Analysis of immunoglobulin heavy chain gene rearrangement using the polymerase chain reaction.

In the July 1, 1991 issue of Blood, Trainor et all reported the results of their method for detecting gene rearrangement in Band T-lymphoproliferative disorders by using the polymerase chain reaction (PCR). The study is based on the assumption that monoclonality of B cells is characterized by a fragment length homogeneity of the amplified third-complementarity-determiningregions (CDR III), ie, a discrete DNA band on electrophoresis, whereas polyclonality is characterized by DNA fragment length heterogeneity, ie, a broad band or a smear on electrophoresis.2 They also reported that their amplification is lineage-specific (75% of specificity for B-cell tumors) showing a 2% to 5% sensitivity. Although more sensitive and specific assays for detecting monoclonality in B-cell malignancies are already available: we would like to comment on some technical aspects of Trainor’s method: (1) The separation of DNA fragments on agarose gel, also using an appropriate concentration for fragment size, cannot discriminate differences of few bases. This is commonly seen, eg, in the pattern of migration of the molecular weight markers commercially available. Because many B-cell clones have a quite similar number of nucleotides in the CDR 111, they will give a discrete band on electrophoresis, mimicking a band composed by only one clone. (2) The efficiency of the PCR is affected by many factors: primer concentration, annealing temperature, buffer composition, number of cycles, and so on. Hence, any variation in the stringency of the reaction can influence the appearance of a DNA band on agarose gel. (3) Starting with a similar approach we amplified the complete assembled variable region (VDJ) by using leader sequence degenerate primers and a JH consensus sequence primer? The known leader sequences showed highly conserved regions and then could be used as 5‘ priming site in the VDJ amplification. When our method was applied to B-cell malignancies a discrete band was always obtained on electrophoresis, but after PCR product cloning and sequencing a mixture of different clones was always detected. From five patient analysis three different results emerged: (1) several identical sequences (tumor VDJ) and polyclonal sequences; (2) polyclonal sequences only; (3) clusters of identical sequences, not corresponding to the neoplastic VDJ later identified with a different method. All of these data supported the hypothesis of a technical artifact, and one possible explanation was that a preferential priming, for some VDJs, could originate during the annealing phase, giving rise to a biased amplification of clones that were not necessarely the neoplastic one (ie, the more represented in the specimen). Based on these findings we elaborated a modified anchor-PCR strategy’ to avoid the troubles due to the preferential amplification that can occur using consensus sequence or degenerate primers. Finally, also excluding the possibility of a preferential priming during the reaction, the presence of a discrete band on electrophoresis cannot be considered a proof of monoclonality unless confirmed by sequence analysis.