Abstract

B-cell chronic lymphocytic leukaemia (B-CLL) is characterized by a highly variable clinical course. TP53 abnormalities in B-CLL are strongly associated with resistance to purine analogues, short survival and large-cell transformation. Several methods can be used to investigate TP53 inactivation: FISH (gene deletion), immunohistochemistry -IHC- (protein overexpression), and DNA sequencing (mutations). The relationship between TP53 deletion and p53 protein overexpression is unclear, and it is uncertain which method is the best to assess TP53 dysfunction and which provides the most useful clinical information. In 92 CLL patients we have correlated the frequency of p53 protein overexpression by IHC, with TP53 deletion status by FISH and subsequently we have examined the response to Fludarabine in these patients. The male/female ratio was 1.35 (52M/40F), with a median age at diagnosis of 54 years (range 25–78 years). The distribution of clinical stage at diagnosis was as follows: 47% (43 cases) stage A, 16% (15 cases) stage A progressive, 14% (13 cases) stage B, 11% (10 cases) stage C, and unknown in 12% (11 cases). Fourteen cases (15%) were untreated and seventy-eight cases (85%) were previously treated. Among the latter, 66 (84%) received Fludarabine, and 9 (11%) received Campath-1H (as a first line of treatment in 5 patients). In all cases peripheral blood samples were used for FISH analysis and IHC was carried out on bone marrow biopsies. Cases were selected on the basis of whether FISH and IHC were analysed at the same time point. Deletions of TP53 (17p13) were detected by FISH (Vysis); a threshold of >20% of cells with p53 deletion was considered clinically significant. For IHC detection a monoclonal antibody (clone BP53-12, Novocastra) was used. Cases were considered positive when p53 stained the majority of lymphocyte nuclei, those cases with few or weakly positive cells in proliferation centres were considered negative. Correlation with FISH (>20% deleted cells) and IHC showed 69 cases (75%) negative for both assays and 6 cases (7%) positive for FISH and IHC. The remaining 17 cases (18%) showed discordant results; 6 cases showed TP53 deletion >20% by FISH but showed no p53 overexpression by IHC, 4 cases had overexpression of p53 and no TP53 deletion, 3 cases had p53 overexpression with TP53 deletion between 10–20%, and 4 cases showed TP53 deletion between 10–20% but no overexpression of p53. This is compatible with a sensitivity of 50% and specificity of 91%. In order to understand the mechanism better, mutation analysis of TP53 gene will be done in discordant cases. All patients positive for both results and a minority of patients (16%) with no p53 abnormalities detected by FISH or IHC were refractory to Fludarabine. Within the discordant group, most of refractory patients (67%) were found in cases with p53 overexpression and TP53 deletion between 10–20%. In conclusion, the combination of both methods (FISH and IHC) may be useful to identify CLL cases with an adverse prognosis and non responders to Fludarabine. A large study with more cases is necessary to clarify the underlying mechanisms involved in those cases with p53 overexpression without TP53 deletion.

Author notes

Disclosure: No relevant conflicts of interest to declare.