Characterization of target genes at the 2p15-16 amplicon in diffuse large B-cell lymphoma

Abstract

Amplification of 2p has been observed as a recurrent alteration in diffuse large B-cell lymphoma (DLBCL). Whereas two candidate oncogenes, REL and BCL11A, have been investigated as targets for 2p amplification, the question remains as to whether the true target gene in the amplicon is REL, BCL11A or both. We previously identified frequent genomic gains of chromosomal 2p in 25 out of 99 DLBCL cases by means of genome-wide array comparative genomic hybridization (CGH). All of these 25 cases included recurrent copy number gain at 2p15-16. In the study presented here, cases were analyzed in greater detail by means of contig bacterial artificial chromosome (BAC) array CGH for the 4.5-Mb region at 2p15-16, which contained 33 BAC clones. We confined the minimal common region to 500-kb in length, where only the candidate oncogene REL, and not BCL11A, is located. Real-time quantitative PCR was carried out to investigate the correlation between genomic gain and expression. It showed a significant correlation for both genes, indicating that these two genes are common targets for the 2p15-16 amplicon. However, given the fact that REL is more frequently amplified than BCL11A, the REL gene may play a more important role than BCL11A in the pathogenesis of DLBCL.

Figure 1

Representative individual genomic profiles of chromosome 2 in diffuse large B‐cell lymphoma. Genome‐wide array‐based comparative genomic hybridization profiles of three cases with 2p amplification: (a) D768, (b) D778 and (c) D792. Dots represent the log₂ ratio of bacterial artificial chromosome/P‐1‐derived artificial chromosome clones, which are shown in order from the p telomere to the q telomere. The vertical arrow above each profile indicates the region of amplification. The threshold for gain and loss was defined as the log₂ ratio of +0.2 and −0.2, respectively.

Figure 2

Illustration of genomic amplification at chromosome 2p. Summary of genome‐wide array‐based comparative genomic hybridization profiles at 2p. Copy number gains were detected in 25 of 99 diffuse large B‐cell lymphoma cases. Thin lines: low copy number gain (+0.2 ≤ log₂ ratio < +1.0); thick lines: high copy number gain (amplification, log₂ ratio ≥ +1.0). Genomic amplifications were observed at 2p15–16 in three cases (D768, D778 and D792). The gray area represents the most common recurrent region in the 25 cases, which is 4.5 Mb in length.

Figure 3

Contig array‐based comparative genomic hybridization (array‐CGH) profiles at 2p15–16 in diffuse large B‐cell lymphoma. Contig array‐CGH containing 33 bacterial artificial chromosome (BAC) clones were constructed and placed contiguously at the most common recurrent region (4.5 Mb) identified with genome‐wide array‐CGH. (a) Genomic profiles of three cases with high copy number gain (log₂ ratio ≥ +1.0) at 2p15–16. (b) Genomic profiles for four cases with low copy number gain (+ 0.2 ≤ log₂ ratio < +1.0) at 2p15–16. Dots represent the log₂ ratio of BAC clones, which are shown in order from the p telomere to the centromere. Vertical lines: log₂ ratio. The thresholds for gain and loss were defined as the log₂ ratio of +0.2 and −0.2, respectively. The BAC clones are all shown contiguously. The underlined BAC clones were used for genome‐wide array‐CGH. The gray area represents the minimum common region in the 25 cases, which is 500‐kb in length between BAC clones RP11–416L21 and RP11–373L24.

Figure 4

Relationship between genomic amplification and expression of REL, BCL11A, PEX13 and LOC344423. (a) Mean log₂ ratio of all bacterial artificial chromosome (BAC) clones containing the REL locus and the BCL11A locus obtained from contig array‐based comparative genomic hybridization (array‐CGH). The thresholds for gain and loss were defined as the log₂ ratio of +0.2 and −0.2, respectively. White bars: REL locus. Black bars: BCL11A locus. (b) Relative expression levels of REL and BCL11A determined by real‐time quantitative–polymerase chain reaction (RQ‐PCR) in cases with 2p15–16 gain and those without any change at 2p15–16 (normal). For relative expression levels, each expression was normalized on the basis of the corresponding G6PDH content. White bars: REL. Black bars: BCL11A. *ND: RQ‐PCR not done for D792 because RNA was not available. (c) Mean log₂ ratio of all BAC clones containing the PEX13 locus and the LOC344423 locus obtained from contig array CGH. The thresholds for gain and loss were defined as the log₂ ratio of +0.2 and −0.2, respectively. Dotted bars: PEX13 locus. Striped bars: LOC344423 locus. (d) Relative expression levels of PEX13 and LOC344423 detected by RQ‐PCR in cases with 2p15–16 gain and of cases without any change at 2p15–16 (normal). For relative expression levels, each expression was normalized on the basis of the corresponding G6PDH content. Dotted bars: PEX13. Striped bars: LOC344423. *ND: RQ‐PCR not done for D792 because RNA was not available.