[1273] Detection of the IGH@-BCL2 Translocation in Follicular Lymphoma by a Novel DNA-Based Looped Ligation Assay (LOLA).

Shuko Harada, Emily Silzle, Ming-Tseh Lin, Christopher D Gocke. Johns Hopkins Medical Institutions, Baltimore, MD

Background: Follicular lymphoma (FL) usually harbors a disease-defining IGH@-BCL2 translocation. Detection of this translocation is complicated by widely-distributed breakpoints in the BCL2 gene, which include the major breakpoint regions 1 and 2 (MBR1, MBR2) and the minor cluster region 1 (mcr1). We have recently developed a ligation-based assay (LOLA) for long range haplotype mapping. This assay can be adapted to detect translocations from DNA samples for a number of diseases that have multiple or widely spaced breakpoints. In this study, we employed LOLA to detect IGH@-BCL2 translocations in the DNA of FL cell lines as well as frozen tissue from FL patients.
Design: LOLA identifies linkage between widely separated genetic loci. Five sets of oligonucleotides were designed to cover all translocations involving MBR1, MBR2, 3'MBR, 5'mcr and mcr1 and at least 40kb around each. DNA was isolated from FL cell lines (SU-DHL4, SU-DHL6 and SU-DHL16) that harbor different BCL2 breakpoints (MBR2, MBR1 and mcr1, respectively), from frozen tumor tissue of 17 newly diagnosed or relapsed FL patients, and from normal spleen, lymph node and bone marrow. DNA (5-500 ng) and oligos were mixed and the ligation reaction was carried out at 60°C for 1 hr. PCR was then performed with primers complimentary to M13 tails on the outermost oligonucleotides and the diagnostic products were identified using capillary electrophoresis (ABI 3130) and GeneMapper software (ABI).
Results: The LOLA produced a specific diagnostic peak at either 125 nt (MBR2, SU-DHL6 cells), 128 nt (MBR1, SU-DHL4), or 124 nt (mcr1, SU-DHL16), depending on the pertinent probe set. A dilution series of SU-DHL6 DNA in normal human DNA (500 ng total) yielded a specific peak at dilutions as low as 1%. The log of peak intensities was linear to the log of SU-DHL6 DNA concentration (R2=0.94). There was no LOLA peak detected with DNA from normal control tissue. Among 17 FL cases, two were excluded due to bad quality of DNA (no internal control peak). Eight cases had a definite LOLA peak–indicating IGH@-BCL2 translocations–and 5 cases had a weak peak. Two cases did not have any LOLA peaks (t(14;18) FISH was also negative).
Conclusions: Our results show that a LOLA to detect translocations is sensitive and specific. In particular, the diagnostic IGH@-BCL2 translocation can be identified in most cases of FL, and the assay permits a rough localization of the breakpoints. This novel assay will be broadly applicable for DNA-based detection of translocations, particularly when breakpoint hotspots are large or multiple.
Category: Hematopathology

Wednesday, March 2, 2011 9:30 AM

Poster Session V # 151, Wednesday Morning

 

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