Comparison of different polymerase chain reaction-based approaches for clonality assessment of immunoglobulin heavy-chain gene rearrangements in B-cell neoplasia

Several frequently applied polymerase chain reaction strategies for analysi s of immunoglobulin heavy-chain gene rearrangements were compared by analyz ing 70 B-cell lymphoproliferative disorders and 24 reactive lymphoid lesion s. Southern blot analysis was used as the "gold standard" for clonality ass essment. For polymerase chain reaction analysis, primers directed against f ramework (FR) 3 (FR3-A and FR3-B), FR2, and FR1 of the variable gene segmen ts and against joining gene segments of the immunoglobulin heavy-chain gene were used. Polymerase chain reaction products were analyzed by high-resolu tion fingerprinting analysis using radiolabeled nucleotides, gene scanning using fluorochrome-labeled primers, and heteroduplex analysis. All of the a ssays generated polyclonal patterns in the reactive tissues. The sensitivit y in detecting monoclonality as defined by Southern blotting varied between 60% (heteroduplex analysis with FR3 primers) and 77% high-resolution finge rprinting analysis with FR2 primers. Comparison of the three FR3 assays rev ealed that gene scanning had the highest sensitivity (69%), probably becaus e it could detect small aber-rant monoclonal amplicons. False-negative resu lts were especially frequent in follicular center lymphoma (n = 20), but al so in diffuse large B-cell lymphoma (n = 18), both renowned for having muta ted variable gene segments, potentially leading to primer mismatching. For example, in follicular center lymphoma, the FR3, FR2, and FR1 region primer sets detected clonality in only 35 to 40, 65, and 50%, respectively. Combi ning these techniques, 17 (85%) of 20 follicular center lymphoma samples sh owed monoclonality. Therefore, especially in follicular center lymphoma, di ffuse large B-cell lymphoma, and, to a lesser extent, in other lymphomas, m ultiple variable gene segment primer sets must be used for a reliable asses sment of clonality. Our results also suggest that gene scanning is somewhat more sensitive than other read-out systems. Heteroduplex analysis, however , is a reliable alternative within a diagnostic setting, avoiding the use o f radioactivity or expensive automated sequencing equipment and fluorochrom e-labeled (oligo)nucleotides.