AUTOMATED HIGH-RESOLUTION PCR FRAGMENT ANALYSIS FOR IDENTIFICATION OFCLONALLY REARRANGED IMMUNOGLOBULIN HEAVY-CHAIN GENES

The development of rapid polymerase chain reaction (PCR) protocols for amplification of rearranged heavy chain immunoglobulin (IgH) gene seq uences has facilitated the identification of clonal IgH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias of B lineage. In the p resent report we have explored the recently described improved strateg y 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 auto mated fluorescence-based strategy for PCR detection of IgH gene rearra ngements. Third complementarity determining region (IgH-CDR3) sequence s were amplified using fluorescent dye labeled consensus primers homol ogous to the corresponding variable (V-H) and joining (J(H)) gene segm ents in combination with a thermostable proofreading DNA polymerase. P CR products were size separated on a high resolution polyacrylamide ge l and analyzed for clonality by exact size determination and fluoresce nce quantification in an automated DNA sequencer. PCR findings obtaine d with the optimized IgH-CDR3-PCR assay showed an overall monoclonalit y detection rate of 97% (97 of 101 cases with B cell neoplasms). The s pecificity was 100% as determined by analysis of 50 controls, all of w hich gave polyclonal PCR results. We found a high rate of monoclonal I SH-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 lgH-CDR3 junctions in 55 out of 59 cases (93 %) with B cell lymphoma and in 42 of 42 (100%) cases with leukemia, im munocytoma and multiple myeloma. The results demonstrate that automate d fluorescence detection of ISH-CDR3-PCR products is an ideal tool for detection of clonal and polyclonal lymphoid B cells. In combination w ith allele-specific primers the procedure may improve current experime ntal approaches to detect occult malginant B cells during initial stag ing and follow-up of NHL and ALL patients.