Abstract: It has been recently shown that RAS mutations, which occur in 11-38% of Chronic Myelomonocytic Leukemia (CMML), do not only act via RAS/MEK/ERK signaling, but contribute to the disease through NLRP3 inflammasome activation (Hamarsheh, Nat Comm 2020). Despite a therapeutic approach based on NLRP3/IL1β axis blockade, as bring to a stem cell transplantation (SCT) has been proposed, data on the efficacy of IL1β inhibitors in hematopoietic neoplasms is limited. A 55 year old man with previous autoinflammatory episodes (constrictive pericarditis) was diagnosed on September 2020 of CMML-1 KRAS G12D (Inter-2). Due to worsening (orchiepidedymitis, pneumonitis, cellulitis), and the impossibility of performing an SCT at that time, on December 02 2020 he started anakinra (a IL1β receptor antagonist) with good response. Due to new episodes of autoinflammation, anakinra was discontinued (12 April 2021) with severe clinical worsening (heart failure) and no response to diuretic/corticosteroid. After anakinra was restarted (04 May 2021), a progressive improvement was seen, allowing a successful pericardiectomy before an SCT. We obtained blood samples from this patient (at different times) and plasma and whole blood samples from 11 and 5 other CMML KRAS mut patients, respectively. We also included CMML patients without KRAS mutations (KRAS wt) (n=8), with sepsis (n=5) and healthy individuals (n=9). Plasma levels of 15 inflammatory cytokines associated with NLRP3 inflammasome and NFkB pathways were measured using a customized MILLIPLEX ® kit. The inflammasome marker activation assays were conducted as previously published (Martínez García JJ, Nature Comm 2019). Compared to healthy controls, KRAS wt CMML patients did not show differences in any cytokine tested, except IL6, while KRAS mut patients showed significantly higher levels of IL1α, IL1ra, IL18, IL12p40 (associated with NLRP3 inflammasome), IL6, IL8 (associated with NFkB pathway) and M-CSF (Fig. 1A B). Compared to KRAS wt CMML patients, those with KRAS mut showed higher levels of cytokines associated with both the NLRP3 and NFkB pathways, reaching statistical significance for those related with NLRP3 inflammasome. We also observed changes in inflammasome related cytokines before and after anakinra (Table 1). This cytokine profile in the plasma made us analyze the oligomerization of ASC as a marker of inflammasome activation in monocytes of KRAS mut CMML. We found that in all cases of KRAS mut CMML patients around 30 to 80% of monocytes presented oligomers of ASC measured by the time of flight assay, while in healthy donors and KRAS wt CMML patients, ASC oligomerization occurred upon NLRP3 inflammasome activation with lipopolysaccharide (LPS) + ATP or Pyrin inflammasome activation with LPS and Clostridium difficile B toxin (TcdB) (Fig. 2A). Ex vivo activation of PBMCs from KRAS mut CMML patients showed that despite the high percentage of cells with ASC oligomers, very low levels of IL1b released from these cells, even when NLRP3 was activated with LPS+ATP (Fig. 2B), suggesting that this inflammasome is activated in vivo and could not be further activated ex vivo. As control, Pyrin inflammasome activation in PBMCs from KRAS mut CMML was able to induce IL1b release similarly to healthy controls (Fig. 2B). We then found that anakinra treatment of the KRAS mut CMML patient followed in this study, resulted in a decrease of the percentage of monocytes with basal active inflammasomes (Fig. 2C). A little ex vivo activation of the NLRP3 inflammasome was obtained when cells were treated with LPS+ATP, while Pyrin inflammasome was activated at normal levels after LPS+TcdB treatment (Fig. 2D). The inflammasome basal activation increased in the monocytes of the KRAS mut CMML patient after anakinra withdraw and during clinical deterioration and restarting anakinra (second arrow) decreased the basal percentage of monocytes with ASC oligomers (Fig. 2C). Since ASC oligomers are associated to pyroptosis via caspase 1 activation and gasdermin D processing, we then analyzed pyroptotic markers in the plasma of the patient during the time. ASC was increased when monocytes presented elevated percentage of ASC oligomers (Fig. 2E), suggesting that ASC detection could be a promising biomarker. Overall, we show that, in vivo, the NLRP3 inflammasome activation of KRAS mut CMML patients may revert with IL1β blockers. ASC could identify those candidates to receive this therapy. PI18/00316 Figure 1 Figure 1. Disclosures Jerez: Novartis: Consultancy; BMS: Consultancy; GILEAD: Research Funding. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau. Hernández-Rivas: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ferrer Marin: Cty: Research Funding; Incyte: Consultancy, Research Funding; Novartis: Speakers Bureau.

Abstract: Introduction. Despite the high proportion of prolonged remissions obtained with ibrutinib in patients with CLL, complete responses (CR) are rarely observed. For the purpose of increasing the deepness of response, ibrutinib has been tested in combination with other drugs that exert a different mechanism of action. Thus, monoclonal antibodies (mAbs) have been concomitantly combined with ibrutinib in untreated or R/R CLLs. Nonetheless, several data derived from both in vitro and clinical studies do not support a synergistic effect of the concomitant administration of ibrutinib with anti-CD20 mAbs. Herein, we present the preliminary results of a multi-center, non-randomized phase 2 study aimed to determine the efficacy and safety of the sequential treatment of CLL patients with ibrutinib followed, in those not attaining CR, by a consolidation phase with ofatumumab (GELLC-7, EudraCT number 2016-004937-26). Patients and methods. Patients aged ≥18 years, physically fit (CIRS score 〈 6) with treatment-naïve CLL were enrolled in this study. Patients received an induction phase consisting of 12 cycles (28-day) of ibrutinib in monotherapy at 420 mg once daily. Patients attaining a CR after this induction phase were kept on ibrutinib until progression. In contrast, patients not obtaining a CR also continued on ibrutinib but received a consolidation treatment with 7 doses of ofatumumab (300 mg D1 and 1000mg D8 of C13, 1000 mg D1 of C14-C18). The primary endpoint of the study was the CR rate assessed after 20 cycles of treatment (2 months after completing ofatumumab consolidation). Results. 84 patients with a median age 69 years (range 38-84 yrs), 71% male, were included in this study. At inclusion, 83.3% had Binet stage B/C, 61% unmutated IGHV status, and 19% high risk genetic aberrations (7.6% 17p deletion and/or TP53mut, and 11.4% 11q deletion). At the interim data cut-off (June 2019), 7 patients had discontinued the study (progression to Richter transformation, n=1; patient withdrawal, n=3; adverse events [AE], n=3, including one G5 AE), 5 of them during the first 12 cycles of treatment. Sixty-seven patients received the induction phase with 12 cycles of ibrutinib, whereas 22 patients completed 20 cycles of treatment and were evaluable for the primary endpoint of the study. After 12 cycles of ibrutinib, 3 patients (4.5%) were in CR, 54 patients (80.5%) in PR, 6 patients (9%) in PR with lymphocytosis, and 4 patients (6%) in SD. In 20 patients receiving the consolidation with ofatumumab an improvement in response was observed, with 8/20 patients (40%) attaining a CR (7 patients converted PR to CR, and one patient SD to CR), whereas the remaining 12 patients were classified as PR. Two patients that were already in CR at cycle 12 maintained the CR under ibrutinib monotherapy. MRD was undetectable in blood ( 〈 10-4 by flow cytometry) only in one of the 22 patients. With a median follow-up of 15 months (2 - 20.2 months), the estimated 12-months PFS and OS was 98%. Grade ≥3 adverse events (AEs) were experienced by 26 patients (31%), whilst 22 serious AEs were observed in 16 patients (19%) (14 infections, 1 febrile neutropenia, 3 dyspnoea, 1 anemia, 1 edema/pleural effusion, 1 renal insufficiency, 1 squamous carcinoma). The most common G3/4 AEs were hematological toxicity (neutropenia [7.1%], anemia [4.5%] , thrombocytopenia [2.4%]) and infections (8.5%). The Gr 5 AE consisted of a severe peripheral edema and pleural effusion leading to death. The great majority of SAEs (67%) and G3/4 AEs (66%) were observed during the first 12 cycles of treatment with ibrutinib monotherapy. Conclusions. The preliminary analysis of the GELLC7 trial showed that the addition of consolidation with ofatumumab after 12 cycles of prior treatment with ibrutinib was well tolerated and elicited a deeper response. These results support the potential role of a sequential therapeutic strategy in CLL, where the addition of a consolidation with mAbs in patients with low tumor burden might improve the quality of the response. Finally, more mature results will be further presented at the meeting. Disclosures Abrisqueta: Roche: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau. González-Barca:Kiowa: Consultancy; Celgene: Consultancy; Takeda: Honoraria; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celtrion: Consultancy. Terol:Roche: Consultancy; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; Astra Zeneca: Consultancy; Gilead: Research Funding. Baltasar Tello:GILEAD: Honoraria; JANSSEN: Consultancy, Honoraria; ABBVIE: Honoraria; ROCHE: Honoraria. de la Serna:Roche, AbbVie, Gilead, Janssen, Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche, AbbVie, Janssen, Gilead: Speakers Bureau. Ramirez Payer:GILEAD SCIENCES: Research Funding. Cordoba:Janssen: Consultancy, Honoraria, Speakers Bureau; Servier: Consultancy, Honoraria, Speakers Bureau; Kyowa-Kirin: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Research Funding, Speakers Bureau; Roche: Honoraria, Speakers Bureau; FUNDACION JIMENEZ DIAZ UNIVERSITY HOSPITAL: Employment; Celgene: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy. Bosch:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Ibrutinib followed by Ofatumumab Consolidation