Cytogenetics And Molecular Cytogenetics

Regular cytogenetic analysis of CLL is confounded by the low proliferative rate of the bulk of the cells and the presence of residual normal T cells. Comprehensive analysis has been performed by a number of dedicated centers worldwide, where conditions for CLL cytogenetics have been optimized. From these studies it is clear that, unlike the related diseases of mature B cells, such as follicular lymphoma or MCL, there is no obvious consistent cytogenetic lesion in CLL.

A number of recurrent abnormalities have been identified, although the molecular consequences of some remain to be unequivocally identified. Practically, these lesions can be readily detected using interphase FISH methods, either using individual BAC clones or using CLL specific BAC arrays.22 However, interphase methods will miss many of the complex cytogenetic events that may occur in CLL.23 A further problem is that many of these events, including both 11q and 17p13 deletions, are secondary and thus only present in a fraction of cells at diagnosis; this may limit the clinical use of BAC arrays, whose sensitivity is not presently adequate to detect such minor changes.

As with acute lymphoblastic leukemia (ALL), detection of these genomic abnormalities is associated with specific prognostic groups and will form the basis for stratified therapy in future clinical trials.24

Del(13)(q14): Loss of a region of 13q14 is the commonest lesion seen in CLL, one allele being lost in about 40% of the cases and both alleles in about 10-20% by interphase FISH analysis. Some of these deletions may be extremely small and may therefore be missed by interphase FISH. Deletions of this region in the absence of other abnormalities are associated with good prognosis disease in CLL. More recently, it has become apparent that deletions of this region may be seen not only in several other B-cell malignancies including MCL, diffuse large B-cell non-Hodgkin's lymphoma (DLBCL), and myeloma, but in solid tumors as well.

These data strongly suggest the presence of a tumor suppressor gene (TSG) within this region. However, despite characterization of many transcripts within the deleted region, the nature of the involved gene remains obscure (see for example Ref. 25 and references therein). According to Knudson's hypothesis for "classical" TSGs, deletion of one allele should be associated with mutation of the other; no mutations have been found in any of the candidate genes.

Similarly, no hypermethylation of the promoter regions, which would also result in reduced expression, has been observed in CLL. Haploinsufficiency, where loss of one allele alone results in a phenotype, may be the answer to this conundrum, but validation will require careful in vivo modeling.26

Another possibility is that the 13q14 deletion may involve micro-RNAs (miRNAs), 19-22 nucleotide long genes that regulate key processes including apoptosis and proliferation.27 Some miRNAs are developmen-tally regulated and some are B-cell specific; some are directly involved in chromosomal translocations, indicating a direct role in the pathogenesis of neoplasia.28 It has been suggested that the target genes in CLL may be two adjacent miRNAs, miRNA15 and 16, clustered together on chromosome 13q14 within the final intron of the DLEU2 gene5 (see also http://www.sanger. Furthermore, the same group has used an miRNA array to demonstrate that specific subgroups of CLL may be associated with specific miRNA "signatures."29'30 However, these data are controversial and need to be confirmed. The possible role of many of the other 206 miRNAs so far identified in the pathogenesis of B-cell malignancies remains to be investigated; it is of some considerable interest that many map to the sites of recurrent DNA damage in malignancy.

Dei(11)(q22.3-q23.1): Deletions of this region are observed in about 12-15% of the cases. These deletions are molecularly variable and often secondary, but tend to be associated with progressive disease occurring in younger men with bulky lymph node disease. The association with poor prognosis may not be present in elderly patients with deletions of this region, about 50% of which appear to involve the gene that causes ataxia-telangectasia, the ATM gene. This gene comprises 63 exons and encodes a huge protein of 3056 amino acids; consequently, mutational analysis is technically difficult. Mutations of ATM are seen in all cases of T-cell prolymphocytic leukemia and may also be found in 50% of CLL patients with 11q deletions. However, some of these "mutations" may in fact represent rare germline polymorphisms.

The ATM protein is an important cell cycle checkpoint kinase that functions as an activator/regulator of a wide variety of downstream proteins, including p53, checkpoint proteins RAD17 and RAD9A, and DNA repair proteins. Loss of ATM functions results in loss of responses to DNA damage, and consequently genomic instability.31

Del(17)(p13): deletion of the short arm of chromosome 17 is seen in about 5-7% of cases of CLL at diagnosis. This is often a secondary abnormality and therefore may be seen in only a fraction of the neo-plastic cells. The target gene of this deletion is cases TP53, although deletion of one allele with no TP53 mutations in the remaining allele may occur, suggesting the presence of another, more telomeric TSG on chromosome 17p.

Abnormalities within the TP53/ATM axis have a profound impact on the biological behavior of CLL; patients with either abnormality, but particularly those with TP53 mutations, fare badly with conventional chemotherapy. Their detection may be an indication for early therapy with agents such as CAM-PATH-1H (alemtuzumab), where elimination of the neoplastic cells does not depend on p53 function. Given the importance of changes in TP53 and ATM to eventual clinical outcome, and the difficulties in assessing both genes, a simple functional assay may be of value. The response to ionizing irradiation in terms of increased p21 expression allows a direct assessment of both p53 and ATM functions.32

Although the consequences of ATM and TP53 inac-tivation are similar, they are by no means identical. Genome-wide expression experiments have indicated that the worse response of patients with TP53 mutations may reflect the loss of p53-dependent apoptotic pathways.33

Trisomy 12: This abnormality occurs in about 15% of CLL patients and again is usually only seen in a fraction of the cells. The percentage of cells with trisomy 12 often does not increase with transformation. The specific molecular consequences of this abnormality, which again is not specific for CLL, are not clear.

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