The detection and clinical significance of TP53 dysfunction in chronic lymphocytic leukaemia.

Abstract

Standard therapy for patients with chronic lymphocytic leukaemia involves the use of DNA damaging drugs. If the cellular DNA damage response has been compromised then these standard therapies will be ineffective at inducing apoptosis. Central to this response is the protein, p53. Loss of the chromosome containing the gene TP53, which encodes the protein p53 and/or mutation of the gene, occurs in approximately 5-10% of newly diagnosed CLL patients. This rises to over 30% in patients who relapse after therapy. In addition another important protein within the DNA damage response, the protein-kinase ATM, may be deleted and/or mutated in a further 20-30% of CLL cases. Loss of the chromosomal loci containing either TP53 or ATM has been shown to predict a poor response to treatment and shorter overall survival. It is now known that detecting loss alone is insufficient as miss-sense or non-sense mutation of these genes alone can lead to an equally poor outcome. Screening these genes for mutations is not carried out routinely and can be technically challenging. In addition to this it is not fully understood to what degree other genes may be involved in dysfunction of the DNA damage response. Functional assays provide an alternative method for testing the integrity of the p53-dependant DNA damage response without needing to focus upon specific genomic abnormalities. A flow-cytometry based assay, utilising etoposide/nutlin3a and developed in our laboratory, has already been shown to detect and discriminate between p53 and ATM abnormalities in a small series of patients. This project aims to assess the clinical value of the etoposide/nutlin3a functional assay for detecting abnormalities of the p53- dependant DNA damage response which would contra-indicate treatment with DNA damaging drugs in samples referred from around the UK . A total of 472 samples from participating centres around the UK have been tested using the functional assay and also assessed for deletion of ATM and p53. The assay is largely unaffected by transport of whole blood samples across the UK if transit time is no greater than 48 hours and the sample is not stored above room temperature. Dysfunctional assay results have been shown to have a highly significant association with deletion of the genes for p53 and ATM. The use of nutlin3a has been shown to discriminate between dysfunctional cases with deletion of ATM or loss/mutation of p53. The assay has been shown to detect patients with mutation of the p53 gene who do not have concurrent deletion of the second allele. A novel dysfunctional response pattern involving p21 expression has been shown to associate with abnormalities of p53 rather than single nucleotide polymorphisms. The results also show that deletion of p53 and particularly ATM does not per se lead to a dysfunctional response as currently detected by the assay. This suggests that other mechanisms or genes are involved in resistance to DNA damage induced apoptosis.