Abstract 5263: Assessment and targeting of the MDM2-p53 network in CLL
Authors: Woodhouse Laura , Crawford Clark , Marshall Scott , Bown Nick , Wallis Jonathan , Summerfield Geoffrey , Willmore Elaine , Lunec John 
Published: August 2015

Abstract
Chronic Lymphocytic Leukaemia (CLL) is the most common adult leukaemia in the western world. It is a highly heterogenic disease with variable disease progression. One of the clearest indicators of poor prognosis in CLL is loss of p53 function via deletion of chromosome 17p13 (del17p) and mutation of TP53. Since TP53 mutation rates rise dramatically in treatment-refractory CLL, identifying TP53 mutations at diagnosis may aid treatment decisions. We have compared next generation (NGS) to Sanger sequencing to determine the optimal method and detection level for TP53 mutation in CLL and assess the clinical significance of small TP53-mutated subclones. Since the incidence of TP53 mutation is relatively low (approximately 10%) when first treatment of CLL is required, targeting the p53 pathway may present a treatment option. To investigate this possibility, primary CLL cells were exposed ex vivo to the MDM2-p53 antagonists Nut-3a, RG-7112 and RG-7388 which prevent the interaction between p53 and its inhibitor protein MDM2 to activate p53 leading to growth inhibition and cell death.
We analysed genomic DNA from the CLL cells of 140 patients, including 36 with multiple samples from different stages of disease progression (median follow-up 10 years), by NGS using a ROCHE 454 GS-FLX pyrosequencing platform. In addition, 90 samples were previously screened for TP53 mutations by Sanger DNA sequencing. Full clinical diagnostic and outcome data was available. To assess the functional status of the MDM2-p53 pathway, the sensitivity of CLL cells to a range of non-genotoxic MDM2-p53 antagonists was evaluated.
Kaplan Meier analysis of NGS data showed that patients with TP53 mutation+del17p had a median overall survival (OS) of 50 months compared to 140 months for wild type TP53 patients (HR = 2.8, CI 1.7-14.6; p = 0.003). 5% of patients harboured a monoallelic TP53 mutation in the absence of del17p, 2.1% had del17p without mutation on the remaining allele, and 9.3% had del17p+TP53 mutation. Interestingly, patients with del17p+TP53 mutation showed poorer survival than patients with TP53 mutation alone (HR 7.2, CI 1.2-14.6; p = 0.02). One patient with TP53 mutation alone has stable disease (10 years, no treatment) since detection of mutation. Four patients had TP53 mutant samples detected by NGS which were not detected by Sanger sequencing. In addition TP53 WT CLL cells showed a cytotoxic response to MDM2-p53 antagonists (RG-7388 LC50 0.4±0.08 μM, n = 11) whereas TP53 mutant CLL cells were 11 fold less sensitive (RG-7388 LC50 4.58±1.36 μM, n = 3) as determined by XTT assay following 48 hour treatment.
NGS is a more sensitive method for detection of TP53 mutations compared with Sanger sequencing. Patients with TP53 mutation alone in the absence of del17p display longer OS compared to patients with del17p+TP53 mutation. In addition, MDM2-p53 antagonists provide a potential future treatment option in TP53 WT CLL.
Citation Format: Laura Woodhouse, Clark Crawford, Scott Marshall, Nick Bown, Jonathan Wallis, Geoffrey Summerfield, Elaine Willmore, John Lunec. Assessment and targeting of the MDM2-p53 network in CLL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5263. doi:10.1158/1538-7445.AM2015-5263