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Detection of the t(14;18) translocation in frozen and formalin-fixed tissue
1Department of Pathology, Sylvius Laboratory, Leiden University, The Netherlands.
Abstract:
As part of a retrospective study into the prevalence of the t(14;18) translocation in B-cell lymphomas, we assessed the suitability of the polymerase chain reaction (PCR) to amplify the t(14;18) major breakpoint region (MBR) in frozen and formalin-fixed tissue. Considering Southern blotting as a standard, the sensitivity of PCR was 81%. Of the various procedures used to extract DNA from paraffin-embedded tissue (PET), proteinase K digestion in the presence of nonionic detergents gave the highest yield and quality of DNA and the most efficient amplification rate. Using this method, excellent amplification rates (100%) were obtained for both the beta-globin control sequence and the MBR t(14;18) for fixed follicular lymphoma specimens collected in the previous 2 to 6 years (n = 27). Of nine older PETs, PCR on six gave inconsistent results, probably because of the poorer-quality substrate used for amplification. Specimens exposed to formol sublimate or formalin-acetic acid-alcohol were as suitable for amplification as tissues fixed in neutral-buffered formalin. The overall incidence of the MBR t(14;18) in all follicular lymphoma specimens as detected by both Southern blotting and PCR was 59% (23 of 39).
Insights
Polymerase chain reaction (PCR) effectively detects the t(14;18) translocation in B-cell lymphomas using DNA from formalin-fixed paraffin-embedded tissues. This method shows high sensitivity and is suitable for diagnosing follicular lymphoma.
Area of Science:
- Hematology
- Molecular Biology
- Oncology
Background:
- The t(14;18) translocation is a hallmark genetic alteration in B-cell lymphomas, particularly follicular lymphoma.
- Accurate detection of this translocation is crucial for diagnosis and prognosis.
- Traditional methods like Southern blotting can be labor-intensive and require fresh tissue.
Purpose of the Study:
- To evaluate the utility of polymerase chain reaction (PCR) for detecting the t(14;18) major breakpoint region (MBR) in both frozen and formalin-fixed tissues.
- To optimize DNA extraction methods from paraffin-embedded tissues (PET) for efficient PCR amplification.
- To determine the prevalence of the t(14;18) MBR in follicular lymphoma specimens.
Main Methods:
- Retrospective analysis of frozen and formalin-fixed tissue samples from B-cell lymphoma patients.
- Comparison of PCR sensitivity against Southern blotting as the gold standard.
- Optimization of DNA extraction protocols from PET, focusing on proteinase K digestion.
- Assessment of DNA quality and amplification efficiency using beta-globin as a control sequence.
- Evaluation of different tissue fixation methods on PCR suitability.
Main Results:
- PCR demonstrated 81% sensitivity for detecting the t(14;18) MBR compared to Southern blotting.
- Proteinase K digestion with nonionic detergents yielded the highest quality DNA from PET, leading to efficient amplification.
- Excellent amplification rates (100%) were achieved for recent PETs (2-6 years old) and the beta-globin control.
- Older PETs yielded inconsistent PCR results, likely due to substrate degradation.
- Various fixation methods (formol sublimate, formalin-acetic acid-alcohol, neutral-buffered formalin) were suitable for PCR.
- The overall incidence of the t(14;18) MBR in follicular lymphoma was 59% (23/39) by combined PCR and Southern blotting.
Conclusions:
- PCR is a sensitive and reliable method for detecting the t(14;18) MBR in formalin-fixed paraffin-embedded tissues.
- Optimized DNA extraction using proteinase K is essential for successful PCR amplification from PET.
- PCR offers a valuable tool for assessing the prevalence of the t(14;18) translocation in B-cell lymphomas, including follicular lymphoma.
- The study confirms the high incidence of the t(14;18) translocation in follicular lymphoma.