Abstract
Despite improvement in treatment strategies, virtually all chronic lymphocytic leukemia (CLL) patients will relapse and experience tumor resistance. The 17p deletion resulting in loss of the TP53 gene, found in up to 20-40% of relapsing patients, is strongly associated with impaired response to genotoxic agents, reduced progression free survival and poor overall survival. The 17p deletion usually coincides with TP53 mutation, leading to the impairment of the p53-associated pathway. In addition, sole TP53 mutations (without 17p deletion) appear also associated with poor outcome in prospective trials. However, TP53 mutation screening is time consuming, can be not exhaustive, and the respective impact of different patterns of TP53 gene impairment on p53 function and prognostic remains unclear. We previously developed a functional assay to detect p53 dysfunction (Le Garff-Tavernier, 2011) and we aim to validate this analysis on a large prospective trial.
Clinical and laboratory data were collected from CLL patients (pts) enrolled in the ICLL001 – BOMP phase II trial of the French CLL intergroup (NCT01612988) evaluating a prephase of ofatumumab (300 mg) followed by 6 monthly courses of BOMP including bendamustine (70 mg/m2 d1-2), ofatumumab 1000 mg TD (d1 and d15 on 1st and 2nd courses) and high dose methylprednisolone (1 g/m2 d1-3) in fit patients with relapsing CLL and IWCLL treatment criteria. In addition to conventional screening, we focused on p53 evaluation. FISH analysis for 17p deletion was done with a 10% cut-off for positive result, TP53 gene mutation screening was performed using Sanger sequencing of the entire coding region (exons 2–11) and the p53 functional status in CLL cells was determined by a flow cytometry assay based on induction of p53 and p21 protein expression after etoposide and nutlin-3a exposition.
Data from the first 55 enrolled pts are available. Sex ratio M/F was 3.3 and median age was 63.8 yrs (44.6-76.4). CLL diagnostic had been done 7,2 (1,9-16,8) years before inclusion. All patients had according to IWCLL criteria an active disease of Binet stage of A (11%), B (57%) and C (32%) respectively. Patients had been previously pretreated with a median of 1 (1-3) lines, including FCR (or FCR-like) in 51 (93%) pts and 22 (42%) pts had experienced high-risk relapses within 24 months post-FCR, with 7 (13%) pts being fludarabine refractory (less than PR after fludarabine regiment and/or response lasting less than 6 months). IGVH gene status was unmutated in 90%, elevated β2-microglobulin (>4) was found in 52%. Karyotypes were complex (≥ 3 abnormalities) in 18/46 (39%) successful cases. Using FISH, we found 15/55 (27%) del17p (median of positive cells 71%, range 10-98), 6/55 (11%) tri12, 18/55 (33%) del11q, 35/55 (64%) del13q. Results of p53 functional assay was available for 52 pts with the following results: normal in 31 pts and abnormal in 21 pts including type A (n=4), type B (n=13) and type C (n=4) dysfunction. Mutation screening was available in 55 pts. No mutation were detected in 38 pts, one significant mutation was detected in 14 pts within exon 5 (n=1), exon 6 (n=2), exon 7 (n=2), exon 8 (n=6), exon 10 (n=1) and intronic splice site (n=2) ; 3 pts had 2 mutations within exons 7 and 8 (n=1), exons 7 and 10 (n=1), exons 5 and 7 (n=1). Among the 52 pts with available functional results we found the 7 following groups (Table). In this study, the sensitivity and specificity of the p53 functional test to detect patient with 17p deletion and/or TP53 mutation was 89.5% (66.9 –99.7) and 87.9% (71.8 – 96.6) respectively.
Response to p53/21 functional assay 17p deletion TP53 mutation n % Group 1 Normal No No 29 56 Group 2 Abnormal Yes Yes 13 25 Group 3 Abnormal Yes No 1 2 Group 4 Abnormal No Yes 3 5.5 Group 5 Abnormal No No 4 7.5 Group 6 Normal Yes No 1 2 Group 7 Normal No Yes 1 2
This study shows that an in vitro p53 functional analysis can predict with an acceptable sensitivity the presence of TP53 gene disruption and could be useful to identify pts with TP53 mutation without 17p deletion. Interestingly, this functional assay coupled with cytogenetic and mutational screening could reveal 3 sub-groups of pts with potential clinical consequences: i) normal p53 function despite a del17p deletion (group 6) ii) normal p53 function despite a TP53 mutation (group 7) and in contrast iii) abnormal p53 function without any TP53 gene disruption (group 5) allowing to describe alternative alterations of p53 pathway.
Disclosures:
Dilhuydy: Roche: Honoraria. Leblond:Roche: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Mundipharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Feugier:Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Tournilhac:MUNDIPHARMA: Consultancy, travel funding Other; GSK: Consultancy, travel funding, travel funding Other; Celgene: Consultancy, teaching, teaching Other.
Impact Of TP53 Gene Promoter Methylation On Chronic Lymphocytic Leukemia Pathogenesis And Progression