In my first report from the IWCLL I want to focus on chromosomes. FISH - fluorescent in-situ hybridization is the mainstay of chromosomal examination in CLL. With this technique florescent probes are used to detect certain DNA sequences in the CLL nucleus. It has the advantage that the cell does not have to divide in order to be examined. and because of this many cells can be examines (typically 200). The disadvantage is that you can only see what you look for - usually deletions at 13q14, 11q23 and 17p13 plus trisomy 12. Sometimes del 6q is also looked for. There were reports at this meeting of the use of other techniques including looking for copy number changes and loss of heterozygosity using high density SNP arrays, and new mitotic agents to induce cell division.
In the SNP array paper from Freiburg, Germany an area at 2p16 that contains came under REL and BCL11A came under suspicion. BCL11A was one of the genes that is differentially expressed in mutated and unmutated patients, identified by the original Rosenwald paper.
Most people cannot get CLL cells to divide properly. A good way of checking whether chromosome banding techniques are successful is to compare the percentage of del 13q14 patients that are detected by banding and FISH. Most people find many more by FISH and by this reckoning only the Haferlach group at Munich and the Oscier group in Bournemouth are any good at chromosome banding in CLL, and both reported their results here. The Munch group largely confirmed the results that had previously been found by FISH, but added the fact that chromosomal translocations occur in about 20% of patients. Most involve either 14q32 or 13q14, and they are usually not balanced - the involve a loss of chromosomal material - effectively they are deletions. They do not have common partners and they do not form fusion genes. They also reported that trisomies of 3, 18 and 19 are seen as well as trisomy 12. Finally, about 16% have complex karyotypes which are usually associated with bad prognosis markers.
The Oscier group reported on karyotyic evolution. this can be detected by demonstrating sub-populations of cells with none, one, two or three abnormalities apparently acquired in a sequential way (eg 10% of cells have normal karyotypes, 27% just del 13q14, 48% of cells del 13q14 plus del 11q23, and the rest del 13q14, del 11q23 and del 17p13). Or they cen be demonstrated by watching for changes on sequential samples taken at interval - this would include someone who had 14% del 13q14 at diagnosis, but 85% del 13q14 four years later. The Oscier group reported on 342 patients. In 103 there was evidence that karyotypic evolution had occurred prior to diagnosis. 314 had a second sample taken after an interval of at least a year. Patients had a median of 4 (range 2-13) samples taken. 189 patients eventually showed evidence of karyotypic evolution (ie 55%). There were specific patterns of evolution. Those with trisomy 12 (which was almost universally a primary event) either acquired a 14q32 translocation (t14;18 or t14;19) or developed further trisomies (of 19, 18 or less commonly 3). Further trisomies only occurred on a background of trisomy 12, and only occurred in those with mutated VH genes.
In patients withdel 13q14 the commonest progression was for the other chromosome to become involved with a similar lesion. These has been some debate as to whether this carries a poor prognosis. Other groups (including the Munich group) have suggested that it might do so although the figures do not reach statistical significance, However, in this series those with the second chromosome involved actually did significantly better than those with only one - possibly because this is a relatively late phenomenon, and the patient has to survive a long time without acquiring a different chromosomal abnormality in order to just have a homozygous deletion at 13q.
The hunt for the gene responsible at 13q14 has been long and apparently fruitless. The missing chromosomal portion was first reported in 1987:
Chromosome abnormalities involving band 13q14 in hematologic malignancies
Margaret Fitchett, Michael J. Griffiths, David G. Oscier, Sharron Johnson and Marina Seabright Cancer Genetics and Cytogenetics Volume 24, Issue 1, January 1987, Pages 143-150.
There have been several attempts to identify the genes on the missing fragment, largely by Dr Oscier's group, but to no avail until the problem attracted the big boys, Carlo Croce and Ricardo Dalla-Favera. They both agreed that there was no gene coding for any tumor-suppressor protein at this site. Croce's wide reading led him to microRNA genes and the discovery that two such genes, miR-16-1/miR-15a were located within the deleted area. miR genes interfere with the expression of messenger RNA, and thus the expression of a target protein. At least one of the targets of these genes is BCL-2, so that if they are missing BCL-2 is over-expressed and, as everyone knows, BCL-2 is over-expressed in CLL. this is thought to at least partly explain the resistance to apoptosis.
The miR genes in CLL are very close to one of the introns for Leu-2, a gene that is not translated into protein. Dalla-Favera decided to investigate what Leu-2 does. He produced evidence that Leu-2 is a long controlling gene. Thus it is not clear whether the effect of the 13q deletion is mediated just through the miR genes or whether the whole Leu-2 gene acts epigenetically to control some important function in B cells and that loss of this function leads to leukemia.