Molecular Characterization of a Chromosomal Translocation Linking CDK6 to the IGK Locus In CD5- Monoclonal B-Cell Lymphocytosis (MBL).

Abstract 1191

Efforts to decipher the biological basis of lymphoid malignancy have recently been accentuated by the discovery of clonal B-cells in otherwise healthy individuals, an entity now referred to as monoclonal B-cell lymphocytosis (MBL). However, while considerable attention has been devoted towards characterizing this pervasive condition, the subgroup of MBL lacking CD5 expression has been largely overlooked. To address this imbalance, our group previously examined a series of cases presenting with persistent but non-progressing CD5- clonal lymphocytosis (Amato D et al, Am J Clin Pathol 2007). Herein, karyotypic analysis revealed a unique case harboring the translocation t(2;7)(p11;q22), a genetic abnormality which has been documented in association with splenic marginal zone lymphoma (SMZL). The present study set out to further characterize the t(2;7) breakpoint of this novel case study in order to test whether the translocation represents a common genetic link between CD5- MBL and SMZL. Using fluorescent in situ hybridization (FISH), the breakpoint on chromosome 7 was initially narrowed down to a 41 kilobase (kb) candidate region at 7q21.2, as inferred by a split signal from the cosmid probe cos130a6. Based on the proximity of the chromosome 2 breakpoint to the immunoglobulin kappa (IGK) locus on 2p11, long-range polymerase chain reaction (L-PCR) was subsequently performed according to the hypothesis that the t(2;7) translocation had arisen from aberrant VJ recombination. Primers targeting conserved sequences within the IGK variable genes were sequentially juxtaposed with primers on chromosome 7 located at 6 kb intervals across the 41 kb candidate region, and L-PCR conducted using both patient and reference DNA as a template. This process gave rise to a single instance of strong patient-specific amplification, thereby indicating successful targeting of the t(2;7) breakpoint. Sanger sequencing of the corresponding PCR product confirmed this result, revealing a breakpoint at 2p11.2 localized to the heptamer recombination signal sequence (RSS) of the IGK variable gene V3-15, alongside a breakpoint at 7q21.2 located 3.6 kb upstream of the transcription start site of the cell cycle regulator cyclin dependent kinase 6 (CDK6). Remarkably, the breakpoint on chromosome 7 showed perfect sequence homology to the immunoglobulin RSS heptamer, and was within 3 base pairs of a breakpoint previously characterized in association with SMZL (Corcoran M et al, Oncogene 1999). Hence, this result implicates the dysregulation of CDK6 during the clonal expansion observed in CD5- MBL, and highlights the potential role of aberrant VJ recombination during the formation of this genetic abnormality. By establishing a clear genetic link between CD5- MBL and SMZL, this study supports the notion of CD5- MBL as a precursor of overt malignancy within a ‘multi-hit’ model of oncogenesis. Finally, the sequence-level characterization of a novel translocation breakpoint by L-PCR strongly validates the potential utility of this approach during future studies of immunoglobulin-associated translocations.