13Q Deletion Anatomy and Disease Progression in Patients with Chronic Lymphocytic Leukemia

13q deletion anatomy and disease progression in patients with chronic lymphocytic leukemia

H Parker1,7, MJJ Rose-Zerilli1,7, A Parker2, T Chaplin3, R Wade4, A Gardiner2, M Grifﬁths5, A Collins6, BD Young3, DG Oscier2,8 and JC Strefford1,8

1Cancer Genomics Group, Cancer Sciences Division, School of Medicine, University of Southampton, Southampton, UK; 2Department of Pathology, Royal Bournemouth Hospital, Bournemouth, UK; 3Medical Oncology Group, Institute of Cancer, St Bartholomew’s Hospital, London, UK; 4Clinical Trial Service Unit, University of Oxford, Oxford, UK; 5West Midlands Regional Genetics Laboratory, Birmingham Women’s Hospital, Birmingham, UK and 6The Genetic Epidemiology and Bioinformatics Research Group, Human Genetics Division, University of Southampton, Southampton, UK

Historically, genes targeted by recurrent chromosomal One such example is deletion of the long arm of chromosome deletions have been identified within the smallest genomic 13, which is the most frequent alteration in chronic lymphocytic region shared in all patients, the minimally deleted region leukemia (CLL). Early G-banding demonstrated that 13q dele- (MDR). However, deletions this small do not occur in all patients 2 and are a simplification of the impact larger heterogeneous tions involved multiple non-overlapping chromosomal regions. deletions have during carcinogenesis. We use the example of Although it was initially suggested that RB1 was the pathogenic 13q14 deletions in chronic lymphocytic leukemia to show that locus,3 homozygous deletions at a more telomeric locus genes outside MDRs are associated with disease progression. were identified.3–5 Physical maps of this region also identified Genomic profiling of 224 patients identified 205 copy number several non-overlapping MDRs,6–8 implicating a multigenic alterations on chromosome 13 in 132 cases. Deletions including tumor suppressor mechanism. Ultimately, a 10 kb transcript-rich DLEU2 were heterogeneous (845 Kb–96.2 Mb) and identified two MDR including exons of two genes, DLEU1 and DLEU2, breakpoint cluster regions within short interspersed nuclear 9 elements proximal to DLEU2 and within long interspersed was identified. Sequence mutations and aberrant methylation nuclear elements/L1 repeats distal to GUCY1B2. After defining a patterns in this region have not been observed.10–12 The deletion class on the basis of size and location, we show that identification of the miR15a/16–1 microRNA cluster within (a) at diagnosis, larger deletions (class II) were associated with this region was a pivotal observation,13 and it is now clear that a significantly increased risk of disease progression (odds this cluster drives a lymphoproliferative phenotype in vivo.14 ratio ¼ 12.3; P ¼ 0.005), (b) in progressive patients, class II deletions were enriched (P ¼ 0.02) and (c) this association was Interestingly, only a fraction of miR15a/16–1 knock-out mice independent of IgVH mutational status, ZAP70 expression and developed B-cell malignancies, whereas the MDR-deleted mice ATM/TP53 deletion. Deletion of a 1 Mb gene cluster (48.2– exhibited more aggressive disease. This observation supports a 49.2 Mb), including SETDB2, PHF11 and RCBTB1, was signifi- multigenic hypothesis, and TRIM13 (DLEU5) and DLEU7 have cantly associated (Po0.01) with disease progression. Here, we been implicated, possibly through altered ubiquitination or show that the deletion of genes outside MDRs can influence NF-KB signaling.15,16 It has been proposed that 13q deletions clinical outcome. Leukemia (2011) 25, 489–497; doi:10.1038/leu.2010.288; could be categorized into two types; Type I deletions target published online 10 December 2010 a region including the MDR, whereas larger Type II deletions Keywords: SNP array; copy number alterations; 13q deletion; CLL; include the RB1 locus and were enriched in treated patients and disease progression those with higher Rai stage.17 The significance of large 13q deletions and RB1 in particular, is further emphasized by their association with elevated genome complexity.18 Introduction It is likely that the biological consequence of a unique deletion anatomy is complex, resulting in the disruption of Chromosomal deletions are recurrent features of human multiple regulatory sequences. Furthermore, this is likely to malignancy and can be critical events for the inactivation be the situation with 13q deletions in other tumor types, such of tumor suppressor genes in human cells. A plethora of genes as lymphoma, multiple myeloma and prostate cancer, as well have been identified in almost every tumor type through the as deletion events in cancer in general. Here, we employ identification and characterization of minimally deleted regions genomic profiling to show that 13q deletion size is associated (MDRs).1 Although MDRs have been critical ‘signposts’ to with disease progression. As a consequence, we propose a novel the location of cancer genes, they are a simplification of the gene cluster centromeric to the 13q MDR that may contribute to architecture and gene content of these deletions in patients. clinical outcome. Indeed, deletions are invariably larger, and multiple non- overlapping MDRs are often described. Materials and methods Correspondence: Dr JC Strefford, Cancer Genomics Group, Cancer Sciences Division, Somers Cancer research Building, Southampton Patients and molecular diagnostic assays General Hospital, Tremona Road, Southampton SO16 6YD, UK. A total of 224 CLL patients diagnosed on the basis of standard E-mail: [email protected] morphologic and immunophenotypic criteria were included in 7These authors contributed equally to this work 8These authors share senior authorship this study and sub-divided into three cohorts (Supplementary Received 18 August 2010; revised 13 October 2010; accepted 8 Table 1). Cohort I comprised 63 patients, where samples were November 2010; published online 10 December 2010 taken at disease presentation from patients with either stable 13q deletion size in CLL H Parker et al 490 disease for at least 5 years (n ¼ 38) or progressive disease within in the absence of paired normal DNA, copy number neutral 3 years (n ¼ 25). Cohort II consisted of 64 unselected patient loss-of-heterozygosity (CNNLOH) was as a region greater than samples taken at disease progression ranging from 0 to 22.5 20 Mb, extending to a telomere. years after diagnosis (median 2.3 years). Cohort III consisted of samples taken at enrollment to the UK CLL4 treatment trial19 from 97 patients treated on the fludarabine and cyclophos- Confirmation with array-based comparative genomic phamide arm, sub-divided on the basis of treatment response; hybridization with either complete (n ¼ 49), partial (n ¼ 40) or no response To confirm the SNP6.0. data, DNA samples from 22 patients (n ¼ 8) to treatment. Informed consent was obtained from all were also profiled using the Human Genome CGH Custom patients in accordance with the Helsinki declaration. Microarray (8 Â 60K, Agilent Technologies, Stockport, UK) Fluorescence in situ hybridization analysis was performed on according the manufacturer’s instructions. Test DNA samples all cases for the presence of established rearrangements using a were sex-matched with normal genomic reference DNA range of commercially available probes (Abbott Diagnostics, (Promega, Southampton, UK) and analysis was performed using Maidenhead, UK; DakoCytomation, Glostrup, Denmark) accor- proprietary software (DNA Analytics, Agilent Technologies). ding to the manufacturers’ instructions. Chromosomal analysis was performed and described according to the International System for Human Cytogenetic Nomenclature.20 ZAP70 and Statistical analysis CD38 expression was determined as previously described,21,22 Statistical analysis was performed using STATA (9.2) on a linux where 10 and 30% positive cells were classed as positive, platform. Logistic regression was employed within the indivi- respectively. IGHV genes were sequenced as previously dual cohorts for binary response variables. Variables examined described23 and a cut-off of X98% germ-line homology was included the disease progression variable (stable or progressive taken to define the unmutated sub-set (Supplementary Materials disease), presence/absence of 13q deletion and class I/II 13q & Methods and Supplementary Table 1). deletion category. Age at diagnosis and gender were included as covariates in all models where available (age at diagnosis was not available for cohort III). In cohorts I and II, overall survival Genomic profiling was defined as time, in months, from diagnosis to death or to the DNA extraction, SNP6.0. array hybridization and data last follow-up date for survivors. In cohort III, overall survival extraction. Genomic DNA was extracted from fluorescent- was defined as time from randomization, on entry, to the CLL4 activated cell sorter-purified CD19 þ lymphocytes, CD19À clinical trial, to death, or to the follow-up date for survivors. granulocytes and buccal swabs using standard approaches, Progression-free survival was only measured in cohort III and and the concentration and purity was assessed using gel was defined as time from randomization to relapse needing electrophoresis and spectrophotometry (NanoDrop ND-1000, further therapy, progression or death or to the follow-up date for Thermo Scientific, Wilmington, NC, USA). DNA was amplified, those with no progression/death. In all cohorts, time to first labeled and hybridized to the Affymetrix SNP6.0 platform treatment was defined as time from diagnosis to therapy, or to according to the Affymetrix Cytogenetics Copy Number proto- the last follow-up date for untreated patients. Before survival col (Affymetrix, Santa Clara, CA, USA) (Supplementary Materials analysis, power calculations were performed and showed that & Methods). Each array was scanned (GeneChip Scanner 3000 our total cohort was powered (0.8) to detect a hazard ratio of 7G, Affymetrix) and processed using proprietary software 2.0 at the 5% level, based on the median overall survival of (GeneChip Operating System software, Affymetrix). The our 13q-deleted patients.24 Cox regression was undertaken for feature-extracted .CEL file was then quality controlled using survival analyses examining the impacts of 13q deletions on the Genotyping Console 2.1 software (Affymetrix), and only overall survival and time to first treatment on the whole sample those achieving manufacturers’ quality cut-off scores were stratified by cohort and on progression-free survival for cohort included in this study. III. Fixed covariates included in the models were IgVH mutation status and 17p deletion along with genetic complexity.

SNP6.0.FData analysis The .CEL files were imported into Partek Genomics Suite (Partek Results Inc., MO, USA) for copy number and loss-of-heterozygosity analysis. Initially, a paired and unpaired analysis of 32 samples CLL patients are characterized by considerable genomic with matched germline DNA was performed. Employing the 270 heterogeneity HapMap Reference baseline (Affymetrix) as a control and Analysis with the SNP 6.0 platform identified 776 acquired excluding germline copy number variants based on published copy number alterations (CNAs) in 224 patients (mean 3.5, data (Database of Genomic Variants, http://projects.tcag.ca/ range 0–45), including 24 CNNLOH events and 33 whole variation/), a concordance of 497% was observed and there chromosome gains and losses (trisomy 12, n ¼ 24) (Supple- was no difference in how individual cases were defined on mentary Table 2). Of the remaining CNAs, deletions were more the basis of genome complexity. As a consequence, the entire frequent than gains (n ¼ 593 vs n ¼ 126). Sub-chromosomal cohort was analyzed using the unpaired approach. The raw CNAs size ranged from 52 kb to 131 Mb. Deletions of ATM and fluorescence intensity values for each array feature were TP53 were observed in 25% (n ¼ 56) and 9% (n ¼ 19) of cases, extracted from the .CEL image file, normalized and aligned respectively. Apparently normal and complex genomes were onto the human genome sequence (Build 36.3). Copy number observed in 18 and 96 patients, respectively, where complexity gains and losses were defined as a deviation of probes from was defined as previously reported (X3 CNAs excluding normal value of 2, within a consecutive genomic window of aneuploidy).18 Microarray data was concordant with diagnostic 50 Kb. Copy number changes were verified by two independent fluorescence in situ hybridization for 96.2% of the tests researchers. The allele ratio was calculated for each sample performed (790/816). Array-CGH analysis confirmed the posi- using the HapMap Allele Reference baseline (Affymetrix), and tion of all the CNAs identified in 22 patients by SNP6.0 analysis.

Leukemia 13q deletion size in CLL H Parker et al 491 Heterogeneous 13q deletions exhibit some clustering located between 49.3 and 49.5 Mb, targeting C13orf1, TRIM13, of breakpoints KCNRG and exons 7–11 of the DLEU2 gene. A 210 kb distal A total of 205 CNAs targeted 13q in 132 cases, including BCR was identified in 46 patients, located between 50.35 and 10 duplications, 185 deletions (monoallelic n ¼ 146, biallelic 50.56 Mb, targeting RNASEH2B and GUCY1B2 (Figures 1c n ¼ 39), 9 CNNLOH and a single case with monosomy 13 and d). Interestingly, class I deletion breakpoints were often (Figure 1, Supplementary Figure 1 and 2, Supplementary Table within the proximal BCR and distal BCR. Next we analyzed 3). A MDR encompassing DLEU2 and the miR15a/16–1 cluster both BCRs for the presence of repetitive DNA elements that may (49454689–49597678bp) was lost in 119 cases (range 84 Kb to provide insight into how these CNA arose. In total, 98 of 181 96.2 Mb). In all, 13 cases had partial deletion of the MDR, breakpoints within these regions were within repeat regions. including (n ¼ 11) or excluding (n ¼ 2) miR15a/16–1 (Figure 1d, The most frequently targeted DNA repeats were short- and long Supplementary Figure 3). A total of 9 cases had CNNLOH interspersed nuclear elements in the proximal and distal BCR, extending to the telomere that ranged from 66.4–96.1 Mb in size respectively (Supplementary Figure 4). Deletion breakpoints and was accompanied by a biallelic deletion of a region were frequently intragenic, accounting for 219 breakpoints encompassing the MDR in all cases. The remaining 30 biallelic within 64 genes including RB1, FNDC3A, KPNA3, DLEU7 and deletions encompassed the MDR and were 0.22–2.98 Mb in INTS6 (Supplementary Figure 5). size. We visually inspected the copy number data and dichotomised deletions into two classes on the basis of size and inclusion of the MDR; both deletion classes Multiple deletions can target 13q but are not associated included the MDR, whereas class I (o2 Mb in size, n ¼ 63) with genome complexity and II (42 Mb in size, n ¼ 68) deletions were less than or Complex patterns of CNA targeting 13q (defined as X3 CNA) greater than the 50th percentile of deletion size, respectively were identified in eight patients. Patients exhibited as many as (Supplementary Table 4). five CNA and focal deletions of the RB1 gene, as well as a In spite of the breakpoint heterogeneity, a 210 kb proximal deletion encompassing the MDR were also observed (n ¼ 3) breakpoint cluster region (BCR) was identified in 31 patients, (Supplementary Figure 6). Interestingly, this complexity appears

N N (i) (ii)(iii) (iv)

Class II Class I RXFP2 Class II Class I Bi Mono EEFIDP3

30 RB1

MRPS31 40

CPB2 C13orf18 LOC730174 PHF11

WDFY2 KPNA3 LOC22015 C13orf1 KCNRG TRIM13/ 60 Genomic position (Mb) position Genomic DLEU2 Mir16-1/15a

LOC10013067 DLEU1 STARP1

70 FAM104A LOC10013126 LOC730239 DLEU7 RNASEH2B

80 GUCY1B2 FAM124A

Figure 1 SNP 6.0 analysis of chromosome 13 identifies deletion heterogeneity and breakpoint clustering. (a) 13q deletion profiles and heatmaps for two patients, representing a class I and class II deletion, respectively (both deletion classes included the MDR, whereas class I are o2 Mb and class II are 42 Mb). Genomic position in Mb is located to the left. In the profiles, deviation to the left, from a normal copy number (N) indicates deletion and the heatmap represents loss, gain and normal copy number as blue, red and gray, respectively. (b) Heatmap of 13q copy number changes in 132 CLL patients. Each column represents one patient. Class I and II deletions are subdivided by the white dashed line. (c) Frequency map showing the proximal and distal breakpoints of all deletions in purple and green, respectively. The locations of genes flanking breakpoint clusters are indicated. (di) Heatmap focusing on the subregion of 13q indicated by the black dotted box in a–c. CNAs and class I/ II definitions are as shown in B. (dii) The size and location of deletions partially targeting the MDR, excluding and including miR15a/16–1 (blue and green lines, respectively), with the location of key genes indicated (diii). (div) Frequency map showing the breakpoint clusters within this region, with mono- and biallelic breakpoints to the right and left, respectively. Each line represents a single breakpoint and is colored to indicate the targeted gene.

Leukemia 13q deletion size in CLL H Parker et al 492 to be confined to chromosome 13. With the exception of a outside of the MDR on 13q as clinically and biologically single case (#258) involving loss of TP53, all these cases relevant (Figures 2a and c and Table 1b). exhibited one or two CNAs on other chromosomes. Recently, it Type II 13q deletions are defined by deletion of RB1,as has been suggested that 13q deletion size is an independent described by Ouilette et al.17 and have previously been found to predictor of genomic complexity.18 To investigate this asso- be enriched in patients who received treatment or had a higher ciation further, we used our 13q deletion classification, clinical stage.17 Therefore, 13q deletion types were assigned to and although there was an association between a class II 13q our cases. By logistic regression, 13q deletion type II was shown deletion and increased genetic complexity in cohort I, this to be associated with disease progression in cohort I (odds was confounded by the high frequency of mono- and biallelic ratio ¼ 6.7; 95% confidence intervals: 1.22–37.25; P ¼ 0.03 deletion of 13q (Supplementary Table 5). This, with the obser- Table 1a). However, this association was not stronger than the vation that in cohorts II and III, a class II deletion was risk of disease progression associated with gender (P ¼ 0.03), not associated with a complex genome, suggests a more subtle and was far less predictive of progression than the class II relationship between 13q deletion size and genome complexity cut-off, strongly supporting the latter as a predictor of disease through disease evolution. progression.

13q deletion size may provide additional clinical In early stage CLL 13q deletion size is associated with information alongside established prognostic markers disease progression In light of our findings that a class II 13q deletion predicts In-line with previous genetic studies of CLL the presence of a progression, we examined whether this is independent of 13q deletion at disease presentation (cohort I) was found to be established prognostic markers. Cohort I cases that progressed more associated with stable disease (odds ratio ¼ 0.16, 95% were more likely to be ZAP70 positive (P ¼ 0.03) and have confidence intervals: 0.04–0.60; P ¼ 0.007). However, 13q an unmutated IgVH gene (P ¼ 0.001). When cohort I cases deletion size in cases that went on to progress, defined as were stratified by the presence of a class II deletion, the requiring treatment within 3 years, were significantly larger association with disease progression was independent from all when compared with cases with stable disease after 5 years prognostic markers tested for, except for CD38 positivity (P ¼ 0.04; median size 3.5 vs. 1.2 Mb, Table 1a). (P ¼ 0.03, Table 2). Cohorts II and/or III supported the cohort I In cohort I, 31 stable cases (82%) had 13q deletions, of which, finding as the presence of a class II deletion was not asso- seven (23%) were class II. In comparison, only 13 progressive ciated with CD38, ZAP70 positivity, 17p or 11q deletion by cases (52%) had a deletion and 9 (69%) were class II. After fluorescence in situ hybridization, IgVH mutation status, white controlling for age at diagnosis and gender, class II deletions blood cell count, beta-2-microglobulin levels and lactose were found to be associated with an increased risk of disease dehydrogenase activity (Table 2). It has previously been progression (odds ratio ¼ 12.3; 95% confidence intervals: demonstrated that CLL cells exhibiting high clonal populations 2.1–72.2; P ¼ 0.005) compared with cases with class I deletions. of 13q-deleted cells (480%) have inferior survival. More- On the basis of this initial finding we went on to investigate over, the presence of a biallelic 13q deletion has been whether class II deletions were enriched in two further CLL associated with a poor outcome in certain studies. Neither of cohorts sampled subsequent to progression. Class II deletions these observations predicted deletion class in our patients were found to be enriched in cohorts II and III when compared (Supplementary Table 6). with cohort I (60 vs. 36%; P ¼ 0.02), supporting a role for 13q Multivariate Cox proportional hazard model analysis, strati- deletion size in progressive disease and also implicating genes fied by cohorts I, II and III, did identify associations between 17p

Table 1 Comparison of the size and frequency of 13q deletions within cohort I (a) and across all cohorts (b)

Disease status (number 13q deletions/total number cases) Stable (31/38) Progressive (13/25) P-value

(a) Disease progression (cohort I) 13q deletion size Mean (Mb) 4.20 10.88 Median (Mb) 1.2 3.50 0.04 Range (Mb) 0.76–31.1 0.24–74.0 13q deletion class Class I 24 (77%) 4 (31%) 0.005 Class II 7 (23%) 9 (69%) 13q deletion type Type I 27 (72%) 6 (46%) 0.03 Type II 7 (26%) 7 (54%)

(b) All cohorts Cohort name (number 13q deletions/total number cases) Cohort I (44/63) Cohort II (39/64) Cohort III (48/97) 13q deletion size Mean (Mb) 6.17 8.26 6.63 Median (Mb) 1.32 2.8 2.50 Range (Mb) 0.24–74.0 0.13–81.90 0.84–38.7

13q deletion class Class I 28 (64%) 14 (36%) 21 (44%) Class II 16 (36%) 25 (64%) 27 (56%)*

13q deletion type Type I 33 (70%) 18 (49%) 26 (58%) Type II 14 (30%) 19 (51%) 19 (42%) *P ¼ 0.02. 2 Â 2 w2-test (Cohort I vs Cohort II + III). Fishers exact one-sided P-value.

Leukemia 13q deletion size in CLL H Parker et al 493

Figure 2 SNP 6.0 analysis of chromosome 13 reveals that the deletion of a subset of genes flanking the MDR is enriched in progressive cases. The genomic position in Mb is located to the left. 13q deletions in cohort I stable (a), cohort I progressive (b) and cohort II and III progressive (c) cases are shown. Black vertical lines indicate the size and location of deletions on 13q. Class I and II deletions in each subgroup are indicated by parentheses above. (d) The percentage of cases within each subgroup (cohort I stable; solid black line, cohort I progressive; dashed black line, Cohort II and III progressive; dot/dashed black line) with deletion of the subregion of 13q highlighted by the black dotted box shown in (a–c). (e) The location of genes within the region highlighted by the black dotted box in (d). (f) Gray shading highlights the enriched deletion of genes flanking the MDR in progressive cases from all cohorts, compared with stable cases. Deletions of regions to the right of the dotted line are significantly enriched in progressive cases (2 Â 2 contingency table w2 P-valueso0.01). deletions, IgVH status and genome complexity and reduced within these deletions, revealed a group of 15 genes outside the survival outcomes, but revealed no significant risk for shorter MDR region that were more frequently deleted in cases with overall survival, time to first treatment or progression-free subsequent progressive disease when compared with the survival in cases with class II 13q deletions (Supplementary deleted genes in cases with stable disease (Po0.01; Figures 2d Table 6). Furthermore, the presence of either a biallelic and f and Table 3). In all, 14 of these genes were in a 1 Mb gene 13q deletion or a deletion of RB1 (Type II deletion) was cluster located between the MDR and RB1 (RB1 deletions not associated with an adverse outcome. In cohort III, we occurred in 54 individuals), suggesting that these genes may be determined whether deletion size predicted response to treat- associated with clinical outcome. Deletion of the same set of ment (fludarabine and cyclophosphamide). Deletions were genes was observed in progressive cohorts II and III. Interest- larger (mean size (partial response/no response) 7.62 versus ingly, this association was further supported by profiling the (complete response) 5.65 Mb) and class II deletions were more second deletion event in cases with biallelic loss. Class II frequent in patients with partial or no response to treatment. deletions of the second allele were exclusively observed in However, the difference in deletion size was not statistically progressive cases, and showed enrichment of the same 1 Mb significant (P ¼ 0.343). cluster of deleted gene (Supplementary Figure 1C and D). This shows that these genes can be completely inactivated by biallelic loss, supporting a tumor suppressor role. Deletion of a gene cluster centromeric to the 13q MDR is more frequent in progressive patients As large 13q deletions at presentation were found to be Discussion associated with disease progression, deletions were mapped onto 13q gene architecture to determine whether there is a Here, we show the development of a novel approach for the commonly deleted gene cluster associated with disease progres- identification of putative cancer genes outside established sion. In cohort I, a detailed analysis of the genes contained MDRs, using enrichment analysis based on clinical outcome

Leukemia 13q deletion size in CLL H Parker et al 494 Table 2 Univariate logistic regression analysis. (a) The Relationship between disease progression and 13q deletion size/prognostic markers. (b) The relationship between class II 13q deletions and prognostic markers in each cohort

Biomarker/variablea OR Standard error Z-score 95% CI P-value

(a) Disease Progression (Cohort I only; n ¼ 44)b Presence of 13q deletion 0.16 0.11 À2.72 0.04–0.60 o0.01 13q deletion class II 12.32 11.11 2.78 2.10–72.19 o0.01 13q deletion Typec 6.73 5.88 2.18 1.22–37.25 0.03 Zap70 positive 4.97 3.65 2.18 1.18–21.0 0.03 CD38 positive 2.55 1.78 1.34 0.65–9.99 0.18 IgVH mutated 0.02 0.02 À3.22 0.00–0.21 o0.01 Del 17p 1.72 2.70 0.35 0.08–37.12 0.73 Del 11q 2.43 3.71 0.58 0.12–48.35 0.56

(b) class II 13q deletion in cohort I (n ¼ 44)b Zap70 positive 0.41 0.48 À0.76 0.04–4.18 0.45 CD38 positive 13.92 16.35 2.24 1.39–139.13 0.03 IgVH mutated 0.59 0.64 À0.49 0.07–4.82 0.63 Del 11q FF F F F Del 17p 1.79 2.63 0.40 0.10–32.00 0.69

(c) Class II 13q deletion in cohort II (n ¼ 39)b Zap70 positive 1.93 1.78 0.71 0.32–11.77 0.48 CD38 positive 0.52 0.48 À0.71 0.09–3.10 0.48 IgVH mutated 0.90 0.86 À0.11 0.14–5.88 0.91 Del 11q 0.71 0.66 À0.37 0.11–4.43 0.71 Del 17p 1.08 1.25 0.07 0.11–10.36 0.94

(d) Class II 13q deletion in cohort III (n ¼ 48)b Zap70 expression 0.72 0.46 À0.51 0.21–2.25 0.61 CD38 expression 2.19 1.47 1.17 0.59–8.19 0.24 IgVH mutated 0.87 0.53 À0.23 0.26–2.88 0.82 Del 11q 1.07 0.80 0.09 0.25–4.64 0.93 Del 17p FF F F F Stage1 0.72 0.34 À0.69 0.28–1.84 0.49 WBC count2 1.00 0.003 0.74 1.00–1.01 0.46 b2M3 0.56 0.47 À0.69 0.11–2.87 0.49 LDH4 1.00 0.002 À0.02 1.00–1.01 0.98 Abbreviations: CI, confidence intervals; OR, odds ratio. Significant P-values are shown in bold. aAge at diagnosis and gender included as covariates where available. bNumber of informative cases. 1,2,3 and 4 were only measured at randomization to the UKCLL4 treatment trial. c13q deletion type as defined by Ouillette et al.17 1Rai stage. 2White blood cell (WBC) count. 3Beta-2-microglobulin (b2M) using previously defined cut-offs (31 X4 mg/l). 4Lactose dehydrogenase (LDH) activity.

criteria. With the application of this approach, we confirm the may be mechanistically dissimilar. However, rare BCRs may heterogeneity of 13q deletion architecture in patients with CLL. emerge from a larger study. Although the MDR (142.9 Kb) we identified is larger than those At diagnosis (cohort I), we show that 13q deletions are previously published,9,13 it more accurately reflects the in vivo significantly larger in patients who subsequently developed situation.17,25 It may also explain the incomplete penetrance of progressive disease. Indeed, class II deletions predict disease a leukemia phenotype observed in the 13q MDR/miR15a/16–1 progression more strongly than previous definitions,17 implicating mouse model.14 The demonstration that the DLEU2 promoter genes positioned between the MDR and RB1. The clinical can control miR15a/16–1 expression is a likely explanation for importance of deletion size is further supported by, (a) their the two cases we found with deletion of the DLEU2 promoter enrichment in samples taken subsequent to disease progression region but not the miR15a/16–1 cluster.26 We refine two BCRs, (Cohort II and III) and (b) their independence of established proximal and distal, to the MDR that may be prone to breakage prognostic markers, such as TP53/ATM deletions and IgVH and recombination in CLL B cells. In light of the BCRs identified mutational status. Contrary to the previous suggestion that deletion here, it is clear that 13q deletions can be grouped thus: class I of DLEU7 may be associated with adverse outcome, we have deletions are confined to a 2 Mb region where breakpoints shown that class I deletions, which contain DLEU7, are not often occur in the two BCRs identified, which in addition to the associated with a poor prognosis.16 Although the stratification of genes within the MDR, include FLJ31945, FAM10A4, BCMS and our patients into three distinct cohorts is necessary to account for DLEU7; class II deletions extended beyond this region in either a sources of biological heterogeneity, this reduces statistical power centromeric and/or telomeric direction, encompassing a large relative to an equivalently sized single-cohort study. Although, our number of additional genes. Rather than being the result of data demonstrates the clinical importance of 13q deletion size, it consistent BCRs, class II deletions displayed highly hetero- was not possible to confirm this with measures of event-free geneous breakpoints suggesting that these classes of deletion survival, suggesting either a lack of power because of a minor

Leukemia 13q deletion size in CLL H Parker et al 495 Table 3 Enrichment of deleted genes in progressive CLL cases (Po0.01)

Genomic Location Gene symbol Description location (Mb) relative to MDR

48.24–49.27 Centromeric LOC338099 Proteasome activator subunit 2 pseudogene 2 LOC647131 Discontinued record in GenBank (20–6–2009) FNDC3A Fibronectin type-III domain-containing protein 3A. RAD17P2 RAD17 homolog (S. pombe) pseudogene 2. COX7CP1 Cytochrome c oxidase subunit VIIc pseudogene 1. LOC387924 2-oxoglutarate and iron-dependent oxygenase domain containing 1 pseudogene.

MLNR Motilin receptor. CAB39 l Calcium-binding protein 39-like. SETDB2 SET domain, bifurcated 2. PHF11 PHD ﬁnger protein 11. RCBTB1 Regulator of chromosome condensation (RCC1) and BTB (POZ) domain containing protein 1.

LOC100131941 Hypothetical pseudogene. ARL11 ADP-ribosylation factor-like protein 11. KPNA3 Karyopherin alpha 3 (importin alpha 4).

50.47–50.54 Telomeric GUCY1B2 Guanylate cyclase 1, soluble beta-2. Abbreviation: MDR, minimally deleted region.

impact on prognosis (HRo2.0) or a more complex relationship that the association is more subtle. Class II deletions were only between 13q deletion heterogeneity and clinical outcome. We associated with a complex genome at disease presentation and identified deleted 13q genes that are strongly associated with our data suggests that this relationship is confounded by the high disease progression at diagnosis (cohort I) and confirmed frequency of mono- and biallelic 13q deletions. The relationship enrichment of these gene deletions in progressive patients (cohorts between 13q and genome complexity might be complicated by II and III). In addition to supporting the proposed role of RB1 in multiple mechanisms contributing to genome instability later in these deletions,17 our approach also identifies a cluster of genes disease that are not present at earlier stages. Also, it may identify positioned between the MDR and RB1. SETDB2 is a methyl- weaknesses in interpreting data derived from large hetero- transferase with a role in chromosome condensation and geneous cohorts of CLL patients that include samples taken segregation.27 Given its capacity for lysine methylation, it is also at different stages of disease evolution and from treated/ possible that SETDB2 may be involved in post-translation control untreated patients. Our findings suggest that care is required of P53 through methylation of lysine 372.28 In light of the when interpreting this type of data, and in fact a more accurate prognostic impact of P53 deregulation, this is an interesting insight into associations between CNA and disease charac- avenue for further study. The deletion of other genes, such as teristics can be obtained from smaller homogeneous subgroups. PHF11, which is expressed in B- and T cells, and controls gene Although our analysis demonstrates that complexity can be expression through interactions with the p65 subunit of NF-kB,29 accurately assessed using unpaired analysis, it is possible that and RCBTB1, which has been implicated in ubiquitination any association with this measure may be confounded by the through its role as a substrate for a CUL3 E3 ligase,30 may also absence of analyzing germ line material. be important genes within the region. Interestingly, the result of a Here, we show that the consequence of 13q deletions in CLL is unique 13q deletion may be the loss of multiple genes that the loss of a variable number of gene loci. Although it has been contribute to the disruption of a small number of key biological proposed that chromosomal deletions, including those targeting pathways, causing a variable degree of pathway dysfunction and a 13q14 in CLL, may drive disease pathogenesis through targeting heterogeneous impact on prognosis. We confirm the presence of multiple genes, this study provides the first clinical associations to CNNLOH targeting chromosome 13, resulting exclusively in the support this hypothesis. Although this observation requires further duplication of a deletion targeting the MDR. Although this validation, additional prognostic information could be provided in suggests that the consequence of the CNNLOH event is loss of the clinical setting using fluorescence in situ hybridization, both copies of genes within the MDR, it resulted in homozygosity multiplex ligation-dependent probe amplification and array-based of the cluster of genes shown to be associated with progressive comparative genomic hybridization to distinguish between disease. These CNNLOH events were detected in patients who informative deletion sizes. These deletions have the potential to progressed and are likely to be valuable for the identification of deregulate multiple genes in a number of key biological pathways sequence changes or epigenetic alterations within this region. that include key regulators of cell cycle control, ubiquitination Our data shows that 13q deletions can be the result of a and NF-kB signaling. This observation has application to cancer complex series of CNAs, which in three patients include focal in general, where recurrent CNAs are equally heterogeneous. deletions targeting RB1 in addition to a loss of the MDR, Although the historical analysis of MDRs has identified genes suggesting the RB1 may contribute to instability of chromosome involved in carcinogenesis, genes outside MDRs that contribute 13. It has been suggested that there may be gene(s) on 13q that to a more aggressive disease will not be identified. Moreover, it is are contributing to complexity in certain CLL patients, as an plausible that the MDRs without an identified pathogenic association between Type II deletions and genome complexity mechanism are the result of inherent instability within these has already been demonstrated.18 However, our study included genomic regions. It is deletion of genes outside the MDR that may more 13q-deleted patients with complex genomes, and suggests provide the cancer cell with a growth advantage. The application

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