Evaluation of chronic lymphocytic leukemia by BAC-based microarray analysis

Abstract

Background: Chronic lymphocytic leukemia (CLL) is a highly variable disease with life expectancies ranging from months to decades. Cytogenetic findings play an integral role in defining the prognostic significance and treatment for individual patients.

Results: We have evaluated 25 clinical cases from a tertiary cancer center that have an established diagnosis of CLL and for which there was prior cytogenetic and/or fluorescence in situ hybridization (FISH) data. We performed microarray-based comparative genomic hybridization (aCGH) using a bacterial artificial chromosome (BAC)-based microarray designed for the detection of known constitutional genetic syndromes. In 15 of the 25 cases, aCGH detected all copy number imbalances identified by prior cytogenetic and/or FISH studies. For the majority of those not detected, the aberrations were present at low levels of mosaicism. Furthermore, for 15 of the 25 cases, additional abnormalities were detected. Four of those cases had deletions that mapped to intervals implicated in inherited predisposition to CLL. For most cases, aCGH was able to detect abnormalities present in as few as 10% of cells. Although changes in ploidy are not easily discernable by aCGH, results for two cases illustrate the detection of additional copy gains and losses present within a mosaic tetraploid cell population.

Conclusions: Our results illustrate the successful evaluation of CLL using a microarray optimized for the interrogation of inherited disorders and the identification of alterations with possible relevance to CLL susceptibility.

Figure 1

Microarray results for case 1. (A) Single-copy loss of probes at 11q14.1q11.23.2, approximately 28.9 Mb in size. This deletion interval includes the ATM locus. Probes are ordered on the x axis according to physical mapping positions, with the most distal p-arm probes on the left and the most distal q-arm probes on the right. The blue line represents the ratios for each clone from the first experiment (control/patient), and the pink line represents the ratios for each clone obtained from the second experiment in which the dyes have been reversed (patient/control). The yellow shaded region shows a deletion at 11p11.2p11.12 with a single BAC clone showing apparent homozygous deletion over the gene PTPRJ. Results are visualized using Genoglyphix (Signature Genomics). (B) Single-copy loss of probes at 13q14.2q14.3, approximately 1.8 Mb in size. This deletion interval includes the RB1 locus. Probes are arranged as in (A).

Figure 2

Microarray characterization of a der(6)del(6)(q15q21)inv(6)(p21.3q23) in case 2. In addition to a del(6)(q15q21) (arrow), single-copy losses were identified at each of the inversion breakpoints. Probes are arranged as in Figure 1A.

Figure 3

Microarray results for cases with 13q14 deletions identified by FISH reveal insufficient coverage for the detection of small deletions. (A) Microarray plot for case 2. For this case, the CLL FISH panel showed ~78.5% of cells with a 13q14 deletion. A deletion was detected by microarray analysis, but the extent of that deletion cannot be defined owing to insufficient coverage on the BAC array. (B) Insufficient coverage for the BAC array does not permit determination of the involvement of MIR16-1 and MIR15A in the deletion shown in part A. (C) Microarray plot for case 25. For this case, the CLL FISH panel showed ~20% of cells with a 13q14 deletion using a probe specific to MIR16-1 and MIR15A. Insufficient BAC coverage on this array is likely responsible for the failed detection of a small deletion. Plots are arranged as in Figure 1A.

Figure 4

Microarray results for case 8 showing discordance between microarray and FISH. (A) Microarray analysis did not detect the deletion at ATM that was identified in 10.5% of cells by FISH. (B) Microarray detected the 13q14 deletion that was present in 44.8% by FISH. Microarray analysis also detected (C) trisomy 4 and (D) trisomy 18. Probes are arranged as in Figure 1A.

Figure 5

Microarray results for case 10 showing discordance between FISH and microarray results. (A) Microarray plot showing trisomy 12, which was in 55.8% of cells by FISH. (B, C) Normal microarray plots for (B) chromosome 11 and (C) chromosome 17. Probes are arranged as in Figure 1A.

Figure 6

Microarray characterization of a sample compromised by mosaic tetraploidy (case 11). Despite the mosaic tetraploidy, deletions and duplications of multiple chromosomal regions were identified. These are outlined in greater detail in Table 1. A whole-genome view is displayed with chromosome 1 on the left and the sex chromosomes on the right. The sex chromosome pattern is consistent with a male specimen run against a female control. Probes are arranged as in Figure 1A.