EXPORTACIONES DEFINTIVAS

As early as 1979, trisomy 12 was defined as the most common cytogenetic abnormality seen in B-cell CLL (Juliusson and Merup, 1998). Early studies used cytogenetic banding techniques to diagnose trisomy 12 and estimated the incidence at approximately 15% (Juliusson et a i, 1990). With the advent of fluorescent in situ hybridisation techniques (FISH), probes for the centromere of chromosome

12 were designed for use on fresh interphase cells and the incidence has now been shown to be slightly higher at 18-20% (Matutes et al, 1996; Garcia-Marco et al.^

1997). A recent review of all publications since 1991 averaged the incidence of trisomy 12 found by FISH analysis to be 22% (Juliusson and Merup, 1998) (see table 1.1).

Using restriction fragment length polymorphisms (discussed below), the extra chromosome 12 seen in trisomy 12 has been shown to derive from the duplication of one autosome with retention of the other and not as a result of a triplication of one autosome with loss of the other (Dierlamn et a l, 1997).

The association of trisomy 12 and atypical lymphocyte morphology has been clearly documented. Matutes et al analysed 544 patients for the presence of trisomy 12 using FISH, as part of the MRC CLL 3 trial (Matutes et a l, 1996). They showed that overall, 83% of cases had typical CLL morphology and 10% had more than 10% prolymphocytes (immature lymphocytes). Out of those patients with trisomy 12 (20%), 31% had more than 10% prolymphocytes and

Chromosomal abnormality Conventional Cytogenetics FISH Molecular Techniques

trisomy 12 15% - Juliusson 1990 18% - Matutes 1996

20% - Garcia-Marco 1997

22% - Juliusson review 1998

deletion 13q 20% - Juliusson 1991 51% - Stilgenbauer 1998 50% - Panayiotidis 1997

31 % - Avet-Loiseau 1996 41 % - Corcoran 1997

deletion l l q 9% - Juliusson 1990 20% - Dohner 1997

16% - Juliusson 1991 16.6% - Schaffner 1998

7% - Juliusson review 1998

deletion 6q 12% - Juliusson 1991 7% - Merup 1994

24% had atypical morphology. She concluded that trisomy 12 defines a subgroup of CLL with more frequent atypical morphology.

In a large, multicentre study of 433 patients with CLL, it was demonstrated that patients with sole trisomy 12 abnormalities had the poorest survival of all the single abnormality subgroups analysed (Juliusson et al, 1990). This was significantly worse than the survival of patients with single 13q abnormalities (P=0.01). This association of trisomy 12 with more progressive disease and a shorter therapy-free survival was then confirmed with the second international compilation of data by the International Working Party on Chromosomes in Chronic Lymphocytic Leukemia (IWCCLL) (Juliusson et a l,

1991).

An interesting feature of patients with trisomy 12 is that the abnormality is not found in all CLL cells. Garcia-Marco et al demonstrated 21-37% of cells showing trisomy of chromosome 12 in an analysis of 6 patients (Garcia-Marco et a l, 1994). A later study by the same group showed the numbers o f cells with the abnormality ranged between patients, from 7%-84% (Garcia-Marco et al, 1997). The median percentage of affected cells from the IWCCLL data was 45% (Juliusson et a l, 1991). This finding of trisomy 12 in a sub population of cells has been suggested to represent clonal evolution of the disease. A recent review stated that no patient had been documented to acquire trisomy 12 over the course of a longitudinal study (Juliusson and Merup, 1998). However, during the course of progressive disease the percentage of cells with trisomy 12 may increase. A FISH study by Garcia-Marco et al contests this, showing one patient to acquire trisomy 12 during the course of their disease with the other 30 patients analysed showing an increasing percentage of cells with trisomy 12 during the course of their illness (Garcia-Marco et a l, 1997).

Another study showed trisomy 12 to be detectable in a proportion of CD34+ stem cells in B-cell CLL patients with known trisomy 12 (Gahn et a l,

1997). This group suggest that CLL may not be a malignancy of mature B cells but may arise due to a stem cell disorder, at least in the subset o f patients with trisomy 12. The biological significance of trisomy 12 within CLL remains unknown.

1 .3 .2 .13q deletions

Deletions of the long arm of chromosome 13 involving band 14 are the commonest structural abnormalities seen in B-cell CLL. Some recent studies suggest that the incidence of 13q deletion, when investigated by FISH and Southern blot analysis, may be greater than the incidence of trisomy 12 (table 1.1). Using conventional cytogenetic techniques, the incidence of 13q deletions reported by the IWCCLL was 20% (Juliusson et al, 1991). Using FISH analysis other groups have reported incidences of 51% (Stilgenbauer et a l, 1998) and 31% (Avet-Loiseau et a l, 1996). A similar frequency is seen with Southern blot analysis. Panayiotidis et al found 13ql4 deletions in 50% of patients (Panayiotidis et a l, 1997) whilst Corcoran et al reported an incidence of 41% (Corcoran et a l, 1998). Approximately 6% of the patients reported by both Corcoran and Panayiotidis show homozygous deletion at this locus. It is thought that a tumour suppressor gene, whose loss or inactivation is crucial in the development of CLL, is located at 13ql4.3

Large scale studies have shown that a single abnormality involving 13q does not proffer a worse prognosis than standard risk B-cell CLL i.e. patients with a normal karyotype (Juliusson et a l, 1990). This contrasts with all other cytogenetic abnormalities described in B-cell CLL to date. If another cytogenetic abnormality co-exists with the 13q changes then the prognosis is worse (Juliusson

e ta l, 1990).

Considering that trisomy 12 and 13q deletions are the most frequent cytogenetic changes seen in CLL, it is interesting to note that they are very rarely seen together within the same tumour. Early studies suggested that the two abnormalities might be mutually exclusive but they have since been reported co­ existing within the same tumour in a small number of cases. Using both conventional cytogenetics and FISH analysis. Mould et a l described 8 patients out of a series of over 400 cases with both trisomy 12 and abnormalities of 13q (Mould et al, 1996). In a similar study o f 600 cases, Navarro et al reported 9% to show abnormalities of chromosome 12 and 13 (Navarro et al, 1998). The IWCCLL study reported 233 patients out of 512 with more than one chromosomal abnormality, of which 10 patients had both structural changes of

13q and trisomy 12. This low incidence was shown to be significantly less than expected (Juliusson et a l, 1991) and Juliusson therefore suggests that the two abnormalities may provide a similar, but unrelated pathogenic influence towards

differentiating B cells (Juliusson and Merup, 1998). If this assumption is correct then acquisition of both abnormalities gives no advantage to a cell progressing towards malignancy.

When DNA from patients was analysed for 13q deletions at the D13S25 locus (see below) using Southern blot and densitometry techniques, comparison with normal controls showed the malignant cells to have 100%, 50% or 0% of the normal values recorded (Jabbar et al., 1995). Investigators drew the conclusion that loss of 13q alleles was a clonal event with the values seen representing no loss (100%), heterozygous loss (50%) or homozygous deletion (0%). Subsequent studies using FISH have cast doubt on this assumption. FISH techniques have an advantage over Southern blotting in this type of analysis in that individual cells within the malignant clone can be analysed. As discussed, Navarro et al found 13q deletions and trisomy 12 within the same patients. In some cases they found that a small number of cells contained three copies of chromosome 12 and another, usually larger, group of cells showed deletion of 13q, suggesting that both abnormalities existed within a proportion of cells (Navarro et a l, 1998). This was verified by another group who used a YAC (933e9) covering the region of interest at 13q to test their patients for deletion of this region using FISH. They found 31% of patients had deletion of the region, with the percentage of cells exhibiting chromosomal loss ranging from 31 to 90% (Avet-Loiseau et al, 1996). Merup et a l reported patients where the major clone showed heterozygous deletion of

13ql4, but where two smaller clones showing either homozygous deletion of 13ql4 or no deletion of 13ql4 could also be detected (Merup et a l, 1998). It should be noted that this group used mitogenic stimulation to culture the B cells and, therefore, normal cells may have been over represented in their samples. It seems likely, therefore, that whilst 13q deletion may exist in the majority of cells in most tumours with 13ql4 deletion (either as a heterozygous or as a homozygous loss), it may not be present in the original cell from which the clonal B lymphocyte population is derived.

Mapping of the deleted region at 13ql4.3 will be described in detail later. It is worth noting that a controversy arose in 1996 suggesting that the important region of deletion at chromosome 13 was not 13ql4 but 13ql2, the site of the tumour suppressor gene BRCA 2 (Garcia-Marco et a l, 1996). Investigators reported the incidence of deletion at this region to be 80%. This would make this the most frequently occurring cytogenetic abnormality in B-cell CLL. This data was derived from interphase FISH analysis. However, FISH analysis from another

group on over 300 patients (Stilgenbauer et al, 1998) and Southern blot data from our group (Panayiotidis et al, 1997) could not confirm this finding in the patients tested. Deletion of 13ql4.3 remains the commonest structural abnormality in most groups of patients reported.

Interestingly, the same region at 13ql4.3 has been shown to be deleted in B-cell non-Hodgkin's lymphoma (Liu et a l, 1995), acute lymphocytic leukaemia (Liu et al, 1995), splenic lymphoma with villous lymphocytes (Garcia-Marco et a l, 1998) and, more recently, myeloid malignancies (La Starza et a l, 1998) and multiple myeloma (Chang et al, 1999). This region has consequently become the focus of much attention.