Korean J Hematol.  2007 Dec;42(4):361-366. 10.5045/kjh.2007.42.4.361.

Polymerase Chain Reaction and Sequencing of Immunoglobulin Heavy Chain Gene Rearrangement in Formalin Fixed, Paraffin-embedded Tissue of Patients with B Cell Lymphoma

• Cho SR
• Park IJ
• Lee MS
• Kim EK
• Lee WG
• Han JH
• Park JE
• Park JS
• Kim HC

• KMID: 2252221
• DOI: http://doi.org/10.5045/kjh.2007.42.4.361

Abstract

BACKGROUND: Immunoglobulin heavy chain (IgH) gene rearrangement has been known to be a useful marker for determining the clonality as well as detecting minimal residual disease in B cell malignancies. This study was performed to establish single polymerase chain reaction (PCR) methods for the detection of IgH gene rearrangements in formalin-fixed, paraffin-embedded tissue of patients with B cell lymphoma and determine the type of JH segments used.
METHODS
We obtained formalin-fixed, paraffin-embedded tissue sections of 44 patients diagnosed with B cell lymphoma at Ajou University Hospital from January 2005 to January 2007 and reviewed medical records retrospectively. After the extraction of DNA, PCR was performed using VH3 and JHPST primers to detect the third complementarity determining region (CDR3) gene of IgH. Sequence analysis of the PCR products was also done in 23 patients.
RESULTS
The CDR3 gene rearrangements were detected in 26 (59%) out of 44 patients with B cell lymphoma. Sequence analysis of the amplified CDR3 gene was successful in 16 (70%) of 23 patients. JH3, JH4, JH5, and JH6 segments were used for CDR3 gene rearrangements in 3 (25%), 4 (33%), 1 (8%), and 4 (33%) patients with diffuse large B cell lymphoma, respectively.
CONCLUSION
Although there are some limitations due to a low sensitivity less than 60%, single PCR using consensus primers could be an effective tool for the detection of CDR3 gene rearrangements in routine laboratory settings. Furthermore, sequence analysis of the CDR3 PCR products will provide basic information necessary for further studies.

Keyword

IgH; Gene rearrangement; Polymerase chain reaction; B cell lymphoma; CDR3; Sequence analysis

MeSH Terms

Complementarity Determining Regions
Consensus
DNA
Formaldehyde*
Gene Rearrangement*
Humans
Immunoglobulin Heavy Chains*
Immunoglobulins*
Lymphoma, B-Cell*
Medical Records
Neoplasm, Residual
Polymerase Chain Reaction*
Retrospective Studies
Sequence Analysis
Complementarity Determining Regions
DNA
Formaldehyde
Immunoglobulin Heavy Chains
Immunoglobulins

Figure

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  Fig. 1 2% agarose gel electrophoresis of the PCR product amplified for CDR3 gene. M, size marker (100bp ladder); 1, positive control; 2∼4, positive samples; 5∼7, positive samples with one or more nonspecific bands; 8, negative control; 9, negative sample.

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