TCR Gene Rearrangement in B-Cell Chronic Lymphocytic Leukaemia.

Background: Chronic lymphocytic leukaemia (B-CLL) is a heterogenous B cell disorder. However, clonal/oligoclonal T cell expansion has also been detected at a low frequency in B-CLL using a range of methodologies including Southern blotting and flow cytometric analysis. The underlying cause of this phenomenon is unclear and it has even been suggested that this clonal T cell receptor (TCR) gene rearrangement occurred in the leukaemic cells, leading to the concept of lineage infidelity. Other suggestions include that the observed T cell clonality may simply be related to the normal aging process or that it may result from the immune response to malignant cells or viral infection. During normal T cell maturation in the thymus, the TCR genes undergo a complex process of V(D)J segment rearrangement to produce a wide repertoire of antigen specificity. As it is an early event in T cell development, TCRγ gene rearrangement is commonly used as a marker of clonality. The TCRβ gene is also routinely investigated due to its greater combinatorial diversity. Lack of sufficiently sensitive detection methodology is often a limiting factor in the discrimination between clonal and polyclonal T-cell populations.

Aims: The aim of this study was to use high resolution heteroduplex gel and fluorescence GeneScan analysis to screen for TCRγ and TCRβ gene rearrangements respectively in a cohort of B-CLL patients and to ascertain if the clonal rearrangement occurred in B cells or T cells.

Methods: B and T cells were purified from peripheral blood from 34 B-CLL patients using a CD19+ or CD3+ magnetic bead system (autoMACS). DNA was extracted from purified B or T cells and the TCRγ and TCRβ genes were amplified by BIOMED-2 multiplex PCR assays (InVivoScribe Technologies). Heteroduplex and GeneScan analysis were then performed to detect clonal TCR gene rearrangments.

Results: Clonal TCR gene rearrangements were detected in11/34 (32%) of cases in purified T cell fractions. No clonal TCR gene rearrangements were found in purified B cell fractions. Clonal TCRβ rearrangements were found in 10/34 (29%) and clonal TCRγ in 8/34 (23%). In most instances a weak clonal pattern was observed. Clonal TCR rearrangements were detected in 7 males and 4 females. Five patients had mutated IGHV genes, while the remaining 6 possessed unmutated IGHV genes. Seven patients presented with more advanced clinical stage (Binet B or C), and 8 patients received treatment.

Summary/conclusions: These results are in agreement with previous work demonstrating that T-cell clonal/oligoclonal expansions occur frequently in B-CLL patients. However this is the first report to demonstrate that the clonal expansions occur in T cells and not in B cells. In most instances a weak clonal pattern was observed which was probably due to minor clonal T-cell populations. It is possible that T-cell clonality may be associated with a poorer prognosis in this disease category. Of interest, we found that 8/11(73%) patients in our B-CLL sub-group with T-cell expansion also had advanced stage disease and/or unfavourable molecular (IGHV gene usage/mutational status) markers. We are currently investigating the possibility of a specific antigenic stimulus resulting in this clonal T-cell expansion.