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VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma
Published on: December 28, 2015
Clonality analysis of B-cell lymphoma in fresh-frozen and paraffin-embedded tissues: the effects of variable
Y T Chen1, K D Whitney, Y Chen
1Department of Pathology, New York Hospital-Cornell Medical Center, New York.
Insights
Polymerase chain reaction (PCR) detects B-cell monoclonality in lymphomas with high sensitivity. Both fresh-frozen and formalin-fixed tissues are comparable for this diagnostic assay.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- B-cell monoclonality is a hallmark of lymphomas.
- Polymerase chain reaction (PCR) is a sensitive molecular technique for detecting gene rearrangements.
Purpose of the Study:
- To evaluate the sensitivity of PCR for detecting B-cell monoclonality in lymphomas.
- To assess the impact of various factors on PCR assay performance.
- To compare fresh-frozen and formalin-fixed tissues for PCR analysis.
Main Methods:
- Analysis of 119 B-cell lymphoma cases with proven IgH gene rearrangements.
- Parallel testing of fresh-frozen and formalin-fixed, paraffin-embedded tissues.
- Application of one-step PCR, seminested PCR, and bcl-2/JH primer amplification.
- Investigation of annealing stringency and false-negative case groups.
Main Results:
- Overall positive rate of 81% (97/119) in fresh-frozen tissues.
- Seminested PCR and adjusted stringency increased detection rates.
- High concordance (89%) between fresh-frozen and formalin-fixed tissues.
- Identification of distinct reasons for false-negative PCR results.
Conclusions:
- PCR is a sensitive method for detecting B-cell monoclonality in lymphomas.
- Fresh and formalin-fixed tissues yield comparable results.
- Optimized PCR protocols can improve diagnostic accuracy.
Abstract:
To investigate the sensitivity of polymerase chain reaction (PCR)-based detection of B-cell monoclonality and the effects of several variables, we analyzed 119 cases of B-cell lymphomas with proven IgH gene rearrangements, testing fresh-frozen and formalin-fixed materials in parallel. Using fresh-frozen tissue, 83 cases (70%) were positive with one-step PCR and high-stringency annealing. Two groups of false-negative cases were identified, Group I (16 cases) showing no PCR products, and Group II (20 cases) showing polyclonal smear patterns. Seminested PCR and/or lowered annealing stringency revealed nine additional positive cases in Group I but none in Group II. Amplification with bcl-2/JH primers resulted in five more positive cases. The overall positive rate in the fresh-frozen category was 81% (97 of 119). Parallel analysis was performed on formalin-fixed, paraffin-embedded material from 61 cases, and a high concordance rate (89%) was observed. The results indicated that fresh and formalin-fixed specimens are comparable in this PCR-based assay, and that the two groups of false-negative results can be accounted for by different reasons. In addition, we reviewed the current literature, discussed the diagnostic applications of this technique, and listed the core elements of a proposed PCR protocol that should be suitable for most laboratories.
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