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[Detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma by hemi-nested PCR]
Hui-Yong Jiang1, San-Quan Zhang, Xi-Qun Han
1Department of Pathology, Nan Fang Hospital, Guangzhou 510515, China.
Insights
A new hemi-nested PCR method improves detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma (DLBCL). This approach minimizes false positives, enhancing diagnostic accuracy for this cancer.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Context:
- Diffuse large B cell lymphoma (DLBCL) is a common non-Hodgkin lymphoma.
- Accurate detection of the bcl-2/IgH gene rearrangement is crucial for DLBCL diagnosis and prognosis.
- Existing PCR methods can yield false positive results due to primer homology.
Purpose:
- To develop and validate a sensitive and specific hemi-nested PCR method for detecting bcl-2/IgH gene rearrangement in DLBCL.
- To assess the credibility and accuracy of the established method compared to traditional approaches.
Summary:
- A hemi-nested PCR technique was designed using specialized software to detect bcl-2/IgH gene rearrangement.
- Fifty-two DLBCL samples and 10 tonsil tissues were analyzed. The hemi-nested PCR identified 5 positive DLBCL cases, all confirmed by sequencing, while ruling out false positives seen with a one-way method.
- Sequencing revealed that false positives from the one-way method showed homology to human genomic sequences.
Impact:
- The hemi-nested PCR method significantly improves the specificity of bcl-2/IgH gene rearrangement detection in DLBCL.
- This enhanced diagnostic accuracy can lead to better patient stratification and treatment decisions.
- The findings highlight the importance of primer design in molecular diagnostics to avoid false positives.
Objectives:
To explore a sensitive and specific method for detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma (DLBCL), and verify the credibility of the established method.
Methods:
bcl-2/IgH hemi-nested PCR primers were designed using the professional primer design software. Fifty-two samples of pathologically diagnosed DLBCL and 10 fresh tonsil tissues were amplified using hemi-nested touch down-PCR to detect bcl-2/IgH gene rearrangement. The PCR products were cloned and sequenced.
Results:
bcl-2/IgH gene rearrangement was detected in 6 of 52 DLBCL samples and 2 of 10 fresh tonsil tissues using one-way method. By using the hemi-nested PCR for the second round amplification, 5 of DLBCL were positive, but all of the fresh tonsil tissues were negative. The positive PCR products were sequenced and analyzed on the Internet, 3 of 8 cases obtained by one-way method were false positive, 5 positive cases amplified using hemi-nested PCR were all bcl-2/IgH gene rearrangement. PCR products of 3 false positive cases were homologous to BAC331191 and LLNLR-245D11 in human chromosome 19 and RP11-498P10 in chromosome 1.
Conclusion:
There are false positive results using common primers for detecting bcl-2/IgH gene rearrangement. The mechanism may be that highly homologous sequences to human genome exist in commonly used primers. The specificity of the diagnosis could be improved by hemi-nested PCR using the combination of primers we designed and the traditional ones.
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