Molecular Biology and Cytogenetics of Chronic Lymphocytic Leukemia

David G. Oscier

Early cytogenetic studies in chronic lymphocytic leukemia (CLL), performed in the 1960s and 1970s, failed to detect clonal cytogenetic abnormalities. This inauspicious start to the investigation of genetic abnormalities in CLL was the consequence of the low spontaneous mitotic rate in CLL and the use of the T-cell mitogen phytohemaglutinin to obtain analyzable metaphases. With the discovery of polyclonal B-cell mitogens, the first cytogenetic abnormality in CLL, trisomy 12, was discovered in 1980.1,116 Subsequent studies have shown clonal cytogenetic abnormalities in approximately 50 percent of cases of CLL.2 The most frequent abnormalities are structural abnormalities of chromosome 13q14 and trisomy of chromosome 12, occurring in 10 to 20 percent of cases. Abnormalities of chromosomes 6q, 11q, 14q, and 17p are each found in less than 5 percent of cases. In patients with a cytogenetic abnormality, 50 percent have a single abnormality, 25 percent have two abnormalities, and the remainder have a complex karyotype. The results obtained using different B-cell mitogens, such as tetrade-canoyl phorbol acetate (TPA) or the Epstein-Barr virus (EBV), have been comparable, although a recent study in which CLL cells were incubated with a variety of different mitogenic combinations, including TPA, interleukin 2, tumor necrosis factor alpha, and Staphylococcus aureus Cowan 1, demonstrated cytogenetic abnormalities in 80 percent of cases.3 Although cytoge-netic analysis has the advantage of being a global technique, small deletions are not detectable and the mitogens employed stimulate normal T cells as well as leukemic B cells. Studies that have combined cytogenetic analysis with immunophenotyping of single cells have clearly shown that the normal mitoses in CLL are frequently derived from the normal T-cell population.4 These problems were overcome with the introduction of interphase fluorescent in situ hybridization (FISH) using either centromeric probes specific for individual chromosomes or locus-specific probes cloned into yeast artificial chromosome (YAC), P1-derived artificial chromosome (PAC), or bacterial artificial chromosome (BAC) vectors. By using combinations of these probes selected to detect the most frequent known abnormalities in CLL, genetic abnormalities can be found in 80 percent of cases of CLL. Comparative genomic hybridization (CGH) is a further FISH technique, which enables a global screen for genetic gains, amplifications, and losses of tumor

DNA. Gains of genetic material on chromosomes 4q and 8q have been identified that would not have been predicted from cytogenetic studies.5,6 The sensitivity of CGH will be greatly enhanced once metaphase chromosomes are replaced as targets by microarrayed DNA fragments.7

Molecular techniques, including Southern analysis and microsatellite analysis, to detect loss of heterozygosity have also been employed in CLL. In general the concordance between the techniques described above is high, although both interphase and metaphase FISH show that the complexity of genetic abnormalities, including the presence of multiple subclones, is greater than can be detected from cytogenetic or microsatellite analysis.8 Although global studies of gene expression are in their infancy, DNA microarray studies are being performed in CLL and differences in gene expression between CLL and other B-cell malignancies and between different subsets of CLL have been identified.9 Proteomic analysis has also shown differences in protein expression between patients with stable or progressive disease.10

The purpose of this chapter is to review the known genetic and epigenetic abnormalities in CLL and to assess their clinical significance in the light of recent data. These show that patients with CLL can be subdivided into two groups with widely differing survivals, depending on the presence or absence of mutations in the immunoglobulin heavy-chain variable-region genes.


Structural abnormalities involving chromosome 13q14 were first noted to be a recurring cytogenetic abnormality in CLL in 1987.11 Subsequent larger cytogenetic studies have shown that abnormalities of 13q14 are found in approximately 20 percent of patients, whereas with use of more sensitive techniques, the incidence of loss is 30 to 60 percent. Two-thirds of patients have a deletion usually involving (13)(q14q22) or (13)(q12q14), and the remainder have a translocation involving q14. The translocations involve a wide variety of different partner chromosomes (Figure 6-1) and are frequently complex. FISH analysis of metaphases with 13q14 translocations invariably show that the translocations are accompanied by genetic loss with variable proximal and distal breakpoints.12

When 13q abnormalities in CLL were first identified, the only known gene assigned to this region was the retinoblas-toma gene (RB1). Loss of RBI was subsequently identified by both Southern and FISH analysis in patients with CLL, including those with a normal karyotype.13 However, no evidence of RB1 gene mutations or low or absent retinoblastoma protein expression was found in cases of heterozygous RB1 loss.14 In addition, genetic loss at more telomeric loci within 13q14 was identified both at a higher frequency than RB1 loss and in cases in which the RB1 locus was intact (Table 6-1). In contrast to other regions of chromosomal deletion found in CLL, homozygous loss at 13q14 is a frequent finding, occurring in 10 to 20 percent of cases, which strongly suggests that one or

Cytogenetics Translocation
Figure 6—1. The reciprocal translocation breakpoints of cases with a translocation involving 13q14 are shown. Solid circles indicate those cases in which the translocation was clonal and open squares indicate cases with a 13q14 translocation as a single-cell abnormality.

more genes that are important in the pathogenesis of CLL lie within this region.

Several groups of investigators have constructed detailed physical maps of 13q14 and have performed deletion mapping using either FISH, Southern, or microsatellite analysis.112-115 A number of overlapping regions of minimal genetic loss have been identified, and these are shown in Figure 6-2. One possible explanation for the differing regions of loss is the presence of a large gene, with several splice forms, covering a large genomic region. Another is that chromosome inversion may accompany genomic deletion in a proportion of cases. This would account for the discontinuous regions of loss that we and others have reported. Finally, more than one gene may be implicated. A 10-kb region of minimal loss close to D13S319 encompasses exons of two separate transcripts termed LEU1 and LEU2.15 The RFP2 gene is located 50 kb centromeric to the minimally deleted region on chromosome 13 and encodes a 407-amino-acid ring-finger protein, which shares homology with a number of transcription factors, including BRCA2.16 Sequence analysis of the corresponding mouse genomic region has revealed strong identity between the human and mouse LEU2 and RFP2 genes.17 Although LEU1, LEU2, and RFP2 may be considered strong candidate genes, no mutations have been found in these genes in patients with heterozygous loss in this region, nor have mutations been discovered in a 347-kb region of loss encompassing the above three genes.18 Significant hypermethylation close to LEU2 occurs in patients with heterozygous loss of LEU2, as compared with patients' cases with homozygous retention of this region, suggesting that loss of function of LEU2 or a neighboring gene may be important in CLL.19

Using interphase FISH, Garcio-Marco et al.20 found loss at 13q12 encompassing BRCA2, in 80 percent of patients with CLL, but other studies employing FISH or Southern blotting were unable to confirm the very high incidence of genetic loss at this locus.21

Table 6-1. Genetic Loss at Chromosome 13q14 in Chronic Lymphocytic Leukemia

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