ALBERT

Description: Introduction Reduced intensity conditioning (RIC) allogeneic stem cell transplantation (ASCT) remains a valuable and potentially curative strategy in high-risk CLL. Post-ASCT relapse remains an important (30-40%) cause of failure, predicted by persisting positive minimal residual disease (MRD) at 12m post-ASCT. ASCT is also associated with a toxic mortality and with the burden of chronic graft versus host disease (cGVHD). We evaluated the efficacy and safety of a preemptive immunointervention (PrIm) based on serial MRD assessment in high-risk CLL. (NCT01849939) Methods Main inclusion criteria were (1) EBMT criteria for ASCT, (2) CLL in PR/CR, (3) mass ≤5cm, (4) age ≤70, (5) SORROR score ≤2 and (6) HLA donor (10/10). RIC regimen included fludarabine 120 mg/m2, IV busulfan 6.4 mg/kg, rabbit ATG 5mg/kg and CsA prophylaxy. Centralized 10-color flow cytometry blood MRD was assessed before and at M1, M2, M3, M4, M5, M6, M9 and M12 post-ASCT. MRD[-] was defined by blood detection 95% donor derived CD3+ cells respectively. MRD evolution followed different patterns (Table 1). At the M3 checkpoint, among evaluable patients, 10 were already MRD[-], and 16 were still MRD[+]. Then 10 pts became MRD[-] and 6 remained MRD[+]. This approach achieved 67% blood MRD[-] at M12 (ITT). When early CSA T&D had failed DLI done in 6 pts did not achieve MRD negativity. Finally, 6 pts (20%) remained MRD[+] at 12m. At 12m FU, the rate of Gr≥2 aGVHD was (11/30) 37% (consecutive to early CsA T&D in 2 pts and to early CsA T&D followed by DLI in 1 pt) and the rate of extensive cGVHD was (5/29) 17% (consecutive to CsA T&D in 1 pt). Hence we observed the occurrence of Gr≥2 aGVHD and extensive cGVHD in 2/8 and 1/8 pts after early CsA T&D. We also observed one case of Gr≥2 aGVHD among the 6 pts who had early CsA T&D followed by DLI. Eighteen severe adverse events were reported, including the 2 aforementioned deaths. These events were not related to the study procedure (PrIm). Discussion These preliminary data highlight the feasibility, safety and efficacy of a standardized PrIm strategy in high-risk CLL. As DLI appears to have limited impact when CsA T&D fails, the preemptive use of new agents should be considered at this point for persistent MRD[+] after 3-6m post-ASCT. Table 1. Table 1 M12 MRD N % MRD[-] pre-ASCT remaining MRD[-] [-] 2 7 MRD[+] (M1-2) spontaneously translating to MRD[-] within 3m [-] 8 27 MRD[+] (M1-3) translating to MRD[-] after early CsA T&Dconcomitant of severe GVHD (≥G2 aGVHD and/or ext cGVHD) [-] 10 8 2 33 MRD[+] (M1-3) remaining MRD[+] despite early CsA T&D followed by DLIdespite early CsA T&D, transient MRD[-], relapse and DLI [+] 6 5 1 20 Non evaluable: toxic deaths (2), EBV LPD (1), graft rejection (1) NE 4 13 Disclosures Tournilhac: GSK: Other: Travel Support, Research Funding; Mundiphrama: Honoraria, Other: Travel Support, Research Funding; Celgene: Other: Travel support; Roche: Other: Travel support, Research Funding; Janssen Cilag: Honoraria, Other: Travel support; Gilead: Other: Travel Funding.

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: Evaluation of MRD after treatment has an increasing importance in CLL treatment. Recent studies have demonstrated that patients achieving MRD below 10-4 have significantly longer OS and/or PFS compared to subjects with positive MRD (Böttcher, JCO 2012). Additionally, in CLL patients treated by FC(R)-based regimen, 4-color flow MRD was found as effective as ASO RQ PCR for MRD evaluation down to the level of 10-4, but below that level, PCR was more sensitive (Böttcher, Leukemia 2009). The aim of our study was to investigate the sensitivity of 8-color Flow-MRD and compare it to ASO RQPCR for disease evaluation at post-treatment time-point. 140 patients with active disease were recruited in a randomized first-line phase II trial evaluating the benefit of the addition of a high-dose rituximab pre-phase to standard FCR (R-dense arm) as compared to standard FCR. Clinical response and MRD were monitored at 3 months after last cycle, Flow-MRD was performed in peripheral blood (PB) and bone marrow (BM), and RQPCR in blood only. IGH ASO RQPCR was performed in an EU-MRD laboratory according to EU-MRD guidelines. Results were expressed as 10-8, corresponding to undetectable MRD, when no specific signal was detected, 10-6 when positivity was detected below quantitative range for the patient, and specific number when residual disease was within the quantitative range. For Flow-MRD we have developed an 8-color 9-antibody panel, the analyses were performed in four centers using harmonization procedures. Flow-MRD was considered undetectable (u-MRD) when less than 10 CLL events were detected or if the percentage of CLL events was below the absolute limit of detection (LOD) of the technique established at 0.5x10-6on normal blood samples. Quantitative range was reached when at least 50 CLL events were detected (Rawstron, Leukemia 2012). 137 patients were randomized and analyzed for clinical response. Approximately 87% had MRD evaluation by Flow, ASO RQPCR or both. U-MRD was observed in 81 patients out of 121 tested by flow in blood (67%) and in 49/109 (45%) in marrow. Samples with u-MRD reached good median LOD of 0.7x10-5 and 10-5in PB and BM respectively. MRD results were concordant in PB and BM (undetectable or positive in both samples) in 84/109 patients. The discordant cases were all positive in BM and negative in PB suggesting a better sensitivity and/or informativity in marrow. Therefore, when considering as Flow-MRD positive the cases with positive MRD in PB and/or BM and as undetectable those with u-MRD in BM, Flow-CR rate was 43%. The 66 cases that were analyzed in PB using ASO RQPCR showed a global PCR-CR rate of 76.5%. Sixty patients were analyzed by both flow cytometry and ASO RQPCR in PB. Results were concordant in 49 patients (82%), either both positive (n=9) or undetectable (n=40), resulting in a good global agreement between the two techniques (Kappa coefficient: 0.50 [0.20-0.73]). The 11 cases with discrepant results are of interest as they highlight the importance of the limit of detection for interpretation of MRD results. Among the 7 patients with u-MRD by ASO RQPCR and positive Flow-MRD, 4 showed a median sensitivity of 5.10-5 by ASO RQPCR and a very low median positivity by Flow 0.0024%. Moreover, all these 7 patients had a positive Flow-MRD in BM. The last 4 discordant cases had negative Flow-MRD and positive ASO RQPCR. In 2 of them a poor LOD in Flow-MRD contrasted with a 10-5sensitivity of ASO RQPCR and positive Flow-MRD was found in BM for 2 of these patients. Finally, clinical response was evaluable in 123 patients with MRD results. Among 65 patients in CR or CRi, 16 (24.6%) were MRD positive and among 55 patients in PR or nPR, 29 (52.7%) were MRD negative. Our 8-color panel for MRD detection by flow cytometry in CLL is applicable for treatment evaluation. This work shows that lowering the limit of detection by one log renders the 8 color flow-MRD as sensitive as ASO RQPCR and that the exploration of bone marrow improves the performances of MRD detection. As previously observed using less sensitive techniques, MRD results do not superimpose clinical response. Finally, a longer follow-up will validate the clinical interest of a one-log gain of sensitivity in MRD detection for prediction of PFS and OS. Disclosures Cartron: Roche: Consultancy, Honoraria. Cymbalista:Roche: Honoraria.