Knowledge of chromosomal deletions has significantly contributed to the identification of tumour suppressor genes. This follows from the observation that deletion of one allele is the first step towards loss of function that can be achieved by the several mechanisms discussed in several section of this thesis. Massive deletions often obliterate entire chromosomes (monosomy) or chromosome arms in tumour tissue. A typical example of this underlying event led to the discovery of the RBI (Knudson, A. G., Jr. 1971).
Such deletions can be identified by cytogenetic analysis, which is sensitive enough to detect chromosomal abnormalities in small population o f cells and if deletion is present in more than 5% of the cell population. Although cytogenetic analysis is highly informative in the majority of the cases, its interpretation may be difficult if the karyotype is complex or multiple clones are present. Failure to produce successful metaphases may occur due to problems associated with low mitotic index or poor chromosome morphology.
The development of fluorescence in situ hybridisation (FISH) has improved the sensitivity, accuracy and reliability of cytogenetic investigation. Interphase FISH, in particular has contributed to the screening of a large number o f non-dividing cells (Harrison, C. J. 2000). The sensitivity and specificity can improve greatly by the use of 2 or 3 probes simultaneously.
However, both cytogenetic and FISH analyses have the problem of being unable to detect small interstitial deletions that might be beyond the resolution of these techniques. Cytogenetic studies have identified non-random 6q deletions in ALL and other tumours including malignant melanoma, renal cell carcinoma, and salivary gland adenocarcinoma (Stenman, 0 . et a l 1989; Hayashi, Y. et a l 1990; Millikin, D. et a l
1991; Morita, R. et a l 1991). FISH studies on 6q deletions helped to identify and the refine the region of minimal deletion on 6q in ALL (Menasce, L. P. et a l 1994; Menasce, L. P. et a l 1994; Sherratt, T. et a l 1997; Jackson, A. et a l 2000; Zhang, Y. et a l 2000).
As methodological problems in the cytogenetic analysis of solid tumours have restrained attempts to apply standard techniques to screening for deleted chromosomal areas, comparative genomic hybridisation (CGH) has more recently proven to be a powerful genome-wide screening method. Comparative genomic hybridisation (CGH) allows DNA copy number losses and gains to be studied in one hybridisation experiment (Kallioniemi, A. et a l 1994). CGH is sensitive for detecting deletions that are 10 to 20 megabases in size (Bentz, M. et a l 1998). However, as cytogenetics and FISH, CGH requires cultured cells in addition to its limited ability of detecting small deletions.
In view of the difficulty in obtaining suitable material for cytogenetics and FISH analysis, because of the limited ability to identify small deletions, but thanks to the progress in genome mapping and sequencing, LOH analysis has also made a great contribution to the identification of region of deletions using closely linked microsatellite markers. LOH analysis has helped to identify regions of deletions and direct the search for candidate tumour suppressor genes to specific area of the human genome. As a consequence, several TSGs have been identified from regions showing frequent LOH in tumours (Fearon, E. R. and Vogelstein, B. 1990) both in solid and haematological cancers.
Several types of polymorphic markers have been used in LOH studies including restriction fragment length polymorphism (RFLP) where LOH can be analysed by Southern blot hybridisation using the chromosome specific RFLP probes (Gaidano, G.
et a l 1992). However the use of RFLP markers is restricted by the need of large amount of DNA, the need for tedious and time consuming Southern hybridisation assays and their relatively low level of informativeness.
Microsatellite markers are short tandem repeats of DNA that are distributed throughout the genome. Repetition of a microsatellite may be based on single base (A)n or poly A, two bases (CA)n or dinucleotide, or three or more bases. Microsatellites may be either monomorphic and show no variation in size between cells or individuals, or polymorphic showing a variety of sizes. They are stable andl very occasionally the length of a microsatellite marker can be seen to change whien it passes from one individual to another. The polymorphic nature of the microsattellite markers together with the use of PGR based technique helped to overcome the problems associated with RFLP. The amount of DNA is very limited and the markers are numerous and most of
them are highly informative. PCR amplification using oligonucleotide primers is rapid an can be automated. Analysis of LOH can be either done by separating isotipically labelled (^^P- or preferably ^^P) PCR products on denaturing polyacrylamide gels followed by autoradiography or one primer of each PCR primer pair can be labelled with a fluorescent dye then after amplification the PCR products are analysed on an automated DNA sequencer using the software programme (Gene Scanning).
LOH was identified only when substantial reduction was measured in the ratio of allele radiographic signal intensities in the tumour sample relative to that in the corresponding normal sample (Takeuchi, S. et a l 1995; Gerard, B. et a l 1997; Merup, M. et a l 1998; Takeuchi, S. e ta l 1998).
Although LOH analysis using microsatellite markers is a powerful technique that identified higher frequencies of chromosomal deletions than the reported frequencies by cytogenetic studies but it still show some limitations.
These limitations include the requirement of tumour cell purity that exceed 70-80% as the presence of contaminating non-tumour DNA may affect the interpretation. In our study on 6q deletions in ALL we have selected our samples with the percentage of blasts of at least 80-90% in cases of the tumour samples and the non-tumour samples correspond to remission specimen collected at time of morphological and clinical complete remission. Additional limitations include uninformative markers with limited polymorphism or lack of markers within a critical region when aiming at accurate identification of deletions.
In the definition o f RMD, interstitial deletions can be difficult to interpret but are common to every study, which has implemented LOH, and 6q deletion analysis is no exception (Gerard, B., Cave, H. et a l 1997; Takeuchi, S. et a l 1998). In the study presented here we have obtained similar pattern of interstitial deletions with some cases showing loss of some markers and retention of others. Only 3 out o f 16 cases showed contiguous deletions and 9 out of 16 cases studied showed loss o f only one marker which was not consistent with one specific microsatellite marker. The presence of such interstitial deletions may be due to the power of LOH technique to pick small deletions that would be missed by cytogenetics.
Another potential interpretation pitfalls may occur when one o f the alleles of a certain micro satellite marker is missing and no other novel fragment aire present. This case is usually not easy to interpret, as this might represent true LOH or MSI in which the shifted allele has migrated with the wild type allele. The NCI workshop recommended
that such a finding should be scored as negative for MSI. However, the frequency that a shifted allele would co-migrate with the remaining wild allele is believed to be low. Also, difficult interpretation is encountered when a specific marker shows both apparent LOH and the presences of novel fragments (MSI). In such situations it is difficult to distinguish whether the apparent allelic loss is due to MSI or it is truly present in addition to MSI. Those markers showing MSI should be scored as non-informative for LOH in tumours with MSI/L, whereas all markers should be scored as non-informative for LOH in tumours with MSI/H (Boland, C. R. et a l 1998).
5.8. Future stu d ies
The results discussed above suggest that cytogenetics alone may be insufficient in the characterisation of 6q deletions. FISH on the other hand may fail to identify small interstitial deletions which may be relevant for the definition of RMD. LOH although very sensitive, may only provide information with regards to the identification of region of minimal deletion, but may be insufficient for the identification o f candidate TSGs and the definition of RMD may be complicated by interstitial deletions.
For a comprehensive evaluation of the extent and role of 6q deletion future studies may therefore include:
1. Expansion of the total number of patients analysed by LOH.
2. Narrow down the region of minimal deletion to smaller genomic region by using novel polymorphic sites identified through human genome project.
3. Sequence analysis of genes such as GRIK2 for the identification of somatic mutations responsible for the inactivation of the gene on the retained allele in patients with heterozygous deletions
4. Analysis of other genes like PRDM l, as it was previously suggested that this gene could play a TSG role in B-NHL tumours but confirmation of this role has not been obtained yet. No mutation analysis has yet been reported for the BLIMP
gene and this investigation should be performed in the future.
5. Investigation on the role played by other candidate genes including.- AIM l (a candidate TSG in malignant melanoma) and NR2F1/TLX gene. The role of the latter gene as a TSG gene has been previously ruled out as based on lack of mutations of the retained allele. However, epigenetic mechanisms should also be further investigated although this may depend on the correct identification of promoter sequences.
6. Expansion on the number of cases showing MSI pattern to achieve a better evaluation on the role of MSI in both childhood and adult ALL as this is a novel finding of this study.
7. Investigation of the MMR in the cases that showed MSI. Investigation of the protein expression of MMR genes by immunohistochemical staining would be possible as monoclonal antibodies to at least 4 MMR genes {MLHl, MSH2, PM Sl and PMS2) are now available. Although no previous reports on using this technique for haematological malignancies with MSI, paraffin embedded sections of trephine bone marrow biopsies may be the suitable material for the future investigations. Newly diagnosed patient’s material should be stored for this type of investigation in the future. In addition mutational and epigenetic studies of MMR genes are required to identify possible mechanisms responsible for MSI. 8. Analyse the sequence of the YAC clones (yR39ECll and yX28F10 presently
partially sequenced) to identify the structural characteristics that may explain the difficulty in the cloning of these regions.
9. Use sequence analysis of the two above YACs to identify candidate genes within these YACs that may be relevant to this investigation.
All the above aspects will be important in the planning of the future studies on chromosome 6 deletion in ALL and may help to redefine the involvement of novel genes in the pathogenesis of acute leukaemia in humans.