Multiplex PCR reaction for the detection and identification of immunoglobulin kappa deleting element rearrangements in B-lineage leukaemias

Immunoglobulin kappa (Ig kappa) gene recombinations can be used - similarly to IgH rearrangements - as clonal markers in B-lineage leukaemias. Based o n the extensive junctional diversity, these rearrangements represent valuab le targets for the analysis of minimal residual disease (MRD). In order to provide a simple method for the rapid detection of leukaemia clone-specific kappa deleting element (Kde) mediated rearrangements, we developed a multi plex PCR reaction that is able to amplify the five most frequent rearrangem ents in one tube. Position of the amplimers were chosen to enable identific ation of the involved segments according to the size of the PCR product. Th is method was tested on 101 B-lineage leukaemias (71 childhood B-cell precu rsor acute lymphoblastic leukaemias (BCP ALL) and 30 chronic lymphocytic le ukaemias (CLL)). 39 and 22 Kde rearrangements could be readily detected in 30 (44%) BCP ALL and 22 (56%) CLL, respectively. 36% of the Kde rearrangeme nts in BCP ALL and 45% in CLL were intron recombination signal sequence (RS S)-Kde rearrangements. The other Kde rearrangements involved the V kappa fa milies: V kappa I in 36% and 50%, V kappa II in 32% and 16.7%. V kappa III in 24% and 25%, and V kappa IV in 8% and 8.3% in BCP ALL and CLL, respectiv ely. The sensitivity of the multiplex system was 10(-2)-10(-3). We compared this multiplex PCR assay with multiple single PCR reactions using differen t sets of primer combinations. Thereby the number and types of rearrangemen ts were confirmed in all cases, Clonality of rearrangements was proven by s equence analysis. Our data show that by this method clonal Kde rearrangemen ts were rapidly detected and precisely identified.