Atypical 11Q Deletions Identified by Array CGH May Be Missed by FISH

Letters to the Editor 1011 1 2 2 2 1 AC Ng , SK Kumar , SJ Russell , SV Rajkumar and MT Drake 2 Axelrod L. Glucocorticoid therapy. Medicine (Baltimore) 1976; 55: 1 Division of Endocrinology, Department of Medicine, 39–65. Mayo Clinic, Rochester, MN, USA and 3 Krasner AS. Glucocorticoid-induced adrenal insufficiency. JAMA 2Division of Hematology, Department of Medicine, 1999; 282: 671–676. Mayo Clinic, Rochester, MN, USA 4 Schlaghecke R, Kornely E, Santen RT, Ridderskamp P. The effect of E-mail: [email protected] long-term glucocorticoid therapy on pituitary-adrenal responses to exogenous corticotropin-releasing hormone. N Engl J Med 1992; 326: 226–230. References 5 Grinspoon SK, Biller BM. Clinical review 62: laboratory assessment of adrenal insufficiency. J Clin Endocrinol Metab 1994; 79: 1 Rajkumar SV, Jacobus S, Callander N, Fonseca R, Vesole D, Williams 923–931. M et al. A randomized trial of lenalidomide plus high-dose 6 Nieman LK. Dynamic evaluation of adrenal hypofunction. dexamethasone (RD) versus lenalidomide plus low-dose dexametha- J Endocrinol Invest 2003; 26 (7 Suppl): 74–82. sone (Rd) in newly diagnosed multiple myeloma (E4A03): a trial 7 Hagg E, Asplund K, Lithner F. Value of basal plasma cortisol assays coordinated by the Eastern Cooperative Oncology Group. Blood in the assessment of pituitary-adrenal insufficiency. Clin Endocrinol (ASH Annual Meeting Abstracts) 2006; 108:799. (Oxf) 1987; 26: 221–226.

Atypical 11q deletions identiﬁed by array CGH may be missed by FISH panels for prognostic markers in chronic lymphocytic leukemia

Leukemia (2009) 23, 1011–1017; doi:10.1038/leu.2008.393; results were because of small clonal cell populations that published online 22 January 2009 comprised o25–30% of the total sample. Interestingly, two CLL cases with cryptic (B1 Mb) 13q14 deletions detected Genomic alterations have increasingly gained importance as by array CGH were missed by both of the 13q14 region prognostic markers in B-cell chronic lymphocytic leukemia (CLL). FISH probes used in this study. These findings suggested that The identification of genetic alterations of prognostic importance array CGH had advantages over FISH for the identification of in CLL is accomplished currently using commercially available certain types of genomic alterations in CLL. In this study, fluorescence in situ hybridization (FISH) panels that can detect the we present four additional CLL cases with atypical 11q most common recurrent aberrations in CLL, involving chromo- deletions, including two cases that were not identified properly somes 11q, 13q, 14q, 17p and whole chromosome 12.1–4 by a commercially available five-probe FISH panel that Although the use of FISH analysis has improved the detection also included an 11q22.3 Vysis LSI ATM probe (Abbott rate of genomic alterations in CLL from B50% using conventional Molecular, Des Plaines, IL, USA). This study is also the first to cytogenetics to 480%,5 there is a need for improved methods to report CLL cases with large 11q deletions that do not include identify prognostic markers that can aid in the risk-stratification of the ATM (Ataxia telangiectasia mutated) gene, suggesting that these patients. The identification of high-risk patients is essential additional genes in this region may be important for the for treatment planning and is of particular importance in early- pathogenesis of CLL. stage, asymptomatic CLL patients, for whom rapid disease Bacterial artificial chromosome array-based array GCH progression and a poor prognosis can be predicted by the was used to detect recurrent genomic alterations in 190 presence of specific genomic abnormalities such as 11q and 17p cases of CLL using a clinically validated array designed to deletions.6–8 Recently, array comparative genomic hybridization interrogate all known CLL prognostic loci.11 This array contains (array CGH) has been gaining acceptance as a diagnostic tool that 179 CLL prognostic marker probes along with a backbone of can also be applied to detect genomic gains and losses of 914 FISH-mapped linearly distributed clones for whole- prognostic importance in CLL, because of the advantages afforded genome coverage at an average resolution of B2.5 Mb. This by simultaneous genome-wide and locus-specific assessment of analysis identified 22 CLL cases with 11q deletions, with 20 theleukemiawithasingleassay.9–14 cases that included the ATM gene in the deleted region. Array CGH is well suited for the identification of genomic However, four of the CLL cases showed atypical 11q deletions, alterations of prognostic importance in CLL, because the most with two cases (Cases 1 and 2) exhibiting a centromeric common and important aberrations are comprised of genomic breakpoint just proximal to the ATM locus, and two cases losses and gains, whereas chromosomal translocations are (Cases 3 and 4) exhibiting proximal breakpoints telomeric to the relatively rare. Although translocations do occur in CLL, their ATM gene (Figure 1). Oligonucleotide-based array CGH prognostic significance is currently controversial. In a number of analysis (Agilent Technologies 44 K or 105 K arrays, Santa Clara, recent studies evaluating the use of array CGH as a clinical tool CA, USA) was also used to confirm the 11q deletions and to map for genomic alteration detection in CLL, array CGH results have the deletion breakpoints in 15 of the 22 chromosome 11q exhibited a high concordance with parallel FISH studies, except deletion cases identified by the BAC array. This analysis when FISH aberrations are present in less than B25–30% of the identified a minimal region of deletion on 11q of B2.94 Mb cells.9–14 In a recent report from our laboratory, we analyzed (Table 1). 174 cases of CLL by bacterial artificial chromosome (BAC)- Bacterial artificial chromosome array CGH analysis of Case 1 based array CGH and found that we could identify correctly the revealed a 2.4 Mb deletion of 13q and an B11 Mb deletion of aberrations identified by FISH 96% of the time.12 The discordant 11q23 involving 19 clones at linear positions 107.57–118.29

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Figure 1 Atypical 11q deletions in four cases of chronic lymphocytic leukemia (CLL) identified by bacterial artificial chromosome (BAC) array comparative genomic hybridization (CGH) analysis. BAC array CGH analysis was performed using the HemeScan microarray (Combimatrix Molecular Diagnostics, Irvine, CA, USA). Differentially Cy3 and Cy5 fluorescence-labeled patient and normal sex-matched reference samples were co-hybridized to the microarray, scanned with a Gene-Pix 4000B microarray scanner and quantified using GenePix Pro microarray image analysis software (Molecular Devices, Sunnyvale, CA, USA). A loss of a particular clone is manifested as the simultaneous deviation of the ratio plots from a modal value of 1.0, with the red ratio plot showing a positive deviation (to the right), whereas the blue ratio plot shows a negative deviation at the same locus (to the left). Conversely, DNA copy number gains show the opposite pattern. A ‘typical’ 11q deletion involving the ATM gene is shown in the panel on the far left. Atypical 11q deletions involving the ATM gene near the proximal breakpoint of the deletion (Cases 1 and 2) and with proximal deletion breakpoints distal to the ATM gene (Cases 3 and 4). Letters to the Editor 1013 Table 1 Sizes and linear positions of atypical 11q deletion cases, were also confirmed by oligonucleotide array analysis and ATM gene locus endpoints, and minimal region of deletion on 11q found to be 8.8, 1.5 and 4.8 Mb in size, respectively. defined by oligonucleotide array CGH analysis of 15 CLL cases Bacterial artificial chromosome array CGH analysis of Case 4 showed a relatively large 18 Mb deletion of 11q23 represented Start Stop Size by the loss of 23 probes (RP11-54P20-RP11-378D7) at linear positions 108.97–124.95 (Figure 1). The two probes covering Case 1 11q del 107 327 853 118 429 797 11.1 Mb Case 2 11q del 107 390 044 116 803 762 9.41 Mb the ATM locus (RP11-56J3 and RP11-27I22) at linear positions Case 3 11q del 111 926 277 117 510 598 5.58 Mb 107.57 and 107.81 were proximal to the breakpoint and not Case 4 11q del 108 775 680 126 456 923 17.68 Mb included in the deletion. In addition to the 11q deletion, BAC ATM gene locus 107 672 911 107 709 905 36 995 bp array CGH revealed complex genomic alterations with losses Minimal region of 111 962 277 114 899 282 2.94 Mb on chromosomes 8p, 8q, 13q, 14q, 17p, 18p, 18q and a gain on deletion on 11q 22q (data not shown). Oligonucleotide array CGH analysis, of CGH, comparative genomic hybridization; CLL, chronic lymphocytic this case, identified a 17.68 Mb deletion extending from leukemia. 11q22.3-q24.2 (Figure 2d and Table 1) and also confirmed the other genomic alterations detected by the BAC array. FISH analysis with a LSI ATM probe did not detect the 11q23 (RP11-56J3-RP11-317A5; Figure 1). Although BAC array CGH deletion, but additional FISH probes confirmed the genomic suggested that the ATM gene might be involved directly in the losses on 13q and 17p. Losses of chromosomes 8p, 8q, 14q, proximal breakpoint region, further analysis using oligo- 18p, 18q and 22q were also not detected by FISH. nucleotide array CGH revealed an 11.1 Mb deletion extending It has previously been assumed that the LSI ATM B500 Kb from 11q22.3-q23.3 that included the CUL5, ACAT, NPAT FISH probe provided adequate coverage for the detection of and ATM gene loci and STS markers D11S1828 and D11S1294 deletions of the 11q22.3q23 region in CLL. The protein encoded near the proximal breakpoint of the deletion (Figures 2a, 3 and by the ATM gene is a cell cycle checkpoint kinase and upstream Table 1). Oligonucleotide array analysis also confirmed the regulator of the p53 tumor suppressor, making it a potentially 2.4 Mb deletion of chromosome 13q14 identified by the BAC important target gene for deletion in cases of CLL with 11q array. FISH analysis of this sample showed loss of LSI 13 (RB1) deletions. Based on the biological role of the ATM gene and and LSI D13S319 on chromosome 13q14, and a LSI ATM probe inclusion of the ATM gene locus, in all the earlier reported cases also detected a deletion at the ATM locus on 11q23 (data not of 11q23 deletions in CLL, FISH analysis using the LSI ATM shown). probe has become the accepted standard for evaluating 11q Bacterial artificial chromosome array CGH analysis of Case 2 status in CLL and other hematological malignancies.15,16 revealed a number of large genomic alterations, including However, the two CLL cases, reported in this study, with whole chromosome losses of chromosomes 1, 6, 13, 22 and X, atypical 11q deletions that did not include the ATM gene, as well as gain of the q-arm of chromosome 12 and loss of the suggest that relying on only a single probe for the detection of a p-arm of chromosome 17. Smaller alterations were also critical CLL prognostic genomic aberration may not be the best observed that included a gain of 5p15.33 and an B9Mb approach. Even recurrent aberrations such as those found in CLL deletion of 11q23 involving 11 clones at linear positions are subject to breakpoint variability and unpredictable genomic 107.57–116.62 (RP11-56J3-CTD-2213H23; Figure 1). Similar rearrangements associated with tumorigenesis and clonal to Case 1, BAC array CGH suggested that the ATM gene might evolution.17 FISH analysis of a large genomic region using a also be involved directly in the proximal breakpoint region. single probe less than a mega base in size, creates a situation in However, further analysis, using oligonucleotide array CGH, which important aberrations may not be identified properly. The revealed an 9.41 Mb deletion extending from 11q22.3-q23.3 genomic region interrogated by a single FISH probe will not with a proximal breakpoint in the centromeric portion of the always coincide with the actual abnormality present, or may not CUL5 gene that included the ACAT, NPAT and ATM gene loci, hybridize at breakpoints in which there is not enough overlap as well as the STS marker D11S1294 near the proximal between the probe and the region containing the genomic breakpoint of the deletion (Figures 2b, 3 and Table 1). aberration. In two of the CLL cases in this study (Cases 3 and 4), Oligonucleotide array analysis also confirmed the other altera- the LSI ATM FISH probe was unable to identify deleted regions tions identified by BAC array CGH. FISH analysis of this sample telomeric to the region on 11q that was interrogated by this showed gain of chromosome 12, loss of LSI 13 (RB1) and LSI probe. D13S319 on chromosome 13, loss of LSI p53 on chromosome This study also defined a 2.94 Mb minimally deleted region 17p, and a LSI ATM probe also detected a deletion at the ATM on 11q in an array CGH analysis of 190 CLL samples that locus on 11q23 (data not shown). However, FISH analysis did included 22 cases with 11q deletions. The minimally deleted not detect the other abnormalities identified in this case by array region contains 18 protein-coding genes (Table 2), and at least CGH on chromosomes 1, 5, 6, 22 and X. three of these genesFZW10, PLZF and TSLC1Fmay represent Bacterial artificial chromosome array CGH analysis of Case 3 alternative targets to ATM for deletion in the 11q23 region. The showed an B5 Mb deletion of 11q23 involving seven clones ZW10 (zeste white 10 homolog) gene is a possible target for at linear positions 112.75–117.04 (CTD-2059P15-RP11- deletion because it encodes a protein involved in mitotic 939C12) that did not include the ATM gene (Figure 1). Deletions checkpoint signaling, and acts to ensure that proper chromo- of chromosomes 3p, 13q and 16p were also identified by the some segregation and stability is present during cell division.18 BAC array. Further characterization of this case, using oligo- This protein has also recently been suggested to be involved in nucleotide array CGH, revealed the 11q deletion to be 5.58 Mb acquired resistance to chemotherapy in melanoma cells.19 in size with a proximal breakpoint distal to the ATM gene locus Another possible target for deletion is the PLZF (promyelocytic (Figure 2c and Table 1). This deletion was B4 Mb distal to the leukemia zinc finger protein) gene. In a variety of cell models, region interrogated by the B500 Kb LSI ATM FISH probe and, expression of this gene has been associated with cell cycle arrest 20,21 therefore, could not be detected by FISH analysis of this region in the G1 phase and eventual apoptosis. The transcriptional of chromosome 11. Deletions of chromosomes 3p, 13q and 16p repressor protein encoded by the PLZF gene maintains cells in a

Leukemia 1014 Leukemia etr oteEditor the to Letters

Figure 2 Oligonucleotide array comparative genomic hybridization (CGH) analysis showing breakpoints of atypical 11q deletions in four chronic lymphocytic leukemia (CLL) cases. Tumor DNA was analyzed using Agilent Human Genome 44 K or 105 K CGH oligonucleotide arrays (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s recommendations. Slides were scanned with an Agilent Scanner, and the data were analyzed with Agilent Feature Extraction and CGH Analytics software. The green and red dots represent the Log2 fluorescence ratios of individual oligonucleotide probes on the microarrays. The red dots represent probes with positive Log2 fluorescence ratios, and the green dots represent probes with negative Log2 fluorescence ratios. Clusters of green probes shifted significantly to the left of zero represent losses, and clusters of red probes shifted significantly to the right of zero represent gains of genetic material. Vertical red bars and the pink- shaded rectangles represent regions determined statistically significant by the software used to analyze the array data (regions of gain or loss of genetic material). Chromosome 11 views are exhibited on the left-side panels and gene views of the proximal deletion breakpoint regions are shown on the right-side panels. The location of the ATM gene is circled in each of the gene view panels. Cases 1 (a) and 2 (b) with proximal 11q deletion breakpoints just centromeric to the ATM gene. Cases 2 (c) 3 (d) showing proximal 11q distal deletion breakpoints distal to the ATM gene. Letters to the Editor 1015 Centromeric Telomeric

D11S1828 D11S1294 107322601 107948738

107384618 107672911 107709905

CUL5 ACAT NPAT ATM

LSI ATM Probe (~500 kb)

Case 1 Case 2 Case 4 Case 3 107327853 107390044 108775680 111926277

Figure 3 LSI ATM FISH probe and gene map at 11q23. The location of the ATM gene on chromosome 11q23 and region interrogated by the B500 kb LSI ATM FISH probe. Approximate centromeric nucleotide positions are shown for each of the proximal breakpoints of the four chronic lymphocytic leukemia (CLL) cases with atypical 11q deletions relative to other genes and STS markers in this region. Nucleotide positions for the genes and STS markers in the ﬁgure were obtained from the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway).

Table 2 Linear positions of genes encompassed by the 2.94 Mb minimally deleted region on 11q

Gene Start Stop Name

1 NCAM1 112 337 205 112 654 368 Neural cell adhesion molecule 1 2 TTC12 112 690 461 112 742 324 Tetratricopeptide repeat domain 12 3 ANKK1 112 763 723 112 776 350 Ankyrin repeat and kinase domain 1 4 DRD2 112 785 527 112 851 091 Dopamine receptor D2 5 TMPRSS5 113 063 483 113 082 305 Transmembrane protease, serine 5 6 ZW10a 113 109 119 113 149 635 Zeste white 10 homolog (Centromere/kinetochore protein) 7 USP28 113 173 807 113 251 466 Ubiquitin speciﬁc protease 28 8 HTR3B 113 280 799 113 322 493 5-hydroxytryptamine (serotonin) receptor 3B 9 HTR3A 113 351 120 113 366 245 5-hydroxytryptamine (serotonin) receptor 3A 10 PLZFa 113 435 641 113 626 608 Promyelocytic leukemia zinc ﬁnger protein 11 NNMT 113 671 745 113 688 448 Nicotinamide N-methyltransferase 12 C11orf71 113 775 003 113 776 443 chromosome 11 open reading frame 71 13 RBM7 113 776 594 113 784 845 RNA binding motif protein 7 14 REXO2 113 815 387 113 826 211 RNA exonuclease 2 homolog 15 FAM55A 113 897 647 113 935 790 Family with sequence similarity 55, member A 16 FAM55D 113 946 523 113 971 694 Family with sequence similarity 55, member D 17 FAM55B 114 054 410 114 082 862 Family with sequence similarity 55, member B 18 TSLC1a 114 549 555 114 880 451 Tumor suppressor in lung cancer 1 (CADM1) (Cell adhesion molecule 1) aBold font denotes possible target genes in non-ATM deleted CLLs.

quiescent state by repressing c-myc expression and preventing possible association of losses of these genes with higher-risk cell cycle progression.22 A third possible target for deletion is subsets of CLL. However, this study suggests that the poor the TSLC1 (tumor suppressor in lung cancer 1) gene. De- prognosis associated with 11q deletions in CLL may be due to regulation of the TSLC1 tumor suppressor gene has been alterations in the expression of a number of genes that contribute described in a number of human malignancies, including to cell growth control and genome integrity rather than owing to neuroblastoma, nasopharyngeal carcinoma, breast cancer, lung only a single gene. This study also indicates that BAC array CGH adenocarcinoma, high-grade gliomas, pancreatic adenocarcino- probes that contain the ZW10, PLZF and TSLC1 genes may be ma, esophageal squamous cell carcinoma, some forms of primary more reliable markers for the detection of 11q23 deletions in CLL gastric cancer and prostate cancer.23–32 The altered expression of than the LSI ATM FISH probe. TSLC1 has been associated with both promoter hypermethyla- Although deletion of the ZW10, PLZF and TSLC1 genes may tion, as well as loss of heterozygosity.25,29,30–32 Each of these contribute to an aggressive clinical course in cases of non-ATM three putative deletion target genes is located clearly within the 11q23-deleted CLL, it could alternatively be possible that 11q23 minimally deleted region on 11q as determined by both BAC and deletions that do not include the ATM gene may represent oligonucleotide array CGH, in all four of the CLL cases with actually a subset of CLL, with a more favorable prognosis than atypical 11q deletions included in this study. The ZW10, PLZF the ATM-deleted cases. Further whole-genome studies cor- and TSLC1 genes were also deleted in all of the other 19 cases relating outcome in CLL cases with atypical 11q23 breakpoints exhibiting 11q deletions in a recent CLL array CGH study from are needed to further risk-stratify these patients. As array CGH our laboratory.11,12 Further studies are needed to evaluate the becomes used more frequently to identify recurrent genomic

Leukemia Letters to the Editor 1016 alterations of prognostic significance in CLL,14 identification of 6 Kay NE, O’Brien SM, Pettit AR, Stilgenbauer S. The role of additional cases exhibiting atypical 11q23 deletions can be prognostic factors in assessing ‘high-risk’ subgroups of expected. Outcome studies for these patients will be critical to patients with chronic lymphocytic leukemia. Leukemia 2007; 21: determine whether non-ATM deleted 11q23 cases should be 1885–1891. 7 Dickinson JD, Smith LM, Sanger WG, Zhou G, Townley P, Lynch stratified in a high-risk category or into a more favorable clinical JC et al. Unique gene expression and clinical characteristics with category. the 11q23 deletion in chronic lymphocytic leukemia. Br J In the short time that high-throughput scanning of the CLL Haematol 2005; 128: 460–461. tumor genome by array CGH has been available for use by 8 Zent CS, Call TG, Hogan WJ, Shanafelt TD, Kay NE. Update on clinical laboratories, it has revealed rapidly the unexpected risk-stratified management for chronic lymphocytic leukemia. Leuk complexity and variability of genomic aberrations in CLL. Lymphoma 2006; 47: 1738–1746. 9 Schwaenen C, Nessling M, Wessendorf S, Salvi T, Wrobel G, Although FISH analysis has contributed tremendously to the Radlwimmer B et al. Automated array-based genomic profiling in molecular cytogenetic understanding of the CLL genome, it is chronic lymphocytic leukemia: development of a clinical tool and clear that many important genomic aberrations may be missed discovery of recurrent genomic alterations. Proc Natl Acad Sci by the currently available FISH prognostic marker panels. The USA 2004; 101: 1039–1044. increasing availability of array CGH for the assessment of CLL 10 Patel A, Kang S-H, Lennon PA, Li YF, Rao PN, Abruzzo L et al. prognostic markers will allow simultaneous genome-wide and Validation of a targeted DNA microarray for clinical evaluation of recurrent abnormalities in chronic lymphocytic leukemia. Am J locus-specific analysis to become standard of care for CLL Hematol 2008; 83: 540–546. patients at the time of diagnosis, as well as for monitoring 11 Gunn SR, Mohammed MS, Mellink CHM, Abruzzo LV, Robetorye existing CLL cases for the acquisition of additional complex RS. The HemeScan test for genomic prognostic marker assessment genomic alterations. in chronic lymphocytic leukemia. Expert Opin Med Diagn 2008; 2: 731–740. 12 Gunn SR, Mohammed MS, Gorre ME, Cotter PD, Kim J, Bahler DW et al. Whole-genome scanning by array comparative genomic Acknowledgements hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia. J Mol Diagn 2008; 10: 442–451. SRG, ELE, MEG and MSM are employees of Combimatrix 13 Sargent R, Jones D, Abruzzo LV, Yao H, Bonderover J, Cisneros M et al. Customized oligonucleotide array-based comparative Molecular Diagnostics. RSR is a clinical consultant for Combima- genomic hybridization as a clinical assay for genomic profiling trix Molecular Diagnostics. MKH is an employee of Clarient of chronic lymphocytic leukemia. J Mol Diagn 2009; 11: 25–34. Diagnostics. 14 Higgins RA, Gunn SR, Robetorye RS. Clinical application of array- based comparative genomic hybridization arrays for the identifica- SR Gunn1,2, MK Hibbard3, SH Ismail2, M Lowery-Nordberg4, tion of prognostically important genetic alterations in chronic CHM Mellink5, DW Bahler6, LV Abruzzo7, EL Enriquez1, lymphocytic leukemia. Mol Diagn Ther 2008; 12: 271–280. 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