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 Leukemia 1012 Leukemia Letters to the Editor 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).