Single-cell analysis of T-cell receptor-gamma rearrangements in large-cell anaplastic lymphoma.

Large-cell anaplastic lymphomas (LCAL) are characterized by their distinctive morphology together with expression of the CD30 antigen. In addition, a chromosomal translocation, t(2;5) (p23; q35), can be detected in most cases. A significant proportion of LCALs carry rearrangements of the T-cell receptor-gamma (TCR-gamma) locus and display a T-cell phenotype. In about a third of the cases, another type of non-Hodgkin-lymphoma precedes LCAL. Early transformations of non-Hodgkin's lymphoma into LCAL might escape clinical detection in a significant number of cases. The existence of clonally related lymphoid cells within the lymph node infiltrates must be claimed in these cases. Recently, a small-cell-predominant variant of LCAL was described in which only few large tumor cells expressing the CD30 antigen are found together with numerous small lymphocytes, which are frequently CD30-. This observation in particular prompted us to investigate the clonal relationship of the tumor cell compartment and admixed small lymphocytes in one case of common LCAL with T-cell genotype. For this purpose, we chose to amplify rearranged TCR-gamma sequences from single cells isolated from immunostained frozen sections by using a micromanipulator. A total of 119 cells were investigated. Amplification products were obtained in 17 of 79 CD3+ cells, 12 of 30 CD30+ cells, and three of 10 CD20+ cells. The nucleotide sequences were determined in 28 cells by nonradioactive sequencing. In 11 CD30+ cells, the predominant rearrangement of TCR-gamma was identified. No clonal diversity was observed. The small CD3+ lymphocytes were unrelated to the anaplastic CD30+ tumor cells. This report describes a method to analyze rearrangements of the TCR-gamma in single cells isolated from immunostained frozen sections. Application of this technique revealed an absence of clonal diversity in a case of LCAL and documented the polyclonal nature of admixed small CD3+ lymphocytes.

Detection of T-cell clonality in paraffin-embedded tissues.

In lymph node diagnosis, difficulties are frequently encountered with the differential diagnosis of reactive and neoplastic T-cell proliferations. Immunohistochemistry is of limited use, and fresh frozen material for DNA studies is not available in many cases. We have established a polymerase chain reaction (PCR) technique to amplify rearranged T-cell receptor (TCR)-gamma sequences from paraffin-embedded material. Our method differs from other techniques previously described in that it uses four sets of family-specific variable (V)gamma primers. Clonality of the investigated T cells is reflected not only by the varying lengths of amplified products but also by differences in the relative amount of rearranged V gamma families. Preliminary studies indicate that this approach can provide information about the presence of predominant T-cell clones within the sample and thus help to classify the lymph node lesion. With this technique we were able to confirm clonality in seven of 12 T-cell lymphomas in paraffin-embedded tissues.

Angioimmunoblastic lymphadenopathy type of T-cell lymphoma and angioimmunoblastic lymphadenopathy: a clinicopathological and molecular biological study of 13 Chinese patients using polymerase chain reaction and paraffin-embedded tissues.

The morphological classification of angioimmunoblastic lymphadenopathy (AILD) or T-cell lymphoma of AILD-type (AILD-TCL) is still a subject of considerable difficulty and controversy. The aim of the current study was to examine the value of clinical, morphological, immunohistochemical variables in paraffin-embedded tissues in predicting the clonality of the respective lesion. Fifteen lymph node biopsies derived from 13 patients from Chengdu, China, were diagnosed as AILD or AILD-TCL and included in this study. The specimens were examined using a panel of monoclonal antibodies and a scoring system of morphological features. Clonality of the paraffin-embedded material was investigated using a novel polymerase chain reaction-technique to amplify rearranged T-cell receptor (TCR)-gamma sequences. Additional experiments were carried out to investigate the presence of clonal rearrangements of the immunoglobulin heavy chain (IgH) locus. We found clonal rearrangements of the TCR-gamma locus in 9 out of 15 lymph node biopsies. In 3 patients, the predominant cell clones carried clonal IgH and TCR-gamma rearrangements whereas 1 patient with polyclonal TCR-gamma pattern displayed IgH-monoclonality. The statistical evaluation of morphological and immunohistochemical data indicated that no single variable was able significantly to predict the clonality of the lesion. Furthermore, demonstrable clonality for the TCR-gamma or the IgH loci of a lesion did not correlate with a bad clinical course. Our data correlate with findings of other studies investigating AILD-TCL in Caucasian populations.