Combined polymerase chain reaction methods to detect c-myc/IgH rearrangement in childhood Burkitt's lymphoma for minimal residual disease analysis

Abstract

BACKGROUND AND OBJECTIVES: The translocation t(8;14)(q24;q32), involving the c-myc gene (8q24) and the immunoglobulin heavy chain (IgH) locus (14q32), represents about 75% of all chromosomal translocations in Burkitt's lymphoma (BL). Due to the large variability of the breakpoint region, only the recently improved long-distance LD-polymerase chain reaction (PCR) allows the specific c-myc/IgH fusion to be identified at the genomic level. The sensitivity of the LD-PCR is only 10(-2) to 10(-3) due to the relatively large size of the amplification products (1 to 10 kbp). We, therefore, established a more sensitive nested PCR with a specific primer combination for each patient based on sequence analysis of the variant breakpoint regions. DESIGN AND METHODS: Using the combined PCR methods, we analyzed bone marrow and peripheral blood without visible blasts at diagnosis from 18 patients with t(8;14)-positive BL. RESULTS: In tests employing dilutions of genomic DNA from the BL cell line CA-46 in the T-cell lymphoma cell line KARPAS-299, which lacks the t(8;14), the sensitivity increased 100-fold, to 10(-5). However, the investigation of 18 c-myc/IgH-positive BL patients with each breakpoint-specific nested PCR showed an inter-patient variability of sensitivity between 10(-3) and 10(-5). Using this assay, the rearrangement was detected in 4/16 bone marrow samples and in 6/15 peripheral blood samples without visible blasts at diagnosis. INTERPRETATION AND CONCLUSIONS: Using the combined PCR methods the detection of c-myc/IgH reaches a level of sensitivity required for the evaluation of minimal residual disease (MRD) in BL patients. Furthermore, the results highlight the importance of verifying the patient-specific sensitivity level for individual MRD monitoring.