Letters to the Editor 1348 up to 80 years of age supporting the use of modern anti-myeloma 6 Turesson I, Kristinsson SY, Bjorkholm M, Landgren O. Clinical trials and population- 16,17 therapy independent of age. based studies to assess survival benefits in multiple myeloma: welcome to the future! Blood 2011; e-pub ahead of print 9 November 2011; http://bloodjournal. hematologylibrary.org/content/118/12/3205/reply. CONFLICT OF INTEREST 7 Turesson I, Velez R, Kristinsson SY, Landgren O. Patterns of improved survival in The authors declare no conflict of interest. patients with multiple myeloma in the twenty-first century: a population-based study. J Clin Oncol 2010; 28: 830–834. SY Kristinsson1,2, WF Anderson3 and O Landgren4 8 Brenner H, Gondos A, Pulte D. Recent major improvement in long-term 1 survival of younger patients with multiple myeloma. Blood 2008; 111: Division of Hematology, Department of Medicine, Karolinska 2521–2526. University Hospital Solna and Karolinska Institutet, 9 Kumar SK, Rajkumar SV, Dispenzieri A, Lacy MQ, Hayman SR, Buadi FK et al. Stockholm, Sweden; Improved survival in multiple myeloma and the impact of novel therapies. Blood 2 Faculty of Medicine, University of Iceland and Department of 2008; 111: 2516–2520. Hematology, Landspitali National University Hospital, 10 Waxman AJ, Mink PJ, Devesa SS, Anderson WF, Weiss BM, Kristinsson SY et al. Reykjavik, Iceland; Racial disparities in incidence and outcome in multiple myeloma: a population- 3Division of Cancer Epidemiology and Genetics, National Cancer based study. Blood 2010; 116: 5501–5506. Institute, NIH, Bethesda, MD, USA and 11 Kumar SK, Dispenzieri A, Lacy MQ, Gertz MA, Buadi FK, Pandey S et al. Continued 4Multiple Myeloma Section, Metabolism Branch, Center for Cancer improvement in survival in multiple myeloma: changes in early mortality and outcomes in older patients. Leukemia 2013; e-pub ahead of print 25 October Research, National Cancer Institute, NIH, Bethesda, MD, USA 2013; doi:10.1038/leu.2013.313. E-mail: [email protected] 12 Surveillance E. End Results (SEER) Programwww.seer.cancer.govSEER*Stat Database: Incidence - SEER 9 Regs Research Data, Nov 2011 Sub (1973-2010) oKatrina/Rita Population Adjustment4 - Linked To County Attributes - Total U.S., 1969-2010 REFERENCES Counties, National Cancer Institute, DCCPS, Surveillance Research Program, 1 Socialstyrelsen. Cancer Incidence in Sweden 2006. Stockholm, Sweden 2007. Surveillance Systems Branch, released April 2013, based on the November 2012 2 Kristinsson SY, Landgren O, Dickman PW, Derolf AR, Bjorkholm M. Patterns of submission. survival in multiple myeloma: a population-based study of patients diagnosed in 13 Fritz AG. International Classification of Diseases for Oncology: ICD-O. 3rd edn. World Sweden from 1973 to 2003. J Clin Oncol 2007; 25: 1993–1999. Health Organization: Geneva, Switzerland, 2000, pp 240, vii. 3 Gay F, Larocca A, Wijermans P, Cavallo F, Rossi D, Schaafsma R et al. 14 Palumbo A, Anderson K. Multiple myeloma. N Engl J Med 2011; 364: Complete response correlates with long-term progression-free and overall 1046–1060. survival in elderly myeloma treated with novel agents: analysis of 1175 patients. 15 Zhan F, Huang Y, Colla S, Stewart JP, Hanamura I, Gupta S et al. The molecular Blood 2011; 117: 3025–3031. classification of multiple myeloma. Blood 2006; 108: 2020–2028. 4 San Miguel JF, Schlag R, Khuageva NK, Dimopoulos MA, Shpilberg O, Kropff M et al. 16 Jakubowiak AJ, Dytfeld D, Griffith KA, Lebovic D, Vesole DH, Jagannath S et al. Bortezomib plus melphalan and prednisone for initial treatment of multiple A phase 1/2 study of carfilzomib in combination with lenalidomide and low-dose myeloma. N Engl J Med 2008; 359: 906–917. dexamethasone as a frontline treatment for multiple myeloma. Blood 2012; 120: 5 Dimopoulos M, Spencer A, Attal M, Prince HM, Harousseau JL, Dmoszynska A et al. 1801–1809. Lenalidomide plus dexamethasone for relapsed or refractory multiple myeloma. 17 Landgren O, Korde N. Treating myeloma: the future is already here! Blood 2012; N Engl J Med 2007; 357: 2123–2132. 120: 1754–1756.

Recurrent genetic defects on 7q in myeloid neoplasms

Leukemia (2014) 28, 1348–1351; doi:10.1038/leu.2014.25 report the recurrent genetic defects in CUX1, LUC7L2 and EZH2, and other results of the comprehensive molecular screen of chr7q. Using metaphase cytogenetics and SNP-array-based karyotyping, loss of heterozygosity (LOH) lesions affecting 7q were identified in Monosomy 7 (-7) and deletion of the long arm (del(7q)) of 171 of 1131 cases (15%), including low-risk myelodysplastic (chr7) belong to the most commonly acquired syndromes (MDS) (14%), high-risk MDS (34%), secondary acute karyotypic abnormalities in myeloid neoplasms and are associated myeloid leukemia (sAML) (18%), MDS/myeloproliferative neo- with poor prognosis. Although somatic mutations or decreased plasms (MPN) (13%), MPN (6%) and primary acute myeloid expression of EZH2 have a role,1–3 the molecular mechanisms leukemia (pAML) (8%) (Supplementary Table S1). Minimal behind chr7 alterations in most cases remain undefined. It is commonly deleted regions (CDRs) were defined as 7q22 likely that other pivotal target contribute to -7/del(7q) (100847518–101872055; CDR1), 7q34 (138190944–139672739; pathogenesis. Somatic mutations may either be unique to del(7q) CDR2) and 7q35-q36 (144707068–148942012; CDR3), as previously hemizygous inactivation, or be shared between 7q diploid and described.4 Analysis of WES results for all exons on chr7 for 428 haploid cases. In addition to the hypothetical mutations in hemizygous cases with different myeloid neoplasms (Figure 1a) identified 490 configuration, del(7q) alone may result in haploinsufficient nonsilent alterations in 306 genes located on chr7 (5% of all expression. Corresponding heterozygous hypomorphic mutations alterations found; Supplementary Table S2). After stringent or epigenetic inactivation of genes located on chr7q may filtering and bioanalytic processing to avoid false positives phenocopy the haploinsufficient expression resulting from (Supplementary Figure S1),5 we narrowed the focus of our del(7q). To identify somatic mutations and haploinsufficiently investigations to ‘tier 1’ mutations; 155 mutated genes were expressed genes located on chr7q, we applied whole-exome found in 25% (17/68) of -7/del(7q) cases, in 100% (6/6) of sequencing (WES) and expression analysis both in deletion cases uniparental disomy for chr7q (UPD7q) and in 30% (107/354) of and in those with an apparently normal diploid chr7. Here we diploid for chr7 (Figure 1b). All mutations were validated by

Accepted article preview online 16 January 2014; advance online publication, 7 February 2014

Leukemia (2014) 1341 – 1379 & 2014 Macmillan Publishers Limited Letters to the Editor 1349 Sanger sequencing and targeted deep sequencing of DNA from In addition to somatic defects in the genes on CDRs of 7q, copy both tumor and germline (CD3 þ T cells or buccal mucosa) cells. number loss of the wild-type genes might result in haploinsuffi- UPD7q was more frequently affected by somatic mutations than ciency and the resultant effects may be similar to the hetero- -7 or del(7q). Notably, 17% (39/228) of the somatic mutations were zygous hypomorphic or loss of function mutations. To investigate located in one of the CDRs with marker genes CUX1 in 7q22, haploinsufficient due to -7/del(7q), we compared LUC7L2 in 7q34, and CUL1 and EZH2 in 7q35-36. relative mRNA levels between cases with and without -7/del(7q). Among many rare somatic events, eight genes (EZH2, CUX1, Underexpressed genes (o À 2 s.d. of 17 healthy controls) were LUC7L2, CFTR, PLXNA4, DYNC1l1, NRCAM and CUL1) corresponding more common in -7/del(7q), in particular within the three CDRs to regions affecting del(7q) were affected by multiple mutations, (Supplementary Figure S3). Furthermore, cases with deletion of totaling 44 events (Supplementary Figure S2). For example, CUX1, LUC7L2 and EZH2 (all located in CDRs) showed a mutations of a core component of E3 ligase complex significantly lower mRNA expression compared with those with CUL16,7 were detected only in cases with -7/del(7q) (N ¼ 2, diploid 7q (Po0.001, o0.001 and o0.05, respectively, Figure 2a). hemizygous mutations). Conversely, somatic mutations of When mutated in case diploid for 7q, these genes were commonly PLXNA4, encoding a component of the neuropilin–plexin complex,8 affected by frameshift and nonsense mutations (Supplementary were observed only in cases with diploid 7q. In cases with diploid Table S3), implying loss of function. Thus, it is likely that either a 7q, we observed 23 different heterozygous alterations, of which hypomorphic mutation, and/or haploinsufficiency may lead to the 63% (five of the eight genes) were also affected in -7/del(7q) downstream pathological consequences. Conversely, there was no or UPD7q (hemizygous/homozygous) (Figure 1b). Previously significant haploinsufficiency identified in the genes located described EZH2 (7q36.1) mutations, located in CDR3, were seen outside CDRs (NRCAM (7q31.1), PLXNA4 (7q32.3) and CFTR in either heterozygous, homozygous or hemizygous configura- (7q31.2) (data not shown). tions. However, EZH2 mutations were most common in UPD7q To elucidate the clinical impact of recurrent deficiency in cases (67%, 4/6), and in only 5% (4/88) of -7/del(7q) cases. The specific genes on chr7, we correlated clinical characteristics to other CDRs also contained genes recurrently affected by mutations either deletion or corresponding somatic inactivating mutations. including CUX1 (7q22, N ¼ 5) and LUC7L2 (7q34, N ¼ 8). Somatic Mutations and deletions in CUX1, LUC7L2 and EZH2 were observed mutations were also identified on 7p (HDAC9, IKZF1 and EGFR), in 11%, 11% and 12% of cases, respectively (Figure 2b) and were which may account for the differences between -7 and del(7q). less prevalent in cases with MPN compared with MDS (P ¼ 0.02,

CDR1 CDR2 CDR3 7q22 7q34 7q35-36

diploid chr7 1

0.75

0.5 mutation ratio 0.25

0 diploid chr7 UPD7 -7/del(7q)

missense nonsense frameshift splice site

mutated gene numbers

6 5 141 2 UPD7 1

-7/del(7q) heterozygous mutation hemizygous mutation hetero/homozygous mutation hetero/hemizygous mutation hetero/hemi/homozygous mutation

acquired genetic change diploid UPD deletion Figure 1. Mutations and LOH on chr7. (a) Mutations of chr7 detected by whole-exome sequencing in the whole cohort (N ¼ 428) are shown in red. LOH on chr7 is demonstrated as follows: a normal diploid, blue and orange; a uniparental disomy (UPD7), double blue lines; and a deletion for chr7, dashed line. Three distinct CDRs, indicated by vertical rectangles, are identified on 7q by mapping of SNP-A karyotyping. (b) Frequency of the cases with mutations in each chr7 status (diploid, UPD and deletion), and mutational types (missense, nonsense, frameshift and splice site) are demonstrated by bar graph and pie chart, respectively (upper panel). Venn diagram shows the number of mutated genes categorized by their configuration of zygosity (heterozygous, homozygous and heterozygous) (lower panel).

& 2014 Macmillan Publishers Limited Leukemia (2014) 1341 – 1379 Letters to the Editor 1350 o0.01 and ¼ 0.02, respectively) and sAML (P ¼ 0.01, o0.01 and We also investigated the potential relationship of somatic ¼ 0.01, respectively). Deletions involving these three gene loci mutation events observed on other to concomitant were more prevalent in high-risk MDS than in low-risk -7/del(7q) and UPD7 (Supplementary Figure S4a). There were clear MDS (Po0.001) and in sAML than in pAML (Po0.001). Consistent differences between both LOH7 groups, in which -7/del(7q) with this finding, mutations in any one of three genes were was more associated with accessory chromosomal events (that is, significantly associated with shorter survival (Po0.0001; HR ¼ 2.55; del(5q), del(17p) or trisomy 8) than cases with UPD7q. 95% CI, 1.98 to 7.64; Figure 2c): CUX1 mutations were linked to Although well-known, frequent mutations in U2AF1, TET2 and compromised survival, as were mutations in LUC7L2 or TP53 were commonly found in both LOH7 groups, some specific EZH2 (HR ¼ 2.32 and HR ¼ 3.16, respectively). Furthermore, the genes, including the CSMD family, were uniquely observed in -7/ presence of low CUX1, LUC7L2 or EZH2 expression correlated del(7q). LOH7 was also associated with somatic mutations in with a significantly shorter survival (P ¼ 0.001; HR ¼ 2.05, 95% CI, SETBP1 (P ¼ 0.02, Supplementary Figure S4b). When we investi- 1.45 to 4.45; Figure 2d) in pAML patients. We performed additional gated concomitant mutations and genetic events in mutation clinical investigations, but no relationship between the low cases with CUX1, LUC7L2 and EZH2, these three most common expression of these three genes and other clinical parameters mutations were not mutually exclusive (Supplementary (for example, age, cytogenetics and blood counts) was found Figure S4c). It is possible that concomitant mutations exert (data not shown). synergetic effects analogous to the defect created by a long

Figure 2. Gene expression of CUX1, LUC7L2 and EZH2.(a) By expression array analysis, -7/del(7q) patients’ samples (n ¼ 9) show significantly decreased expressions of all CUX1, LUC7L2 and EZH2 genes relative to those from MDS patients who possess normal diploid chr7 (n ¼ 174). Statistics analysis was performed using Student’s t-test with a two-sided test. *Po0.05, **Po0.001, N.S.; not significant. (b) Frequency of deletion and somatic mutation in CUX1, LUC7L2 and EZH2. In the cohort of patients with myeloid malignancies, SNP array karyotyping and metaphase cytogenetics (n ¼ 1559) were applied for the detection of 7q deletion affecting CUX1, LUC7L2 and EZH2 loci. Mutations of each gene were searched in 428 cases by whole-exome and validation sequencing. Frequency of the deletions and mutations was calculated in each disease phenotypes. MDS-low includes refractory cytopenia with unilineage dysplasia, refractory cytopenia with multilineage dysplasia, MDS with isolated del5q, MDS unclassifiable and refractory anemia with ring sideroblasts. MDS-high includes refractory anemia with excess blasts. *Po0.001 (w2-test). (c) Overall survival was compared among patients with mutations (mt) of CUX1, LUC7L2 or EZH2, wild type (wt) and del(7q). (d) Overall survival was compared between AML patients with low expression of EZH2, CUX1 and EZH2 and those with normal expression of the corresponding genes. P-values presented correspond to the Cox regression between the groups indicated. n.s.; not significant.

Leukemia (2014) 1341 – 1379 & 2014 Macmillan Publishers Limited Letters to the Editor 1351 deletion on chr7q (involving multiple CDRs). TET2 mutations were 2First Department of Internal Medicine, Faculty of Medical Sciences, most prevalent in CUX1 or EZH2 mutant cases (Po0.001, University of Fukui, Fukui, Japan; Supplementary Figure S4d), suggesting that TET2 lesions consti- 3Cancer Genomics Project, Graduate School of Medicine, tute the driver events. In addition to LUC7L2, we found mutations The University of Tokyo, Tokyo, Japan; in other spliceosomal genes (U2AF1 or U2AF2)inCUX1 or EZH2 4Department of Pathology and Tumor Biology, Graduate School of mutation cases, which were also mutated in cases of LOH7q Medicine, Kyoto University, Kyoto Japan; without these mutations. Of note is that we could not find any 5Department of Genetics and Genome Sciences, Case Western concomitant TP53 mutations in cases with mutated CUX1, LUC7L2 Reserve University, Cleveland, OH, USA; and EZH2. In sum, our findings indicate that diverse additional 6Laboratory of DNA Information Analysis, Center, molecular events might have a significant role in hypomorphic Institute of Medical Science, The University of Tokyo, Tokyo, Japan; gene defects on chr7. 7Laboratory of Sequence Analysis, Human Genome Center, Institute Our results demonstrate the various associations of the LOH7q of Medical Science, The University of Tokyo, Tokyo, Japan; (including UPD) with or without concomitant mutations involving 8Albert Einstein College of Medicine, Bronx, NY, USA; genes located in 7q region. Regardless of their causes (deletion or 9Divisions of Hematology and Hematological Malignancy, normal diploid chr7) or heterozygous hypomorphic mutation, Departments of Internal Medicine and Oncology, Johns Hopkins haploinsufficient expression of CUX1, LUC7L2 and EZH2 is University School of Medicine, Baltimore, MD, USA; associated with a poor survival suggesting that hypomorphic 10Leukemia Program, Cleveland Clinic Taussig Cancer Institute, mutations and haploinsufficiency both lead to deficient function Cleveland, OH, USA and of these genes in -7/del(7q) myeloid neoplasms. Hypomorphic 11Department of Hematologic Oncology and Blood Disorders, EZH2 mutations (located in SET domain) found in myeloid Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA neoplasms have been implicated in premature termination or E-mail: [email protected] direct abrogation of histone methyltransferase activity. It remains 12These authors contributed equally to this work. unclear why EZH2 mutations occur more commonly in the context of UPD7q rather than -7/del(7q),2 but likely, as reported here and 9 recently, loss of EZH2 function may occur through multiple REFERENCES pathways, including homozygous mutations with UPD7q, 1 Ernst T, Chase AJ, Score J, Hidalgo-Curtis CE, Bryant C, Jones AV et al. Inactivating haploinsufficiency and through phenocopy by splicesome mutations of the histone methyltransferase gene EZH2 in myeloid disorders. mutations. The function and likely the consequences of LUC7L2 Nat Genet 2010; 42: 722–726. mutations are distinct from EZH2; LUC7L2 is one of the splicesome 2 Makishima H, Jankowska AM, Tiu RV, Szpurka H, Sugimoto Y, Hu Z et al. subunits interacting with U1 snRNP to recognize non-consensus Novel homo- and hemizygous mutations in EZH2 in myeloid malignancies. splice donor sites.10,11 We identified eight mutant cases, Leukemia 2010; 24: 1799–1804. interestingly, mostly LUC7L2 mutations resulted in premature 3 Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tonnissen ER et al. stop codons. The biological effect of wild-type and mutant LUC7L2 Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic on myeloid neoplasms has not been studied. CUX1 as a syndromes. Nat Genet 2010; 42: 665–667. factor regulates a large number of genes and 4 Jerez A, Sugimoto Y, Makishima H, Verma A, Jankowska AM, Przychodzen B et al. Loss of heterozygosity in 7q myeloid disorders: clinical associations and genomic microRNAs involved in DNA replication, DNA damage response 12,13 pathogenesis. Blood 119: 6109–6117. and progression. In agreement with clinical 5 Gomez-Segui I, Makishima H, Jerez A, Yoshida K, Przychodzen B, Miyano S et al. observations, including not only MDS/MPN overlap but also Novel recurrent mutations in the RAS-LIKE GTP-Binding gene RIT1 in myeloid 14 MPN cases, Cux1 transgenic mice developed an MPN-like malignancies. Leukemia 2013; 27: 1943–1946. myeloid leukemia with massive expansion of neutrophils.15 6 Kipreos ET, Lander LE, Wing JP, He WW, Hedgecock EM. cul-1 is required for cell In conclusion, no single gene defect explains the pathogenesis cycle exit in C. elegans and identifies a novel gene family. Cell 1996; 85: 829–839. of LOH, but some specific molecular events, including recurrent 7 Marti A, Wirbelauer C, Scheffner M, Krek W. Interaction between ubiquitin- somatic mutations in CUX1, LUC7L2 and EZH2, are recurrent in -7/ ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation. del(7q). While del(7q) results in low expression in these genes, Nat Cell Biol 1999; 1: 14–19. 8 Tamagnone L, Artigiani S, Chen H, He Z, Ming GI, Song H et al. Plexins are a large down modulation and hypomorphic mutations are also seen in family of receptors for transmembrane, secreted, and GPI-anchored semaphorins some cases diploid for 7q and all are uniformly associated with in vertebrates. Cell 1999; 99: 71–80. poor survival. 9 Khan SN, Jankowska AM, Mahfouz R, Dunbar AJ, Sugimoto Y, Hosono N et al. Multiple mechanisms deregulate EZH2 and histone H3 lysine 27 epigenetic changes in myeloid malignancies. Leukemia 2013; 27: 1301–1309. CONFLICT OF INTEREST 10 Howell VM, Jones JM, Bergren SK, Li L, Billi AC, Avenarius MR et al. Evidence for a The authors declare no conflict of interest. direct role of the disease modifier SCNM1 in splicing. Hum Mol Genet 2007; 16: 2506–2516. 11 Singh H, Lane AA, Correll M, Przychodzen B, Sykes DB, Stone RM et al. ACKNOWLEDGEMENTS Putative RNA-splicing gene LUC7L2 on 7q34 represents a candidate gene in We thank the Cancer Genome Atlas (TCGA) for providing access to the whole-genome pathogenesis of myeloid malignancies. Blood Cancer J 2013; 3:e117. sequencing results described in the text. This work is supported by the National 12 Nepveu A. Role of the multifunctional CDP/Cut/Cux homeodomain transcription Institutes of Health (NIH; Bethesda, MD, USA) grants RO1 HL082983 (JPM), U54 factor in regulating differentiation, cell growth and development. Gene 2001; 270: RR019391(JPM and MAS) and K24 HL077522(JPM); AA & MDS International Foundation 1–15. (Rockville, MD, USA; HM) and Scott Hamilton CARES grant (Cleveland, OH, USA; HM). 13 Hulea L, Nepveu A. CUX1 transcription factors: from biochemical activities and cell-based assays to mouse models and human diseases. Gene 2012; 497: 18–26. N Hosono1,2, H Makishima1,12, A Jerez1, K Yoshida3,4, 14 Thoennissen NH, Lasho T, Thoennissen GB, Ogawa S, Tefferi A, Koeffler HP. Novel B Przychodzen1, S McMahon5, Y Shiraishi6, K Chiba6, H Tanaka7, CUX1 missense mutation in association with 7q- at leukemic transformation of MPN. Am J Hematol 2011; 86: 703–705. S Miyano6,7, M Sanada3,4,IGo´mez-Seguı´1, AK Verma8, 9 10,11 3,4 1,11,12 15 Cadieux C, Fournier S, Peterson AC, Bedard C, Bedell BJ, Nepveu A. Transgenic MA McDevitt , MA Sekeres ,SOgawa and JP Maciejewski mice expressing the p75 CCAAT-displacement protein/Cut homeobox isoform 1 Department of Translational Hematology and Oncology Research, develop a myeloproliferative disease-like myeloid leukemia. Cancer Res 2006; 66: Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA; 9492–9501.

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

& 2014 Macmillan Publishers Limited Leukemia (2014) 1341 – 1379