ALBERT

Description: Background Disruptions of the TP53 tumor suppressor pathway by TP53 gene mutations and/or by17p deletions resulting in loss of the TP53 locus are clearly associated with poor survival and chromosomal instability in chronic lymphocytic leukemia (CLL). Chromosomal instability is promoted by telomere dysfunction, and the TP53 pathway is involved in the monitoring of telomere integrity. Aim The purpose of the present study was to describe the changes in main telomeric parameters, which can affect telomere integrity, associated with TP53 mutations and 17p deletions and to evaluate their potential prognostic value. Patients and Methods We performed a comparison between a group of TP53 disrupted CLL patients (n= 24) and a group of TP53 wild-type CLL patients (n= 61). Median age was 70 [39-89] years, M/F ratio was 2.4/1. At the time of sampling, 35 patients were in Binet stage A, 19 patients in stage B and 31 patients in stage C. A total of 50 patients were subsequently treated by immunochemotherapy (FCR n= 30, other n= 8), alemtuzumab (n= 8) or alkylating agent-based regimens (n= 4). TP53 gene mutation screening was performed using Sanger sequencing of the entire coding region (exons 2–11). Cytogenetic analysis (karyotype and FISH) were performed to evaluate the chromosomal instability and detect the marks of telomeric fragility. Quantitative PCR approaches were used to measure the relative telomere length and to evaluate expression levels of shelterin genes (TRF1, TRF2, POT1, RAP1, TPP1, and TIN2) and telomerase (hTERT). Results The patients showing a TP53 mutation without a 17p deletion and the patients carrying a 17p deletion with or without TP53 mutation appear to display the same level of telomeric instability. These cases are characterized by significant telomeric erosion (P

Description: Key Points Targeted NGS of relapsed/refractory CLL reveals a high incidence of concurrent mutations that mostly affect the TP53, ATM, and SF3B1 genes. Concurrent mutations of the TP53, ATM, and/or SF3B1 genes confer short survival in patients with relapsed/refractory CLL.

Description: Introduction: The 17p deletion (del(17p)) resulting in loss of the TP53 gene is associated with impaired response to genotoxic agents and has an impact on PFS following BTK inhibitor and possibly also venetoclax. The del(17p) usually coincides with TP53 mutation, leading to the impairment of the p53-associated pathway. Sole TP53 mutations appear also associated with poor outcome in prospective trials. The iwCLL guidelines recommend to look for del(17p) and TP53 mutation before each line of treatment. An original approach is the functional assay, which highlights the functional abnormalities of p53 whether it is a TP53 gene disruption (del(17p) and/or TP53 mutation) or a defect of another actor in the p53 pathway. We aim to validate this functional assay on a prospective trial and to study the impact of p53 status on the clinical response regardless of the biological method. Methods: Clinical and biological data were collected from 74 CLL patients (pts) enrolled in the BOMP phase II trial of the French Innovative Leukemia Organization (FILO) (NCT01612988) evaluating 6 monthly courses of BOMP including bendamustine, ofatumumab and high dose methylprednisolone in fit pts with relapsing CLL. In addition to conventional screening, we focused on p53 evaluation at time of inclusion. FISH analysis for del(17p) was done with a 5% cut-off for positive result. TP53 gene mutation screening was performed by Sanger sequencing of the coding region (exons 2-11). A targeted NGS screening (19 genes including TP53, Illumina MiSeq) was also performed. The p53 functional status was determined by a flow cytometry assay based on induction of p53 and p21 protein expression after etoposide and nutlin-3 exposition, as previously described (Le Garff-Tavernier M., 2011), which allows the detection of 3 types of p53 dysfunction (A, B and C), irrespective of an ATM default. Clinical response was evaluated by PFS, OS and TTNT Kaplan-Meier analyses (MedCalc stat). Results: Data from the whole cohort are available. Median age was 64 yrs. Pts had a median of 1 (1-3) lines of treatment previous to this trial, including FCR in 〉90%. Concerning p53 evaluation, a del(17p) was found in 30% of cases by FISH (22/73 pts with a median of 68% positive cells, range 10-98). The percentage of p53 abnormalities increased to 41% when TP53 mutations were screened (30/73 pts with 1 to 8 mutations, median VAF 10 %, range 1.6-90). Results from the p53 functional assay were available for 69 pts showing the highest level of p53 abnormalities. Indeed, p53 dysfunction was observed in 48% of pts (33/69) including type A (n=11), type B (n=17) and type C (n=5) dysfunction. Thus, the sensitivity and specificity of the p53 functional assay to detect pts with del(17p) and/or TP53 mutation were of 87% and 84% respectively (n=68 pts for which the 3 tests were available). Interestingly, discordant results were observed in 10 pts: 4 pts with a functional p53 despite a TP53 gene disruption (3 with TP53 mutation only and 1 with del(17p) only) and conversely 6 pts with a p53 dysfunction (all with type B dysfunction) but without any TP53 gene disruption, suggesting alternative alterations of the p53 pathway. The only similarity for those latter pts is the occurrence of at least one ATM abnormality (del(11q) and/or ATM mutation). The combination of the 3 assays defines 3 groups: (1) "intact p53" (no TP53 disruption and functional p53, n=32), (2) "altered p53" (TP53 disruption and p53 dysfunction, n=26) and (3) "discordant p53" (n=10). PFS and TTNT were higher in pts without (n=38) compared to those with TP53 gene disruption (n=30) (p=0.04 for both). The OS, even though not significant, presented a similar trend. When considering the functional status, a similar profile is observed but with a better discrimination between pts with normal p53 function (n=36) and pts with p53 dysfunction (n=32) (p=0.002 and 0.003, respectively). Combining the 3 assays, PFS and TTNT of the group 3 "discordant p53" profiles' appeared intermediate (Figure 1). Conclusion: This study shows that a p53 functional analysis can predict with an acceptable sensitivity the presence of a TP53 gene disruption. Interestingly, this functional assay coupled with cytogenetic and mutational screening could reveal a sub-group of pts with discordant results for which PFS and TTNT appeared intermediate. Evaluation of other discordant cases is mandatory to confirm these results and could lead to a wider use of this global functional approach. Figure 1. Figure 1. Disclosures Feugier: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sylvain:Gilead: Other: scientific advisor board. Schuh:Giles, Roche, Janssen, AbbVie: Honoraria. Guieze:abbvie: Honoraria; janssen: Honoraria; gilead: Honoraria. Leblond:Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Sandoz: Honoraria; Amgen: Honoraria.

Description: Background: Previous studies using next-generation sequencing (NGS) have led to the identification of a number of genes mutated frequently in CLL. Recent publications focus on the most recurrently mutated genes (TP53, SF3B1 and NOTCH1) which tend to be mutually exclusive. Large series of untreated patients have shown that these mutations have a prognostic impact. Relapse may be associated with more frequent mutational events. Further investigation of relapsed CLL genomes within a clinical trial setting using a comprehensive NGS gene panel is required. Methods: Using targeted NGS we determined the mutational spectrum of 118 refractory/relapsing CLL patients enrolled in one French and two UK prospective trials (ICLL01 from the French intergroup GCFLLC/MW-GOELAMS, NCRNCLL201, NCRNCLL202 respectively). Eighty percent of patients had an unmutated IGHV status and 21 (18%) patients carried a 17p deletion. Sequencing libraries were composed of a panel of nine recurrently mutated genes in CLL (i.e.TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3 and MYD88) and run on the Illumina MiSeq instrument (Illumina Inc). On average 14.1 M reads were obtained per run of which 96.8% were identified reflecting an acceptable signal to noise ratio. Yield was 4.1 Gb and 95.9% of reads were above Q30 across 6 MiSeq runs. Data was analysed using our in-house bioinformatics pipeline consisting of a combination of two different aligners (Custom Amplicon Alignment, Illumina Inc and Stampy, Wellcome Trust centre for Human Genetics), two variant callers (GATK, Broad Institute and Platypus, Wellcome Trust centre for Human Genetics) and a stringent filtering process in order to detect SNVs and indels with a variant allele frequency down to 7%. Results: We identified a total of 196 mutations (mean=1.7/sample) in 95 (80%) patients: 138 missense mutations, 41 substitutions/indels, 12 nonsense and 5 splicing mutations. TP53, SF3B1 and ATM mutations occurred frequently in 29 (24.6%), 33 (28%) and 29 (24.6%) patients, respectively. Eighteen (15.3%) patients harbored a NOTCH1 mutation matching the range of reported frequency. Mutations in the other genes sequenced were distributed as follows: XPO1 mutations in 17 (14.4%), SAMHD1 mutations in 12 (10.2%), MED12 mutations in 10 (8.5%), BIRC3 mutations in 6 (5.1%) and MYD88 mutations in 3 (2.5%) patients. Twenty-three (20%) patients did not have any mutations present (Figure 1, cluster #1). A total of 51 (43%) patients had one gene mutated (Figure 1, cluster #2) and the remaining 44 (37%) patients had two or more genes mutated (Figure 1, clusters #3 & #4). Recurrent combinations of mutations (affecting more than 5% of patients) were found in a group of 23 (20%) patients. These combinations of mutations comprised of at least two of the following genes: TP53, SF3B1 and ATM (Figure 1, cluster #3, so called multiple-hit (MH) profile). Remarkably, mutations in these 3 genes were found significantly more frequently associated than in isolation. We then investigated the potential clinical relevance of the MH profile. This profile was associated with poorer ORR than the remaining cohort (43% vs 80%, P

Description: 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.