TP53 Deletion in CLL: The Significance of Borderline Level Deletion

Numerous studies have shown the prognostic importance of TP53 deletion in CLL. Assessing TP53 deletion prior to initiating therapy is good clinical practice and many laboratories now offer testing. However, there is no harmonisation of methodology and little agreement about ‘Best Practice’. Information about cut-off values is scanty in published series. The best guide to date is the LRF CLL4 trial that showed a cut-off of >20% TP53 deletion as clinically significant. This value relates to a single institution and may not be applicable to other centres using alternative sample preparation methods. We reviewed the TP53 FISH results seen in our institution and looked at the cases with levels of TP53 deletion <20% to determine their outcome. 1,424 CLL cases (Jan 2003-June 2008) were tested for deletion of TP53 using FISH (Abbott Molecular CLL probe set). Cases were referred from the Yorkshire and Humberside Cancer Network region. There were 1,020 bone marrow aspirates, 308 peripheral blood samples and 95 lymph node/tissue biopsies. CLL diagnosis was confirmed by multi-parametric flow cytometry. The criteria for FISH was the sample contained >10% B cells and had smears/dabs of suitable quality. Minor modification of standard manufacturers protocols were used to reflect the nature of the samples. Slides were examined using a Zeiss microscope and images were captured using ISIS3 (MetaSystems). Deletion analysis was microscopically scored by counting 100 lymphocytes. Normal result is defined as <5% of lymphocytes with one signal. This definition is based on our laboratory validation of the probe sets. TP53 deletion is defined as >15% of lymphocytes with one signal. Scores >5% but <15% are borderline and an additional 200 cells are scored. Results were checked by a second observer and discrepant results are referred to a third observer. In this series of CLL +12 was seen in 16.5%, mono-allelic 13q14 deletion in 43.1%, bi-allelic 13q14 deletion in 9.2%, ATM deletion in 14.2% and TP53 deletion in 11.7% of cases. Borderline results for TP53 deletion were seen in 76/1424 (5.3%) cases. No further samples were received for 38/76 (50%) cases; 18/76 (24%) were treated to CR/MRD level and CLL counts were too low for repeat FISH. 20/76 (26%) cases had additional FISH carried out, 6/20 (30%) normalised, 12/20 (60%) remained the same and 2/20 (10%) progressed to >20% TP53 deletion at 8 (25% TP53 deletion) months and 18 (>95% TP53 deletion) months. Although the technical aspects of the FISH are straightforward, and results are reproducible, the interpretation can be problematic. A small proportion of these patients show marked expansion of the TP53 clones within short time periods. Rapid clinical progression was observed in these patients. Where repeat testing was possible 2/3 had the same borderline score, these patients continue on a ‘watch & wait’ policy; the remaining 1/3 normalised their TP53 result. This suggests that there are technical quality issues with the sample. CLL cells have a tendency to disintegrate due to their fragility because of this significant subpopulations may not survive manipulation and thus evade scrutiny. A CLL patient in this series who had borderline TP53 deletion on the PB smear was found to have a TP53 level of 40% by the local referring laboratory using their cytogenetic technique. FISH on smears has an advantage over other methodologies as all CLL cells are present. There is a need for improved cross-disciplinary guidelines, and further collaborative harmonisation studies are needed. Our results show that there is no clear cut-off for a TP53 deletion result and that any result over the 5% technical cut-off needs to be reviewed. A proportion of borderline cases reflect technical difficulties of handling CLL samples but in many cases they genuinely detect a small clone of TP53 deleted cells that may expand. Careful follow-up is needed for all cases with borderline TP53 deletion results.