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Figure S2C displays probability of progression-free survival over time in subgroups of patients with and without bulky disease

Purpose:In the phase III DUO trial, duvelisib, an oral dual PI3K-δ,γ inhibitor, demonstrated significantly improved efficacy versus ofatumumab [median (m) progression-free survival (PFS), 13.3 vs. 9.9 months (HR, 0.52; P < 0.0001); overall response rate [ORR], 74% vs. 45% (P < 0.0001)], with a manageable safety profile in patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). We report results from patients with progressive disease (PD) after ofatumumab who crossed over to duvelisib in the DUO trial.Patients and Methods:Patients with radiographically confirmed PD after ofatumumab received duvelisib 25 mg twice daily in 28-day cycles until PD, intolerance, death, or study withdrawal. The primary endpoint was ORR per investigator. Secondary endpoints included duration of response (DOR), PFS, and safety.Results:As of December 14, 2018, 90 ofatumumab-treated patients in the DUO trial prior to crossover had an ORR of 29%, mDOR of 10.4 months, and mPFS of 9.4 months. After crossover, 77% of patients (69/90) achieved a response, with an mDOR of 14.9 months and mPFS of 15.7 months. Patients with del(17p) and/or TP53 mutations had similar outcomes [ORR, 77% (20/26); mPFS, 14.7 months]. Notably, 73% of patients (47/64) with disease previously refractory to ofatumumab achieved a response. The most frequent any-grade/grade 3/4 treatment-emergent adverse events were diarrhea (47%/23%), neutropenia (26%/23%), pyrexia (24%/4%), cutaneous reactions (23%/4%), and thrombocytopenia (10%/6%).Conclusions:Duvelisib demonstrated high response rates with good durability and a manageable safety profile in patients with R/R CLL/SLL who progressed on ofatumumab, including patients with high-risk disease and disease previously refractory to ofatumumab.

Supplemental Figure Legend

Distribution of IGHV4-34 CLL according to IGHV mutational status and subset membership.

Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication.Experimental Design: In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34–expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes.Results: We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly (P < 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively).Conclusions: Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Clin Cancer Res; 23(17); 5292–301. ©2017 AACR.

Figure S3 displays probability of overall survival over time in the study population

•Table S1 shows a summary of prior anticancer therapies received by all patients in the study •Table S2 shows best overall response, overall response rate, and duration of response by investigator in the study population of patients while receiving ofatumumab in the parent DUO study before crossover •Table S3 shows rates of all serious TEAEs and individual hematologic and non-hematologic serious TEAEs in {greater than or equal to} 2% of patients in the study population • Table S4 shows rates of all TEAEs leading to death in patients in the study population •Table S5 shows a summary of patient disposition information

Figure S1 displays probability of progression-free survival over time in the study population and in the subgroup of patients with del(17p) and/or TP53 mutations that received ofatumumab while participating in the parent DUO study before crossover

Univariable and multivariable analysis for TTFT within M-CLL.

The MURANO trial (A Study to Evaluate the Benefit of Venetoclax Plus Rituximab Compared With Bendamustine Plus Rituximab in Participants With Relapsed or Refractory Chronic Lymphocytic Leukemia [CLL]; ClinicalTrials.gov identifier #NCT02005471) reported superior progression-free survival (PFS) and overall survival (OS) with venetoclax-rituximab (VenR) vs bendamustine-rituximab (BR) in relapsed/refractory (R/R) CLL. Patients were randomized to 2 years of VenR (n = 194; rituximab for the first 6 months) or 6 months of BR (n = 195). Although undetectable minimal residual disease (uMRD) was achieved more often with VenR, the long-term implications of uMRD with this fixed-duration, chemotherapy-free regimen have not been explored. We report MRD kinetics and updated outcomes with 5 years’ follow-up. Survival benefits with VenR vs BR were sustained (median PFS [95% confidence interval]: 53.6 [48.4, 57.0] vs 17.0 [15.5, 21.7] months, respectively, P < .0001; 5-year OS [95% confidence interval]: 82.1% [76.4, 87.8] vs 62.2% [54.8, 69.6], P < .0001). VenR was superior to BR, regardless of cytogenetic category. VenR-treated patients with uMRD at end of treatment (EOT; n = 83) had superior OS vs those with high-MRD+ (n = 12): 3-year post-EOT survival rates were 95.3% vs 72.9% (P = .039). In those with uMRD at EOT, median time to MRD conversion was 19.4 months. Of 47 patients with documented MRD conversion, 19 developed progressive disease (PD); median time from conversion to PD was 25.2 months. A population-based logistic growth model indicated slower MRD median doubling time post-EOT with VenR (93 days) vs BR (53 days; P = 1.2 × 10−7). No new safety signals were identified. Sustained survival, uMRD benefits, and durable responses support 2-year fixed-duration VenR treatment in R/R CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL. Key Points: • In a series of 29 856 CLL patients, the incidence of BcR stereotypy peaked at 41%. • Higher-order relations exist between stereotyped subsets, particularly for those from U-CLL, for which satellite subsets were identified.

The Complement 1 trial investigated the efficacy and safety of ofatumumab + chlorambucil with chlorambucil monotherapy in patients with previously untreated chronic lymphocytic leukaemia (CLL). On long-term follow-up in the chemoimmunotherapy arm vs. the chemotherapy arm there was an estimated 12% (not significant) and 39% risk reduction in overall survival and progression-free survival, respectively. A high rate (61%) of treatment with next-line therapies in both the treatment arms may dilute any potential OS difference and confound the interpretation of the OS results. Addition of ofatumumab to chlorambucil demonstrated clinical benefit and tolerability as a frontline treatment option in patients unfit for fludarabine-containing therapy, with no new safety concerns.

Ibrutinib revolutionized the CLL treatment approach and prognosis demonstrating its efficacy and safety even at extended follow-up. During the last few years, several next-generation inhibitors have been developed to overcome the occurrence of toxicity or resistance in patients on continuous treatment. In a head-to-head comparison of two phase III trials, both acalabrutinib and zanubrutinib demonstrated a lower incidence of adverse events in respect to ibrutinib. Nevertheless, resistance mutations remain a concern with continuous therapy and were demonstrated with both first- and next-generation covalent inhibitors. Reversible inhibitors showed efficacy independently of previous treatment and the presence of BTK mutations. Other strategies are currently under development in CLL, especially for high-risk patients, and include BTK inhibitor combinations with BCl2 inhibitors with or without anti-CD20 monoclonal antibodies. Finally, new mechanisms for BTK inhibition are under investigations in patients progressing with both covalent and non-covalent BTK and BCl2 inhibitors. Here we summarize and discuss results from main experiences on irreversible and reversable BTK inhibitors in CLL.

The importance of coexisting conditions in chronic lymphocytic leukemia (CLL) outcome has been increasingly recognized over the past years. The role of comorbidities to predict patients’ vulnerability toward immunochemotherapy has been well establish, especially since some of the tools commonly used to evaluate patients’ fitness were employed to determine treatment eligibility in randomized trials. Nevertheless, is it still unclear how much fitness weights on treatment outcome with targeted agents and which assessment should be preferred. There are key differences in the toxicity profile between novel agents that are getting much more evident in retrospective, real-life experiences, rather than clinical trials. Therefore, an individual patient's comorbid medical conditions may be a deciding factor in therapy selection. Here, we analyze main evidence in literature on the predicting value of comorbidity assessment on outcome and management of CLL patients receiving novel agents.

Background. The PI3Kδ inhibitor idelalisib given in combination with rituximab in relapsed/refractory chronic lymphocytic leukemia (R/R CLL) has been associated with an objective response in 81% of cases with a median PFS of 20.3 months in a phase-3 clinical trial (Sharman et al, JCO 2019). Because the efficacy of new drugs reported in clinical trials may not translate into similar results in the day-to-day practice, we performed an observational retrospective-prospective study on the efficacy and safety of idelalisib and rituximab (IR) in R/R CLL patients treated outside of clinical trials in Italian centers belonging to the GIMEMA group. Methods. R/R CLL patients treated with IR outside of clinical trials between July 2014 and May 2017 were enrolled. Treatment was initiated at symptomatic progression; treatment response and disease progression were assessed according to the NCI criteria. The data were extracted from the medical files and entered into case record forms (CRF) by each treating physician. Computerized and manual consistency checks were performed by the GIMEMA data center on newly entered forms and queries were issued in case of inconsistencies. The primary endpoint was progression-free survival (PFS), secondary endpoints were overall response rate (ORR), the percentage of patients on treatment at 12, 24 and 36 months, overall survival (OS) and response to the subsequent anti-leukemic treatment. Results. 149 R/R CLL patients from 35 Italian centers were included in the study. The baseline characteristics are shown in Table 1. At a median follow-up of 39.4 months, the median PFS was 22.9 months (95% CI 20.5-28.8). In univariate analysis, a shorter PFS was associated with ≥3 lines of therapy (p=0.0175) (Fig. 1a). The ORR (CR + PR) was 72.5%. Age ≤70 yrs (p=0.037) and a PS grade 3 during RI treatment were neutropenia (35% of cases), gastrointestinal disorders (17%), infections (17%) and transaminitis (6%). Conclusions. The results hereby observed show that the efficacy of IR in a cohort in a real-life RR/CLL with a long follow-up is superimposable to those reported in the pivotal clinical trial, suggesting that the patient population who received IR in the day-to-day clinical practice mirrored that enrolled in the trial. Advanced clinical stage and 3 or more previous lines of therapy were associated with shorter PFS and OS. Interestingly, PFS and objective measures of efficacy such as the proportion of patients still on treatment at 12, 24 and 36 months, as well as OS, were not influenced by the TP53 status. Furthermore, the majority of patents responded to the subsequent anti-leukemic treatment and relatively durable responses were observed in patients who discontinued IR due to toxicity. Disclosures Rigolin: Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Visentin:Janssen: Honoraria; Gilead: Honoraria; Abbvie: Honoraria. Coscia:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Farina:Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Gaidano:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astrazeneca: Membership on an entity's Board of Directors or advisory committees; Sunesys: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Murru:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Varettoni:Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Other: Travel/accommodations/expenses; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel/accommodations/expenses. Sportoletti:Janssen: Honoraria; AbbVie: Honoraria. Laurenti:Janssen: Honoraria; Gilead: Honoraria; AbbVie: Honoraria; Roche: Honoraria. Marasca:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria. Marchetti:Takeda: Other: Sponsored meetings; Gilead: Consultancy; Novartis: Speakers Bureau; Amgen: Speakers Bureau; AbbVie: Other: Sponsored meetings; Pfeizer: Other: Sponsored meetings. Mauro:Jannsen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Octopharma: Consultancy; Astrazeneca: Membership on an entity's Board of Directors or advisory committees. Molica:Roche: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Pane:Amgen: Consultancy, Other: Travel Expenses, Speakers Bureau; AbbVie: Consultancy, Other: Travel Expenses, Speakers Bureau; Daiichi Sankyo: Consultancy, Other: Travel Expenses; Jazz Pharmaceuticals: Consultancy, Other: travel expenses, Speakers Bureau; Novartis pharma SAS: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Other: Travel Expenses; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gozzetti:Janssen: Honoraria, Research Funding; Amgen: Honoraria; Takeda: Honoraria. Galieni:Celgene: Honoraria; Takeda: Honoraria; AbbVie: Honoraria; Janssen: Honoraria. Krampera:Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Trentin:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Octapharma: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria. Montillo:AbbVie: Honoraria, Speakers Bureau; F. Hoffmann-La Roche: Honoraria, Research Funding; Janssen: Honoraria, Speakers Bureau; Verastem: Honoraria; Gilead: Honoraria, Speakers Bureau; Astra Zeneca: Honoraria. Foà:Incyte: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees. Cuneo:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra Zeneca: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Introduction: The randomized Phase III MURANO study (NCT02005471) compared fixed-duration VenR with standard bendamustine-rituximab (BR) in R/R CLL. Deep responses with uMRD were associated with superior progression-free survival (PFS) of VenR vs BR with 48 months (mo) follow-up (f/u). We now report long-term MRD kinetics and updated efficacy outcomes, including re-exposure to VenR (to be presented), with a 5 year (yr) median follow-up (clinical cutoff date May 8, 2020). Methods: As published, pts were randomized to VenR (Ven 400 mg daily for 2 yrs + standard dose R for the first 6 mo) or B (70 mg/m2)R (6 mo). A sub-study was introduced in 2018, allowing pts who developed progressive disease (PD) following Tx with BR or VenR to receive the MURANO VenR regimen. PFS was based on investigator assessment. Peripheral blood MRD was analyzed centrally by allele-specific oligonucleotide polymerase chain reaction and/or flow cytometry. Pts were categorized by MRD status as previously reported, using Results: 389 pts were enrolled (VenR, n=194; BR, n=195). With a median f/u of 59.2 (range, 0-71.5) mo, the PFS benefit with VenR over BR was sustained (HR, 0.19 [95% CI: 0.15-0.26]; p Improved OS outcome was observed among the VenR pts that reached EOT without PD and had uMRD (83/118) compared with those with MRD (35/118), with 3-yr post-EOT survival estimates of 95.3% (95% CI: 90.0-100.0) vs 85.0% (95% CI: 72.8-97.2), respectively (Figure 1). Of the pts with uMRD at EOT, 32/83 had not shown PD and remained uMRD at the 5-yr update, 4/83 had PD without prior confirmed MRD conversion and 47/83 had MRD conversion. Median time to MRD conversion from EOT was 19.4 (95% CI: 8.7-28.3) mo. Of the 47/83 pts with confirmed MRD conversion, 19 subsequently developed PD by International Workshop on CLL criteria with a median time to PD from MRD conversion of 25.2 (95% CI: 19.4-30.4) mo. These 19 pts exhibited more rapidly increasing rates of MRD post-EOT than pts that had MRD conversion but were PD-free (Figure 2). Among pts that were uMRD at EOT, the baseline presence of del(17p), GC and unmutated immunoglobulin heavy chain gene (IGVH) were each associated with increased risk of MRD conversion and subsequent PD post-EOT (Table 1). All 4 pts with del(17p) experienced MRD conversion with subsequent PD. 8/18 (44%) pts with GC vs 8/40 (20%) pts without GC eventually converted to MRD and developed PD. The rate of MRD conversion with eventual PD was also higher among those with unmutated IGVH (21/56; 37%) than those without (1/23; 4%). Once uMRD at EOT was achieved, pts without del(17p) or GC, or with mutated IGVH, were more likely to maintain uMRD or experience MRD conversion without subsequent PD at this follow-up (Table 1). No new safety signals were identified. Excluding non-melanoma skin cancers, 2 second primary malignancies (VenR [acute myeloid leukemia and multiple myeloma]) were reported since the previous update. Rates of Richter transformation remained balanced between treatment arms (7 on VenR, 6 on BR). Following PD on the main study, 29 pts were enrolled in the sub-study (re-treatment; n=21, crossover; n=8). Further data on their biologic profile, updated response rates, and MRD in the re-treatment cohort will be presented. Conclusions: Five-yr data from MURANO demonstrate sustained PFS and OS benefit with VenR vs BR. In the VenR cohort, uMRD at EOT is associated with improved OS. Unmutated IGVH, del(17p) and GC (≥3 CNV) are associated with higher rates of MRD conversion and subsequent PD after attaining uMRD at EOT. Overall, a substantial proportion of pts who completed Ven Tx retained uMRD 36 mo after treatment cessation, displaying durable response following 2-yr fixed-duration VenR. Disclosures Kater: Janssen: Research Funding; Celgene: Research Funding; Abbvie: Research Funding; Roche: Research Funding; Genentech: Research Funding. Kipps:Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VelosBio: Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees. Eichhorst:F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; ArQule: Consultancy, Honoraria, Other: travel support, Research Funding; Oxford Biomedica: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: travel support, Research Funding; BeiGene: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding. Hillmen:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding, Speakers Bureau; Astra Zeneca: Honoraria; F. Hoffmann-La Roche: Honoraria, Research Funding; Pharmacyclics: Research Funding; Gilead: Research Funding. D'Rozario:F. Hoffmann-La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Owen:AbbVie, F. Hoffmann-La Roche, Janssen, Astrazeneca, Merck, Servier, Novartis, Teva: Honoraria. Assouline:AbbVie: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Research Funding. Lamanna:MingSight: Other: Institutional research grants, Research Funding; Astra Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional research grants, Research Funding; Oncternal, Verastem, TG Therapeutics: Other: Institutional research grants, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Juno: Other: Institutional research grants, Research Funding; Loxo: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional research grants, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bei-Gene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional research grants, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional research grants, Research Funding; Octapharma: Research Funding; Columbia University Medical Center: Current Employment; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Robak:Abbvie, Pharmacyclics, Gilead, GlaxoSmithKline, Novartis, Janssen, Roche, Acerta, AstraZeneca, BioGene, UCB: Research Funding; Abbvie, Pharmacyclics, Novartis, Janssen, Acerta, AstraZeneca, BioGene, UCB, Sandoz: Honoraria; Sandoz, Janssen, AbbVie, Roche, Gilead: Consultancy. de la Serna:F. Hoffmann-La Roche, Abbvie, Pharmacyclics, Gilead, GlaxoSmithKline, Novartis, Janssen, Roche, Acerta, AstraZeneca, BioGene, UCB: Research Funding; Abbvie, AstraZeneca: Other: Travel, Accommodations, Expenses; Abbvie, Janssen: Speakers Bureau; Gilead, AstraZeneca, Abbvie, Janssen, Sandoz, F. Hoffmann-La Roche: Consultancy; Abbvie, Pharmacyclics, Novartis, Janssen, Acerta, AstraZeneca, BioGene, UCB, Sandoz: Honoraria. Jaeger:F. Hoffmann-La Roche: Honoraria, Research Funding. Cartron:Celgene: Consultancy, Honoraria; F. Hoffmann-La Roche: Consultancy, Honoraria; Sanofi: Honoraria; Gilead: Honoraria; Jansen: Honoraria; Abbvie: Honoraria. Montillo:Gilead: Honoraria, Speakers Bureau; AbbVie: Honoraria, Speakers Bureau; F. Hoffmann-La Roche: Honoraria, Research Funding; Janssen: Honoraria, Speakers Bureau; Astra Zeneca: Honoraria; Verastem: Honoraria. Mellink:Genentech, Inc: Research Funding; Amsterdam University Medical Centre: Current Employment. Chyla:AbbVie: Current Employment, Current equity holder in publicly-traded company. Wilson:Roche Products Limited: Current Employment. Wu:Genentech, Inc.: Current Employment, Current equity holder in publicly-traded company; F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Jiang:F. Hoffmann-La Roche: Current equity holder in publicly-traded company; Genentech, Inc.: Current Employment. Lefebure:F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Boyer:Roche: Current Employment, Current equity holder in publicly-traded company, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Seymour:Mei Pharma: Consultancy, Honoraria; Nurix: Honoraria; Morphosys: Consultancy, Honoraria; Gilead: Consultancy; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Ibrutinib, a once-daily oral inhibitor of Bruton tyrosine kinase, has greatly improved outcomes for patients with chronic lymphocytic leukemia (CLL). The phase 3 RESONATE trial, which compared single-agent ibrutinib to ofatumumab in high-risk, relapsed patients with CLL, provided support for approval of ibrutinib in the United States and Europe. We describe long-term follow-up of patients treated in RESONATE, where continued superiority of progression-free survival (PFS) (hazard ratio [HR], 0.133; 95% confidence interval [CI], 0.099-0.178) was observed. Overall survival benefit continues (HR, 0.591; 95% CI, 0.378-0.926), although with decreased magnitude relative to that seen before crossover to ibrutinib was implemented for patients on ofatumumab (HR, 0.426; 95% CI, 0.220-0.823). Notably, overall response to ibrutinib increased over time, with 91% of patients attaining a response. The PFS benefit with ibrutinib was independent of baseline risk factors, although patients with ≥2 prior therapies had shorter PFS than those with

Context: Ibrutinib inhibits BTK with the most typical response in CLL being partial remission (PR) with measurable minimal residual disease (MRD) in blood. Remission is usually maintained until development of genetically-driven resistance. Objective: Mechanism of adaptation and survival in MRD. Patients and Methods: Pre-treatment CLL cells and under-treatment MRD were systematically collected from 33 high-risk CLL patients. Samples were characterized by high-dimensional, single-cell flow cytometry; bulk genomic, transcriptomic, and chromatin accessibility profiling; and single-cell RNA-seq profiling. Results: The genetic composition of MRD remained constant across timepoints, indicating that ibrutinib did not have any impact in shaping its genomic diversity. Ibrutinib induced chromatin dynamics in MRD resulting in a predominantly closed state. Chromatin regions that became more accessible under ibrutinib were enriched of binding sites for transcription factor that lay downstream ERK signaling. Regions that turned into a less accessible state were enriched for the binding sites of TF emanating from the BCR. At the transcriptomic level, most of the differentially expressed genes between pre- and post-treatment samples were downregulated in MRD, while only a fraction was upregulated. IL4, NF-kB, and metabolism and proliferation signatures were downregulated in MRD, while MAPK and RAS signatures were upregulated. CLL spleen samples from TCL1 mice under ibrutinib treatment showed an upregulation of MAPK pathway genes compared to untreated mice. Single-cell transcriptomes revealed a distinct post-treatment cell population driven by MAPK activity that functionally pre-existed in a fraction of CLL cells of baseline samples before ibrutinib start. At the signaling level, ibrutinib had no effect on the integrity of RAS-BRAF-MAPK-ERK pathway protein expression upon crosslinking of the BCR with anti-IgM but not after stimulation of TLR9 or CD40. Functional validation on primary samples by western blotting shows that RAS/ERK can be activated by BCR stimulation, irrespective of ibrutinib treatment in ex vivo experiments. Pharmacological inhibition of MEK (trametinib) and ERK (ulixertinib) synergized with ibrutinib, leading to decreased cell viability. Conclusions: MRD under ibrutinib adapts its phenotype in an epigenetic way to maintain functional competence of BCR signaling via the MAPK pathway, which turned to be a vulnerability of MRD persisting under ibrutinib.

Patients with relapsed/refractory chronic lymphocytic leukemia (R/R CLL) whose treatment failed with a Bruton's tyrosine kinase inhibitor have poor outcomes. We investigated tafasitamab plus idelalisib (cohort A) or venetoclax (cohort B) in this patient population in a phase II study (NCT02639910). In total, 24 patients were enrolled (cohort A: n = 11, median time on study, 7.4 months; cohort B: n = 13, median time on study, 15.6 months). The most common treatment-emergent adverse event (TEAE) in cohort A was anemia (63.6%) and in cohort B was infusion-related reaction (53.8%). The most common severe TEAE was neutropenia (cohort A: 45.5%; cohort B: 46.2%). The best overall response rate was 90.9% (cohort A) and 76.9% (cohort B). Undetectable minimal residual disease in peripheral blood was achieved in 2/8 patients (cohort A) and 6/7 patients (cohort B). Overall, these results suggest that anti-CD19 antibody-based combinations may be important in the treatment of patients with CLL.

Key points Age per se does not influence outcome in CLL patients on ibrutinib, whereas CIRS score is predictive of treatment management, PFS, and EFS.ECOG-PS and neutropenia resulted as the only baseline parameters affecting overall survival., Visual Abstract, Functional reserve of organs and systems is known to be relevant in predicting immunochemotherapy tolerance. Age and comorbidities, assessed by the cumulative illness rating scale (CIRS), have been used to address chemotherapy intensity. In the ibrutinib era, it is still unclear whether age, CIRS, and Eastern Cooperative Oncology Group performance status (ECOG-PS) retain their predictive role on treatment vulnerability. In this series of 712 patients with chronic lymphocytic leukemia (CLL) treated with ibrutinib outside clinical trials, baseline ECOG-PS and neutropenia resulted as the most accurate predictors of treatment feasibility and outcomes. Age did not independently influence survival and ibrutinib tolerance, indicating that not age per se, but age-related conditions, may affect drug management. We confirmed the role of CIRS > 6 as a predictor of a poorer progression- and event-free survival (PFS, EFS). The presence of a severe comorbidity was significantly associated with permanent dose reductions (PDRs), not translating into worse outcomes. As expected, del(17p) and/or TP53mut and previous therapies affected PFS, EFS, and overall survival. No study so far has analyzed the influence of concomitant medications and CYP3A inhibitors with ibrutinib. In our series, these factors had no impact, although CYP3A4 inhibitors use correlated with Cox regression analysis, with an increased risk of PDR. Despite the limitation of its retrospective nature, this large study confirmed the role of ECOG-PS as the most accurate predictor of ibrutinib feasibility and outcomes, and importantly, neutropenia emerged as a relevant tool influencing patients’ vulnerability. Although CIRS > 6 retained a significant impact on PFS and EFS, its value should be confirmed by prospective studies.

Autoimmune hemolytic anemias (AIHAs) are rare and heterogeneous disorders characterized by the destruction of red blood cells through warm or cold antibodies. There is currently no licensed treatment for AIHA. Due to the paucity of clinical trials, recommendations on diagnosis and therapy have often been based on expert opinions and some national guidelines. Here we report the recommendations of the First International Consensus Group, who met with the aim to review currently available data and to provide standardized diagnostic criteria and therapeutic approaches as well as an overview of novel therapies. Exact diagnostic workup is important because symptoms, course of disease, and therapeutic management relate to the type of antibody involved. Monospecific direct antiglobulin test is considered mandatory in the diagnostic workup, and any causes of secondary AIHA have to be diagnosed. Corticosteroids remain first-line therapy for warm-AIHA, while the addition of rituximab should be considered early in severe cases and if no prompt response to steroids is achieved. Rituximab with or without bendamustine should be used in the first line for patients with cold agglutinin disease requiring therapy. We identified a need to establish an international AIHA network. Future recommendations should be based on prospective clinical trials whenever possible.

Introduction Although CCAAT/enhancer binding protein alpha double mutated (CEBPADM) acute myeloid leukemia (AML) is considered a low-risk form of AML according to 2017 ELN recommendations, relapse remains a major cause of death. To assess the broader prognostic impact of other cancer-associated genes, we sequenced a panel of 40 myeloid disorders-related genes in a 25 patient cohort. Methods 16 CEBPADM AML diagnosis samples along with 9 CEBPAsingle mutated (SM) were analyzed by targeted next-generation sequencing (Ion Torrent) using Oncomine Myeloid Research Assay. 4 CEBPADM and 2 CEBPASM AML relapse samples were analyzed as well. All patients received intensive chemotherapy according to 2017 ELN recommendations. Results With a median follow-up of 3.2 years (range 0.4-12) 5y OS was 61% and 14% for CEBPADM and CEBPASM patients respectively. Overall, a median of 3 concurrent mutations were present at diagnosis, slightly more in CEBPA SM patients (4 vs 3 in CEBPA DMpatients). The number of somatic mutations influenced both PFS and OS (p = 0.04 and p < 0.01 respectively) independently of CEBPA mutational status. Each single unitary increase in the number of mutations increased the hazard for death of 27.7% (95% CI: -1.4-+65; p = 0.064) while passing from 4 to 5 mutations increased the odds of death by 367%. 5y OS in patients with 5 or more concurrent mutation was 14.3% vs 61.8% in patients with less than 5 co-mutated genes; 5y PFS was 0% vs 38.6%. Mutational landscape of CEBPADMand CEBPASMAML differed significantly, with GATA2, FLT3, DNMT3A and TET2 being the most frequently mutated genes in CEBPADM vs NPM1, FLT3, DNMT3A and WT1 in CEBPASM patients. NPM1(77.8% vs 6.7%; p < 0.01) and ASXL1 mutations (44.4% vs 0%; p = 0.02) were more frequent in CEBPASM patients, confirming they are mutually exclusive with CEBPA biallelic lesions. DNA methylation was the most frequently mutated pathway in biallelic patients (87%) while chromatin/cohesin complex (88%) was the most frequently mutated one in CEBPA monoallelic patients. Mutations of CEBPA, NPM1, DNMT3A, WT1, STAG2, TET2, ASXL1, IDH2, SRSF2, CALR, PRPF8, NF1, TP53, RUNX1 had the highest median variant allele frequency (VAF), more often representing founding mutations. GATA2, IDH1, KRAS, BCOR, MPL, IKZ2F1 and PTPN11 had a more borderline median VAF, variably being clonal or subclonal. Mutations in the 3 tyrosine kinases genes FLT3-ITD , CSF3R, NRAS were only subclonal. Mutations in WT1 and FLT3 were associated with increased relapse rate (p = 0.02 and p = 0.01 respectively), while patients with GATA2 mutations had a strong trend towards better 5y OS (83% vs 32%, p = 0.053). We also identified a not previously described allelic variant in the SH2B3 gene (ATGGGG/A INDEL) with an overall prevalence in our population of 58.3% (46.7% of CEBPA DM and 77.8% of CEBPA SMpatients). Patients with the SH2B3allelic variant had a significantly lower bone marrow blast percentage at diagnosis (p = 0.014) and a strong trend towards a higher number of concurrent mutations (p = 0.056). Moreover, when present at diagnosis, SH2B3 variant persisted at relapse with the same VAF. By real time PCR we demonstrated that this SH2B3 allelic variant leads to a dramatic reduction of the corresponding transcript. This gene encodes for a negative regulator of many crucial signaling pathways (SCF/c-KIT, erythropoietin/JAK2, thrombopoietin/MPL WT/ W515L, JAK2 WT/ V617F, GM-CSFR and FLT3-WT/ITD) of the hematopoietic stem cell. Matched diagnosis and relapse samples analysis suggested different features of clonal evolution: while mutations of SH2B3, WT1, DNMT3A, NPM1, and IDH1 consistently persisted at relapse, CEBPA and GATA2 mutations were unstable during disease course. ZRSR2 and PRPF8 mutations were found in relapse samples only. Summary Our study offers insights into the genetic landscape of CEBPADM mutated AML as compared to CEBPASM AML, highlighting the contribution of NGS to risk stratification. In fact, our data show that the number and the type of concurrent mutation has a prognostic impact, possibly identifying patients eligible to first line allogeneic stem cell transplantation. We identified an allelic variant of SH2B3 that had never been functionally characterized nor associated with AML and that could represent a marker for genetic instability and a potential new target in AML treatment strategies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

We performed a comparative gene expression profiling (GEP) study on B-cells and plasma cells of Waldenström Macroglobulinemia (WM), IgM monoclonal gammopathies of undetermined significance (IgMMGUS), and normal individuals (CTRLs) to identify GEP changes as reliable predictors of progression of IgMMGUS to WM. We analyzed bone marrow B-cells and plasma cells from 36 WM patients, 13 IgMMGUS subjects, and 7 CTRLs by Affymetrix microarray, respectively (Table 1). GEP experiments were performed on the CD19+ and CD138+ cells using GeneChip-HGU133 Plus 2.0. Data were preprocessed and normalized by Robust Multi-Array Average and ComBat. Selection of the different expressed genes was performed separately for CD19+ and CD138+ cells, using Significance Analysis of Microarrays (SAM) on the 3 groups and a false discovery rate threshold of 5%, followed, for significance comparisons, by a pair-wise SAM test corrected for multiple testing. We focused on the comparison of the CD19+ cells of WM vs. IgMMGUS vs. CTRLs which highlighted 2038 unique genes whereas the same comparison of the CD138+ cells determined 29 unique genes (Trojani et.al. Cancers 2021). Among the 2038 DEGs, 115 genes were grouped in KEGG pathways involved in Wnt-signaling, BCR-signaling, calcium signaling, hematopoietic cell antigens, cell adhesion, adherens junctions, coagulation cascade, platelet activation, cytokine receptor, and signaling pathways responsible for cell cycle, apoptosis, and survival. Interestingly, most of the 115 DEGs in B-cells were different expressed in WM vs. IgMMGUS and CTRLs. Only 9/115 DEGs were significantly different expressed in WM vs. CTRLs and in IgMMGUS vs. CTRLs, but no significant expression changes were noted between WM and IgMMGUS (Table 2). To further inspect the similarities and the differences among WM and IgMMGUS, we computed the Euclidean pair-wise distance between subjects and, using this distance as weight, constructed a minimum spanning tree (MST) (Figure 1). Considerably, four probesets identified ADRB2 (transmembrane Beta adrenergic receptor) which was up regulated in WM and IgMMGUS compared to CTRLs. The over expression of ADRB2 was also demonstrated in Mantle Cell Lymphoma cell lines and in Diffuse large B-cell lymphoma (DLBCL) lymphocytes compared to normal B-cells (doi10.1016/j.cellsig.2017.08.002), and in most malignancies (doi10.1007/s11033-021-06250-y) . As far as we know, ADAM23 (ADAM Metallopeptidase Domain23) has not been found in WM, whereas we suggest its possible role in WM patients with Sjogren's syndrome (SS). ADAM23 plays a role in the peripheral neuropathy by controlling the activity of potassium channels in SS (doi10.1007/s10067-016-3499-z). Some authors found that sensory/motor neuropathies were associated with MGUS patients (doi10.1017/s0317167100011483). We strongly believe that the down regulation of ADAM23 in WM and IgMMGUS has a good chance to be associated with clinical neuropathy in WM and IgMMGUS. RASGRP3 (RAS Guanyl Releasing Protein3) and PIK3AP1 (Phosphoinositide 3 Kinase Adaptor Protein1) play crucial roles in BCR signaling pathway: PIK3AP1 activates the PI3K-Akt signaling while RASGRP3 stimulates MAPK signaling pathway. The deregulation of LEF1 (Lymphoid Enhancer Binding Factor1) and genes of Wnt-pathway were previously demonstrated in B-cell disorders and multiple myeloma (doi10.1007/s00277-017-3207-3, doi10.1016/j.pathol.2019.09.009). According to these studies, we showed the under expression of LEF1 in WM and IgMMGUS compared to CTRLs. We identified the down regulation of EZH2 (Enhancer Of Zeste2 Polycomb Repressive Complex2Subunit) in WM and IgMMGUS compared to CTRLs. EZH2 is involved in Follicular lymphoma and DLBCL (doi10.1080/2162402X.2017.1321184). CDHR3 (Cadherin Related Family Member3), CHEK1 (Checkpoint Kinase1), and HIST1H1B (Histone-H1.5) were over expressed in CTRLs compared to IgMMGUS and WM. In conclusion, the common gene-set in WM and IgMMGUS could suggest two-hit hypothesis. First, the gene-set could play a role in the risk of progression of IgMMGUS to WM. Until now, all the IgMMGUS subjects have not been transformed in WM or other NHL, but they have been monitored every 6 months, and their possible transformation to lymphoma could highlight new insights. The second hypothesis suggests their involvement in the biological processes of leukemogenesis in WM and IgMMGUS which will be further investigated. Figure 1 Figure 1. Disclosures Tedeschi: AbbVie: Honoraria, Speakers Bureau; AstraZeneca: Honoraria, Speakers Bureau; Beigene: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau.

In the phase 3 RESONATE study, ibrutinib demonstrated superior progression-free survival (PFS), overall survival (OS) and overall response rate (ORR) compared with ofatumumab in relapsed/refractory CLL patients with high-risk prognostic factors. We report updated results from RESONATE in these traditionally chemotherapy resistant high-risk genomic subgroups at a median follow-up of 19 months. Mutations were detected by Foundation One Heme Panel. Baseline mutations in the ibrutinib arm included TP53 (51%), SF3B1 (31%), NOTCH1 (28%), ATM (19%) and BIRC3 (14%). Median PFS was not reached, with 74% of patients randomized to ibrutinib alive and progression-free at 24 months. The improved efficacy of ibrutinib vs ofatumumab continues in all prognostic subgroups including del17p and del11q. No significant difference within the ibrutinib arm was observed for PFS across most genomic subtypes, although a subset carrying both TP53 mutation and del17p had reduced PFS compared with patients with neither abnormality. Reduced PFS or OS was not evident in patients with only del17p. PFS was significantly better for ibrutinib-treated patients in second-line vs later lines of therapy. The robust clinical activity of ibrutinib continues to show ongoing efficacy and acceptable safety consistent with prior reports, independent of various known high-risk mutations.

Ibrutinib, an oral inhibitor of Bruton's tyrosine kinase (BTK), at a once-daily dose of 420 mg achieved BTK active-site occupancy in patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) that was maintained at 24 hours. It is unknown if intermittent interruption of ibrutinib therapy contributes to altered clinical outcomes. We therefore evaluated the effect of ibrutinib dose adherence on patient outcomes in the phase 3 RESONATE trial. The overall mean dose intensity (DI) was 95% with median treatment duration of similar to 9 months. Pharmacokinetic assessment of ibrutinib exposure at 420-mg dose suggested similar exposure regardless of patient weight or age. As assessed by independent review committee, patients with higher DI experienced longer median progression-free survival (PFS) compared with those with lower DI regardless of del17p and/or TP53 status. Of 79 patients requiring a drug hold, treatment was restarted at the original dose in 73 (92%) patients. Mean duration of a missed-dose event was 18.7 days (range, 8-56). Patients missing >= 8 consecutive days of ibrutinib had a shorter median PFS vs those missing