Improved long-distance polymerase chain reaction for the detection of t(8;14)(q24;q32) in Burkitt's lymphomas

Abstract

The t(8;14)(q24;q32), involving MYC gene (8q24) and the immunoglobulin heavy chain (IgH) locus (14q32), represents about 75% of all translocations in Burkitt's lymphoma (BL). Due to the great variability of the breakpoint region, a standard polymerase chain reaction assay is not sufficient for the detection of this chromosomal translocation. The availability of new and more efficient DNA polymerases that allow the amplification of genomic fragments many kilobase-pairs long, makes it possible to identify the t(8;14) in BL cells by long-distance polymerase chain reaction (LD-PCR). We have established a simplified and efficient LD-PCR for the detection of t(8;14)(q24;q32) that relies on the use of one primer specific for MYC exon II combined, in different reactions, with four primers for the IgH locus: three for the constant regions Cmu, Cgamma, and Calpha, and one for the joining region (JH). We first studied seven BL cell lines and optimized LD-PCR reaction for analysis of tumor specimens. Five of seven cell lines were positive for the t(8;14), whereas two lines derived from endemic BL were negative, as expected. Of 15 biopsies obtained from pediatric BL and subsequently analyzed, 13 (87%) were positive for the translocation detected by LD-PCR and showed a product ranging in size from 2.0 to 9.5 kb. Cmu region was involved in 6 cases, Cgamma and Calpha in 2 cases each, and JH in 3 cases. Interestingly, 2 of the tumors positive for JH showed a second, larger PCR product with the Calpha- and Cgamma-specific primer, respectively. We established that our LD-PCR method could detect 10(-3) BL cells within a population of hematopoietic cells lacking the translocation. In conclusion, our LD-PCR method represents a fast, highly sensitive, and specific tool to study sporadic BL and to detect minimal disease and residual disease in patients affected by t(8;14)-positive lymphomas.

Figure 1.

Schematic representation of t(8;14)(q24;q32) in sporadic BL. The breakpoint on chromosome 8q24 usually occurs in the region of exon 1 or intron 1 of the MYC gene, which is juxtaposed to the immunoglobulin heavy-chain (IgH) gene on chromosome 14q32, in opposite orientation. In the chromosome 14q+ scheme, arrows indicate the regions recognized by each primer used in our study.

Figure 2.

LD-PCR analysis of BL cell lines. DNA from 7 BL cell lines was processed in the LD-PCR assay for the detection of t(8;14). The cell lines BL41, CA46, DG75, RAJI, and RAMOS showed a PCR product ranging from 1.5 to 7.6 kb in an ethidium bromide-stained agarose gel (A). They represent the positive controls for all four of the primer combinations selected for our study. Hybridization to a MYC-specific digoxigenin-labeled oligonucleotide probe (MYC/06) demonstrates the specificity of the reaction (B).

Figure 3.

Sensitivity of the t(8;14) LD-PCR for the detection of BL cells. Increasing dilution of BL41 cells were prepared in acute T lymphoblastic leukemia cells (Jurkat). DNA was extracted and used as a template for LD-PCR. The PCR product was visible in ethidium bromide-stained gels at a ratio of BL41/Jurkat cells as low as 10⁻³ (A). Southern blot and subsequent hybridization to a MYC specific digoxigenin labeled oligonucleotide probe (MYC/06) increased the detectability of the signal (B).

Figure 4.

LD-PCR analysis of BL tumor samples. Fifteen pediatric BL tumors were studied by LD-PCR and the products were analyzed by gel electrophoresis on agarose gels and subsequently stained with ethidium bromide. The results for a representative group of patients are illustrated in A. Specificity of the PCR products was demonstrated by hybridization to a MYC-specific oligonucleotide probe (MYC/06) after Southern blotting (B).

Figure 5.

LD-PCR analysis of case B14. The t(8;14) translocation was studied by LD-PCR combining, in different reactions, the MYC/04 primer with the Cγ/02, Cμ/03, Cα/01, and JH primers, respectively. A PCR product was obtained with two different primer couples. The tPA primers were used to confirm the amplifiability of the DNA.