Immunoglobulin and t-cell receptor gene analysis in the diagnosis of lymphoid malignancies

Southern blotting and PCR analysis allow a detailed evaluation of the immunoglobulin (Ig) and Tcell receptor (TCFt) gene configuration in lymphoproliferations based on the combinatorial and junctional diversity of the gene .rearrangements, respectively. If appropriate probe/restriction enzyme combinations and appropriate primer sets are employed, both techniques can be used for diagnostic clonality studies. PCR-based analysis of Ig and TCP genes can only be performed in a reliable way if amplification is followed by PCR product analysis to discern between polyclonality and monoclonality. In the vast majority of suspect lymphoproliferations the IgH and TCRβ genes can be analyzed for clonality assessment via Southern blotting. If inconclusive results are obtained, the Igte and TCRygene complexes can be studied in a second step. PCR analyses generally focus on junctional regions of rearranged IgH and TCRy genes, as these are rearranged in the majority of B- and T-cell malignancies, respectively, and require only a limited number of primer sets. The possibilities and limitations of the application of both techniques are discussed in this review.</p

Immunoglobulin and t-cell receptor gene analysis in the diagnosis of lymphoid malignancies

Southern blotting and PCR analysis allow a detailed evaluation of the immunoglobulin (Ig) and Tcell receptor (TCFt) gene configuration in lymphoproliferations based on the combinatorial and junctional diversity of the gene .rearrangements, respectively. If appropriate probe/restriction enzyme combinations and appropriate primer sets are employed, both techniques can be used for diagnostic clonality studies. PCR-based analysis of Ig and TCP genes can only be performed in a reliable way if amplification is followed by PCR product analysis to discern between polyclonality and monoclonality. In the vast majority of suspect lymphoproliferations the IgH and TCRβ genes can be analyzed for clonality assessment via Southern blotting. If inconclusive results are obtained, the Igte and TCRygene complexes can be studied in a second step. PCR analyses generally focus on junctional regions of rearranged IgH and TCRy genes, as these are rearranged in the majority of B- and T-cell malignancies, respectively, and require only a limited number of primer sets. The possibilities and limitations of the application of both techniques are discussed in this review.</p

Flow cytometric analysis of the Vβ repertoire in healthy controls

Background: Analysis of the T-cell receptor (TCR)-Vβ repertoire has been used for studying selective T-cell responses in autoimmune disease, alloreactivity in transplantation, and protective immunity against microbial and tumor antigens. For the interpretation of these studies, we need information about the Vβ repertoire usage in healthy individuals. Methods: We analyzed blood T-lymphocyte (sub)populations of 36 healthy controls (age range: from neonates to 86 years) with a carefully selected most complete panel of 22 Vβ monoclonal antibodies, which together recognized 70-75% of all blood TCRαβ+ T lymphocytes. Subsequently, we developed a six-tube test kit with selected Vβ antibody combinations for easy and rapid detection of single ('clonal') Vβ domain usage in large T-cell expansions. Results: The mean values of the Vβ repertoire usage were stable during aging in blood TCRαβ+ T lymphocytes as well as in the CD4+ and CD8+ T-cell subsets, although the standard deviations increased in the elderly. The increased standard deviations were caused by the occurrence of oligoclonal T-cell expansions in the elderly, mainly consisting of CD8+ T lymphocytes. The 15 detected T-cell expansions did not reach 40% of total TCRαβ+ T lymphocytes and represented less than 0.4 x 109 cells per liter in our study. Vβ usage of the CD4+ and CD8+ subsets was comparable for most tested Vβ domains, but significant differences (P &lt; 0.01) between the two subsets were found for Vβ2, Vβ5.1, Vβ6.7, Vβ9.1, and Vβ22 (higher in CD4+), as well as for Vβ1, Vβ7.1, Vβ14, and Vβ23 (higher in CD8+). Finally, single Vβ domain expression in large T-cell expansions can indeed be detected by the six-tube test kit. Conclusions: The results of our study can now be used as reference values in studies on distortions of the Vβ repertoire in disease states. The six-tube test kit can be used for detection of single Vβ domain expression in large T-cell expansions (&gt;2.0 x 109/l), which are clinically suspicious of T-cell leukemia. (C) 2000 Wiley-Liss, Inc.</p

Consistent B Cell Receptor Immunoglobulin Features Between Siblings in Familial Chronic Lymphocytic Leukemia

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21R110 subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21R110 subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family