Immunoglobulin (Ig) gene somatic hypermutation (SHM) is a prognostic factor in small B-cell lymphomas, as demonstrated by studies reporting that hypermutated chronic lymphocytic leukemia (CLL) and splenic marginal zone lymphoma (SMZL) cases show a better prognosis, while hypermutated mantle cell lymphoma (MCL) cases display specific clinicopathological features (leukemic course, increased survival). However the mechanisms and markers of SHM are poorly characterized, with the partial exception of the role of the genes ZAP70 and AID. With the purpose of identifying SHM surrogate markers in small B-cell lymphomas, we analyzed IgVH mutational status and expression profiles of 93 small B-cell lymphoma patient samples including SMZL (24 cases), MCL (33 cases) and CLL (36 cases). Patients were classified into two groups: high SHM (>5% mutations) and low SHM (<5% mutations). T-test analysis with 100,000 permutations was performed and 39 genes were identified whose expression is significantly different (p<0.005, FDR<0.05) between high and low SHM groups. Furthermore, patterns associated with ongoing SHM in patient samples were also analyzed. To dissect the molecular mechanisms of Ig hypermutation and validate the observed findings, SHM was induced in a model system and compared to results in patient samples. SHM was induced in the BL2 cell line by treating the cells with IL4 and CD40. After 24 hours, active SHM was confirmed by western blot using an anti-AID monoclonal antibody on treated and untreated cells. These same cells were analyzed using microarrays to identify the genes which were induced or downregulated during the SHM activation. A total of 29 genes (18 upregulated and 11 downregulated) were identified which are up/down-regulated in the cell line model during induction of SHM (expression change >0.4 Log2 scale) and are also differentially expressed between cases with high and low SHM (p<0.001, FDR<0.1). In all analyses, the upregulated genes identified are implicated in transcription, DNA repair and replication and chromosome maintenance, correlating well with previous hypotheses indicating the necessity of active transcription for SHM. Based on these observations, a group of 10 key genes, implicated in DNA repair, replication and transcription, were selected and protein expression was analyzed in a set of Tissue Micro Arrays containing 150 paraffin embedded small B-cell lymphoma cases, for which clinical data is available and SHM status is known. Provisional results confirm that many of these genes including cell cycle regulators of the cyclin dependent kinase family are associated with SHM and may be useful as surrogate markers for analysis of IgVH SHM in patient samples using paraffin-embedded tissue samples.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]