Abstract
Malignant and reactive lymphoproliferations in most cases can be distinguished by histology and immunohistology alone; however, in T-cell lymphoproliferations and lymphoproliferations of unknown origin, histology often is inconclusive, and there is no reliable protein marker of malignancy. At the genomic level T-cell neoplasms clonally rearrange T-cell receptor (TCR) genes that can serve as clonal markers. In comparison with Southern blot analysis, polymerase chain reaction (PCR) techniques increasingly are being used to detect these rearrangements because PCRs are rapid, easy to perform, and can be used to amplify poor-quality deoxyribonucleic acid (DNA) from paraffin-embedded formalin-fixed biopsies. Nevertheless there are a number of problems associated with the detection of gene rearrangements using PCR. The foremost of these is improper primer annealing that will lead to false-negative or false-positive PCR results that may arise from poor primer design, especially for TCRB genes with an extensive variable (V), diversity (D), and joined (J) gene repertoire. There also can be difficulty in discriminating between clonal and polyclonal PCR products unless specific methods such as heteroduplex analysis or gene scanning are used. In this chapter, we describe methods, derived from a recent European collaborative BIOMED-2 program, for the detection of TCRG, B, and D rearrangements. TCRB VJ and DJ gene rearrangements are detected using 23 VB primers, 13 JB primers, and 2 DB primers in 3 multiplex tubes. TCRG VJ gene rearrangements are detected with four VG and two JG primers in two multiplex tubes, and TCRD VJ, VD, DJ, and DD rearrangements are detected with six VD primers, four JD primers, and two DD primers in one multiplex tube. Gaussian distributions of polyclonal PCR products are seen at specific size ranges for both TCRB and TCRG. Interpretation of TCRD rearrangements is more complex because of the wide range in PCR product size and often low numbers of TCRGD cells in target tissue. For all loci, some indication of gene usage can be ascertained by labeling the primers with different fluorochromes and gene scan analysis of PCR products. Furthermore the complementarity of the TCR loci affords an unprecedented high clonal detection rate. In addition, we also describe a set of control gene primers designed to amplify amplicons of 100, 200, 300, 400, and 600 bp for the assessment of the integrity and amplifiability of DNA. This set of primers is particularly useful for the assessment of DNA extracted from paraffin-embedded biopsies.
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References
Tumours of haematopoietic and lymphoid tissues, in WHO Classification of Tumours, (Jaffe, E. S., Harris, N. L., Stein, H., Vardiman, J. W., eds). IARC Press, Lyon, France, 2001.
van Dongen, J. J. M., and Wolvers-Tettero, I. L. M. (1991) Analysis of immunoglobulin and T-cell receptor genes. Part II: possibilities and limitations in the diagnosis and management of lymphoproliferative diseases and related disorders. Clin. Chim. Acta 198, 93ā174.
Griesser, H. (1995) Gene rearrangements and chromosomal translocations in T-cell lymphoma-diagnostic applications and their limits. Virchows Arch. 426, 323ā338.
Slack, D. N., McCarthy, K. P., Wiedemanm, L. M., et al. (1993) Evaluation of sensitivity, specificity, and reproducibility of an optimised method for detecting clonal rearrangements of immunolglobulin and T-cell receptor genes in formalin fixed, paraffin embedded sections. Diag. Mol. Pathol. 2, 223ā232.
McCarthy, K. P., Sloane, J. P., Kabarowski, J. H. S., et al. (1992) A simplified method of detection of clonal rearragements of the T-cell receptor Ī³ chain gene. Diagn. Mol. Pathol. 1, 173ā179.
Langerak, A. W., Szczepanski, T., van der Burg, M., et al. (1997) Heteroduplex PCR analysis of rearranged T-cell receptor genes for clonality assessment in suspect T-cell proliferations. Leukemia 11, 2192ā2199.
Kneba, M., Boilz, I., Linke, B., et al. (1995) Analysis of rearranged T-cell receptor beta-chains by polymerase chain reaction (PCR) DNA sequencing and automated high resolution PCR fragment analysis. Blood 86, 3930ā3937.
Delabesse, E., Burtin, M. L., Millien, C., et al. (2000) Rapid, multifluorescent TCRG Vgamma and Jgamma typing application to T-cell acute lymphoblastic leukaemia and to the detection of minor clonal populations. Leukaemia 14, 1143ā1152.
Lefranc, M-P. (2000) Locus maps and genomic repertoire of the human T-cell repertpoire. The Immunoloogist. 8, 72ā78.
Langerak, A. W., van den Beemd, R., Wolvers-Tettero, I. L., et al. (2001) Molecular and flow cytometric analysis of the Vbeta repertoire for clonality assessment in mature TCRalphabeta T-cell proliferations. Blood 98, 165ā73.
van Dongen, J. J. M., Langerak, A. W., San Miguel, J. F., et al. (2001) PCR-based clonality studies for early diagnosis of lymphoproliferative disorders: Report of the BIOMED-2 concerted action. Blood 98, p505a (Abstract 543).
van Dongen, J. J. M., Langerak, A. W., BrĆ¼ggeman, M., et al. (2003) Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene rearrangements in suspect lymphoproliferations. Report of the BIOMED-2 concerted Action BMH4-CT98-3936. Leukaemia 17, 2257ā2317.
Clevers, H., Alarcon, B., Wileman, T. et al. (1988) The T-cell receptor/CD3 complex: a dynamic protein ensemble. Annu. Rev. Immunol. 6, 629ā662.
Alt, F. W., Oltz, E. M., Young, F., et al. (1992) VDJ recombination. Immunol. Today 13, 306ā314.
Acknowledgment
This work was supported by a BIOMED-2 Concerted Action (BMH4-CT98-3936). We thank all colleagues involved in this program, especially Professor J. J. M. van Dongen, Dr. A. Langerak, Professor E. R. Macintyre, Dr. F. L. Lavender, Dr. M. BrĆ¼ggeman, Dr. H. E. White, and Dr. H. van Krieken. This work was also supported by the Leukaemia Research Fund (LRF).
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Hodges, E., Williams, A.P., Harris, S., Smith, J.L. (2005). T-Cell Receptor Molecular Diagnosis of T-Cell Lymphoma. In: Illidge, T., Johnson, P.W.M. (eds) Lymphoma. Methods in Molecular Medicineā¢, vol 115. Humana Press. https://doi.org/10.1385/1-59259-936-2:197
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DOI: https://doi.org/10.1385/1-59259-936-2:197
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