Short Remission Research Articles

Lipopolymer/siRNA complexes engineered for optimal molecular and functional response with chemotherapy in FLT3-mutated acute myeloid leukemia

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled. RNA interference (RNAi), mediated by short interfering RNA (siRNA), presents a mechanistically distinct therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. This study explored the use of a non-viral approach for delivery of FLT3 siRNA (siFLT3) in FLT3-ITD positive AML cell lines and primary cells as well as the feasibility of combining this treatment with drugs currently used in the clinic. Treatment of AML cell lines with FLT3 siRNA nanocomplexes resulted in prominent reduction in cell proliferation rates and induction of apoptosis. Quantitative analysis of relative mRNA transcript levels revealed downregulation of the FLT3 gene, which was accompanied by a similar decline in FLT3 protein levels. Moreover, an impact on leukemic stem cells was observed in a small pool of primary AML samples through significantly reduced colony numbers. An absence of a molecular response post-treatment with lipopolymer/siFLT3 complexes in peripheral blood mononuclear cells, obtained from healthy individuals, denoted a passive selectivity of the complexes towards malignant cells. The effect of combining lipopolymer/siFLT3 complexes with daunorubucin and FLT3 targeting TKI gilteritinib led to a significant augmentation of anti-leukemic activity. These findings demonstrate the promising potential of RNAi implemented with lipopolymer complexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML. Statement of significanceWe show that a clinically validated target, the FLT3 gene, can be eradicated in leukemia cells using non-viral RNAi. We validated these lipopolymers as effective vehicles to deliver nucleic acids to leukemic cells. The potency of the lipopolymers was superior to that of the ‘gold-standard’ delivery agent, lipid nanoparticles (LNPs), which are not effective in leukemia cells at clinically relevant doses. Mechanistic studies were undertaken to probe structure-function relationships for effective biomaterial formulations. Cellular and molecular responses to siRNA treatment have been characterized in cell models, including leukemia patient-derived cells. The use of the siRNA therapy with clinically used chemotherapy was demonstrated.

Analysis of remissions in patients with combined addiction to psychostimulants and other psychoactive substances and comorbid psychiatric pathology after rehabilitation

Despite the annual increase in the number of patients with psychostimulant addiction and the presence of comorbid psychiatric pathology, the question of finding effective therapies for these disorders remains open. When developing therapeutic remission, specialists often do not take into account the peculiarities of its duration and quality in these patients. The purpose of the study is to assess the duration and quality of therapeutic remissions in patients with addiction to psychostimulants, combined use of other psychoactive substances, and comorbid psychiatric pathology. Materials and methods. 325 patients with psychostimulant addiction, who underwent a course of medical and social rehabilitation in the Moscow Research and Practical Centre for Narcology of the Moscow Healthcare Department, were examined. Methods: clinical-psychopathological, psychometric ("Addiction Severity Index: Treatnet Version"), anamnestic, catamnestic, and statistical. The results of the study. It was found that the longest remissions were noted in patients only with psychostimulant addiction. Patients with combined addiction to psychostimulants and the presence of comorbid psychiatric pathology are characterized by short remissions. Patients only with psychostimulant addiction have the best dynamics of remission quality. Conclusion. The revealed clinical features of patients, together with quantitative and qualitative features of remissions, will help to increase the effectiveness of care for these patients.

Значение клинико-динамических параметров пациентов с сочетанной зависимостью от психоактивных веществ на этапе медицинской реабилитации

According to official statistics, the number of patients with combined addiction of psychoactive substances increases annually. At the same time, the effectiveness of therapy for such disorders remains at a low level. Aim. To study the clinical characteristics of patients with combined addiction of psychoactive substances in dynamics to improve the effectiveness of rehabilitation. Material and methods. 204 patients with combined addiction of psychoactive substances, men and women aged 18 to 50 years, who underwent inpatient treatment at the Moscow Scientific and Practical Center of Narcology, were examined. The patients were divided into 4 groups addiction on the combinations of psychoactive substances. Methods: clinical-psychopathological, anamnestic, psychometric (Visual-analog scale of pathological drug addiction, Montgomery–Asberg depression assessment scale, Beck depression severity self-assessment scale), statistical. The patients were examined on the 8–10th day and the 20–30th day of therapy. Further, the patients were followed by catamnestic observation for 12 months. Their mental status, quality and duration of remissions were assessed. Results. In patients with combined addiction of cannabinoids and psychostimulants and alcohol and psychostimulants, a lower percentage of hereditary burden, less pronounced antisocial personality traits, and a lower intensity of pathological attraction to psychoactive substance, especially behavioral and affective components, were revealed compared with patients with combined addiction of opioids and psychostimulants and addiction of several psychoactive substances. Patients with combined addiction of opioids and psychostimulants and several psychoactive substances at once had shorter remissions. Conclusion. Among patients with combined addiction of psychoactive substances, associations were found between the premorbid characteristics of patients, clinical features and quality, and duration of remissions. This will create a personalized approach to the rehabilitation of such patients and increase the effectiveness of therapy.

Delphi consensus recommendations on treatment for advanced-stage marginal zone lymphoma in South Korea.

Due to the lack of treatment guidelines for the management of advanced-stage marginal zone lymphoma (MZL), only one chemoimmunotherapy-cyclophosphamide, vincristine, and prednisone plus rituximab (R-CVP)-is reimbursed in the first-line setting in South Korea. The aim of this study was to develop a consensus-based recommendation for the treatment of patients with advanced-stage MZL. Twelve hematologist oncologists participated in a two-round Delphi process to identify consensus on the management of patients with advanced-stage MZL in South Korea. Physicians rated their level of agreement with each statement on a four-point Likert scale. Statements were divided into two sections: definitions used in clinical practice and clinical management of patients with advanced-stage MZL. Consensus was reached for 23 of 33 (69.7%) and 5 of 13 statements (38.5%) in rounds 1 and 2, respectively. There was strong consensus (91.7%) that advanced-stage MZL subtypes are defined according to the Lugano staging system. First-line systemic treatment should be prescribed for patients with symptomatic advanced-stage MZL. Although there was unanimous agreement that R-CVP is the standard first-line treatment for advanced-stage MZL, physicians also agreed that bendamustine with rituximab (BR) has greater efficacy than R-CVP as first-line treatment (91.7%). For the treatment of relapsed/refractory advanced-stage MZL, BR and R-CVP can be repeated in patients with short (< 24 months) and long remission periods (≥ 24 months), respectively. This study provides insights on the management of patients with advanced-stage MZL in South Korea. This may enhance clinical decision-making, thus improving patient outcomes.

Polatuzumab vedotin in diffuse large B-cell lymphoma therapy: Literature review and experience of the Oncohematology Division of the Lapino Clinical Hospital

Study of molecular and genetic features of diffuse large B-cell lymphoma (DLBCL) suggests differential approach to treatment of this aggressive B-cell disorder characterized by short remission after each subsequent therapy line. Therefore, more effective regimens should be used at earlier stages. Identification of biological markers and their integration into prognostic scales can help to personalize therapy, especially in patients with high risk of quick progression and increase survival.Our own experience of using immunoconjugate polatuzumab vedotin in therapy of patients with DLBCL. The use of Pola-BR combination in a patient with refractory non-GCB DLBCL allowed to achieve full remission alongside satisfactory tolerability and absence of significant adverse events. An evaluation of the Pola-R-CHP regimen in treatment of a patient with newly diagnosed generalized GCB DLBCL is presented.

Cognitive Alterations Associated with Remission and Alcohol Dependence Severity in Ethnically Diverse Patients of Siberia.

Impaired cognition in individuals with alcohol dependence may be associated with increased relapse risk. It has been recorded in more than half of patients during six months after treatment. In certain ethnic groups, for example, Tuvinians, the indigenous people of Siberia, relapses occur in extremely short periods of one to three months after treatment. An approach currently used to alcohol dependence treatment may be less effective for these patients. The study aimed to investigate cognitive sequelae in indigenous Tuvinian patients with alcohol dependence. The sample included 166 patients, 74 of indigenous ethnicity (Tuvinians) and 92 non-indigenous white patients. Data on inhibitory control, cognitive flexibility, attention, and working memory were collected from all the patients and processed using cluster analysis. The clustering data were then complemented by indicators of disorder dynamics, impulsivity, and emotion regulation. The clustering procedure revealed groups with severe cognitive sequelae. More than four-fold attention decrease was found in 43.5% of non-indigenous patients, and more impaired cognitive flexibility was revealed among 60.8% of indigenous patients. Groups with severe cognitive sequelae had higher impulsivity, maladaptive emotion regulation, more hospitalizations, faster disease progression, and shorter remissions. The latter was significantly reduced to 90 days on average in the severe group of indigenous patients versus 135 days of remission in the non-indigenous severe group. Results obtained may advance tailored intervention in alcohol-dependent patients of the indigenous Tuvinian ethnicity. While little is still known about the alcohol dependence course and consequences in the indigenous Tuvinians of Siberia, this study contributes to the global mental health data on alcohol abuse and dependence in indigenous communities.

Covalent-103: A phase 1, open-label, dose-escalation and expansion study of BMF-500, an oral covalent FLT3 inhibitor, in adults with acute leukemia (AL).

TPS6589 Background: FLT3 mutations occur in 25-35% of AML and result from an internal tandem duplication (ITD) of amino acids in the juxtamembrane domain of FLT3 kinase or point mutations in the tyrosine kinase domain (TKD). FLT3-ITD mutations are associated with increased relapse, shorter remission, and decreased disease-free and overall survival. BMF-500 is a novel oral, highly potent and selective covalent inhibitor of FLT3 including wild-type (WT), ITD, TKD, and resistance mutations (e.g., gatekeeper F691). BMF-500 has high affinity for FLT3, lack of cKIT inhibition, and sustained cell-killing despite drug washout (Law et al., ASH 2022 Abst 2756). BMF-500 shows sustained tumor regression and improved survival in subcutaneous and disseminated xenografts of FLT3-m AML. Methods: COVALENT-103 (NCT05918692) is a multicenter, first-in-human study evaluating the safety, tolerability, and antileukemic activity of escalating doses of daily BMF-500 in patients with relapsed or refractory (R/R) AL, including AML, ALL, or MPAL, with or without FLT3-m. The trial has 2 arms that dose escalate in parallel: Arm A (without) and Arm B (with) concomitant use of a CYP3A4 inhibitor. Using an accelerated titration design (ATD), doses of BMF-500 are escalated in single-subject cohorts until one subject experiences ≥Grade 2 related adverse event or dose-limiting toxicity (DLT). At that point, the cohort will switch to a classical “3 +3” design. Patients with WT FLT3 AL may enroll up to 33% per arm. Treatment continues in 28-day cycles until progression or intolerability. Expansion cohorts will enroll additional patients to obtain further safety and efficacy data. Patients must be refractory, relapsed or have progressed on or following discontinuation of the most recent anti-cancer therapy or be ineligible for any approved standard of care, including hematopoietic stem cell transplant (HSCT). FLT3-positive AML patients must have received a FLT3 inhibitor approved for R/R FLT3-m AML. Additional inclusion criteria include ECOG PS ≤2, adequate organ function, and documented FLT3 mutation status. Key exclusion criteria include known CNS disease, clinically significant cardiovascular disease, and WBC &gt;50,000/µL (uncontrollable with cytoreductive therapy). The primary objective is to evaluate safety and tolerability and determine the optimal biological dose (OBD)/ recommended Phase 2 dose (RP2D) of BMF-500 monotherapy based on available pharmacokinetic/pharmacodynamic (PK/PD), safety and efficacy data. Secondary objectives include characterization of the PK/PD of BMF-500, and assessment of its antitumor activity per modified Cheson (2003) criteria or NCCN Clinical Practice Guidelines (ALL Version 1.2022) as determined by the investigator. The study was initiated in July 2023, is currently in dose escalation, and we plan to enroll approximately 110 patients total. Clinical trial information: NCT05918692 .

A potential best-in-class BCMA-CD19 bispecific CART with advanced safety by self-inhibiting IFNG signaling during CRS.

2502 Background: CART cells have demonstrated remarkable clinical efficacy in treating hematological cancers. However, CRS remains as a major challenge in clinical application, with multiple cytokines significantly elevated. IL6 signaling blockade by Tocilizumab has become a standard treatment to relieve CRS in patients. Our previous studies suggested that CAR T secreting IL6 antagonist could effectively maintain IL6 at very low levels during CRS. Furthermore, we observed that a huge quantity of tumor cells in one patient was significantly reduced after CART infusion, but a low level of IFNG with only grade 2 CRS was present. Therefore, we hypothesized that high level of IFNG might not be necessary to CART clinical efficacy and direct blockade of IFNG signaling by CART secreting an IFNG antagonist (designated as SAFET) serves an interesting approach to effectively reduce CRS toxicity. Although IFNG is usually thought important in T cell cytotoxicity, our results indicated that IFNG KO or autonomous secretion of IFNG antagonist did not affect the killing activity of CART cells. Methods: This pilot phase 1 study of SAFET BCMA/CD19 BiCART self-inhibiting IFNG signaling is a single arm study conducted in China, enrolling refractory/relapsed multiple myeloma patients. The patients had received more than 3 lines of prior therapies including at least a proteasome inhibitor and an immunomodulatory agent and were refractory to the last line of treatment. Lymphodepletion was performed with fludarabine and cyclophosphamide prior to the CART infusion. Following 2-14 days of rest, patients received a single infusion of SAFET BCMA/CD19 BiCART, at the dose of 0.4-1.0 × 108 CAR+ T cells/patient. The primary objectives of this study were to evaluate the safety and efficacy of SAFET BCMA/CD19 BiCART. The pharmacokinetics of SAFET BCMA/CD19 BiCART was investigated by quantitative PCR based detection of CAR vector copies in peripheral blood. Minimal residual disease (MRD) negativity was assessed by standardized multicolor flow cytometry analysis of bone marrow aspirate. Results: 10/11 R/R MM patients achieved CR and 1 achieved VGPR. Among the 10 CR patients, 7 remained CR after treatment with a median PFS 858 days; 1 patient showed relapse at Days 215; 2 patients showed MRD positive relapse at Days 637 and 733, and then received KRd and/or Kd treatment to achieve SD and MRD negative CR. Interestingly, the remarkable short and long term remission suggested that CART self-inhibiting IFNG signaling did not impair the CART clinical efficacy. Notably, minimal CRS was observed, including 6 grade 0, 2 grade 1, 2 grade 2, and 1 grade 3 who displayed mild transient hypotension for only 1 day while there were 79.5% blast tumor cells in the bone marrow before CART therapy. Conclusions: These results suggest BCMA-CD19 bispecific CART self-inhibiting IFNG signaling is promising in translation to a best-in-class treatment for MM. Clinical trial information: ChiCTR2000032124.

369 Investigating the impact of bariatric surgery on metabolic mechanisms that promote obesity-associated inflammation in subjects with and without Type 2 Diabetes

OBJECTIVES/GOALS: This project will provide novel insights into mechanism(s) by which differences in inflammation develop &amp; resolve, or fail to resolve, in metabolically different groups of bariatric surgery patients determined by Type 2 Diabetes status. My work may uncover unique differences between cohorts, encouraging development of personalized medicine. METHODS/STUDY POPULATION: I analyzed human blood samples collected before and 3, 6, &amp; 12 months after bariatric surgery at the University of Kentucky through an established tissue bank. Subjects hadnormal glucose tolerance, pre-diabetes, or Type 2 Diabetes, based on HbA1c%. Iisolated peripheral blood mono nuclear cells &amp; will comparecytokine profiles among cohorts across all time points. I will define &amp; perturbmetabolic differencesin immune cells among cohorts &amp; across time via isotope tracing, fuel source limitation, and metabolite inhibition. This will determine causal relationships between cytokine profiles &amp; immune cell metabolism. RESULTS/ANTICIPATED RESULTS: I anticipatecytokine profiles, a functional output of immune cells, will differ among cohorts pre-surgery, and that this difference will diminish post-surgery. Differencesmay be insignificant by the 12 month time point. I also anticipate differences in fuel usage and metabolite productionin immune cells among cohorts pre-surgery, and that these differences only partially resolve post-surgery to poise immune cells for continued chronic inflammatory action. I hypothesize that T2D status has a lasting impact on immune cell function and fuel usage patterns, and will continue to supportchronic inflammation following short term T2D remission and longer-term weight loss. DISCUSSION/SIGNIFICANCE: There has been an alarming increase in obesity and its comorbidities over recent decades, and inflammation is a known supporter of T2D. The anticipated rewiring of immune cell metabolism post-surgery, if incomplete, may poise subjects forweight regain and T2D recurrence.

Investigator choice of standard therapy versus sequential novel therapy arms in the treatment of relapsed follicular lymphoma (REFRACT): study protocol for a multi-centre, open-label, randomised, phase II platform trial

BackgroundRelapsed or refractory follicular lymphoma (rrFL) is an incurable disease associated with shorter remissions and survival after each line of standard therapy. Many promising novel, chemotherapy-free therapies are in development, but few are licensed as their role in current treatment pathways is poorly defined.MethodsThe REFRACT trial is an investigator-initiated, UK National Cancer Research Institute, open-label, multi-centre, randomised phase II platform trial aimed at accelerating clinical development of novel therapies by addressing evidence gaps. The first of the three sequential novel therapy arms is epcoritamab plus lenalidomide, to be compared with investigator choice standard therapy (ICT). Patients aged 18 years or older with biopsy proven relapsed or refractory CD20 positive, grade 1-3a follicular lymphoma and assessable disease by PET-CT are eligible. The primary outcome is complete metabolic response by PET-CT at 24 weeks using the Deauville 5-point scale and Lugano 2014 criteria. Secondary outcomes include overall metabolic response, progression-free survival, overall survival, duration of response, and quality of life assessed by EQ-5D-5 L and FACT-Lym. The trial employs an innovative Bayesian design with a target sample size of 284 patients: 95 in the ICT arm and 189 in the novel therapy arms.DiscussionWhilst there are many promising novel drugs in early clinical development for rrFL, understanding the relative efficacy and safety of these agents, and their place in modern treatment pathways, is limited by a lack of randomised trials and dearth of published outcomes for standard regimens to act as historic controls. Therefore, the aim of REFRACT is to provide an efficient platform to evaluate novel agents against standard therapies for rrFL. The adaptive Bayesian power prior methodology design will minimise patient numbers and accelerate trial delivery.Trial registrationClinicalTrials.gov: NCT05848765; 08-May-2023.EudraCT2022-000677-75; 10-Feb-2022.

High-multiplex single-cell imaging analysis reveals tumor immune contexture associated with clinical outcomes after CAR T cell therapy

Chimeric antigen receptor (CAR) T-cell therapy has made great progress in treating lymphoma, yet patient outcomes still vary greatly. The lymphoma microenvironment may be an important factor in the efficacy of CAR T therapy. In this study, we designed a highly multiplexed imaging mass cytometry (IMC) panel to simultaneously quantify 31 biomarkers from 13 patients with relapsed/refractory DLBCL who received CAR19/22 T-cell therapy. A total of twenty sections were sampled before CAR T-cell infusion or after infusion when relapse occurred. 35 cell clusters were identified, annotated and subsequently redefined into 10 metaclusters. The CD4+ T-cell fraction was positively associated with remission duration. Significantly higher Ki67, CD57, and TIM3 levels and lower CD69 levels on T cells, especially the CD8+/CD4+ Tem and Te cell subsets, were seen in patients with poor outcomes. Cellular neighborhood containing more immune cells was associated with longer remission. Fibroblasts and vascular endothelial cells resided much closer to tumor cells in patients with poor response and short remission after CAR T therapy. Our work comprehensively and systematically dissects the relationship between cell composition, state, and spatial arrangement in the DLBCL microenvironment and the outcomes of CAR T-cell therapy, which is beneficial to predict CAR T therapy efficacy.

Comparative pharmacogenetic study of disulfiram or cyanamide efficacy for alcohol dependence: the key role of dopamine neurotransmission gene polymorphisms

The actual direction of increasing the efficacy of alcohol dependence (AD) treatment is the search for opportunities for individualization of therapy using pharmacogenetic markers to stratify patients in order to select the most optimal therapeutic tactics.Aims. To test an associations of possible pharmacogenetic markers with indicators of the efficacy of disulfiram and cyanamide to stabilize remission in patients with AD.Materials and methods. A pharmacogenetic study was conducted on the basis of a double-blind, randomized, comparative, placebo-controlled clinical study of the efficacy and tolerability of disulfiram and cyanamide in the treatment of alcohol dependence syndrome. The main outcome: the duration of retention of patients in the treatment program (in remission), and withdrawal from the treatment program for any reason was considered a negative outcome. Secondary outcomes: time to relapse to alcohol use and time to recurrence to AD. 150 patients with AD (ICD-10 criteria) (av. age - 40.65±1.09 y.o., 19.3% females) were randomly assigned to one of three treatment groups (50 subjects in each): Disulfiram, Cyanamid and Placebo. All patients had weekly (12 weeks) visits to research clinic for brief counselling session. The genetic panel of the study consisted of 15 polymorphic loci in 9 genes: dopamine receptors 2 (DRD2) and 4 (DRD4) types, transmembrane dopamine transporter (DAT), enzymes dopamine-beta-hydroxylase (DBH) and catechol-ortho-methyl-transferase, as well as a two polymorphisms in the genes of the endogenous opioid system and the aldehyde dehydrogenase enzyme gene cluster.Results. For disulfiram, the DBH rs1108580 is associated with a longer remission (p=0.053, trend), and DRD4 VNTR 48 bp is associated with a shorter remission (p=0.006). For cyanamide, DAT VNTR 40 bp was associated with shorter remission (p=0.006) and rapid recurrence to AD (p=0.045). DAT rs27072 has an effect simultaneously in two treatment groups, while the direction of the effect is opposite. For cyanamide, the marker is slightly associated with a longer remission (p = 0.082, trend), a longer time to relapse (p = 0.063, trend) and a longer time to recurrence to AD (p = 0.083, trend). For placebo, DAT rs27072, on the contrary, is associated with a shorter time to to recurrence to AD (p = 0.066, trend). For placebo, DRD2 rs1799732 was associated with a shorter remission (p = 0.001), a shorter time to relapse (p = 0.018), and a shorter time to recurrence to AD (p = 0.001).Conclusion. Preliminary pharmacogenetic markers of the efficacy of alcohol dependence treatment have been identified in genes that control dopaminergic neurotransmission. After independent validation, the obtained genetic markers may be used for pharmacogenetic stratification of patients in order to select the optimal treatment options for alcohol dependence.

Enhanced potency of immunotherapy against B-cell precursor acute lymphoblastic leukemia by combination of an Fc-engineered CD19 antibody and CD47 blockade.

CD19-directed immunotherapy has become a cornerstone in the therapy of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). CD19-directed cellular and antibody-based therapeutics have entered therapy of primary and relapsed disease and contributed to improved outcomes in relapsed disease and lower therapy toxicity. However, efficacy remains limited in many cases due to a lack of therapy response, short remission phases, or antigen escape. Here, BCP-ALL cell lines, patient-derived xenograft (PDX) samples, human macrophages, and an in vivo transplantation model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were used to examine the therapeutic potency of a CD19 antibody Fc-engineered for improved effector cell recruitment (CD19-DE) and antibody-dependent cellular phagocytosis (ADCP), in combination with a novel modified CD47 antibody (Hu5F9-IgG2σ). For the in vivo model, only samples refractory to CD19-DE monotherapy were chosen. Hu5F9-IgG2σ enhanced ADCP by CD19-DE in various BCP-ALL cell line models with varying CD19 surface expression and cytogenetic backgrounds, two of which contained the KMT2A-AFF1 fusion. Also, the antibody combination was efficient in inducing ADCP by human macrophages in pediatric PDX samples with and adult samples with and without KMT2A-rearrangement in vitro. In a randomized phase 2-like PDX trial using seven KMT2A-rearranged BCP-ALL samples in NSG mice, the CD19/CD47 antibody combination proved highly efficient. Our findings support that the efficacy of Fc-engineered CD19 antibodies may be substantially enhanced by a combination with CD47 blockade. This suggests that the combination may be a promising therapy option for BCP-ALL, especially in relapsed patients and/or patients refractory to CD19-directed therapy.

MEK-kinase Inhibitor as a treatment option for hairy cell leukemia

Introduction. Hairy cell leukemia (HCL) is a chronic indolent B–cell lymphoproliferative disease with a good response to treatment in most cases. However, despite successful treatment, there remains a group of patients with a resistant/recurrent course of the disease, a short remission after treatment, and with contraindications to standard therapy. The use of the RASRAF- MEK-ERK pathway inhibitor in the V600E marker mutation of the BRAF gene may prove to be an effective treatment option for complicated/resistant cases of HCL.Aim — to present the results of the use MEK-kinase inhibitor trametinib in the treatment of HCL.Main findings. The MEK-kinase inhibitor trametinib was used at a dose of 1 mg/day for 3 months in three patients. In two patients trametinib was used as preliminary stage before the main course of treatment with cladribine. In one patient with a resistant/relapsing course of HCL, trametinib monotherapy made it possible to achieve a good partial remission, and the treatment continues without a decrease in the quality of life.Conclusion. Trametinib can be used as a preliminary stage before analogous purine treatment in patients with HCL without the BRAF V600E mutation, in a case of deep neutropenia or infectious complications, and as the main antitumor therapy in patients with resistant/recurrent HCL. Trametinib is effective in the absence of the MAP2K1 mutations. Trametinib monotherapy can be effective at a reduced dosage (1 mg/day or 1 mg every other day).

Poor Outcome of Patients with Acute Myeloid Leukemia in First Relapse Underscores the Need for Effective Initial Treatment

Introduction: The incorporation of novel targeted agents into combination regimens used in the treatment of patients with acute myeloid leukemia (AML) has improved their outcomes. However, disease relapse is still challenging and associated with a poor response rate, limited therapeutic options and poor outcome. Predictors of outcome for patients in first relapse include the duration of first remission, cytogenetic risk at diagnosis, age and whether they underwent an allogeneic stem cell transplantation (alloSCT) in first remission. The incorporation of genetic aberrations detectable at diagnosis has been useful in further stratifying prognosis, but their predictive value at the time of relapse is not well established. Herein we analyze a large cohort of frontline-treated AML patients, focusing on the outcomes of patients presenting a first disease relapse. Methods: This is a retrospective single-center study including patients diagnosed with AML from April 2017 to October 2022. Risk assessment and response to treatment were defined according to the ELN 2022 guidelines. Mutations were assessed using an 81-gene NGS panel. Survival analyses were estimated with the Kaplan-Meier method, using the log-rank test for comparison. Univariate and multivariate analysis were performed using Cox proportional hazards regression. Results: A total of 875 patients with AML were included in the study, with a median age of 65 years old (range,18-95). According to the ELN risk category, 171 (20%) patients had favorable disease, 200 (23%) patients intermediate and 473 (54%) adverse. Patients received intensive treatment (IT; 40% of patients, 41% of whom with the addition of venetoclax) or low intensity treatments (LIT; 60% of patients, 72% with venetoclax). The overall response rate including complete remission (CR)/CR with incomplete hematologic recovery (CRi) was 73%. Among responding patients, 201 (32%) underwent alloSCT in first remission. After a median follow-up of 25 months, the median OS was 53 months for patients receiving IT and 11 months for patients receiving LIT (Figure 1). The median OS for favorable, intermediate and adverse risk patients was not achieved, 24 and 11 months, respectively. During this follow up time, 337 patients were alive and maintaining CR. 197 patients have relapsed after having achieved CR/CRi, 36 of them after undergoing alloSCT. Available data for analysis was available in 164 of these patients. The median age at relapse was 67 (21-95) and 57 (35%) received prior IT whereas 107 (65%) received LIT. At diagnosis, 15%, 16% and 69% were favorable, intermediate and adverse risk, respectively. The median time from best response to relapse was 7.5 (range,1-35) and 6.3 (range, 1-49) months for patients treated with IT and LIT, respectively. At the time of relapse, 36% of patients had a complex karyotype, 4% had MECOM gene rearrangement and 3% had KMT2A gene rearrangement. The most common mutations at time of relapse were TP53 (34%), DNMT3A (34%), TET2 (23%) and RUNX1 (19%). After relapse, 32 (20%) and 132 (80%) received salvage IT and LIT treatment, respectively. The CR/CRi rate for IT and LIT were 31% and 27%, respectively. The mOS from relapse was 3.4 and 5.5 months for patients with salvage IT and LIT, respectively (p=0.6). Age at relapse (mOS of 6.5 vs 5.1 months for patients age &amp;lt;60 and ≥60, respectively [p=0.1]), type of induction therapy (mOS of 6.6 vs 4.9 for patients receiving IT and LIT, respectively [p=0.07]), or risk classification at relapse (mOS of 4.9, 6.3 and 5.1 months for favorable, intermediate and adverse risk, respectively [p=0.7]) did not stratify patients. Duration of first remission longer than 12 months was associated with a better OS (mOS 8.1 vs 4.3 months, p=0.008). Univariate and multivariate analysis was performed, including clinical and biological variables at diagnosis and relapse (Table 1). Importantly, high white blood cell count at relapse (HR 2.1), short first remission (HR 1.6), complex karyotype at relapse (HR 1.7) and KMT2A rearrangement at relapse (HR 3.8) were independent predictors of outcome after relapse. Conclusion: Although outcomes of patients with AML have improved, disease relapse remains a major obstacle with poor outcomes despite the availability of new salvage strategies. Highly effective induction and maintenance regimens are needed to reduce the risk of relapse.

Phenotypic Analysis of Baseline T cell Subsets and Third Generation CD20 Targeted CAR T cell Products for Treatment of Relapsed/Refractory Follicular Lymphoma

Background: Chimeric antigen receptor (CAR) T cell therapy achieves high response rates in the treatment of follicular lymphoma (FL), with two FDA-approved therapies to date. However, some patients (pts) fail to achieve a complete response and others experience early relapse, and the reasons for this are unclear. One potential explanation for treatment failure is poor persistence or exhaustion of CAR T cells. Indeed, in other settings of CAR T cell (CAR-T) therapy, phenotypic differences in the CAR-T product and even pre-manufacturing T cells can predict duration of anti-tumor CAR-T function and antigen-responsive cytotoxicity. Methods: We previously reported promising efficacy and safety in a cohort of pts with relapsed/refractory FL treated with a fully-human 3 rd generation CD20-targeted CAR (Shadman et al., ICML 2023). In pt samples from this trial, we quantified CD4 + and CD8 + T cell subsets in screening blood samples at study entry (n=20), based on CD45RA, CD45RO, CCR7, CD62L, CD27, and/or CD95 expression (naïve [T N], central memory [T CM], effector memory, stem cell memory, effector, etc.) We also analyzed various combinations of markers from exhaustion (PD-1, Tim-3, Lag-3, CD39, and CD57) and memory (CD45RA, CD45RO, CCR7, CD62L, and CD127) panels in CAR-T products (measured 1 day prior to infusion) (n=19; 1 pt had insufficient cells in the infusion product [IP] to analyze). These were recorded as both percent expression (of total CD4 + or CD8 + cells) compared to isotype controls and calculated absolute cell counts per µl at apheresis or in IP. We performed a univariate analysis of hypothesis-driven biomarkers (7 at baseline, 10 in IP) (Table 1) capturing combinations of T N / T CM markers (screening and IP) and absence of exhaustion markers (IP only) with endpoints of complete remission (CR) at 6 months, progression-free survival (PFS), CAR-T expansion (area under the curve [AUC] and peak), and cytokine release syndrome (CRS) (no pts developed neurologic toxicity). Due to the small sample size, we performed multivariable analysis using a novel high dimensional inference (HDI) approach to rank potential biomarkers of interest with primary endpoint CR at 6 months and secondary endpoint CAR-T cell AUC. HDI provides “de-biased” estimates for coefficients, confidence intervals, and p-values without refitting, which is easier to interpret. Biomarker combinations comprising &amp;lt;1% cells were excluded. Results: We did not find any significant association between baseline or IP hypothesis-driven markers and CR at 6 months or CRS in univariate analysis. We noted a possible association with baseline absolute counts of CD45RO +CD62L +CD8 + cells for PFS; however, this was largely driven by an outlier pt with a short remission due to CD20-negative relapse. Baseline % of CD45RA +CD62L + in CD8 + cells was associated with higher peak CAR-T expansion, and infused absolute counts of CD8 + CAR T cells with CD45RA +CD62L +, CD45RO +CD62L +, CD39 -PD-1 -, or Lag3 -PD-1 - phenotypes were found to be associated with both higher CAR-T AUC and peak CAR-T expansion. The multivariable HDI analysis revealed a rank list of markers of interest associated with CR at 6 months and CAR-T AUC, with the top 5 combinations for each association shown in Table 2. Conclusion: We found that higher infused numbers of CD8 + CAR T cells with T N or T CM phenotypes, or low expression of certain exhaustion phenotype marker combinations, were associated with improved CAR-T expansion. We did not find any notable phenotypic signatures at baseline or in the IP that were predictive of prolonged CR, PFS, or toxicity, though the analysis was limited by a relatively small sample size. In FL, where a response to CAR-T therapy is almost universal, pre-manufacture and IP T cell phenotypes appear to have a lower importance for achieving an initial response than what has been reported in other settings with lower response rates (chronic lymphocytic leukemia, large B cell lymphoma). Our lack of clear association between T cell phenotypes and prolonged CR or PFS suggests that later treatment failures in FL may be driven primarily by factors other than T cell fitness, such as tumor biology. However, our exploratory multivariable analysis did identify some potential phenotypic signatures that may prove to be predictive with further validation, and future studies incorporating duration of B cell aplasia may show that phenotypes are important for functional CAR T cell persistence.

High-Dose Methotrexate, Ibrutinib and Temozolomide (MIT) for the Treatment of Newly Diagnosed Primary Central Nervous System Lymphoma: A Multi-Center Prospective Phase II Study

Abstract Background: Primary central nervous system lymphoma (PCNSL) is a rare and aggressive subtype of B-cell lymphoma with poor survival. Genetic alterations often occur in the chronic active B-cell receptor signaling which mediate the response to BTK inhibition (BTKi) in PCNSL. However, the efficacy of traditional high-dose methotrexate-based regimen as a first-line treatment was suboptimal with a short effective remission time and low response rate. Therefore, we initiated this phase II trial to investigate the efficacy of novel induction combination of high-dose methotrexate (HD-MTX) plus ibrutinib and temozolomide (MIT), in the treatment of newly diagnosed PCNSL for the first time (ClinicalTrials.gov identifier: NCT04514393). Methods: Patients aged 18-75 years, newly diagnosed PCNSL and at least one measurable or evaluable lesion were enrolled. The MIT induction treatment included six cycles of HD-MTX (3.5 g/m 2, every three weeks), ibrutinib (560 mg/d, after HD-MTX clearance), and temozolomide (150 mg/m2 d1-d5, every three weeks). After the completion of the six cycles of MIT induction treatment, ASCT consolidation (only for patients &amp;lt; 65 years) or daily ibrutinib maintenance (560 mg/d) were administered up to two years, or until disease progression, intolerable toxicity, or death. Treatment response was evaluated by brain MRI and FDG-PET and/or cerebrospinal fluid (CSF) examination every two cycles of MIT treatment. The primary objective of this study was to evaluate the overall response rate (ORR) of MIT induction treatment. The safety profile of MIT was also investigated. At the same time, next generation sequencing (NGS) of baseline tissues and dynamic monitoring with CSF/ plasma ctDNA were performed by Nanjing Geneseeq Technology Inc. Results: Between July 2021 and October 2022, thirty-three patients were enrolled in this open-label, multi-center phase II prospective clinical study. The median age was 56 years (range: 27-75) and 17 of them were male. Eye involvement occurred in 5 patients (15%). Twenty-five patients (76%) had multiple cerebral lesions. The best ORR was 94% and complete response rate was 73%. The median follow-up was 17.5 months (range: 5.8-21.3 months) for survivors. Twenty-three patients underwent ibrutinib maintenance after 6 cycles of induction therapy. The swimmer plot of the 33 patients was shown in Figure 1. The 1-year progression-free survival was 66.4% (95%CI: 58.1-74.7%) and overall survival was 90.5% (95%CI: 85.2-95.8%) (Figure 2). Treatment-related toxicities were acceptable, with grade 3-4 neutropenia occurring in 9%, thrombocytopenia in 6%, subdural hematoma (SH) in 6%, and atrial fibrillation in 3%. The 2 patients with SH recovered following discontinuation of ibrutinib. No fungal infections and treatment-related deaths were observed. NGS of Tissue/CSF was tested in 30 patients, 19 (63%) of whom were classified as MCD subtype, 2 (7%) as BN2 subtype. The mutational profiles of baseline tissue and/or CSF samples revealed that PIM1 and MYD88 mutations were present in 75.9% and 65.5% of the entire cohort followed by BTG2 (55.2%) and CD79B (51.7%). Patients with dynamic CSF ctDNA monitoring during MIT treatment were analyzed (Figure 1). The consistence of ctDNA clearance in CSF/plasma samples and imaging complete remission was observed. Patients with clearance of ctDNA in the CSF after 2 cycles of MIT treatment achieved better PFS. Conclusions: The novel induction treatment of MIT achieved encouraging responses in newly diagnosed PCNSL patients with acceptable toxicities. The early clearance of ctDNA in CSF may be related to favorable survival. Disclosures No relevant conflicts of interest to declare.

Phase 1 Trial of Orelabrutinib in Combination with Rituximab, Methotrexate, and Dexamethasone in Patients with Newly Diagnosed Primary CNS Lymphoma Implementing Bayesian Design for Dose-Seeking

Background: Primary central nervous system lymphoma (PCNSL) is an aggressive extranodal subtype of non-Hodgkin's lymphoma (NHL). High-dose methotrexate (HD-MTX)-based regimens are the first-line treatment for PCNSL, but remain suboptimal due to response instability and short effective remission duration. Orelabrutinib is a novel, potent, highly selective BTK inhibitor with a high CSF concentration. This study aims to investigate optimal combination dose of Orelabrutinib in combination with Rituximab, Methotrexate, and Dexamethasone (ORMD) as well as safety and efficacy of the regimen. Methods: This was a phase 1, investigator-initiated, dose-escalation study of ORMD, being conducted at Huashan Hospital, Fudan University (NCT05036577). Eligible patients must have histopathologically confirmed PCNSL-DLBCL by biopsy of brain lesions and be aged 18-75 years with adequate organ functions. Patients were treated for 6-8 cycles of induction therapy with 21 days per cycle, receiving rituximab (375mg/m 2 on day 1), dexamethasone (10-15mg on d1-d4), MTX (d2, 3.5g/m 2 or 5g/m 2), and orelabrutinib (once daily, after MTX clearance, 150mg/d, or 200mg/d), followed by orelabrutinib maintenance up to one year among CR/CRu patients. The primary objective was to determine the maximum tolerated dose (MTD) of the combination of orelabrutinib and MTX with R and D and investigate the safety and tolerability of this regimen using Bayesian Optimal Interval (BOIN) waterfall design to determine rule of dose escalation and movement among dose combination matrix to identify MTD contour. DLT was defined by the occurrence of severe toxicities during the first cycle: any grade 4 hematologic toxicity, grade 3 febrile neutropenia and grade 3 thrombocytopenia with hemorrhage, or any grade 3 non-hematologic toxicity that failed to respond to supportive therapy and possibly related to orelabrutinib and/or MTX (assessed according to NCI CTCAE V5.0). The secondary objectives included ORR/CR（u） and PFS/OS. Results: From October 2021 to July 2023, the study completed enrollment of all 13 patients. The median age was 60 years, and other basic characteristics are shown in Figure 1. All the patients had completed at least one cycle of ORMD treatment and were evaluable for DLT. The median cycle of ORMD was seven (range: 2.5-8). No DLT occurred among first 3 patients in cohort 1 (Orelabrutinib: 150mg/MTX: 3.5g/m 2) and dose escalation was allowed to cohort 2 (Orelabrutinib: 200mg/MTX: 3.5g/m 2). In cohort 2, 5 patients were dosed in total, and Gr 3 lung infection was observed in patient #4 during cycle 1, was deemed as a DLT. Another four patients were enrolled in the same cohort according to BOIN design algorithm and no DLT was identified, allowing to escalate to cohort 3 (Orelabrutinib:200mg/MTX:5g/m 2). In cohort 3, 5 patients were dosed in total, and Gr 3 elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was observed in patient #9 during cycle 1, was deemed as a DLT. The causality of this AE was certain to methotrexate due to folate metabolic gene polymorphism in the assessment of the investigator. The toxicity probability from 3 dose combo cohort is 0.13, 0.13 and 0.21, all of which is below predefined threshold 0.3. So MTD is not reached and 200mg+5g/m 2 dose combo remains to be RP2D candidate. No Gr 4 AE had been observed. The most common adverse events were lymphopenia (Gr 3 in 8 case), neutropenia(Gr 3 in 2 case), Covid-19(Gr 3 in 2 case) and hyperglycemia (Gr 3 in 2 case). No atrial fibrillation or bleeding was observed by the cut-off date. ORR and CR/CRu in 12 patients possessed at least one efficacy evaluation were both 83.3%, the PFS and OS rate were 56.6% (95% CI, 29.6% ,100%) and 100%(95% CI, 100% ,100%) at 1 year. Among nine patients completed ≥6 cycles of induction therapy, ORR and CR/CRu were both 100%. PFS and OS rate were 75.0% (95% CI, 42.6% ,100%) and 100%(95% CI, 100% ,100%) at 1 year. All patients in remission achieved CR/CRu, the high rate of deep remission may be related to the addition of orelabrutinib. Conclusions: As a novel therapeutic combination of induction therapy, ORMD therapy has shown deep remission potential as well as a predictable and manageable safety profile in patients with newly diagnosed PCNSL. MTD contour not reached. The combination of this induction therapy with consolidation therapies such as ASCT deserves further investigation and may provide greater survival benefit for patients.

The Impact of Soluble BCMA and BCMA Gain on Anti-BCMA Immunotherapies in Multiple Myeloma

B cell maturation antigen (BCMA) targeting chimeric antigen receptor T cells (CAR T) and bispecific T cell engagers (TCE) have remarkable efficacy in multiple myeloma (MM). While BCMA antigen escape, driven by TNFRSF17 deletions or mutations, is a mediator of anti-BCMA CAR T/ TCE resistance, the majority of patients retain BCMA surface expression at progression. Furthermore, patients with high disease burden and extramedullary disease associated with poorer response to anti-BCMA therapies, have high levels of soluble BCMA (sBCMA). The mechanisms mediating this resistance, and in particular the contribution of sBCMA, have not been fully elucidated. In order to assess the effect of sBCMA on anti-BCMA CAR T function, we measured CAR T cells activation with an in-house manufactured anti-BCMA CAR transduced with an NFAT-GFP reporter gene after co-culture with increasing concentrations of sBCMA (0-2500 ng/mL). sBCMA bound to CAR scFv in a dose dependent manner, effectively competing with the fluorophore tagged BCMA peptide binding. Despite their stable engagement, sBCMA failed to induce the expression of GFP, CD69, CD25, or 41BB on CAR T cells. To further examine the functional effect of sBCMA on CAR T cytotoxicity, we generated isogenic OPM2 cell lines with distinct BCMA expression profiles: OPM2 over-expressing BCMA (OPM2_BCMA high) generated by lentiviral transduction of TNFRSF17, and Cas9 BCMA knockout OPM2 (OPM2_BCMA -/-). OPM2_BCMA high cells exhibited one-log-fold increase in surface BCMA expression and 10-fold increase in sBCMA and were more resistant to CAR T mediated lysis. In addition, in co-culture assays, elevated sBCMA impaired OPM2 target cell lysis by anti-BCMA CAR T in a dose dependent-manner. To gain insight into the effect of chronic sBCMA exposure on CAR T, we exposed anti-BCMA CAR T to OPM2 cells secretome in the absence of direct cell to cell contact in a trans-well co-culture system. On day 24, CAR T in co-culture with OPM2_BCMA high (sBCMA levels of 2343 ng/mL) significantly contracted with no viable cells, while CAR T in culture with parental OPM2 (sBCMA level of 267 ng/mL) did persist with up-regulated TIM3 and TIGIT. In contrast CAR T cultured with OPM2_BCMA -/- remained viable with no expression of exhaustion markers. In examining the impact of sBCMA on TCE activity, sBCMA level as low as 50 ng/mL in vitro impaired anti-BCMA TCE binding to K562 cells transduced to stably express BCMA (K562_wtBCMA) . Consistent results were found in cytotoxicity assays wherein the addition of 2 ng/mL of sBCMA was sufficient to induce a reduction in anti-BCMA TCE mediated cytotoxicity of K562_wtBCMA cells by healthy donor peripheral blood mononuclear cells. To identify genomic events that lead to elevated sBCMA expression and subsequent resistance to CAR T/TCE, we conducted bulk whole genome sequencing (100X) on CD138+ bone marrow MM cells obtained from 40 MM patients treated with anti-BCMA CAR T/ TCE. Structural variants (SVs) at or adjacent to TNFRSF17 locus were identified in four patients. These included i) focal copy number gain of TNFRSF17 (3 copies) in a patient who progressed within 3 months post anti-BCMA TCE, ii) subclonal duplication of TNFRSF17 locus in another patient (n=1), as well as iii) focal copy number losses 5‘ or 3‘ to the TNFRSF17 gene locus (n=2). In addition to SVs affecting TNFRSF17 locus, we also identified mutations involving TNFRSF17 transcriptional regulators. Notably, MM clones at progression 4 months post anti-BCMA TCE (n=1) harboured copy number gains (3 copies) of POU2AF1 gene on chr11q23. POU2AF1 encodes a key transcriptional regulator of TNFRSF17 expression. Indeed, sBCMA level from this patient was significantly elevated at relapse at a level (638 ng/mL) correlated with resistance to anti-BCMA TCE. In addition, we identified translocation involving MAP3K14 encoding the NF-kB regulator NIK in a patient with short remission post anti-BCMA CAR T. Of interest, analysis of the CoMMpass dataset revealed significantly higher BCMA transcripts in patients with translocations involving MAP3K14 locus. Our findings highlight the impact of sBCMA levels and its chronicity of exposure on anti-BCMA TCE and CAR T activities in MM. We further identified structural genomic events driving TNFRSF17 over-expression and their correlation to sBCMA levels facilitating tumoral immunotherapeutic escape.

Time-Limited Therapy with Zanubrutinib Plus Rituximab in Treatment-Naïve Chronic Lymphocytic Leukemia: Early Results of an Ongoing Phase 2 Trial

Introduction: The Bruton tyrosine kinase inhibitor (BTKi) zanubrutinib has recently been approved for treatment of chronic lymphocytic leukemia (CLL). Zanubrutinib induces durable remissions in most patients with CLL, and treatment is typically given as continuous monotherapy, until disease progression or toxicities prohibit further use. We developed time-limited therapy with zanubrutinib in combination with rituximab as an alternative to long-term BTKi monotherapy. Here, we report early results from the first 17 treatment-naïve patients with CLL who enrolled in this ongoing phase 2 trial (NCT04458610) between July 2020 and April 2023 at MD Anderson Cancer Center. Patients and Study Design: Patients with treatment-naïve CLL requiring therapy per iwCLL criteria receive zanubrutinib 160 mg orally twice daily and rituximab (375 mg/m 2 intravenously) on days 1, 8, 15, 22 of cycle 1, and then on day 1 of cycles 2 - 6 (one cycle is 28 days). Patients who achieve a complete remission (CR) by iwCLL 2018 criteria after 6 cycles of combination therapy continue with zanubrutinib alone for another 6 cycles. Patients who achieve a partial remission (PR) or stable disease (SD) after 6 cycles continue for another 6 cycles of the combination. Patients who achieve a CR after 12 cycles will receive 6 additional cycles of zanubrutinib alone, and then stop treatment. Patients who achieve a PR or SD after 12 cycles will continue zanubrutinib alone for 12 cycles and the stop therapy after a total of 24 cycles. After this time-limited therapy, patients transition to observation and can resume therapy if they relapse and fulfill iwCLL criteria for salvage therapy. The primary objective of the trial is to determine the proportion of patients who have treatment-free remission 6 months after discontinuation of zanubrutinib. Secondary objectives are to determine the length of treatment-free remission, identify clinical factors associated with long versus short remissions, and to evaluate the efficacy of re-treatment with zanubrutinib plus rituximab in patients who relapse. Results: The median age of the patients was 68 years (range, 54 - 77 years), 47% had unmutated IgHV and 41% advance stage disease (RAI stage III and IV). The median baseline absolute lymphocyte count (ALC) and β2 microglobulin were 51.9x10 9/L (range, 1.7 - 231.0x10 9/L) and 3.9 mg/L (range, 2.5 - 6.8 mg/L), respectively. After a median follow-up of 13 months (range, 0.5 - 29 months), all patients remain on study, and the estimated one-year progression-free (PFS) and overall survival (OS) is 100%. Seven patients completed therapy (1 patient after 12 cycles, 6 patients after 24 cycles) and have moved on to observation (median observation time post-therapy 4.5 months). So far, we have not observed any progression events after treatment discontinuation. Overall response rate is 100%, with 9% CR and 91% PR after 6 months (n=11), 11% CR and 89% PR after 12 months (n=9), and 33% CR and 67% PR after 24 months of therapy (n=6). The median levels of bone marrow involvement by CLL cells, quantified by flow cytometry, declined from 81.6% at baseline (range, 25.8 - 93.2%, n=16) to 20.3% after 6 cycles (range, 0.07 - 82%, n=11, p&amp;lt;0.0001), to 12.1% after 12 cycles (range, 0.04 - 55.60%, n=9, p&amp;lt;0.0001), and to 7.23% after 24 cycles (range, 0.2 - 41.4%, n=6, p=0.001). Grade ≥3 adverse events (AE) were observed in 9 patients (52.9%), which included hypertension (n=4) and decreased neutrophil counts (n=3). The most frequent grade 1-2 AE were decreased neutrophil (59%) and platelet counts (47%), bruising (41%), chills, increased creatinine, arterial hypertension, nausea (35% each), fatigue, and headache (29% each). No atrial fibrillation events were observed. Conclusion: This early analysis indicates that time-limited therapy with zanubrutinib and rituximab is well tolerated and induces remissions with a major reduction in bone marrow disease burden in patients with treatment-naïve CLL. The first 7 patients who stopped therapy so far have not progressed, but longer follow-up in more patients is needed to determine the durability of responses after therapy discontinuation, and how those compare to more intensive CLL combination regimens.