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PCR-based clonality analysis in diffuse large B-cell lymphoma using BIOMED-2 primers of IgH (FR3) on formalin-fixed
Farid Kosari1, Fatemeh Shishehbor, Hiva Saffar
1Department of Pathology, Dr. Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran. Faridkosari@yahoo.com
Insights
This study optimized the BIOMED-2 polymerase chain reaction (PCR) protocol for detecting B-cell clonality in diffuse large B-cell lymphoma (DLBCL) using immunoglobulin heavy chain (IgH) framework region 3 (FR3) primers. The modified assay achieved a 62.8% detection rate in FFPE tissues, improving diagnostic accuracy.
Area of Science:
- Hematology
- Molecular Biology
- Oncology
Background:
- The BIOMED-2 polymerase chain reaction (PCR) protocol is standard for lymphoma clonality assessment.
- Diffuse large B-cell lymphoma (DLBCL) poses challenges for PCR due to high somatic hypermutation rates in immunoglobulin (Ig) domains.
- Detecting B-cell clonality in formalin-fixed paraffin-embedded (FFPE) DLBCL tissues requires optimized methods.
Purpose of the Study:
- To evaluate the efficacy of immunoglobulin heavy chain (IgH) framework region 3 (FR3) primers for detecting B-cell clonality in DLBCL.
- To optimize a PCR-based assay for FFPE tissues, addressing DNA degradation issues.
- To assess the impact of sample storage duration on DNA quality and clonality detection rates.
Main Methods:
- Assessed 100 DLBCL FFPE samples diagnosed between 2005 and 2011.
- Employed a modified PCR amplification method using IgH (FR3) primers optimized for FFPE tissue.
- Incorporated a filter for enhanced DNA purification, particularly in negative cases.
Main Results:
- The modified IgH (FR3) assay achieved a clonal detection rate of 62.8%.
- DNA quality was significantly better in FFPE samples stored for less than five years compared to older blocks (P < 0.001).
- While a trend towards higher clonality in recent samples was observed, it was not statistically significant.
Conclusions:
- The optimized IgH (FR3) primer assay, with an additional DNA purification step, provides a considerable rate of B-cell clonality detection in DLBCL.
- This method effectively mitigates the adverse impact of DNA degradation in FFPE tissues.
- The findings support the utility of this modified PCR approach for improved DLBCL diagnosis.
Introduction:
BIOMED-2 multiplex polymerase chain reaction (PCR) protocol is a widely accepted tool for evaluation of clonality in lymphoma diagnosis. Diffuse large B-cell lymphoma (DLBCL) is the most common non- Hodgkin,s lymphoma and displays a special challenge for PCR-based clonality analysis due to a high frequency of somatic hypermutation in rearranged immunoglobulin (Ig) domains. In this study, we evaluated detection of B- cell clonality in DLBCL by using Ig heavy chain (IgH) framework region 3 (FR3) primers in formalin- fixed paraffin- embedded (FFPE) tissue.
Method:
FFPE samples from 100 cases diagnosed as DLBCL in the period of 2005 through 2011 were assessed in this study. Clonality of IgH (FR3) was evaluated by PCR amplification method that was optimized for FFPE tissue.
Results:
The clonal detection rate was (62.8%) with IgH (FR3) assay after modification, using filter for better DNA purification on negative cases. DNA quality in FFPE samples stored in recent five years were significantly better than older paraffin blocks (P < 0.001). Although a higher rate of clonality was observed in more recent group, it was not statistically significant.
Conclusion:
By using IgH (FR3) primers followed by one additional filter tube for better DNA purification, we could achieve a considerable rate of clonality with little adverse impact of DNA degradation.
