Simultaneous evaluation of T- and B-cell clonality, t(11;14) and t(14;18), in a single reaction by a four-color multiplex polymerase chain reaction assay and automated high-resolution fragment analysis: a method for the rapid molecular diagnosis of lymphoproliferative disorders applicable to fresh frozen and formalin-fixed, paraffin-embedded tissues, blood, and bone marrow aspirates.
Current polymerase chain reaction (PCR) methods for the molecular diagnosis of B- and T-cell lymphomas by determination of clonality of immunoglobulin heavy chain (IgH) and T-cell receptor-gamma rearrangements and by detection of the chromosomal translocations t(14;18) and t(11;14), require several laborious and costly PCR assays for each of these diagnostic tests. We have developed a multiplex PCR assay for the simultaneous determination of B- and T-cell clonality and the detection of the chromosomal translocations t(14;18) and t(11;14) in a single reaction, using four-color fluorescence and automated high-resolution fragment analysis. The 26 primers combined in the multiplex PCR correspond to the sequences of >90% of the 69 variables and 6 join IgH genes and 100% of the T-cell receptor-gamma variables and join genes that could participate in the respective rearrangements. In addition, they detect the major and the minor breakpoint regions of the t(14;18) and the major breakpoint region of the t(11;14), and amplify the beta-globin gene as an internal control. The specificity of the multiplex PCR was confirmed by analysis of 39 T-cell lymphomas and 58 B-cell lymphomas, including 11 mantle cell lymphomas bearing the t(11;14) and 25 follicular lymphomas bearing the t(14;18), with known rearrangements and/or translocations. Fifteen samples of reactive lymphadenitis remained negative.
Meier
V S
VS
Institute for Pathology, University of Basel, Basel, Switzerland. v.meier@unibas.ch
Rufle
A
A
Gudat
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F
D001402:B-Lymphocytes / Q000378:metabolism* / Q000473:pathology; D001483:Base Sequence; D001707:Biopsy, Needle; D001853:Bone Marrow / Q000473:pathology; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D002883:Chromosomes, Human, Pair 14 / Q000235:genetics; D002887:Chromosomes, Human, Pair 18 / Q000235:genetics; D002999:Clone Cells; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D005404:Fixatives; D005557:Formaldehyde; D005629:Frozen Sections; D015321:Gene Rearrangement; D019675:Genes, T-Cell Receptor gamma / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D019169:Jurkat Cells; D016399:Lymphoma, T-Cell / Q000235:genetics / Q000473:pathology; D008232:Lymphoproliferative Disorders / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013601:T-Lymphocytes / Q000378:metabolism* / Q000473:pathology; D016707:Tissue Fixation; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured
D005404:Fixatives; D007143:Immunoglobulin Heavy Chains; D005557:Formaldehyde; D004247:DNA