Identification of the tumor cells in peripheral T-cell lymphomas by combined polymerase chain reaction-based T-cell receptor beta spectrotyping and immunohistological detection with T-cell receptor beta chain variable region segment-specific antibodies

Abstract

Most nodal peripheral T-cell lymphomas (PTCL) originate from alphabeta-T cells, and they often contain reactive T cells that may hamper immunophenotyping. To specifically identify the neoplastic population in immunohistochemically stained slides, we assessed the heterogeneity of the T-cell receptor beta chain variable region (TCRVbeta). This region contains 65 gene segments, of which only one is expressed after rearrangement. To investigate PTCL, we developed a polymerase chain reaction assay to define the clonally rearranged TCRVbeta segment. Detecting the corresponding epitope with segment-specific antibodies enabled identification of tumor cells among the T cells. The TCRVbeta segment of the tumor cells was defined in 13 of 13 PTCL not otherwise specified and 11 of 13 angioimmunoblastic T-cell lymphomas. Antibodies corresponding to the respective TCRVbeta segment of the tumor were available for seven cases from each group. After applying these antibodies in combination with antibodies against CD3, CD5, CD4, CD8, and cytotoxic molecules, double stains were evaluated by confocal laser scanning microscopy. In 9 of 14 cases, less than 50% of T cells expressed the clonally rearranged TCRVbeta segment. Phenotypes defined in double stains differed from those obtained by conventional immunohistochemistry in 11 of 14 cases. The combination of TCRVbeta polymerase chain reaction and immunohistochemistry may facilitate more reliable detection and characterization of tumor cells in PTCL.

Figure 1

Graphical display of fluorescent TCRβ PCR products. Monoclonal PTCL-NOS (a; case 6), oligoclonal AILT (b; case 19), and polyclonal control (c; peripheral blood from a healthy donor) analyzed with the GeneScan software. For each case, the three highest peaks are given. They define the Rn value (see text). Note the different scales on the y axes. Relative fluorescence intensities (y axis) are plotted as a function of PCR fragment size in nucleotides (x axis) for each PCR product.

Figure 2

Definition of the tumor cell phenotype in PTCL-NOS (case 3). a: H&E; b: CD5 stains only small inconspicuous T cells, the large atypical cells are negative. CD4 (c) and CD8 (d) single stains detect about an equal number of T cells, but the perivascular CD8⁺ cells (d) appear to be large and atypical; TCRVβ14 (red) immunoreactivity does not co-localize with either CD4 (green; e) or CD8 (green; f) in a triple stain (shown as double stains with both CD4 and CD8 in green, for better visualization).

Figure 3

Definition of the tumor cell phenotype in AILT (case 21). a: H&E; b: CD3 stains both small inconspicuous and large atypical T cells. CD4 (c) shows a weak expression in clear cells, whereas CD8 (d) shows a strong expression in few cells including activated T cells. TCRVβ8.1 (red) immunoreactivity co-localizes in varying intensities with CD4 (green, e), but not with CD8 (green, f).