IGH Gene Rearrangement Analysis
IGH gene rearrangement analysis is used to confirm lineage and clonality. Clonality testing can be performed for IGH, IGLK or IGLL genes. The most commonly used method is PCR analysis for the IGH gene using primers for
Table 34-7. Tests for Lymphoproliferations of Immunodeficiency
Immunoglobulin (IGH, IGLK or IGLL) gene rearrangement analysis
T-cell receptor (TCR) gene rearrangements
Epstein-Barr virus (EBV/HHV-4) long-terminal repeat analysis
Epstein-Barr virus detection or quantitation Kaposi sarcoma-associated herpesvirus detection
Oncogenes/Tumor Suppressor Genes (MYC, RAS, TP53, BCL6)
Fluorescence in situ hybridization (FISH) Chromogenic in situ hybridization (CISH) Southern blot analysis
Single-strand conformation polymorphism (SSCP) Sequence analysis framework 3 (FR3) of the variable region and for the joining region. Primers that recognize framework 1 or 2 of the variable region are useful in some instances to exclude false-negative results. PCR is followed by size fractionation by gel or capillary electrophoresis. Primers that recognize rearrangements for the IGLK or IGLL genes also can be used, although these have higher rates of false-negative results. The presence of IGH gene rearrangements also can be determined by Southern blot analysis (SBA), which is particularly useful for testing lymphoma types derived from germinal center or postgerminal center B cells, due to the high false-negative rate seen with PCR as a consequence of somatic hypermutation of the IGH VDJ junction regions. This high false-negative rate is particularly common in PTLD, PEL, and some HIV-related IB lymphomas. However, SBA is much more labor-intensive than PCR analysis, has a longer turnaround time, requires fresh or frozen tissue, and is no longer performed routinely in most clinical molecular laboratories. Alternative methods for immunoglobulin clonality assessment are in situ hybridization, immunohisto chemistry, or flow cytometric analysis for monotypic light-chain expression.
While the vast majority of immunodeficiency-associated lymphomas are of B-cell origin, rare cases with T- or null-cell phenotype occur, which warrant T-cell receptor (TCR) gene rearrangement analysis to define cell lineage. The most common assay is PCR for the TCRG gene rearrangements. See chapter 33 for more detail on these assays.
Clonality of EBV-associated lymphomas can be determined using SBA for the terminal repeat region of the EBV genome. Like other herpesviruses, infectious EBV particles contain a linear double-stranded DNA viral genome that circularizes upon entry into the cell. The linear viral genome contains a variable number of tandem ~500 base pair repeats at each terminus, and upon circularization the number of tandem repeats is fixed. Circular episomes replicate with each cellular division, maintaining the same number of repeats in each infected cell. In a polyclonal population, where EBV independently infected many different cells, a range of repeat sizes will be present. In contrast, if all the cells in a population are derived from a single EBV-infected cell, only one size of terminal repeats will be present. A Southern blot assay that takes advantage of this biology was originally developed by Raab-Traub and Flynn.24 A restriction enzyme that cuts outside the terminal repeat region is used to digest genomic DNA, followed by gel electrophoresis, transfer to a membrane, and hybridization with a probe to the terminal repeat region. A polyclonal population produces a ladder or smear, and a monoclonal population produces a single band. While SBA is not done routinely for diagnostic pur-poses,it can be extremely useful for determination of mon-oclonality in EBV-associated lymphomas, as it is more sensitive than SBA for immunoglobulin gene rearrangements because of the presence of multiple copies of the EBV genome in each infected cell.
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