Background: The t(8;14) translocation in Burkitt’s lymphoma (BL) was the first
non-random cytogenetic lesion to be described in lymphoproliferative
disorders. This lesion occurs in 75-85% of all BL cases. However, the
breakpoints in this cytogenetic lesion are very variable and far apart such that
the t(8;14) translocation is not always amenable to standard polymerase chain
reaction analysis. This is mainly due to the inability of the Thermus aquaticus
(Taq) polymerase enzyme to synthesize long DNA products. Long range
polymerase chain reaction (LD-PCR) with a high fidelity polymerase enzyme
mix capable of longer PCR product synthesis has recently become available.
In early studies, LD-PCR appeared to be capable of amplifying the t(8;14)
translocation in the majority of published sporadic Burkitt’s lymphoma
analyses. The utility of t(8;14) translocation LD-PCR for routine use in the
diagnosis of BL in our setting has not yet been studied. The aim of this study
was to establish and optimize the t(8;14) LD-PCR technique and to apply it in
the retrospective analysis of all BL diagnosed in the University of the
Witwatersrand teaching hospitals in a ten year period from January 1994 to
December 2003.
Materials and methods: High molecular weight non-degraded DNA was extracted from control cell lines as well as stored, unstained bone marrow
slides remaining after routine diagnostic workup of previously identified
Burkitt’s lymphoma patients. Three hundred nanograms of patient and control
DNA were amplified with the LD-PCR high fidelity polymerase enzyme mix
under reaction conditions which were optimized using the tissue plasminogen activator (tPA) gene as well as known Burkitt’s lymphoma cell lines as controls.
Each control and patient DNA sample was amplified with tPA primers as well
as four pairs of MYC/IgH primer sets. The resulting amplicons were size
fractionated on an agarose gel and visualized with ethidium bromide under
ultraviolet (UV) light. The fractionated DNA fragment sizes were compared to
those of the t(8;14) translocation positive controls, tPA controls and known
DNA molecular weight markers.
Results: One hundred and ten Burkitt’s lymphoma diagnoses were made in
the three teaching hospitals of the University of the Witwatersrand from
January 1994 to December 2003. Bone marrow involvement by BL was
present in 84 of these cases. Archival bone marrow slides were available in 74
of the 84 BL patients. Intact high molecular DNA on which the t(8;14) LD-PCR
analysis could be performed was present in 41 of the 74 BL patients. In the
presence of appropriate controls, an t(8;14) translocation specific product was
demonstrable by t(8;14) LD-PCR analysis in only 6 of 41 BL patients.
Conclusion: In this t(8;14) LD-PCR retrospective analysis of a large number
known Burkitt’s lymphomas, the diagnostic yield in carefully selected patients was extremely poor. With five primer pairs required per BL sample analysis,
this technique was found too labour intensive and costly in our hands making it
unsuitable for routine diagnostic use. The reasons for the poor diagnostic yield
remains unclear and may need to be explored in future studies. Emerging
alternative techniques for the diagnosis of BL such as fluorescence in situ
hybridization and microarray gene expression analyses may prove to be better
diagnostic tools than LD-PCR in its current form.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/5766 |
| Date | 20 October 2008 |
| Creators | Mahlangu, Johnny Ndoni |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
Page generated in 0.002 seconds