetic disorder and hematological disease, among others (Supplementary Figure 1). Looking in detail at the list of mutated genes within the Ingenuity pathway analysis hematological disease pathway, we observed that they have Genes found to be mutated among the three cases subjected to ingenuity functions involved in DNA methylation (TET1 and TET2), chromatin pathways analysis (IPA) (Ingenuity Systems, Redwood City, CA, USA) and remodeling (ASXL1) and RNA splicing (U2AF1); all of which have used as a starting point for building biological networks. previously been reported as mutated in myeloid neoplasms. These data suggested that BPDCN has a mutational profile similar to that Targeted resequencing of other well-defined myeloid leukemias. Therefore, we decided to We designed a custom panel of 38 genes (Supplementary Table 3) using use a targeted re-sequencing approach to study these three the SureDesign Tool (Agilent). Ion semiconductor sequencing on the Ion pathways as well as transcription factors, protein kinase and Torrent PGM was performed using the HaloPlex target enrichment system ubiquitination genes involved in myeloid malignancies in an (Agilent) in 25 BPDCN tumors. Briefly, patients’ DNAs were digested by a extension cohort. cocktail of restriction enzymes and hybridized to specific probes incorporating Ion Torrent-specific sequence motifs. Hybridized molecules were captured with magnetic beads, amplified using indexed primers, Screening for somatic mutations in regions recurrently altered in templated onto Ion Sphere Particles (Ion One Touch 200 Template Kit v2 myeloid neoplasm by targeted next-generation sequencing DL), and, finally, sequenced with the Ion PGM Sequencing 200 Kit on an Ion To explore these findings, we applied a comprehensive and cost- 316 chip. Data were analyzed with Variant Caller v2.2.3-31149 using default effective target resequencing approach to indentify mutations in parameters (all Life Technologies, Carlsbad, CA, USA). the coding sequences of 38 selected genes, which are prone to Additionally, FLT3-ITD detection was carried out by conventional PCR in harbor mutations involved in leukemogenesis (n 25) and have 26 ¼ all samples as previously reported. been identified in our previous analysis (n ¼ 13) (Supplementary Table 3). This panel is composed of genes from nine categories: Sanger sequencing validation DNA methylation (5 genes), chromatin remodeling (4 genes), We used PCR amplification and direct DNA sequencing to validate transcription factors (10 genes), splicing machinery candidate variants. Because of the sensitivity of Sanger, sequence variants (5 genes), protein kinase activity (6 genes) and ubiquitination that were reported in less than 20% of the reads could not be included in (4 genes), nucleophosmin (1 gene), RAS family (2 genes) and this validation phase. The purified products were subsequently sequenced tumor suppressor (1 gene). We included a total of 39 BPDCN using the automatic sequencer ABI 3730xl (PE Applied Biosystems, Foster tumor DNAs of which, after quality control for library preparation,

Leukemia (2014) 823 – 829 & 2014 Macmillan Publishers Limited BPDCN coding genome J Menezes et al 825

Figure 1. Identification of somatic exonic variants in BPDCN by WES. (a) Sequencing workflow and bioinformatics pipeline for identification of somatic mutations. After alignment of the sequencing reads from DNA samples of BPDCN and normal cells to the human reference genome, a series of filters were applied to discard reads that were not usable for the downstream purpose of somatic mutation discovery. Sequence variants fulfilling the additional criteria were subjected to Sanger sequencing validation; (b) distribution of the numbers and categories of somatically acquired exonic mutations and indels among the three cases studied; (c) Venn diagram showing that there is no common mutated gene between patients (exonic, frameshift, stop gain/loss and no synonymous mutations-deleterious; QC4200).

25 cases were suitable for analysis. On average, 98% of reads Table 1. SNV and indel filtering steps for identification of somatic mapped uniquely to the target sequences, and we obtained 93% exonic variants in BPDCN mean target coverage with an average read depth of 471 reads per base. Filter Case 1 Case 2 Case 3 After filtering out changes in the intronic regions and polymorphisms (present in dbSNP132), we identified a total of Total 3306 3839 9683 141 variants in all sequenced exons of the 25 BPDCN patients, Somatic 509 590 1505 an average of 5.64 mutations per case. After discarding alterations Exonic 162 133 544 a in noncoding RNAs, the 50 or 30 untranscribed regions, and Consequence 63 59 240 synonymous mutations, only 90 single nucleotide variants and Quality Control 28 8 49 indels resulted in an amino acid change, frameshift or stop codon Abbreviations: BPDCN, blastic plasmacytoid dendritic cell neoplasm; SNV, (Supplementary Table 4). We selected 52 single nucleotide single nucleotide variant. aFrameshift, stop gain and non-synonymous- variants/indels, based on the variant frequency, for validation by deleterious. Sanger sequencing and confirmed 91% of the variants. In addition, with this resequencing strategy, we were able to detect all the alterations found in the three patients studied by WES, confirming two mutations were found in exon 11. The majority of ASXL1 the suitability of the panel for discovering genetic defects in the mutations were frameshift (n ¼ 6). Alterations in the NPM1 gene extension cohort. were found in exons 6 and 12. One of them was homozygous and Based on the pathogenic alterations, we observed mutations in two were present in more than one individual. All the mutations 29 genes and a mean of 3.6 mutations per case. The extreme cases observed in NRAS were single AA changes, with three being in the were one patient with 7 and another two patients with no known codon 12 and only one in codon 146 (Table 2). These data pathogenic alterations in the 38 studied genes (Figure 2a). TET2 are in line with the mutational profiles reported for acute myeloid was the most frequently mutated gene (36%), followed by ASXL1 leukemia and other myeloid neoplasms.27 (32%), NRAS (20%) and NPM1 (20%), the IKAROS family (20%) and ZEB2 and IKZF3, transcription factors previously not reported as ZEB2 (16%). In TET2, we identified ten heterozygous variants being mutated in human leukemias, were found to contain (4 frameshift, 4 nonsense and 2 missense changes) spread across deleterious mutations in our BPDCN DNAs. ZEB2 codes for a the coding exons of the gene in nine patients. The frameshift zinc-finger/homeodomain protein that normally functions as a p.Ile490Tyrfs*14 was found in two individuals and one patient had DNA-binding transcriptional repressor. We found two frameshift two different TET2 mutations. ASXL1 presented heterozygous mutations and one single AA change in ZEB2 (Table 2). Finally, mutations, mostly clustered in exon 12, in six patients. However, IKZF3 and other members of the IKAROS family of genes were also

& 2014 Macmillan Publishers Limited Leukemia (2014) 823 – 829 BPDCN coding genome J Menezes et al 826

Figure 2. Recurrently mutated genes and their interaction in BPDCN. (a) Twenty-five total cases of BPDCN were screened for somatic variants in 13 genes identified by WES and in 25 genes previously reported to be mutated in myeloid malignancies (a total of 38 genes). Mutations are represented in red. We observed mutations in 29 genes; among them, TET2 is the most frequently mutated (36%), followed by ASXL1 (32%), NRAS (20%) and NPM1 (20%), IKAROS family (20%) and ZEB2 (16%). (b) The circos diagram depicts the relative frequency and pairwise co-occurrence of mutations in the BPDCN patients. The length of the arc corresponds with the frequency of mutations in the first gene, and the width of the ribbon corresponds to the percentage of patients who also had a mutation in the second gene. Pairwise co-occurrence of mutations is denoted only once, beginning with the first gene in the clockwise direction. Mutations in TET1-2 and IDH1-2 are mutually exclusive, as previously reported in other myeloid malignancies.

Table 2. The mutated genes in BPDCN revealed by target re-sequencing

Annotated gene Mutation type Position Allele change AA change Case numbers Frequency with mutation (%)

TET2 Nonsense 106157044 C4T p.Gln649* 1/25 9/25 (36%) Frameshift 106164901 A4ACGCTCACCAAT p.Arg1262Serfs*8 1/25 Single AA change 106197378 A4G p.His1904Arg 1/25 Single AA change 106180838 G4T p.Cys1289Phe 1/25 Frameshift 106196345 T4TA p.Tyr1560* 1/25 Frameshift 106156566 C4CT p.Ile490Tyrfs*14 2/25 Nonsense 106196415 C4A p.Ser1583* 1/25 Frameshift 106158403 AATAATTTT4A p.Asn1103Argfs*24 1/25 Nonsense 106164832 G4T p.Glu1234* 1/25 Nonsense 106193892 C4T p.Arg1452* 1/25 ASXL1 Frameshift 31022978 T4TA p.Thr822Asnfs*11 1/25 8/25 (32%) Frameshift 31024844 G4GT p.Leu1444Serfs*3 1/25 Frameshift 31022898 TC4T p.Trp796Glyfs*22 1/25 Frameshift 31025066 AG4A p.Gly1518Alafs*23 1/25 Frameshift 31021157 TTGTG4T p.Cys387Serfs*74 1/25 Nonsense 31021162 T4A p.Cys387* 1/25 Frameshift 31023135 CCT4C p.Pro874Hisfs*5 1/25 Nonsense 31022550 G4T p.Gly679* 1/25 Frameshift 31024021 C4CT p.Leu1170Phefs*12 1/25 NPM1 Frameshift 170837544 T4TCTGC p.Trp288Cysfs*12 2/25 5/25 (20%) Nonsense 170819932 T4TTAG p.Ala159Argfs*35 1/25 Frameshift 170819952 AC4A p.Asp165Glufs*28 1/25 Single AA change 170819930 C4T p.Leu158Phe 2/25 NRAS Single AA change 115258747 C4T p.Gly12Asp 2/25 5/25 (20%) Single AA change 115258748 C4G p.Gly12Arg 1/25 Single AA change 115252204 C4T p.Ala146Thr 1/25 Single AA change 115258747 C4G p.Gly12Asp 1/25 IKZF1 Frameshift 50450305 CA4C p.Ile164Serfs*29 1/25 5/25 (20%) IKZF2 Single AA change 213921691 G4A p.Ala91Val 1/25 IKZF3 Single AA change 37922621 C4T p.Glu318Lys 1/25 Frameshift 37922598 C4CG p.Thr326Hisfs*25 2/25 ZEB2 Single AA change 145157495 A4C p.Leu420Arg 1/25 4/25 (16%) Frameshift 145156878 C4CG p.Gly626Arg 2/25 Frameshift 145161489 T4TC p.Asp268Argfs*12 1/25 HOXB9 Single AA change 46700467 C4T p.Arg183His 1/25 1/25 (4%) UBE2G2 Single AA change 46197270 T4A p.Asp63Val 1/25 1/25 (4%)

Leukemia (2014) 823 – 829 & 2014 Macmillan Publishers Limited BPDCN coding genome J Menezes et al 827 recurrently mutated in BPDCN. One patient had a frameshift subgroups based on the functional classes for the outcome of mutation in IKZF1, another had a single AA change in IKZF2 and analysis. Among the combinations with clinical interest, the worst three patients had alterations in IKZF3: one single AA change and clinically significant changes were those affecting the RAS family, one frameshift in two cases. Twenty percent of our BPDCN TP53 and other transcription factors (median 15 months versus patients harbored mutations in IKAROS family genes, suggesting a 99 months for the non-mutated cases; P ¼ 0.013) (Figure 3b). potential role of these genes in this specific type of myeloid These data suggest that mutations in aberrant methylation genes leukemia (Supplementary Figure 2). represent a poor prognostic factor and are probably involved in the pathogenesis of BPDCN. Functional categorization of mutated genes We used circus diagrams to assess patterns of mutual exclusivity DISCUSSION and co-occurrence between sets of genes. We identify mutually exclusive alterations that affect TET1/2 and IDH1/2; TET1/2 and Because BPDCN is a rare disease that was recognized as a distinct entity only recently, information regarding genomic and genetic DNMT3A; and between IKAROS family genes. Additionally, muta- 14 tions in ASXL1 and DNMT3A rarely co-occurred in our samples, biomarkers is rather limited. Lucioni et al. studied 21 BPDCN with only one harboring mutation in both. A rare co-occurrence patients by comparative genomic hybridization arrays and found was also observed between the splicing genes with only one case complete or partial chromosomal losses with common deleted regions involving the CDKN2A/CDKN2B, RB1, CDKN1B, LATS2 and having mutations in both SF3B1 and SRSF2 (Figure 2b and 15 Supplementary Figure 3). IKZF1 loci. Jardin et al. reported 54 and 38% of TET2 and TP53 We also grouped mutations into larger sets or pathways and mutations, respectively, in the 13 BPDCN individuals studied. examined patterns of mutual exclusivity and co-occurrence In this study, we present, for the first time, a comprehensive between these groups (Figure 2a and Supplementary Figure 4). analysis of BPDCN combining next-generation sequencing with Out of the 25 samples, 23 (92%) contained at least one mutation clinical characteristics and clinical outcomes of 25 patients. in one of the nine categories. The nine categories and how Although we did not find a common alteration that could frequently they were mutated are: aberrant methylation (48%), explain the pathogenesis of BPDCN in the three patients studied chromatin remodeling (52%), transcription factors (44%), splicing by WES, we identified deleterious mutations in IKZF3, HOXB9, machinery (36%), protein kinase activity (16%), ubiquitination UBE2G2 and ZEB2 for the first time in human leukemia and more (12%), nucleophosmin (20%), RAS family (28%) and tumor common alterations such as TET1/2, ASXL1 and U2AF1 previously suppressor—TP53 (8%). Half of the tumors had mutations reported as mutated in other myeloid neoplasms. affecting the methylation profile and chromatin remodeling The best test of the relevance of individual mutations pathways, and 20% had mutations in genes in both pathways. for pathogenesis (in the absence of functional validation) is their recurrence in other samples or other cancers. TET2 mutations featured in our series appear similar to those observed in myeloid Clinical relevance of aberrant methylation genes mutations neoplasms: aberrations are mainly frameshift or nonsense and The analysis of survival data from the study cohort revealed have been observed with the same frequencies (36–58%).28 several significant differences among the patients. First, we found However, our TET2 and TP53 mutation frequency are lower that the patients with mutations in genes in methylation pathways compared with the BPDCN cases from Jardin et al.15 Similar to had a significantly reduced OS rate compared with individuals other hematological malignancies, the relevance of TET2 without mutations in these genes (median 11 months versus 79 mutations remains undetermined in this disease, which is months; P ¼ 0.047, Figure 3a). On the other hand, patients with characterized by a peculiar combination of various genomic mutations in ETV6 (P ¼ 0.022), TP53 (P ¼ 0.003) and N/K RAS alterations. Truncated exon 12 mutations in ASXL1 have recently (P ¼ 0.015) had either a statistically significant worse prognosis been described in 11% of patients with myelodysplastic (Supplementary Figure 5) or showed a trend in the same direction syndromes, 43% of those with chronic myelomonocytic (P ¼ 0.073) when a mutation of IKZF1/2/3 was present. Taking into leukemia, 7% with primary and 47% with secondary acute consideration the limited sample size, we also combined the myeloid leukemia (AML).29 The NPM1 mutations have been

Figure 3. Kaplan–Meier overall survival (OS) curves. (a) We observed a signiﬁcant difference in the OS according to the mutational status of genes included in the DNA methylation class (P ¼ 0.047; mut: n ¼ 7 and 11 months; wt: n ¼ 10 and 79 months); (b) We observed a signiﬁcant difference in the OS according to the mutational status of genes included in the transcription factor class/TP53/RAS (P ¼ 0.013; mut: n ¼ 10 and 15 months; wt: n ¼ 7 and 99 months).

& 2014 Macmillan Publishers Limited Leukemia (2014) 823 – 829 BPDCN coding genome J Menezes et al 828 shown to be involved in 23.5% of AML patients and activating RAS Bank Unit, Spanish National Cancer Research Centre. This work was supported in part mutations were found to be present in 10.7%.30,31 by grants INTRASALUD PI12/0425 and Red Tema´tica de Investigacioń Cooperativa en Notably, only one BPDCN case displayed JAK2 and other Cańcer (RTICC) RD12/0036/0037 to JCC from Instituto de Salud Carlos III and FLT3-ITD mutations, reinforcing that BPDCN and AML or myelo- Ministerio de Economia y Competitividad; and INTRASALUD PI12/01047 to JVC. JM is proliferative disorders may be genetically distinct. Pointing out to recipient of a La Caixa International PhD Fellowship. these differences, we found mutations in genes never shown to be involved in human leukemia. First, the IKAROS gene family AUTHOR CONTRIBUTIONS encodes zinc-finger transcription factors that regulate gene expression via chromatin remodeling in lymphoid development JM designed and performed the research, analyzed and interpreted the data, and differentiation. In fact, IKZF1/2/3 mutations, present as and wrote the manuscript. FA and MW performed the targeted next-generation deletions, are prevalent in acute lymphoblastic leukemia.29,32 sequencing and sequence data analysis. GGL and DGP performed the analysis As 20% of our BPDCN patients harbored mutations in IKAROS of sequence data for whole-exome sequencing. RNS and SA analyzed and family genes, a potential role of these genes in normal or interpreted the data. MJL performed the FLT3-ITD screening. JGTC, IB, JVC, abnormal myelopoiesis should be considered. Secondly, although SMM, MAP, JMH, RA, MJC, LFB and MGV reviewed the pathologic data and mutations in ZEB2 have not been reported in human cancer, ZEB2 confirmed the diagnosis. JCC designed and directed the research and revised protein interacts with several members of the SMAD protein the manuscript, which all authors have approved. family that have been shown to have important roles in normal and malignant hematopoiesis. Finally, a preliminary report indicated that ZEB2 was overexpressed in leukemia patients with URLS MLL gene rearrangements.33 FastQC: http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/; VEP: http:// Recently, a series of mutually exclusive mutations that www.ensembl.org/tools.html; SIFT: http://sift-jcvi.org; PolyPhen-2: http:// affect TET2, IDH1 or IDH2 mutations, and potentially other as of genetics.bwh.harvard.edu/pph2/index.shtml; SureDesign Tool: https://earray. yet unidentified disease alleles, was reported.27,34 We observed chem.agilent.com/suredesign/; SRA database: http://www.ncbi.nlm.nih.gov/sra; the same pattern of mutual exclusiveness in our series and IPA: http://www.ingenuity.com/products/ipa; Circos: http://circos.ca/. additionally between IKAROS genes. Correlation between epigenetic modifier mutations and prognosis has been controversial. TET2 mutations are associated with ACCESSION CODES poor prognosis in normal karyotype-AML,35 but with no clear Next-generation sequences have been deposited in the NCBI Sequence Read association in myelodysplastic syndromes36 or myeloproliferative Archive (SRA), pending accession details. neoplasm.37 Alterations in IDH1/2 are associated with a favorable or negligible effect on prognosis, depending on the type of mutation in AML.38,39 No importance was observed in myelo- REFERENCES dysplastic syndromes36 or myeloproliferative neoplasm.37,40 1 Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A et al. The Several studies show that DNMT3A mutations are associated 2008 revision of the World Health Organization (WHO) classification of myeloid with worsened OS in patients with AML41,42 and an adverse neoplasms and acute leukemia: rationale and important changes. Blood 2009; prognosis in myelodysplastic syndromes.43 In our series of 114: 937–951. BPDCN patients, we found that patients with mutations in genes 2 Herling M, Jones D. CD4 þ /CD56 þ hematodermic tumor: the features of an evolving entity and its relationship to dendritic cells. Am J Clin Pathol 2007; 127: in methylation pathways had a significantly reduced OS as 687–700. compared with individuals without mutations in these genes. 3 Willemze R, Jaffe ES, Burg G, Cerroni L, Berti E, Swerdlow SH et al. WHO-EORTC These data suggest that mutations in DNA methylation genes classification for cutaneous lymphomas. Blood 2005; 105: 3768–3785. represent a poor prognostic factor that are likely to be involved in 4 Cella M, Jarrossay D, Facchetti F, Alebardi O, Nakajima H, Lanzavecchia A et al. the pathogenesis of BPDCN. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large Changing the paradigm of what is known about BPDCN, our amounts of type I interferon. Nat Med 1999; 5: 919–923. data suggest that this dendritic cell leukemia has a mutational 5 Chaperot L, Bendriss N, Manches O, Gressin R, Maynadie M, Trimoreau F et al. profile strikingly similar to that of other well-defined myeloid Identification of a leukemic counterpart of the plasmacytoid dendritic cells. Blood leukemias. Despite the numerous advances in our understanding 2001; 97: 3210–3217. 6 Petrella T, Comeau MR, Maynadie M, Couillault G, De Muret A, Maliszewski CR et al. of the genetics and epigenetics of myeloid malignancies, only ’Agranular CD4 þ CD56 þ hematodermic neoplasm’ (blastic NK-cell lymphoma) limited examples of clinical translation of these findings into new originates from a population of CD56 þ precursor cells related to plasmacytoid therapeutic approaches have been implemented. However, it has monocytes. Am J Surg Pathol 2002; 26: 852–862. now become clear that the epigenetic modifiers provide new 7 Pilichowska ME, Fleming MD, Pinkus JL, Pinkus GS. CD4 þ /CD56 þ hematodermic targets for therapeutic intervention and that targeting neoplasm ("blastic natural killer cell lymphoma"): neoplastic cells express the these enzymatic activities are currently being explored from a immature dendritic cell marker BDCA-2 and produce interferon. Am J Clin Pathol therapeutic standpoint in several types of leukemia.44,45 Our study 2007; 128: 445–453. provides, for the first time, molecular data that suggest that a 8 Feuillard J, Jacob MC, Valensi F, Maynadie M, Gressin R, Chaperot L et al. complete and innovative change is required in the treatment Clinical and biologic features of CD4( þ )CD56( þ ) malignancies. Blood 2002; 99: 1556–1563. approach adopted for patients with BPDCN, including the 9 Petrella T, Bagot M, Willemze R, Beylot-Barry M, Vergier B, Delaunay M et al. administration of drugs that target aberrant methylation, Blastic NK-cell lymphomas (agranular CD4 þ CD56 þ hematodermic neoplasms): chromatin remodeling, specific transcription factors and aberrant a review. Am J Clin Pathol 2005; 123: 662–675. splicing. 10 Reichard KK, Burks EJ, Foucar MK, Wilson CS, Viswanatha DS, Hozier JC et al. CD4( þ ) CD56( þ ) lineage-negative malignancies are rare tumors of plasmacytoid dendritic cells. Am J Surg Pathol 2005; 29: 1274–1283. CONFLICT OF INTEREST 11 Lee JK, Schiller G. Blastic plasmacytoid dendritic cell neoplasm. Clin Adv Hematol Oncol 2013; 10: 60–62. The authors declare no conflict of interest. 12 Marafioti T, Paterson JC, Ballabio E, Reichard KK, Tedoldi S, Hollowood K et al. Novel markers of normal and neoplastic human plasmacytoid dendritic cells. Blood 2008; 111: 3778–3792. ACKNOWLEDGEMENTS 13 Montes-Moreno S, Ramos-Medina R, Martinez-Lopez A, Barrionuevo Cornejo C, We thank all the co-workers in our laboratory for their excellent technical assistance. Parra Cubillos A, Quintana-Truyenque S et al. SPIB, a novel immunohistochemical We especially thank the help of Manuel Morente and their team from the Tumour marker for human blastic plasmacytoid dendritic cell neoplasms: characterization

Leukemia (2014) 823 – 829 & 2014 Macmillan Publishers Limited BPDCN coding genome J Menezes et al 829 of its expression in major hematolymphoid neoplasms. Blood 2013; 121: 29 Tefferi A. Novel mutations and their functional and clinical relevance in myelo- 643–647. proliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia 14 Lucioni M, Novara F, Fiandrino G, Riboni R, Fanoni D, Arra M et al. Twenty-one 2010; 24: 1128–1138. cases of blastic plasmacytoid dendritic cell neoplasm: focus on biallelic locus 30 Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K et al. Recurring 9p21.3 deletion. Blood 2011; 118: 4591–4594. mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 15 Jardin F, Ruminy P, Parmentier F, Troussard X, Vaida I, Stamatoullas A et al. TET2 2009; 361: 1058–1066. and TP53 mutations are frequently observed in blastic plasmacytoid dendritic cell 31 Ley TJ, Miller C, Ding L, Raphael BJ, Mungall AJ, Robertson A et al. Genomic and neoplasm. Br J Haematol 2011; 153: 413–416. epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med 16 Reimer P, Rudiger T, Kraemer D, Kunzmann V, Weissinger F, Zettl A et al. What is 2013; 368: 2059–2074. CD4 þ CD56 þ malignancy and how should it be treated? Bone Marrow 32 Holmfeldt L, Wei L, Diaz-Flores E, Walsh M, Zhang J, Ding L et al. The Transplant 2003; 32: 637–646. genomic landscape of hypodiploid acute lymphoblastic leukemia. Nat Genet 17 Assaf C, Gellrich S, Whittaker S, Robson A, Cerroni L, Massone C et al. 2013; 45: 242–252. CD56-positive haematological neoplasms of the skin: a multicentre study of the 33 Caudell D, Harper DP, Novak RL, Pierce RM, Slape C, Wolff L et al. Retroviral Cutaneous Lymphoma Project Group of the European Organisation for Research insertional mutagenesis identifies Zeb2 activation as a novel leukemogenic col- and Treatment of Cancer. J Clin Pathol 2007; 60: 981–989. laborating event in CALM-AF10 transgenic mice. Blood 2010; 115: 1194–1203. 18 Dalle S, Beylot-Barry M, Bagot M, Lipsker D, Machet L, Joly P et al. Blastic 34 Shih AH, Abdel-Wahab O, Patel JP, Levine RL. The role of mutations in epigenetic plasmacytoid dendritic cell neoplasm: is transplantation the treatment of choice? regulators in myeloid malignancies. Nat Rev Cancer 2012; 12: 599–612. Br J Dermatol 2010; 162: 74–79. 35 Metzeler KH, Maharry K, Radmacher MD, Mrozek K, Margeson D, Becker H et al. 19 Roos-Weil D, Dietrich S, Boumendil A, Polge E, Bron D, Carreras E et al. TET2 mutations improve the new European LeukemiaNet risk classification of Stem cell transplantation can provide durable disease control in blastic acute myeloid leukemia: a Cancer and Leukemia Group B study. J Clin Oncol 2011; plasmacytoid dendritic cell neoplasm: a retrospective study from 29: 1373–1381. the European Group for Blood and Marrow Transplantation. Blood 2013; 121: 36 Bejar R, Stevenson K, Abdel-Wahab O, Galili N, Nilsson B, Garcia-Manero G et al. 440–446. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med 20 Rubio-Camarillo M, Gomez-Lopez G, Fernandez JM, Valencia A, Pisano DG. 2011; 364: 2496–2506. RUbioSeq: a suite of parallelized pipelines to automate exome variation and 37 Tefferi A, Pardanani A, Lim KH, Abdel-Wahab O, Lasho TL, Patel J et al. TET2 bisulfite-seq analyses. Bioinformatics 2013; 29: 1687–1989. mutations and their clinical correlates in polycythemia vera, essential thrombo- 21 Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler cythemia and myelofibrosis. Leukemia 2009; 23: 905–911. transform. Bioinformatics 2009; 25: 1754–1760. 38 Green CL, Evans CM, Hills RK, Burnett AK, Linch DC, Gale RE. The prognostic 22 Homer N, Merriman B, Nelson SF. BFAST: an alignment tool for large scale gen- significance of IDH1 mutations in younger adult patients with acute myeloid ome resequencing. PLoS One 2009; 4:e7767. leukemia is dependent on FLT3/ITD status. Blood 2010; 116: 2779–2782. 23 DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C et al. A frame- 39 Green CL, Evans CM, Zhao L, Hills RK, Burnett AK, Linch DC et al. The prognostic work for variation discovery and genotyping using next-generation DNA significance of IDH2 mutations in AML depends on the location of the mutation. sequencing data. Nat Genet 2011; 43: 491–498. Blood 2011; 118: 409–412. 24 Jones S, Wang TL, Shih IeM, Mao TL, Nakayama K, Roden R et al. Frequent 40 Tefferi A, Jimma T, Sulai NH, Lasho TL, Finke CM, Knudson RA et al. IDH mutations mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma. in primary myelofibrosis predict leukemic transformation and shortened survival: Science 2010; 330: 228–231. clinical evidence for leukemogenic collaboration with JAK2V617F. Leukemia 2012; 25 McLaren W, Pritchard B, Rios D, Chen Y, Flicek P, Cunningham F. Deriving the 26: 475–480. consequences of genomic variants with the Ensembl API and SNP Effect Predictor. 41 Ley TJ, Ding L, Walter MJ, McLellan MD, Lamprecht T, Larson DE et al. DNMT3A Bioinformatics 2010; 26: 2069–2070. mutations in acute myeloid leukemia. N Engl J Med 2010; 363: 2424–2433. 26 Fernandez-Mercado M, Yip BH, Pellagatti A, Davies C, Larrayoz MJ, Kondo T et al. 42 Yan XJ, Xu J, Gu ZH, Pan CM, Lu G, Shen Y et al. Exome sequencing identifies Mutation patterns of 16 genes in primary and secondary acute myeloid leukemia somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic (AML) with normal cytogenetics. PLoS One 2012; 7: e42334. leukemia. Nat Genet 2011; 43: 309–315. 27 Cancer Genome Atlas Research Network. Genomic and epigenomic 43WalterMJ,DingL,ShenD,ShaoJ,GrillotM,McLellanMet al. Recurrent DNMT3A landscapes of adult de novo acute myeloid leukemia. N Engl J Med 2013; 368: mutations in patients with myelodysplastic syndromes. Leukemia 2011; 25: 1153–1158. 2059–2074. 44 James LI, Frye SV. Targeting chromatin readers. Clin Pharmacol Ther 2013; 93: 28 Abdel-Wahab O, Manshouri T, Patel J, Harris K, Yao J, Hedvat C et al. 312–314. Genetic analysis of transforming events that convert chronic myeloproliferative 45 Burridge S. Target watch: drugging the epigenome. Nat Rev Drug Discov 2013; 12: neoplasms to leukemias. Cancer Res 2010; 70: 447–452. 92–93.

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