Cytogenetics

In 1914, Boveri (133) described chromosomal changes in tumor cells. In 1960, Nowell and Hungerford (134) identified the first tumor-specific chromosomal abnormality, the Philadelphia (Ph) chromosome, a minute chromosome in chronic myeloid leukemia. After the development of chromosome banding Caspersson et al. (135,136) identified the Ph chromosome as an abbreviated chromosome 22.

When chromosome banding became available, the chromosomes were enumerated from 1 (the largest) to 22 (the shortest). The sex chromosomes were designated X and Y. The shortest arm of the chromosome is designated p (shown above the centromere in figures), and the long arm is termed q.

Differences in the number of copies were presented with a minus sign for monosomy and a plus for trisomy. Other abnormalities are indicated by letters. The chromosomes involved in the abnormality are placed in parentheses in numerical order separated by a semicolon (;). Following in a second parentheses are the specific breakpoint bands for each chromosome, also separated by a semicolon. Translocations are designated with a "t" before the first parentheses, deletions with a "del," interstitial deletions, with "int del," duplications with "dup," and inversions with "inv." Abnormal chromosomes with the same arm on both sides of the centromere, isochromosomes, are designated as "iso." To be defined as a clonal change, two metaphases with the same abnormality or extra chromosome, or three metaphases with the loss of the same chromosome, are required.

Cytogenetic techniques require metaphase cells of good quality. Rapidly proliferating tumors, such as those in acute leukemia, usually present spontaneous mitosis. Cells from indolent disease such as CLL have a very low mitotic index, and in vivo activation with mitogens is required to induce mitosis in tumor cells from these diseases. To evaluate constitutional abnormalities, the established technique is to culture blood cells with phytohemagglutinin (PHA), which is a T-cell activator. Thus, when this was done in the early 1970s, the conclusion was that there were no chromosomal abnormalities in CLL. As late as 1975, Crossen (137), in viewing 100 metaphases from 20 CLL patients concluded that 97 of them had a normal banding pattern. The remaining three, all from one patient, had a pattern similar to those seen in aging.

As noted just above, CLL have a very low mitotic index, and activation with mitogens is required to induce mitosis. When PHA, a potent T-cell activator, is used on bone marrow cultures, or when peripheral blood samples from patients with CLL have been analyzed, only normal cells were available for analysis. As late as 1975, the conclusion was that there were no chromosomal abnormalities in CLL. Most cells from patients had a normal chromosome number and chromo-

some morphology. A slight increase in aneuploid cells was seen, but none were considered cell lines because they were isolated occurrences. The finding of normal banding patterns in CLL suggested that chromosomal changes were not a feature of this leukemia. Crossen (137) did recognize that there might be a problem with PHA not stimulating B-cells. Using dextran sulfate, lipopolysaccharide from E. coli, purified protein derivative (PPD) from tuberculin, and rabbit IgG antihuman B2-microglobulin, Robert et al. (138) documented the first successful activation of B-cells in 1978. In addition to these early B-cell-activating agents, pokeweed mitogen (PWM), concanavalin A, tetradecanoyl phorbol acetate (TPA), cytochalasin B, antihuman IgM, B-cell growth factor, calcium ionophore, and anti-CD40 antibody have been used in an attempt to generate mitotic figures from CLL samples. After stimulation with Epstein-Barr virus, Gahrton et al. (139) first reported trisomy 12 in a CLL patient in 1979 and subsequently in a fairly large proportion (15%) of cases examined (140,141).

The use of newer molecular genetics techniques has contributed greatly to advances in the understanding of CLL. Fluorescence in situ hybridization (FISH) can detect changes in interphase as well as metaphase cells. PCR analysis allows chromosomal breakpoints to be identified. Comparative genomic hybridization, developed in 1992 (142), has the ability to survey the whole genome in a single hybridization and can detect and map relative DNA sequence copy number between genomes. It can distinguish changes as low as several kilobases up to as many as a few megabases. These techniques have identified a number of new chromosomal aberrations as well as previously known aberrations in previously normal-seeming cases.

While it is still not quantified, the fraction of CLL patients with identifiable genetic aberrations has increased with the use of molecular genetic techniques. Using conventional cytogenetic techniques, Jarosova et al. (143) found that 17% of nonstimulated bone marrow samples of 88 CLL patients had abnormalities. Comparative genomic hybridization and FISH revealed chromosomal changes in an additional 33 individuals, bringing the total with abnormalities to 57%. In 2000, Dohner et al. (144) detected genomic aberrations using FISH in 82% of 325 patients with CLL.

Juliusson and Gahrton (145-147), Juliusson et al. (148), and Dohner et al. (144) summarize the status of cytogenetics in CLL. First discovered in the 1980s, the most frequent genetic aberration in CLL now appears to be a deletion in 13q14 (149-151). A 13q14 deletion may confer protection. Another frequent genetic aberration is a deletion in 11q (152-155). It is associated with disease progression and reduced survival (156,157). The area may contain the neural cell adhesion molecule, or the ataxia telangiectasia (AT) gene may be mutated (ATM). Dohner et al. (158) notes that these patients tend to have massive lymphadenopathy and do not do well clinically. Trisomy 12 appears in between 7 and 25% of the CLL cases, and is said to be associated with atypical CLL (159). Another common aberration is a 17p deletion. The short arm of chromosome 17 is the site of the TP53 tumor suppressor gene. G-banding analysis demonstrated abnormalities of chromosome 17 in 13 of these 17 patients, leading to the loss of band 17p13. These 17 patients exhibited a monoallelic p53 gene deletion, as demonstrated by FISH (160). The median survival time from the date of diagnosis of patients with a p53 gene deletion was 2.1 yr compared with 10.3 yr in the patients without the deletion (p < 0.001). None of12 patients with a deletion responded to therapy with either fludarabine (n = 7) or pentostatin (n = 5), whereas 20 of 36 (56%) patients without a deletion achieved a remission (p < 0.001). A 6q deletion may be found in approx 6% of CLL cases (161-163). The pathogenic significance has not yet been identified. Figure 41 is a convenient summary of the distribution of known cytogenetic abnormalities in CLL, and Figs. 42 and 43 show survival and disease progression data in CLL patients as a function of their cytogenetic abnormality.

Fig. 41. This is a frequency distribution of the common cytogenetic lesions in CLL. Approximately one-third of the patients will have more than one abnormality, and approx 15% will have a normal karyotype. Given the frequency of the 13q14 deletion in CLL, a more detailed summary of this deletion is shown in Fig. 44. FISH, fluorescence in situ hybridization. (From ref. 164, p. 353, Stilgenbauer et al., 2001 by courtesy of Dekker, Inc.)

Fig. 41. This is a frequency distribution of the common cytogenetic lesions in CLL. Approximately one-third of the patients will have more than one abnormality, and approx 15% will have a normal karyotype. Given the frequency of the 13q14 deletion in CLL, a more detailed summary of this deletion is shown in Fig. 44. FISH, fluorescence in situ hybridization. (From ref. 164, p. 353, Stilgenbauer et al., 2001 by courtesy of Dekker, Inc.)

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