6524.Full.Pdf

Published OnlineFirst August 2, 2019; DOI: 10.1158/1078-0432.CCR-19-0725

Translational Cancer Mechanisms and Therapy Clinical Cancer Research Genetic Characterization and Prognostic Relevance of Acquired Uniparental Disomies in Cytogenetically Normal Acute Myeloid Leukemia Christopher J. Walker1, Jessica Kohlschmidt1,2, Ann-Kathrin Eisfeld1, Krzysztof Mrozek 1, Sandya Liyanarachchi1, Chi Song3, Deedra Nicolet1,2, James S. Blachly1, Marius Bill1, Dimitrios Papaioannou1, Christopher C. Oakes1, Brian Giacopelli1, Luke K. Genutis1, Sophia E. Maharry1, Shelley Orwick1, Kellie J. Archer1,3, Bayard L. Powell4, Jonathan E. Kolitz5, Geoffrey L. Uy6, Eunice S. Wang7, Andrew J. Carroll8, Richard M. Stone9, John C. Byrd1, Albert de la Chapelle1, and Clara D. Bloomﬁeld1

Abstract

Purpose: Uniparental disomy (UPD) is a way cancer cells on chromosome arms 13q (7.5% of patients), 6p (2.8%), and duplicate a mutated gene, causing loss of heterozygosity 11p (2.8%). Many UPDs significantly cooccurred with muta- (LOH). Patients with cytogenetically normal acute myeloid tions in genes they encompassed, including 13q UPD with leukemia (CN-AML) do not have microscopically detectable FLT3-internal tandem duplication (FLT3-ITD; P < 0.001), and chromosome abnormalities, but can harbor UPDs. We exam- 11p UPD with WT1 mutations (P ¼ 0.02). Among patients ined the prognostic significance of UPDs and frequency of younger than 60 years, UPD of 11p was associated with longer LOH in patients with CN-AML. overall survival (OS) and 13q UPD with shorter disease-free Experimental Design: We examined the frequency and survival (DFS) and OS. In multivariable models that prognostic significance of UPDs in a set of 425 adult patients accounted for known prognostic markers, including FLT3-ITD with de novo CN-AML who were previously sequenced for and WT1 mutations, UPD of 13q maintained association with 81 genes typically mutated in cancer. Associations of UPDs shorter DFS, and UPD of 11p maintained association with with outcome were analyzed in the 315 patients with CN-AML longer OS. younger than 60 years. Conclusions: LOHmediatedbyUPDisarecurrentfeature Results: We detected 127 UPDs in 109 patients. Most UPDs of CN-AML. Detection of UPDs of 13q and 11p might were large and typically encompassed all or most of the be useful for genetic risk stratification of patients with affected chromosome arm. The most common UPDs occurred CN-AML.

1The Ohio State University Comprehensive Cancer Center, Columbus, Ohio. 2Alliance Statistics and Data Center, The Ohio State University Comprehensive Introduction Cancer Center, Columbus, Ohio. 3Division of Biostatistics, College of Public Acute myeloid leukemia (AML) is a genetically heterogeneous 4 Health, The Ohio State University, Columbus, Ohio. Wake Forest Baptist disease. The prognosis of patients with AML is strongly influenced Comprehensive Cancer Center, Winston-Salem, North Carolina. 5Monter Cancer Center, Zucker School of Medicine at Hofstra/Northwell, Lake Success, New by chromosomal aberrations and gene mutations, and patients fi York. 6Washington University School of Medicine in St. Louis, Siteman Cancer are prognostically strati ed on the basis of the results of gene Center, St. Louis, Missouri. 7Roswell Park Comprehensive Cancer Center, Buffalo, sequencing and cytogenetic analysis (1–3). Although genetic risk New York. 8University of Alabama at Birmingham, Birmingham, Alabama. stratification of patients with AML has been well studied, there 9Dana-Farber Cancer Institute, Boston, Massachusetts. remains room for improvement, especially for the cytogenetically Note: Supplementary data for this article are available at Clinical Cancer normal (CN) group which comprises 40%–45% of adult patients. Research Online (http://clincancerres.aacrjournals.org/). For patients with CN-AML, the 2017 European LeukemiaNet Clinicaltrials.gov identifiers: NCT00085124 (CALGB-10201), NCT00742625 (ELN) guidelines determine genetic risk stratification entirely by (CALGB-10502), NCT00742625 (CALGB-10503), NCT00651261 (CALGB- gene mutations [i.e., mutation of NPM1, RUNX1, ASXL1, and 10603), NCT00006363 (CALGB-19808), NCT00900224 (CALGB-20202), TP53; biallelic CEBPA mutations; and FLT3-internal tandem NCT00048958 (CALGB-8461), NCT00899223 (CALGB-9665), and duplication (ITD) allelic ratio]. NCT00003190 (CALGB-9720). Patients with CN-AML by definition do not have chromosomal Corresponding Authors: Christopher J. Walker, The Ohio State University abnormalities detectable by karyotyping (3), but these patients Comprehensive Cancer Center, Biomedical Research Tower, Room 894, often harbor acquired uniparental disomies (UPDs, also called 460 W. 12th Ave, Columbus, OH 43210. Phone: 614-688-4463; Fax: 614-685- copy-neutral loss of heterozygosity; refs. 4–10), which are somatic 0211; E-mail: [email protected]; and Clara D. Bloomfield, clara.bloomfi[email protected] losses of a chromosome or segment with duplication of the homologous chromosome or segment. In cancer development, Clin Cancer Res 2019;25:6524–31 UPDs have been shown to mediate loss of heterozygosity (LOH) doi: 10.1158/1078-0432.CCR-19-0725 of mutated oncogenes and tumor suppressor genes by duplicating 2019 American Association for Cancer Research. the mutant alleles thus resulting in homozygous mutations.

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UPDs, Mutations, and Outcome in CN-AML

Hispanic, 2% of cases and 1% of controls; Native American, <1% Translational Relevance of cases and controls; Native Hawaiian, <1% of cases and controls; Acquired uniparental disomy (UPD) is recognized as a and middle-eastern <1% of cases and controls. common mechanism by which cancer cells can achieve homozygous mutations in oncogenes or tumor suppressor genes. UPD and copy number alteration detection UPDs frequently occur in patients with cytogenetically normal All samples were genotyped with Infinium Omni-1 Quad- acute myeloid leukemia (CN-AML), and specific UPDs are bead arrays (Illumina) by deCODE Genetics as described reportedly associated with patient outcome. Our study reports previously (34, 35). For quality control, samples with <94% the largest CN-AML patient set screened for UPDs, to our genotyping yield were excluded, and variants were excluded if knowledge. Consistent with previous reports, we found that they had <94% yield, showed significant differences among UPDs often cooccur with mutations in genes they encompass genotyping batches, or if they significantly (P < 10 6)deviated resulting in loss of heterozygosity, including UPD of 13q with from Hardy–Weinberg equilibrium. GenomeStudio 2.0 soft- FLT3 internal tandem duplication and UPD of 11p with WT1 ware (Illumina) and the cnvPartition plugin (v3.2.0) were used mutations. In patients with CN-AML younger than 60 years, to detect UPDs and copy number alterations (CNA) with we found UPD of 13q and UPD of 11p were associated with minimum probe count set to 10. All calls were manually shorter and longer survival, respectively, even in multivariable reviewed by examination of LogR ratio and B-allele frequency models that accounted for known prognostic markers. These plots. Nexus Copy Number v10 (BioDiscovery) was used to results imply that genetic risk stratification of younger patients visualize UPDs. To differentiate between common inherited with CN-AML could be improved by the inclusion of UPD regions of homozygosity present in germline DNA and somat- testing. ically acquired UPDs, 1,798 nonleukemic control samples were genotyped. Thirty-three different inherited regions of homozygosity, all of which were smaller than 2 Mb in size, were present in 1% of samples, and were excluded from the UPD analysis The prognostic relevance of UPD in CN-AML has not been (Supplementary Table S1). These nonleukemic samples did not adequately investigated; however, there is evidence that specific contain any detectable CNAs or large UPDs. UPDs can impact AML patient outcome (11–20). To better define the utility of UPDs for prognostic risk stratification of patients DNA sequencing with CN-AML, we herein screened a large cohort of adult patients Patient DNA obtained from pretreatment bone marrow or with de novo CN-AML for UPDs and investigated their associations blood samples was sequenced for the following 80 protein- with patient survival and relationship with recurrent gene coding genes using two separate TruSeq Custom Amplicon panels mutations. (Illumina) as described previously (36): AKT1, ARAF, ASXL1, ATM, AXL, BCL2, BCOR, BCORL1, BRAF, BRD4, BRINP3, BTK, CBL, CCND1, CCND2, CSNK1A1, CTNNB1, DNMT3A, ETV6, Materials and Methods EZH2, FBXW7, FLT3, GATA1, GATA2, GSK3B, HIST1H1E, Patients and cytogenetic analysis HNRNPK, IDH1, IDH2, IKZF1, IKZF3, IL7R, JAK1, JAK2, JAK3, Studies were conducted using samples obtained from a cohort KIT, KLHL6, KMT2A, KRAS, MAPK1, MAPK3, MED12, MYD88, of 425 adults with de novo AML aged 17–79 years (median 52 NF1, NOTCH1, NPM1, NRAS, PHF6, PIK3CD, PIK3CG, PLCG2, years), with 315 patients younger than 60 years. Patients with PLEKHG5, PRKCB, PRKD3, PTEN, PTPN11, RAD21, RAF1, acute promyelocytic leukemia, AML secondary to myelodysplas- RUNX1, SAMHD1, SETBP1, SF1, SF3A1, SF3B1, SMARCA2, tic syndromes, or therapy-related AML, and patients who received SMC1A, SMC3, SRSF2, STAG2, SYK, TET2, TGM7, TP53, TYK2, allogeneic hematopoietic stem cell transplantation in first com- U2AF1, U2AF2, WT1, XPO1, ZMYM3, and ZRSR2. Libraries were plete remission (CR) were not included in the study. This study prepared according to the manufacturer's instructions, pooled, was limited to patients with CN-AML. Pretreatment cytogenetic and run on a MiSeq instrument using MiSeq v3 Reagent Kits analyses of bone marrow samples of all patients were performed (Illumina). Sequences were aligned to the hg19 genome build by the Cancer and Leukemia Group B (CALGB)-approved insti- with the Illumina Isis Banded Smith-Waterman aligner. Small tutional laboratories using short-term (24–48 hours) unstimu- indel variants and single-nucleotide variants were called using lated cultures, and all karyotypes were centrally reviewed (21). In VarScan2 and MuTect, respectively. The Mucor program was used each case, 20 metaphase cells were analyzed and no clonal as a baseline for integrative mutation assessment (37). The variant abnormality was found. All patients were similarly treated on allele fraction (VAF) cutoff was set to 0.1. Variants were consid- CALGB trials (21–33) and did not die within 30 days (see ered mutations if they were nonsynonymous and not present in Supplementary Methods for details). Study protocols were in the dbSNP v142 database or 1000 Genomes database. All called accordance with the Declaration of Helsinki and approved by variants underwent visual inspection of the aligned reads using the institutional review boards. All individuals in this study Integrative Genomics Viewer. We excluded variants sequenced provided written informed consent. with fewer than 15 reads; variants that only occurred in one read DNA samples from blood of 1,798 nonleukemic individuals direction (if covered by forward and reverse reads); variants in recruited in Columbus, Ohio who had never been diagnosed with regions marked by low phred-score bases or low-mapping score any cancer, were used as negative controls for somatic UPD reads; variants that occurred in all samples; and samples with detection. All healthy donors provided written informed consent. generally poor quality sequencing for the entire panel. Samples The ethnicity of the cases and controls were similar as follows: were considered nonevaluable for a specific gene if 85% of the white European, 90% of cases and 91% of controls; African, 6% of amplicons covering the target regions within the coding sequence cases and 4% of controls; Asian, 2% of cases and 3% of controls; of the gene were sequenced to a depth of <15 reads. Detection

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Walker et al.

of FLT3 and determination of the allelic ratio was done with (DFS) and overall survival (OS) using a limited backward RT-PCR as described previously (38). In addition to the 80 gene selection procedure. Variables that were significant at the like- sequencing panel, testing for CEBPA mutations was performed lihood ratio test P < 0.20 from univariable models were con- with Sanger sequencing as described previously (39). sidered in the multivariable analysis (MVA; ref. 40). Variables considered in univariable models were as follows: age, extra- Statistical analyses medullary involvement, hemoglobin levels, percentage of Associations between UPDs, clinical characteristics, and gene blood and bone marrow blasts, platelet count, race, sex, white mutations were tested using Fisher exact test for categorical blood cell (WBC) count, UPD of 11p, UPD of 13q, FLT3-ITD variables and the Kruskal–Wallis test for continuous variables. status, FLT3-tyrosine kinase domain mutation status (FLT3- UPDs and gene mutations present in at least 8 patients were TKD), biallelic mutation of CEBPA status, and mutation status included in outcome analyses. We used the log-rank test to test for of ASXL1, BCOR, DNMT3A, GATA2, IDH1, IDH2, NPM1, significant associations between categorical variables and surviv- NRAS, PTPN11, RAD21, RUNX1, SMC1A, SMC3, TET2, WT1, al, with Kaplan–Meier curves for illustration. and ZRSR2. Data collection and statistical analyses were per- We constructed multivariable logistic regression models to formed by the Alliance Statistics and Data Center. Analyses analyze the probability of CR attainment and multivariable were performed using SAS 9.4 and TIBCO Spotfire Sþ 8.2, with Cox proportional hazards models for disease-free survival the database locked on July 5, 2018.

Figure 1. UPDs in 425 adult patients with de novo CN-AML. Each orange line represents a single UPD in a single patient. The locations of recurrently mutated genes (mutated in at least 2% of patients) are shown.

6526 Clin Cancer Res; 25(21) November 1, 2019 Clinical Cancer Research

UPDs, Mutations, and Outcome in CN-AML

Results Similarly, patients with mutations in the RUNX1 gene (located at 21q22.12) had UPD of 21q significantly more often than patients We assessed a cohort of 425 adult patients with de novo CN-AML with wild-type RUNX1 (P < 0.001), and patients with EZH2 for UPDs in the autosomes using genotyping arrays. There were (located at 7q36.1) mutations had UPD of 7q significantly more 171 UPDs detected in 116 patients (Fig. 1; Supplementary often than those with wild-type EZH2 (P < 0.001; Table 1). Table S2). Many UPDs encompassed entire chromosome arms, Likewise, there were significant associations between mutation and the chromosome arms that most frequently contained UPDs of CBL (located at 11q23.3) with UPD of 11q (P ¼ 0.01); were 13q (present in 32 patients), 11p (12 patients), and 6p (12 mutation of WT1 (located at 11p13) with UPD of 11p (P ¼ patients; Fig. 1). To begin to assess the clinical relevance of these 0.02); biallelic mutation of CEBPA (located at 19q13.11) with UPDs, we grouped UPDs by chromosome arm and determined UPD of 19q (P ¼ 0.02); mutation of SF3B1 (located at 2q33.1) the associations between the most common UPDs and patient with UPD of 2q (P ¼ 0.03); and mutation of TET2 (located at baseline clinical characteristics. UPD of 13q was associated with 4q24) with UPD of 4q (P ¼ 0.04, Table 1). Expectedly, the higher WBC counts (median, 52.5 vs. 29.6 109/L; P ¼ 0.02), vast majority of gene mutations that cooccurred with UPDs had blood blasts (77% vs. 58%; P ¼ 0.004), and bone marrow blasts VAFs >0.5 (Supplementary Table S5). (80% vs. 69%; P ¼ 0.005); UPD of 11p was associated with lower platelet counts (median, 41 vs. 60 109/L; P ¼ 0.03) and higher UPD of 11p is associated with improved outcome and UPD of blood blasts (85% vs. 60%; P ¼ 0.002); and UPD of 6p was 13q with poor outcome associated with higher bone marrow blasts (median, 86% vs. Because of differences in treatment intensity between older and 70%; P ¼ 0.02; Supplementary Table S3). younger adult patients with AML enrolled onto CALGB/Alliance treatment protocols, outcome studies are typically performed UPDs are associated with mutations in genes they encompass separately in younger (<60 years of age) and older patients We were able to assess associations between UPDs and gene (60 years of age). Between the 315 younger patients and the mutations as these patients with CN-AML were previously 110 older patients in our study, the sample size for examining sequenced for mutations in 81 cancer and/or leukemia- associations with CR status, DFS, and OS for 11p UPD and 13q associated genes (Supplementary Table S4; ref. 36). Many UPDs UPD was adequate only for the younger patients. were found to cooccur in the same patients with mutations in We found that UPD of 11p was associated with longer OS (P ¼ genes they encompassed. Specifically, patients with FLT3-ITD 0.02; Fig. 2A), and UPD of 13q was associated with both shorter (located at 13q12.2) more frequently harbored UPDs of 13q DFS (P < 0.001) and shorter OS (P < 0.001; Fig. 2B and C) in compared with patients without FLT3-ITD (P < 0.001; Table 1). younger patients. MVA was used to examine the effect of UPDs on outcome in the context of known prognostic markers. For OS, the P ¼ Table 1. Number of adult patients with CN-AML who had cooccuring mutations risk of death was lower for patients with 11p UPD ( 0.04) after P ¼ FLT3 P < and UPDs adjusting for age ( 0.005), -ITD ( 0.001), and muta- FLT3 P ¼ DNMT3A P ¼ RUNX1 P < UPD tions in -TKD ( 0.04), ( 0.003), ( Gene (location) Mutation status Yes No Pa 0.001), and WT1 (P < 0.001; Table 2). For DFS, patients with 13q FLT3-ITD Present 30 129 <0.001 UPD had a higher risk of relapse or death (P ¼ 0.009) after (13q12.2) Absent 2 263 adjusting for hemoglobin levels (P ¼ 0.02), FLT3-ITD status (P < RUNX1 Mutated 4 33 <0.001 0.001), and mutations in the DNMT3A (P < 0.001), RUNX1 (P ¼ (21q22.12) Wild-type 2 386 0.002), and WT1 (P ¼ 0.03) genes (Table 2). EZH2 Mutated 3 10 <0.001 (7q36.1) Wild-type 4 408 CBLb Mutated 2 6 0.01 UPD of 13q is associated with shorter DFS in patients with (11q23.3) Wild-type 6 411 FLT3-ITD WT1 Mutated 4 38 0.02 Because both FLT3-ITD and 13q UPD were associated with (11p13) Wild-type 8 375 shorter DFS, and cooccurred in the same patients, we sought CEBPAc Mutated 2 56 0.02 to determine whether 13q UPD had any additional utility as a (19q13.11) Wild-type 0 349 FLT3 SF3B1 Mutated 1 10 0.03 prognostic marker independent of its association with -ITD (2q33.1) Wild-type 0 414 by performing outcome analyses for 13q UPD in only the 112 TET2d Mutated 3 59 0.04 younger patients with CN-AML who harbored FLT3-ITD. Patients (4q24) Wild-type 3 360 with 13q UPD still had significantly shorter DFS (P ¼ 0.004) in the IDH2 Mutated 2 56 0.09 FLT3-ITD–positive group, demonstrating that 13q UPD status is (15q26.1) Wild-type 2 365 fi DNMT3A useful for prognostic strati cation even when considering the Mutated 3 164 0.68 FLT3 (2p23.3) Wild-type 3 255 known prognostic marker -ITD (Fig. 2D). Expectedly, all of FLT3 FLT3 NOTE: Associations between UPDs and gene mutations are shown for all genes the patients with both -ITD and 13q UPD had a -ITD > FLT3 high for which at least 1 patient had a cooccurring mutation and UPD. For each gene, allelic ratio 0.5 (i.e., -ITD ), supporting the view that the the numbers of patients with and without a UPD that encompasses the gene are UPD caused LOH for FLT3-ITD (Supplementary Table S6). listed, stratified by mutation status. Abbreviations: FLT3-ITD, internal tandem duplication of the FLT3 gene. Small CNAs are present in patients with CN-AML a P Fisher exact test was used to calculate values. In addition to UPD analysis, the genotyping data from these bOne patient with a UPD on 11q that did not encompass CBL is counted as not having an 11q UPD. patients allowed us to screen for microdeletions too small to be cOnly patients with biallelic CEBPA mutations are included. detected microscopically by karyotyping. Among the 425 patients dTwo patients with UPDs on 4q that did not encompass TET2 are counted as not with CN-AML, there were 28 deletions and 3 amplifications that having 4q UPDs. ranged in size from approximately 100 kb to approximately 19

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Walker et al.

Figure 2. Associations between UPDs and outcome in younger patients (age <60 years) with CN-AML. A, OS of patients with and without 11p UPD. OS (B) and DFS (C) of patients with and without 13q UPD. D, DFS of patients who have FLT3-ITD and either harbor 13q UPD or do not.

Mb (Supplementary Table S7). Most of the CNAs were observed 13q UPDs with AML patient outcome (including CN-AML; in only one sample each, with six occurring in 2 patients each refs. 5, 13, 14, 16, 49) and again highlights the importance (Supplementary Table S7). Because of the infrequency with which of UPDs in CN-AML. We believe our sample set is the largest these CNAs were observed, we were unable to assess their associa- series of patients with CN-AML screened for UPDs to date, and tions with clinical or molecular characteristics. the finding that both 11p and 13q UPDs were separately associated with patient outcome in multivariable models sug- gests that these UPDs are clinically relevant prognostic markers Discussion foryoungerpatientswithCN-AMLtreatedwithstandard"7þ3" The use of genotyping arrays to assess UPD in AML was first induction therapy. described over a decade ago, and subsequent studies have Biologically, UPDs are a mechanism by which a cancer cell established that UPDs occur in approximately 20% of patients can duplicate an activating mutation in an oncogene or elim- with CN-AML (5, 13). In the time since these initial studies inatethewild-typecopyofatumorsuppressorgenetoaugment assessing the frequency and prognostic relevance of UPDs in the mutation-associated phenotype, such as multiplying an AML, the focus of genetic risk stratification has largely shifted increase in growth advantage caused by a loss-of-function beyond cytogenetics to gene mutations, with some additional mutation in a tumor suppressor gene. Many of the recurrent studies exploring the prognostic relevance of expression signa- UPDs identified in our study were associated with pathogenic tures and epigenetic changes (41–48). Our work validates mutations in genes residing on the same chromosome arms earlier efforts that reported associations between 11p and including FLT3 (13q), RUNX1 (21q), EZH2 (7q), CBL (11q), WT1 (11p), CEBPA (19q), SF3B1 (2q), and TET2 (4q) (Table 1). Table 2. Multivariable analyses for DFS and OS performed in younger (age Although 12 patients had UPDs that encompassed most of <60 years) patients with CN-AML chromosome 6p, we did not identify any recurrently mutated DFS (n ¼ 266) OS (n ¼ 315) genes on 6p. Notably the only gene located on chromosome 6p Variables P HR (95% CI) P HR (95% CI) on our sequencing panel was HIST1H1E, which was only Age, continuous 0.005 1.20 (1.06–1.37) mutated in two of the 425 patients with CN-AML, neither of DNMT3A, mut vs. wt <0.001 1.94 (1.41–2.67) 0.003 1.58 (1.17–2.14) whom had a UPD of 6p. FLT3-ITD, yes vs. no <0.001 2.31 (1.67–3.19) <0.001 2.08 (1.55–2.80) fi FLT3 – Our ndings validate previous reports of 7q UPDs causing -TKD, mut vs. wt 0.04 0.52 (0.28 0.97) EZH2 HG, continuous 0.02 0.91 (0.84–0.98) biallelic inactivation of the tumor suppressor gene, RUNX1, mut vs. wt 0.002 2.82 (1.44–5.52) <0.001 3.82 (2.17–6.75) although both oncogenic and tumor suppressor roles have been UPD of 11p, yes vs. no 0.04 0.35 (0.13–0.98) ascribed to EZH2 in AML and its progression from myelodys- UPD of 13q, yes vs. no 0.009 2.10 (1.20–3.67) plastic syndromes (50, 51). Likewise, UPD-mediated LOH in WT1, mut vs. wt 0.03 1.70 (1.04–2.77) <0.001 2.62 (1.70–4.03) RUNX1-mutated patients with AML has been recently described NOTE: A HR >1(<1) corresponds to a higher (lower) risk for first category listed of and was shown to be associated with poor outcome (52, 53). We a dichotomous variable or higher values of a continuous variable. A limited were unable to assess the relationship between UPD of 21q and backward selection technique was used to build the final models with variables outcome in our samples due to insufficient numbers. We found that were significant at the likelihood ratio test P <0.20 from univariable models WT1 for each outcome endpoint. For DFS, those variables were as follows: HG, 11p UPD was associated with mutation status, and also platelet count, white blood cell count, UPD of 11p, UPD of 13q, FLT3-ITD status, associated with improved DFS. However, WT1 mutation is a biallelic mutation of CEBPA status, and mutation status of BCOR, DNMT3A, known prognostic marker associated with poor outcome (54, 55). FLT3-TKD, GATA2, RUNX1, SMC1A, and WT1. For OS, those variables were as In our MVA for OS, WT1 mutations and 11p UPD were associated FLT3 follows: age, HG, white blood cell count, UPD of 11p, UPD of 13q, -ITD status, with shorter and longer OS, respectively, indicating it is likely that biallelic mutation of CEBPA status, and mutation status of DNMT3A, FLT3-TKD, 11p UPDs mediate an effect on OS independently from their GATA2, RUNX1, SMC1A, WT1,andZRSR2. WT1 Abbreviations: CI, confidence interval; HG, hemoglobin levels; FLT3-ITD, internal association with mutations. tandem duplication of the FLT3 gene; mut, mutated; n, number; FLT3-TKD, We found that patients with FLT3-ITD who also had 13q UPD tyrosine kinase domain mutation in the FLT3 gene; wt, wild type. had a FLT3-ITDhigh allelic ratio, which is consistent with 13q UPD

6528 Clin Cancer Res; 25(21) November 1, 2019 Clinical Cancer Research

UPDs, Mutations, and Outcome in CN-AML

as a common mechanism for acquisition of FLT3-ITDhigh (56). Authors' Contributions Our study also validates reports that FLT3-ITDhigh is associated Conception and design: C.J. Walker, A.-K. Eisfeld, K. Mrozek, J.C. Byrd, A. de la with poor outcome, and supports the inclusion of FLT3-ITDhigh in Chapelle, C.D. Bloomfield the 2017 ELN genetic risk classification system (57). We found Development of methodology: C.J. Walker, L.K. Genutis Acquisition of data (provided animals, acquired and managed patients, that among the younger patients with CN-AML who harbor FLT3- provided facilities, etc.): C.J. Walker, K. Mrozek, D. Papaioannou, ITD, 13q UPD was still associated with poor outcome. Finally, B. Giacopelli, L.K. Genutis, B.L. Powell, G.L. Uy, E.S. Wang, R.M. Stone, 13q UPD was associated with poor outcome in a MVA that C.D. Bloomfield included FLT3-ITD. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Our analysis of CNAs did not identify any frequently occurring computational analysis): C.J. Walker, J. Kohlschmidt, A.-K. Eisfeld, K. Mrozek, CNAs in these cases. We hypothesized that patients with CN-AML S. Liyanarachchi, C. Song, D. Nicolet, J.S. Blachly, M. Bill, D. Papaioannou, C.C. Oakes, B. Giacopelli, S. Orwick, K.J. Archer, A.J. Carroll might harbor micro-CNAs smaller than 5 Mb in critical genes, Writing, review, and/or revision of the manuscript: C.J. Walker, which would not be detectable microscopically by karyotyping. J. Kohlschmidt, A.-K. Eisfeld, K. Mrozek, D. Nicolet, J.S. Blachly, M. Bill, Several recurrent CNAs have previously been reported in CN-AML C.C. Oakes, B. Giacopelli, L.K. Genutis, B.L. Powell, J.E. Kolitz, G.L. Uy, E.S. but they are generally rare. These include deletions encompassing Wang, A.J. Carroll, R.M. Stone, J.C. Byrd, A. de la Chapelle, C.D. Bloomfield TET2 (4q24), ETV6 (12p12.3), TP53 (17p13.1), and NF1 Administrative, technical, or material support (i.e., reporting or organizing (17q11.2), and amplifications encompassing MYC (8q23.2) and data, constructing databases): C.J. Walker, K. Mrozek, J.S. Blachly, S.E. Maharry, S. Orwick, J.C. Byrd KMT2A (11q23.3) (7, 13, 58, 59). Notably we detected deletion of Study supervision: C.J. Walker, A. de la Chapelle, C.D. Bloomfield NF1 in 2 patients and deletion of TET2 in 1 patient (Supplemen- tary Table S7). However, our results indicate that micro-CNAs are Acknowledgments not a defining feature of CN-AML. The authors would like to acknowledge Leslie Davidson, Tammy Woike, Our data provide an overview of recurrent UPDs found in CN- Jan Lockman, and Barbara Fersch for administrative assistance; Donna AML and show UPDs are frequently associated with mutations in Bucci and Wacharaphon Vongchucherd of the CALGB/Alliance Leukemia genes they encompass, leading to LOH. Together our work Tissue Bank at The Ohio State University Comprehensive Cancer Center validates the prognostic importance of 13q UPD and 11p UPD (Columbus, OH) for sample processing and storage services; and Lisa J. Sterling and Christine Finks for data management. This study was sup- in patients with CN-AML, and indicates these UPDs could be ported by the NCI for the NIH under Award Numbers U10CA180821, clinically relevant for risk stratification of younger patients with de U10CA180882, and U24CA196171 (to the Alliance for Clinical Trials in novo CN-AML treated with standard induction therapy. Oncology); P30CA016058, U10CA180833, U10CA180850, U10CA180861, U10CA180866, U10CA180867, and UG1CA233338; the Leukemia Clinical fl Research Foundation; the Warren D. Brown Foundation; and by an alloca- Disclosure of Potential Con icts of Interest tion of computing resources from The Ohio Supercomputer Center. This J.S. Blachly is a consultant/advisory board member for AbbVie, AstraZeneca, study was also supported, in part, by funds from Novartis (CALGB-10603). and KITE Pharma. G.L. Uy is a consultant/advisory board member for Pfizer, Astellas, Jazz Pharmaceuticals, and Glycomimetics. No potential conflicts of The costs of publication of this article were defrayed in part by the payment of interest were disclosed by the other authors. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Disclaimer The content is solely the responsibility of the authors and does not neces- Received March 1, 2019; revised June 6, 2019; accepted July 30, 2019; sarily represent the official views of the NIH. published first August 2, 2019.

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www.aacrjournals.org Clin Cancer Res; 25(21) November 1, 2019 6531

Genetic Characterization and Prognostic Relevance of Acquired Uniparental Disomies in Cytogenetically Normal Acute Myeloid Leukemia

Christopher J. Walker, Jessica Kohlschmidt, Ann-Kathrin Eisfeld, et al.

Clin Cancer Res 2019;25:6524-6531. Published OnlineFirst August 2, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-0725

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