STRUCTURE OF BCL-1 AND IGH-CDR3 REARRANGEMENTS AS CLONAL MARKERS IN MANTLE CELL LYMPHOMAS

Mantle cell lymphoma represent a clinicopathologically distinct entity of malignant non-Hodgkin's lymphoma (NHL) and are characterized by a specific chromosomal translocation t(11;14)(q13;q32) involving the cyc lin D1 gene also designated as bcl-1/PRAD1 gene on chromosome 11 and t he heavy chain immunoglobulin joining region on chromosome 14. We have established a PCR method to amplify t(11;14) junctional sequences in DNA from fresh frozen and paraffin-embedded tissue by bcl-1-specific p rimers in combination with a consensus immunoglobulin J(H) primer. A t otal of 65 cases histologically classified as mantle cell lymphoma (MC L) were analyzed for the presence of a t(11;14) translocation and mono clonal IgH-CDR3 rearrangements. From 26 patients with classical MCL an d three cases with the anaplastic variant of MCL fresh frozen biopsy m aterial was available for DNA extraction. We detected a bcl-1/J(H) rea rrangement in 12 out of 29 samples (41%). In 36 cases paraffin-embedde d lymph node tissue was the only source of DNA. In this material we fo und a bcl-1/J(H) rearrangement in six out of 31 samples with intact DN A (20%). To confirm the specificity of the PCR and to determine the bc l-1/J(H) junctional region sequences as clone-specific marker in indiv idual patients we characterized the junctional DNA sequences by direct PCR sequencing in 16 cases. Interestingly we found that six bcl-1/J(H ) junctions harbored D-H segments in their N regions indicating that b cl-1/J, rearrangements can occur in a later stage of B cell ontogeny d uring which the complete V-H to D-H-J(H) joining or V-H-replacement ta kes place. To investigate the suitability of IgH-CDR3 as sensitive mol ecular marker far those MCL patients in which a t(11;14) translocation can not easily be amplified, we additionally analysed 60 cases for th e presence of monoclonally rearranged IgH genes by IgH-CDR3-PCR. A mon oclonal IgH-CDR3 PCR product could be identified in 24 out of 29 fresh frozen samples (79%) whereas only 11 out of 31 samples (36%) with par affin derived DNA were positive. We demonstrate that automated fluores cence detection of monoclonal IgH-CDR3 PCR products allows the rapid a nd sensitive monitoring of minimal residual disease also in cases that lack a PCR amplifiable t(11;14) translocation. In combination with al lele-specific primers the procedure may improve current experimental a pproaches for detection of occult MCL cells at initial staging and res idual disease during and after therapy.