Expression of FOXP1 in a Poor Prognostic Sub-Group of DLBCL Is Associated with Deregulated Expression of AID.

We have previously shown that strong, uniform expression of the transcription factor FOXP1 is associated with a poor outcome in a sub-group of activated (non-germinal centre) diffuse large B-cell lymphoma (DLBCL).

To further define this poor prognostic group we have compared gene expression profiles of 10 cases which had either FOXP1 uniform, positive expression (n=6) or were FOXP1 negative (n=4). For all cases, a non-Germinal Centre (GC) B cell phenotype, namely CD10 negative, BCL6 negative, MUM1 positive, BCL2 positive, was matched as closely as possible between the two groups, with FOXP1 expression being the solitary differential factor. Gene expression profiling using Affymetrix U133 plus 2.0 chips was performed with RNA extracted from the 10 patient samples and differentially expressed genes were identified between the 2 groups following normalisation of the results. FOXP1 positive lymphoma cells were associated with a distinct expression pattern with activation-induced deaminase (AID) being the gene determined as having the biggest difference in expression, namely an approximately 60-fold increase in expression in FOXP1 positive samples. AID is associated with both the somatic hypermutation and class switch recombination processes of immunoglobulin heavy chain (IgH) genes of normal B-cells, being primarily expressed at the GC stage of B-cell development. An RT-PCR undertaken for AID expression of the 8 cases, where further RNA was still available, confirmed that AID expression was present in FOXP1 positive cases (n=5) but either absent or very weakly expressed in FOXP1 negative cases (n=3). PCR amplification of the IgH variable (V) gene of the sample from RNA, where successful was followed by sequence analysis which showed expected levels of mutation of at least 5% variation from germline IgH V genes. Intraclonal variation of the IgH V region genes was also examined in both FOXP1 positive and negative samples, there was no correlation between the expression of AID and the occurrence of SHM or even evidence of ongoing mutation. Reports have variably suggested that AID expression is confined to GC-type DLBCL, or is heterogeneous between GC and non GC-type (activated) DLBCL. Here we have demonstrated expression of AID in association with over-expression of FOXP1 in cases that are of post-GC origin. This would suggest that AID is deregulated, being expressed beyond the normal GC stage of B-cell differentiation.

We hypothesise that FOXP1 positive DLBCL arise from cells at a specific phase of B-cell development. Normal B-cells expressing FOXP1 are found predominately in the mantle zone with a small number of cells in the GC. Ectopic expression of AID has been linked with tumour formation in mice, implying AID has oncogenic potential. Continued (or post-GC) deregulated expression of AID in these DLBCL cells may be a significant pathogenic event associated with these tumours. Normally AID is tightly regulated in B-cells, one vital function of this regulation may be to prevent lymphomagenesis. It is therefore conceivable that overexpression of FOXP1 in this poor prognostic subgroup of DLBCL results in deregulated expression of AID and is therefore a major contributing factor to lymphomagenesis.