The raison d'être of B cells is the manufacture of immunoglobulin (Ig). Ig embedded in the surface membrane has the clear function of recognizing and responding to exogenous antigens. Recognition is via the variable (V) regions, which differ in sequence from one B-cell to another, and provide a complete catalog of potential antigen-combining sites. Cutting and pasting the component gene segments produces the extreme diversity of the B-cell receptor (51,52). This recombinatorial process, which occurs in the bone marrow, is mediated by proteins encoded by the recombination-activating genes RAG1 and RAG2 (53).
For the heavy chain of Ig, selection takes place from the potentially functional genes of the unrearranged repertoire with 51 VH genes divided into 7 families (VH1-VH7), 27 D genes, and 6 JH genes. The junctions of VH to D and D to JH are imprecise, with the deletion by exonucleases of templated nucleotides or the insertion by terminal deoxytransferase (TdT) of nontemplated nucleotides in a random manner (54). This introduces a further huge diversity into the shape of the Ig molecule, especially as the D segment can be read in any of the three frames (55). The consequence is that the third complementarity-determining region (CDR3) of any given lymphocyte is virtually unique and provides a clonal signature for any tumor deriving from it.
The heavy chain genes also have a low degree of allelic polymorphism that has not been fully mapped (56). The recombination of the constant regions (CH and CL) occurs following transcription; splicing of the transcribed RNA leads to a functional mRNA that can be translated into a ^ heavy chain and k or X light chain proteins that combine to form whole IgM.
The usage of the 51 VH genes is not random. Brezinschek et al. (57) analyzed the gene usage by individual normal B-cells in the blood of three individuals. Overusage of the VH3 family was seen. The V3-23 gene was most commonly used, followed by V3-30.3, V3-30, and V3-07. One possible reason for this predominance is the duplication of these segments in some haplotypes.
Rearrangement of the light chain variable region genes occurs in a similar manner, involving single-step recombinations of Vk/Jk or VX/JX gene segments but with no D segments. The B-cell receptor of pre-B-cells combines Ig heavy chains with the surrogate light chain encoded by the VpreB and X5/14.1 genes (58). Successful rearrangement of the light chain genes extinguishes expression of the pre-B-cell complex and suppresses further rearrangement. There are several checkpoints in B-cell maturation. Failure to produce a functional immunoglobulin induces apoptosis, as does an autoreactive specificity. Escape from apoptosis can occur by rearranging the other allele, most commonly of the light chain. This process is known as receptor editing. Occasionally B-cells express both k and X light chains, indicative of failure of allelic exclusion.
On completion of this maturation, the B-cell leaves the bone marrow for the periphery, where it may encounter antigen. It then undergoes affinity maturation, usually in the germinal centers of the peripheral lymphoid organs. Here, somatic mutation is induced under the influence of CD40+ T-cells, cytokines, and antigen-bearing follicular dendritic cells (59). The rate of introduction of base pair changes is on the order of 10-4-10-3 per generation. The mutations tend to cluster in the CDRs, possibly for structural reasons and possibly because of antigen selection.
A further genetic arrangement is necessary for Ig class switching from IgM plus IgD to IgG, IgA, or IgE. This process generally also occurs in the germinal center, although it may not be confined there (59). The choice of isotype is cytokine determined (60). The array of constant region genes at the heavy chain locus each has a 5' switch region that consists of tandem repeats.
Isotype switching conventionally occurs between two switch regions, looping out the intervening constant region genes, although RNA splicing, leading to the generation of multiple isotypes, may also occur (61).
Cells leaving the germinal center become either plasma cells or memory cells, the choice being directed by different cytokines. The plasma cells migrate mainly to bone marrow, but also to spleen, lymph nodes, and the mucosa-associated lymphoid tissue. Memory cells form part of the circulating pool but are also found in peripheral lymphoid organs and in the marginal zone of the spleen (62).
The peripheral blood B-cells of normal individuals comprise 60% naïve cells with unmutated IgVH genes and 40% memory cells that carry somatically mutated IgVH genes and express surface CD27 (63). Only a small proportion of naïve cells express CD5 (64), although this proportion is higher in early life. Most of circulating memory cells express surface IgM. (Some also express IgD.) Only a minority show evidence of class switching.
Because CLL cells express CD5, many authorities had accepted that CLL cells derived from the minor population of CD5+ naïve B-cells. Early sequences of the IgVH genes of tumor cells from patients with CLL found them to be in germline configuration (65-67), tending to confirm their origin from a naïve B-cell. However, reports began to appear in the literature detailing cases with evidence of somatic mutation, culminating in 1994 with a review of the literature by Schroeder and Dighiero (68), which found that 36/75 reported cases had IgVH genes with less than 98% sequence homology to the appropriate germline gene. The figure of 98% was chosen because polymorphisms, which are quite common in VH genes, can account for that degree of disparity (69). Schroeder and Dighiero (68) suspected that CLL might be a heterogeneous disorder but were unable to cull from the literature the comprehensive clinical details needed to establish this. They did report, however, that some of the cases with mutated VH genes were CD5-negative and that there were a disproportionate number with class-switched IgVH genes. These were not all cases of classical CLL.
More recently, a multicenter study of 64 patients with surface IgM+, CD5+ CLL also found two groups of roughly equal numbers with mutated and unmutated VH genes (70). Although no clinical details were available otherwise to distinguish the two subsets, the authors were able to confirm the observation of Schroeder and Dighiero (68) that the presence or absence of somatic mutations was associated with the use of particular VH genes.
In 1997 our group examined the VH genes of 22 patients with classical B-cell CLL segregated according to karyotype. Tumors with trisomy 12 had unmutated VH genes, but those with 13q14 abnormalities detected by conventional cytogenetics had evidence of somatic mutations (71). Since it has been previously shown that CLL patients with trisomy 12 have a poorer survival than those with abnormalities at 13q14 (26), this pointed to an association between clinical status and degree of somatic mutation.
The suspicion that the clinical heterogeneity of CLL might have a biological basis led us to extend this study. We examined the Ig VH gene sequences in a series of 84 patients with classical B-cell CLL attending our hematology clinic and compared our results with various clinical characteristics of the patients and their survival. The striking finding to emerge was that the presence of IgVH gene mutations placed the CLL patient in a disease group with a clearly better prognosis (4).
Patients with unmutated IgVH genes had a median survival of 9.7 yr, whereas those with mutated IgVH genes had a median survival of 24.4 yr. At the same time, Damle et al. (5), in a series
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