Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes.

The development of rapid polymerase chain reaction (PCR) protocols for amplification of rearranged heavy chain immunoglobulin (IgH) gene sequences has facilitated the identification of clonal IgH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias of B lineage. In the present report we have explored the recently described improved strategy for assessment of clonality of rearranged immunoglobulin heavy chain (IgH) genes in more detail in a series of 101 B cell malignancies and 50 polyclonal controls. The assay is based on an IgH-PCR with an automated fluorescence-based strategy for PCR detection of IgH gene rearrangements. Third complementarity determining region (IgH-CDR3) sequences were amplified using fluorescent dye labeled consensus primers homologous to the corresponding variable (V[H]) and joining (J[H]) gene segments in combination with a thermostable proofreading DNA polymerase. PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. PCR findings obtained with the optimized IgH-CDR3-PCR assay showed an overall monoclonality detection rate of 97% (97 of 101 cases with B cell neoplasms). The specificity was 100% as determined by analysis of 50 controls, all of which gave polyclonal PCR results. We found a high rate of monoclonal IgH-CDR3-PCR results not only in the leukemias and diffuse lymphoma but also in the group of follicular lymphoma, where a high rate of false negative results is frequently reported in the literature. In summary, we identified monoclonal IgH-CDR3 junctions in 55 out of 59 cases (93%) with B cell lymphoma and in 42 of 42 (100%) cases with leukemia, immunocytoma and multiple myeloma. The results demonstrate that automated fluorescence detection of IgH-CDR3-PCR products is an ideal tool for detection of clonal and polyclonal lymphoid B cells. In combination with allele-specific primers the procedure may improve current experimental approaches to detect occult malginant B cells during initial staging and follow-up of NHL and ALL patients.

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 cyclin D1 gene also designated as bcl-1/PRAD1 gene on chromosome 11 and the 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 primers in combination with a consensus immunoglobulin JH primer. A total of 65 cases histologically classified as mantle cell lymphoma (MCL) were analyzed for the presence of a t(11;14) translocation and monoclonal IgH-CDR3 rearrangements. From 26 patients with classical MCL and three cases with the anaplastic variant of MCL fresh frozen biopsy material was available for DNA extraction. We detected a bcl-1/JH rearrangement in 12 out of 29 samples (41%). In 36 cases paraffin-embedded lymph node tissue was the only source of DNA. In this material we found a bcl-1/JH rearrangement in six out of 31 samples with intact DNA (20%). To confirm the specificity of the PCR and to determine the bcl-1/JH junctional region sequences as clone-specific marker in individual patients we characterized the junctional DNA sequences by direct PCR sequencing in 16 cases. Interestingly we found that six bcl-1/JH junctions harbored DH segments in their N regions indicating that bcl-1/JH rearrangements can occur in a later stage of B cell ontogeny during which the complete VH to DH-JH joining or VH-replacement takes place. To investigate the suitability of IgH-CDR3 as sensitive molecular marker for those MCL patients in which a t(11;14) translocation can not easily be amplified, we additionally analysed 60 cases for the presence of monoclonally rearranged IgH genes by IgH-CDR3-PCR. A monoclonal 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 paraffin-derived DNA were positive. We demonstrate that automated fluorescence detection of monoclonal IgH-CDR3 PCR products allows the rapid and sensitive monitoring of minimal residual disease also in cases that lack a PCR amplifiable t(11;14) translocation. In combination with allele-specific primers the procedure may improve current experimental approaches for detection of occult MCL cells at initial staging and residual disease during and after therapy.