Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma

Abstract

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.

Figure 1

(a) Correlation of BCL6 (MBR) translocation with BCL6 transcript level. The activated B-cell-like (ABC) subgroup had a significant increase in mRNA expression in the translocated cases as compared with non-translocated cases (P=0.045), but no increase was observed in the other subgroups. (b) Correlation of BCL6 translocation with BCL6 protein expression. There is a higher percentage of cases with detectable protein expression in the translocated cases in the ABC subgroup but it does not reach statistical significance. The other subgroups contain too few translocated cases for evaluation.

Figure 2

Correlation of BCL6 mutation status with (a) BCL6 transcript level, and (b) BCL6 protein expression. No significant association of BCL6 mRNA or protein expression was observed with mutation status in any of the DLBCL subgroups. DLBCL, diffuse large B-cell lymphoma.

Figure 3

Spectrum of BCL6 mutations along the major mutation cluster (MMC) in intron 1. BCL6 mutations were clustered within the MMC, in which three regions were observed to have an increased frequency of mutations. This phenomenon was observed in the GCB subgroup (B), but not readily discernable in the ABC subgroup, except for the third cluster (470–513). Insertions and substitutions are indicated by lines above the baseline, whereas deletions are represented below the baseline. Grey lines indicate single-base deletion or insertion and black lines indicate a single-base substitution. GCB, germinal center B-cell-like.

Figure 4

Correlation between BCL6 mRNA and protein expression. The ABC subgroup showed a significant association of BCL6 mRNA with protein expression, but not the GCB or the PM subgroup- (data not shown). *The immunoperoxidase assay indicated the percentage positive cells for BCL6. ABC, activated B-cell-like; GCB, germinal center B-cell-like.

Figure 5

Association of BCL6 expression and BCL6 target repression. (a) BCL6 mRNA expression is associated with BCL6 target gene repression but the pattern is variable among the different subgroups. An inverse relationship is observed between BCL6 mRNA level and BCL6 target gene expression. (b) BCL6 protein expression is associated with repression of BCL6 target genes. Again, the pattern of repression varies with the subtypes of DLBCL. 30% and 50% (not shown) cutoff for protein expression showed same pattern. DLBCL, diffuse large B-cell lymphoma.

Figure 6

Relationship between BCL6 translocation and BCL6 target gene or translocation partner gene expression. Most of the BCL6 target genes showed increased rather than decreased expression in both the ABC and GCB subgroups (for example, Id2, Blimp-1, CD80, MIP-αIP-10 and P53). Some of the known BCL6 translocation partner genes (IL-21R, MBNL, Pim-1 kinase, JAW1, CD71 and eIFF4G1) were highly expressed in non-IgH/BCL6 translocated cases. GCB, germinal center B-cell-like.

Figure 7

Association of (a) BCL6 protein, and (b) mRNA expression, with overall survival (OS) in the entire group of DLBCL. BCL6 protein expression or high mRNA expression is associated with a favorable outcome. (c) BCL6 protein expression or (d) high mRNA expression is also associated with a favorable outcome in patients with low IPI scores (0–2). DLBCL, diffuse large B-cell lymphoma.