High frequency of t(14;18)-translocation breakpoints outside of major breakpoint and minor cluster regions in follicular lymphomas: improved polymerase chain reaction protocols for their detection

Abstract

The detection of t(14;18) translocations is widely used for the diagnosis and monitoring of follicular lymphomas displaying a high prevalence for this aberration. Cytogenetics, Southern blotting, and polymerase chain reaction (PCR) are commonly used techniques. It is generally believed that the vast majority of the breakpoints occurs on chromosome 18 in the major breakpoint region (mbr) and the minor cluster region (mcr). Yet, by improving long-distance PCR protocols we identified half of the breakpoints outside of these clusters. Our study included biopsies from 59 patients with follicular lymphoma. Seventy-one percent carried translocations detectable with our long-distance PCR protocol. The novel primer sets were derived from the hitherto uncharacterized 25-kb-long stretch between mbr and mcr that we have sequenced for this purpose. Sequence analysis of the novel breakpoints reveals a wide distribution between mbr and mcr displaying some clustering 16 kb downstream from the BCL2 gene. By including a primer for this intermediate cluster region in standard PCRs we could also improve the detection of t(14;18) translocations in formalin-fixed and paraffin-embedded biopsies. Our new PCRs are highly sensitive, easy to perform, and thus well suited for routine analysis of t(14;18) translocations for the primary diagnosis of follicular lymphoma and surveillance of minimal residual disease.

Figure 1.

PCR cloning of the genomic region spanning mbr and mcr. A: Schematic representation of the 3′BCL2 gene locus. Black bars on top indicate the localization of previously determined sequences (A, M14745; B, AB010948; C, AB010949; D, sequence from Ngan et al 12 ). Two large fragments containing the unknown sequences were amplified with LD-PCR (primer pairs A2-up/A2-low and B4-up/B4-low, respectively). B: Gel electrophoresis of LD-PCR products derived from human genomic DNA. Two percent of the reaction volumes were run on a 0.5% agarose gel and stained with ethidium bromide.

Figure 2.

Detection of t(14;18) translocations with standard PCR analysis. Representative examples of the initial screening of patients’ samples with standard PCRs using s-MBR/s-JH and s-mcr/s-JH primer pairs, respectively. Twenty percent of the amplification volumes were loaded on a 2% agarose gel stained with ethidium bromide. Direct sequencing of the PCR products confirmed breakpoints in the mbr (patient 14) and mcr (patient 21). No amplification product was obtained from the sample of patient 36 despite the presence of a t(14;18) translocation (for breakpoint determination see Table 2 ▶ and Figure 4 ▶ ). A control amplification of a 268-bp genomic fragment was included in each analysis to verify the quality of the DNA extracts. Furthermore, negative reagent controls were performed each time to exclude contamination.

Figure 3.

Localization of primers used in long-distance and modified standard PCRs for the detection of t(14;18) translocations. Position of upper primers in close vicinity to the BCL2 gene locus on chromosome 18 are shown together with their lower strand primer partners localized in the IGH Eμ enhancer region or the JH elements on chromosome 14. Long-distance PCR screening was initially done with primer set A and extended with primer set B (lower primer: Eμ-low). Improved standard PCR analysis included s-MBR, s-mcr, and the new s-icr upper primer together with a JH consensus primer (s-JH).

Figure 4.

Representative examples of t(14;18) LD-PCR analysis. A: Products of LD-PCRs with primer sets A and B were run on 0.5% agarose gels and stained with ethidium bromide. The DNA quality of each extract was tested in a reaction amplifying a 13.3-kb (set A) or a 11.5-kb control fragment (set B). Depending on breakpoint location a unique amplification pattern was observed and the approximate position of the breakpoint on chromosome 18 was established. Direct sequencing of the LD-PCR products revealed in patient 21 a breakpoint in the mcr region, in patient 32 a localization 9.5 kb downstream from MBR, and in patient 36 a translocation with involvement of the icr. B: The neoplastic cells of all three patients expressed Bcl-2 as revealed by immunohistochemistry. Scale bar, 50 μm.

Figure 5.

Distribution of t(14;18) breakpoints on chromosome 18. The exact positions of the breakpoints on the 3′BCL2 genomic locus were determined by sequencing of the PCR products. Black arrowheads indicate breakpoints being only detectable by LD-PCR. Breakpoints within the MBR and mcr clusters, which were also amplified by standard PCRs, are shown with gray arrowheads. In certain regions clustering of breakpoints was observed. In those cases, the numbers of breakpoint in close vicinity to each other are indicate above the arrowheads.

Figure 6.

Improved standard PCR analysis for the detection of t(14;18) translocation in formalin-fixed tissues. By supplementing the standard MBR- and mcr-specific PCR analysis with the s-icr/JH primer pair, frequently occurring breakpoints within the icr can be detected in extracts even from formalin-fixed and paraffin-embedded biopsies (example: patient 33; 2% agarose gel electrophoresis, ethidium bromide staining).

Figure 7.

Frequency of t(14;18) translocations detected in follicular lymphomas by standard and LD-PCR assays. In comparison to the standard PCR protocols using MBR and mcr primers, our LD-PCR assay greatly increases the detection rate of t(14;18) translocations in follicular lymphoma samples from European patients (from 36 to 71%). Some improvement of the standard PCR can be achieved by including mbr-, mcr-, and icr-specific primers in the analysis (from 36 to 46%). Numbers in the columns indicate number of patients.