ABSTRACT
Principles and techniques for determining B cell and T cell 755 clonality: antigen receptor gene rearrangements and the generation of receptor diversity
Techniques used in the determination of lymphoid clonality 756 Cautions and considerations in data interpretation 757 Detection and clinical significance of chromosomal 758
translocations and other genetic anomalies in the lymphoid neoplasms
FISH and PCR techniques for detecting translocations 759 in lymphoid tumors
Common genetic abnormalities useful in the diagnosis and 760 differential diagnosis of B lineage lymphoid neoplasms
Common genetic abnormalities useful in the diagnosis and 764 differential diagnosis of T lineage lymphoid neoplasms
Tumor genetics, minimal residual disease and 765 prognosis prediction
Precursor B lymphoblastic leukemia/lymphoma 765 B cell chronic lymphocytic leukemia 768 Multiple myeloma 768 Concluding remarks and future directions 769 Key points 769 References 769
In many, if not most, cases of lymphoid hyperplasia or neoplasia, the morphologic expertise of the hematopathologist supplemented by selective ancillary investigative techniques, is sufficient to establish a conclusive diagnosis. Often, the formal demonstration of monoclonality in a neoplastic lymphoid population is not required for this purpose, such as in the setting of a lymph node biopsy harboring typical diffuse large B cell lymphoma. In practice however, there are seemingly more exceptions than rules in clinical and histopathologic presentations of the lymphoproliferative diseases, and the demonstration of clonality to facilitate the diagnosis of a lymphoid malignancy is an important tool in the hematopathology laboratory. To this end, flow cytometry of cell suspensions, or immunoperoxidase and in situ hybridization (ISH) methods on fixed tissue sections, can often successfully delineate restricted immunoglobulin light chain expression by
Nonetheless, there exist a number of scenarios in routine hematopathology practice in which the detection of clonal lymphocyte populations can best be achieved by molecular methods (Box 43.1). Of note, standard molecular clonality assays can be supplemented by laboratory investigations for common chromosomal translocations and their corresponding gene fusion abnormalities, which are prevalent in many types of non-Hodgkin lymphomas and lymphoid leukemias. In this way, the molecular hematopathologist has at his or her disposal a formidable set of tools to:
● distinguish between benign and malignant lymphoproliferations
● assist in the accurate subclassification of the lymphoid diseases
● identify markers to monitor therapeutic efficacy and disease recurrence
● provide the technological platforms for incorporating novel advances in the molecular pathogenesis of
The immune receptors present on B and T lymphocytes are generated by a process of somatic rearrangement of the antigen receptor genes, occurring in early precursor B and T cells of the bone marrow and thymus respectively. For B cells, this process is initiated at the immunoglobulin heavy chain locus (IGH) located on chromosome 14(q32), in turn leading to production of the heavy chain peptide component of the immunoglobulin molecule.1,2 Following sequentially, the κ light chain gene (IGK) on chromosome 2(p12) is next rearranged, and if these attempts fail at both κ alleles, rearrangement then proceeds at the λ gene (IGL) located on chromosome 22(q11). In this way, the hierarchical recombination of the light chain genes ensures only one light chain type is expressed by a given B cell, along with its respective heavy chain. The complete immunoglobulin protein is a tetramer of paired identical heavy and light chains and is displayed on the cell surface as part of the B cell receptor signaling complex. Ordered gene segment recombination also underlies the formation of the heterodimeric T cell receptors, which are of either αβ or γδ type. In thymic T cells, rearrangement of the δ gene (TRD) on chromosome 14(q11) is initiated in concert with similar activity at the T cell receptor γ gene (TRG), located on chromosome 7(p15). However, δ gene rearrangements are seldom productive, thus excluding expression of a complete γδ surface T cell receptor on the majority of developing T lymphocytes. The cascade of T cell receptor gene rearrangements therefore proceeds nearly synchronously at the alpha (TRA) and beta (TRB) loci, located on chromosomes 14(q11) and 7(q34), respectively. As the TRD gene is in fact embedded within the alpha locus, it is most often deleted in a site-specific manner during rearrangement of the TRA gene. The potential for functional alpha and beta T cell receptor gene rearrangements is robust, explaining in part the observed distribution of αβ T cells (95 percent) versus γδ T cells
receptor proteins on B and T cells involve the coordinated scission, splicing and rejoining of DNA coding regions known as V (variable), D (diversity) and J (joining) segments as schematically illustrated for the IGH locus in Figure 43.1. This activity is mediated by the recombination activating gene products (RAG1, RAG2) and several proteins involved in DNA repair.1 While V and J segments are common to all antigen receptor genes, D elements are not present at the TRA, TRG, and immunoglobulin light chain gene loci. The individual V-(D-) and J segments are selected randomly, but are precisely identified over very large regions of intervening DNA by the placement of recombination signal sequences (RSS), characterized by a 3 palindromic heptamer sequence separated from a nonamer sequence by 12 or 23 nucleotide spacers.1 Assembly of the rearranged V-D-J or V-J segment is followed by splicing to a C (constant) region exon, to produce a potential DNA coding sequence for an immunoglobulin or T cell receptor protein. Additional important events in this process include the addition of random, nontemplated nucleotides (N nucleotides) at the junctions of V-(D)-J segments via the action of the enzyme terminal deoxynucleotidyl transferase (TdT), and some degree of ‘trimming’ of nucleotides at the junctional ends by DNA exonucleases1 (Fig. 43.1).
