Chronic Lymphocytic Leukemia Progression Research Articles

Early reappearance of intraclonal proliferative subpopulations in ibrutinib-resistant chronic lymphocytic leukemia.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib represents an effective strategy for treatment of chronic lymphocytic leukemia (CLL), nevertheless about 30% of patients eventually undergo disease progression. Here we investigated by flow cytometry the long-term modulation of the CLL CXCR4dim/CD5bright proliferative fraction (PF), its correlation with therapeutic outcome and emergence of ibrutinib resistance. By longitudinal tracking, the PF, initially suppressed by ibrutinib, reappeared upon early disease progression, without association with lymphocyte count or serum beta-2-microglobulin. Somatic mutations of BTK/PLCG2, detected in 57% of progressing cases, were significantly enriched in PF with a 3-fold greater allele frequency than the non-PF fraction, suggesting a BTK/PLCG2-mutated reservoir resident within the proliferative compartments. PF increase was also present in BTK/PLCG2-unmutated cases at progression, indicating that PF evaluation could represent a marker of CLL progression under ibrutinib. Furthermore, we evidence different transcriptomic profiles of PF at progression in cases with or without BTK/PLCG2 mutations, suggestive of a reactivation of B-cell receptor signaling or the emergence of bypass signaling through MYC and/or Toll-Like-Receptor-9. Clinically, longitudinal monitoring of the CXCR4dim/CD5bright PF by flow cytometry may provide a simple tool helping to intercept CLL progression under ibrutinib therapy.

#2991 Two cases of chronic lymphocytic leukemia associated immunotactoid glomerulopathy: variable clinical presentation, close hematological collaboration

Abstract Background and Aims Immunotactoid glomerulopathy (ITG) is a very rare disease, observable in less than 0.1% of native kidney biopsy samples. It is most often associated with hematological diseases. We confirmed ITG in two cases among our 850 kidney biopsy patients in the past 9 years. In both patients, chronic lymphocytic leukemia (CLL) was the underlying cause. Method Case 1, a 56-year-old man was admitted due to severely increased proteinuria (2 g/day) with preserved renal function, while case 2, a 57-year-old-man was referred with rapidly decreasing eGFR (20 ml/min/1.73m2) with nephrotic syndrome (serum albumin level was 24 g/l). In case 1, no previous history of hematological disease was known, in case 2, CLL - that did not require treatment - was diagnosed half a year before admission. He still had no kidney involvement even 3 months after the CLL diagnosis. We performed kidney biopsy. Light microscopy revealed mesangial proliferative-atypical membranous pattern in case 1, and membranoproliferative pattern in case 2. Immunofluorescence microscopy showed IgG1-lambda deposition, electron microscopy confirmed the organised deposits, microtubular structures with a diameter of 44 nanometers in both cases. Neither monoclonal gammopathy nor systemic immune disease was confirmed. In case 2, low titer ANA positivity, low C3 and high anti-C1q antibody titer was detected, lupus nephritis was ruled out. Previously, lymphocyte count was slightly increased in case 1, CLL was confirmed after kidney biopsy. The immunoglobulin heavy chain variable region gene somatic hypermutation status was borderline in case 1, while in the other case it was unmutated. Fluorescein in situ hybridisation revealed trisomy 12 in both cases. In case 1, chemotherapy was not used due to Rai stage 0, and with appropriate antihypertensive treatment, we could preserve kidney function, and proteinuria was persistently low (below 1 g/day). In case 2, due to Rai stage 1, and progressive renal and lymph node involvement, immunochemotherapy was started (obinutuzumab-venetoclax). Three months later eGFR stabilized at 27 ml/min/1.73 m2, proteinuria decreased below 1 g/day, the C3 level normalized. Conclusion ITG can develop in the early stages of CLL, but it can also indicate CLL progression. The light microscopy histological pattern and, in parallel, the clinical appearance can be of varying severity. Immunochemotherapy resulting reduction of CLL tumor burden and cessation of complement damage, both play a role of improving renal status. Our cases also draw attention to the need for close collaboration with hematologists.

IDENTIFYING RISK FACTORS FOR TREATMENT FAILURE IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS RECEIVING BENDAMUSTINE±RITUXIMAB THERAPY

Introduction. The initial assessment of prognosis is of particular importance in determining the management of patients with B-cell chronic lymphocytic leukemia and the choice of specific therapy. The aim is to analyze the risk factors for failure to achieve a complete response to the bendamustine±rituximab treatment regimen in patients with B-cell chronic lymphocytic leukemia. Materials and methods. Twenty-two patients undergoing treatment for B-cell chronic lymphocytic leukemia (B-CLL) progression were included in the study. Response to treatment was assessed on the 56th day, categorized as complete, partial, or no response. Patients were divided into three groups based on their response: group I (n=6) included patients who showed a complete response after receiving bendamustine±rituximab; group II (n=11) included patients who achieved a partial response; and group III (n=5) consisted of patients with no response. Risk factors for chemotherapy resistance, as well as general and biochemical blood counts, were evaluated and subjected to statistical analysis. Results: Stage III (C) and IV (C) according to the Rai, Binet classification were recorded in 33.2% of patients with a complete response to chemotherapy, in 36.4% of patients with a partial response to chemotherapy, and in 60% of patients with no response to specific treatment. A decrease in hemoglobin level below 100 g/L was recorded only in patients with a partial response and no response, namely in 4 (36%) patients of group II and 1 (20%) patient of group III. On day 56, in patients of group II who received a partial response, there was a direct correlation between the level of leukocytes in the hemogram and the activity of aspartate aminotransferase (r=0.71; p=0.01) and the content of total bilirubin (r=0.63; p=0.03) in the biochemical blood test. After 2 courses of chemotherapy in patients of group III, a direct correlation of high strength between the level of leukocytes in the hemogram and the activity of aspartate aminotransferase (r=0.93; p=0.02) and alanine aminotransferase (r=0.93; p=0.02) in the biochemical blood test was found. The bendamustine±rituximab chemotherapy regimen has a high safety profile.

Mutational profile of previously treated patients with chronic lymphocytic leukemia progressing on acalabrutinib or ibrutinib

AbstractChronic lymphocytic leukemia (CLL) progression during Bruton tyrosine kinase (BTK) inhibitor treatment is typically characterized by emergent B-cell receptor pathway mutations. Using peripheral blood samples from patients with relapsed/refractory CLL in ELEVATE-RR (NCT02477696; median 2 prior therapies), we report clonal evolution data for patients progressing on acalabrutinib or ibrutinib (median follow-up, 41 months). Paired (baseline and progression) samples were available for 47 (excluding 1 Richter) acalabrutinib-treated and 30 (excluding 6 Richter) ibrutinib-treated patients. At progression, emergent BTK mutations were observed in 31 acalabrutinib-treated (66%) and 11 ibrutinib-treated patients (37%; median variant allele fraction [VAF], 16.1% vs 15.6%, respectively). BTK C481S mutations were most common in both groups; T474I (n = 9; 8 co-occurring with C481) and the novel E41V mutation within the pleckstrin homology domain of BTK (n = 1) occurred with acalabrutinib, whereas neither mutation occurred with ibrutinib. L528W and A428D comutations presented in 1 ibrutinib-treated patient. Preexisting TP53 mutations were present in 25 acalabrutinib-treated (53.2%) and 16 ibrutinib-treated patients (53.3%) at screening. Emergent TP53 mutations occurred with acalabrutinib and ibrutinib (13% vs 7%; median VAF, 6.0% vs 37.3%, respectively). Six acalabrutinib-treated patients and 1 ibrutinib-treated patient had emergent TP53/BTK comutations. Emergent PLCG2 mutations occurred in 3 acalabrutinib-treated (6%) and 6 ibrutinib-treated patients (20%). One acalabrutinib-treated patient and 4 ibrutinib-treated patients had emergent BTK/PLCG2 comutations. Although common BTK C481 mutations were observed with both treatments, patterns of mutation and comutation frequency, mutation VAF, and uncommon BTK variants varied with acalabrutinib (T474I and E41V) and ibrutinib (L528W and A428D) in this patient population. The trial was registered at www.ClinicalTrials.gov as #NCT02477696.

Lessons learned from the Eµ-TCL1 mouse model of CLL

The Eµ-TCL1 mouse model has been used for over 20 years to study the pathobiology of chronic lymphocytic leukemia (CLL) and for preclinical testing of novel therapies. A CLL-like disease develops with increasing age in these mice due to a B cell specific overexpression of human TCL1. The reliability of this model to mirror human CLL is controversially discussed, as none of the known driver mutations identified in patients are found in Eµ-TCL1 mice. It has to be acknowledged that this mouse model was key to develop targeted therapies that aim at inhibiting the constitutive B cell receptor (BCR) signaling, a main driver of CLL. Inhibitors of BCR signaling became standard-of-care for a large proportion of patients with CLL as they are highly effective. The Eµ-TCL1 model further advanced our understanding of CLL biology owed to studies that crossed this mouse line with various transgenic mouse models and demonstrated the relevance of CLL-cell intrinsic and -extrinsic drivers of disease. These studies were instrumental in showing the relevance of the tumor microenvironment in the lymphoid tissues for disease progression and immune escape in CLL. It became clear that CLL cells shape and rely on stromal and immune cells, and that immune suppressive mechanisms and T cell exhaustion contribute to CLL progression. Based on this knowledge, new immunotherapy strategies were clinically tested for CLL, but so far with disappointing results. As some of these therapies were effective in the Eµ-TCL1 mouse model, the question arose concerning the translatability of preclinical studies in these mice. The aim of this review is to summarize lessons we have learnt over the last decades by studying CLL-like disease in the Eµ-TCL1 mouse model. The article focuses on pitfalls and limitations of the model, as well as the gained knowledge and potential of using this model for the development of novel treatment strategies to achieve the goal of curing patients with CLL.

Mouse models of CLL: In vivo modeling of disease initiation, progression, and transformation to Richter transformation

Chronic lymphocytic leukemia (CLL) is a highly complex disease characterized by the proliferation of CD5+ B cells in lymphoid tissues. Current modern treatments have brought significant clinical benefits to CLL patients. However, there are still unmet needs. Patients relapse on Bruton's tyrosine kinase inhibitors and BCL2 inhibitors and often develop more aggressive diseases including Richter transformation (RT), an incurable complication of up to ∼10% patients. This evidence underscores the need for improved immunotherapies, combination treatment strategies, and predictive biomarkers. A mouse model that can recapitulate human CLL disease and certain components of the tumor immune microenvironment represents a promising preclinical tool for such purposes. In this review, we provide an overview of CRISPR-engineered and xenograft mouse models utilizing either cell lines, or primary CLL cells suitable for studies of key events driving the disease onset, progression and transformation of CLL. We also review how CRISPR/Cas9 established mouse models carrying loss-of-function lesions allow one to study key mutations driving disease progression. Finally, we discuss how next generation humanized mice might improve to generation of faithful xenograft mouse models of human CLL.

Abstract 3849: Frontline ibrutinib-based therapy enhances CART cell functions in patients with CLL: Analysis of the phase 3 randomized clinical trial ECOG-ACRIN E1912

Abstract Introduction: Despite the remarkable activity of CD19-directed chimeric antigen receptor T cell therapy (CART19), durable remissions remain low, especially in chronic lymphocytic leukemia (CLL). Ibrutinib is an oral irreversible inhibitor of Bruton’s tyrosine kinase and is a first line therapy for CLL. Preclinical and early clinical data indicate that ibrutinib plus CART19 may be synergistic through inhibition of IL2-inducible T cell kinase. To systematically study the effects of prior ibrutinib treatment on subsequent CART functions, we utilized blood samples from the pivotal clinical trial E1912 (NCT02048813). E1912 was a phase 3 randomized trial comparing treatment with ibrutinib/rituximab (IR) to fludarabine/cyclophosphamide/rituximab (FCR) in previously untreated patients with CLL, which led to the FDA approval of ibrutinib as a first-line therapy in CLL. Methods: We evaluated the ex vivo expansion and effector functions of T cells and CART19 generated from patients (n=12) with CLL progression following treatment with IR or FCR. We generated CART19 from longitudinal samples obtained at baseline (pretreatment), 6 months post-treatment, 12 months post-treatment, and disease progression. Isolated T cells were lentivirally transduced to express CD28- or 41BB-costimulated CAR19. To assess antigen specific killing and proliferation, untransduced T cells or CART19 were co-cultured at a 1:1 ratio with the CD19+ cell line, JeKo-1, as a surrogate for CLL cells. Results: Ex vivo T cell expansion increased from an average of 1.5-fold at baseline to 6.7-fold at 6 months, 11.6-fold at 12 months, and 16.9-fold at progression, following ibrutinib-based therapy (p = 0.02, 0.04, and 0.04, respectively). Improved T cell expansion was independent of disease burden (p = 0.89, 0.73, and 0.54 for expansion vs B cell count at 6 months, 12 months, and progression). T cell expansion significantly worsened following treatment with FCR-based therapy compared to ibrutinib-based therapy (0.6- vs 11.6-fold at 12 months and 0.9- vs 16.9-fold at progression; p = 0.031 and 0.039, respectively). Starting at 6 months post-IR, CART19 demonstrated improved antitumor activity and antigen-specific proliferation against JeKo-1. There was no significant difference in the expansion of 41BB- vs CD28-costimulated CART19 following ibrutinib treatment. However, 41BB-CART19 showed enhanced antigen-specific proliferation upon stimulation with JeKo-1 (p = 0.008, 0.02, and 0.02 at 6 months, 12 months, and progression). Conclusion: Our results indicate significant improvement in T cell expansion and CART19 cell functions in patients with CLL following frontline ibrutinib-based therapy, and worsening T cell functions after FCR treatment. Improvement in T cell functions is independent of malignant B cell eradication and is more significant in 41BB-costimulated CART19. Citation Format: Truc Ngoc Huynh, Victoria Wang, Tait Shanafelt, Elizabeth L. Siegler, Carli M. Stewart, Kun Yun, Long K. Mai, Olivia Sirpilla, James H. Girsch, Claudia Manriquez-Roman, Jennifer M. Feigin, Makena Rodriguez, Brooke L. Kimball, Hong Xia, Reona L. Sakemura, Ismail Can, Omar L. Gutierrez-Ruiz, Ekene J. Ogbodo, Erin E. Tapper, Lionel A. Fonkoua, Mehrdad T. Hefazi, Michael W. Ruff, Sameer Parikh, Neil E. Kay, Saad J. Kenderian. Frontline ibrutinib-based therapy enhances CART cell functions in patients with CLL: Analysis of the phase 3 randomized clinical trial ECOG-ACRIN E1912 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3849.

The Effect of Mind-Body Intervention on Lymphocyte Doubling Time and Treatment-Free Survival in Treatment-Naïve Chronic Lymphocytic Leukemia Patients

Introduction: Mind-body intervention (MBI) serves as a supportive aid in oncology. We hypothesized that MBI could impact the progression of chronic lymphocytic leukemia (CLL) in the “watch and wait” (w&w) phase. Methods: We conducted a non-randomized, prospective controlled study between the years 2020 and 2022 on 76 treatment-naïve CLL patients in the w&w phase. Thirty-seven patients were included in the intervention arm and received MBI, while 39 patients were included in the control group. The primary and secondary endpoints were prolongation of lymphocyte doubling time (LDT) and treatment-free survival (TFS). LDT was compared at 0, 180, 360, and 540 days using paired t tests. TFS was compared between the intervention and control groups using the log-rank test. Cox proportional hazards models were used to calculate the hazard ratio (HR) and 95% confidence interval (CI) for TFS in the intervention group compared to the control, stratified by the study covariates. Results: MBI prolonged LDT at all time points, including at day 360 (median of 2.47 years; CI 1.05–3.9; p = 0.001). TFS at 18 months was longer in the intervention group compared to the control group (HR 0.23; CI 0.06–0.79, p = 0.01). Conclusions: MBI was associated with prolonged LDT and TFS in patients with CLL in the w&w phase. These results provide a basis for a larger randomized controlled trial.

An unappreciated cell survival-independent role for BAFF initiating chronic lymphocytic leukemia.

Chronic Lymphocytic Leukemia (CLL) is characterized by the expansion of CD19+ CD5+ B cells but its origin remains debated. Mutated CLL may originate from post-germinal center B cells and unmutated CLL from CD5+ mature B cell precursors. Irrespective of precursor types, events initiating CLL remain unknown. The cytokines BAFF and APRIL each play a significant role in CLL cell survival and accumulation, but their involvement in disease initiation remains unclear. We generated novel CLL models lacking BAFF or APRIL. In vivo experiments were conducted to explore the impact of BAFF or APRIL loss on leukemia initiation, progression, and dissemination. Additionally, RNA-seq and quantitative real-time PCR were performed to unveil the transcriptomic signature influenced by BAFF in CLL. The direct role of BAFF in controlling the expression of tumor-promoting genes was further assessed in patient-derived primary CLL cells ex-vivo. Our findings demonstrate a crucial role for BAFF, but not APRIL, in the initiation and dissemination of CLL cells. In the absence of BAFF or its receptor BAFF-R, the TCL1 transgene only increases CLL cell numbers in the peritoneal cavity, without dissemination into the periphery. While BAFF binding to BAFF-R is dispensable for peritoneal CLL cell survival, it is necessary to activate a tumor-promoting gene program, potentially linked to CLL initiation and progression. This direct role of BAFF in controlling the expression of tumor-promoting genes was confirmed in patient-derived primary CLL cells ex-vivo. Our study, involving both mouse and human CLL cells, suggests that BAFF might initiate CLL through mechanisms independent of cell survival. Combining current CLL therapies with BAFF inhibition could offer a dual benefit by reducing peripheral tumor burden and suppressing transformed CLL cell output.

Эффективность ибрутиниба в первой линии терапии у пациентов из группы высокого риска и во второй, третьей линиях при резистентном течении хронического лимфоцитарного лейкоза

Risk stratification appears to be the most valid criterion in decision-making regarding optimal specific therapy in chronic lymphocytic leukemia (CLL). The CLL International Prognostic Index takes account of unfavorable cytogenetic abnormalities (del(17p)/del(11q) and/or TP53 gene mutations) as well as the mutation status of immunoglobulin heavy chain variable (IGHV) region genes. Unmutated V(H)-status is commonly associated with such prognostically unfavorable genetic markers as del(17p)/del(11q), trisomy 12, and TP53 mutation. The combination of unmutated V(H)-status with unfavorable karyotype abnormalities (del(17p)/del(11q)) negatively affects the prognosis and overall survival rate. Besides, in high-risk CLL, the efficacy of therapy is rather low, and the development of refractoriness is possible. Targeted therapy (Bruton tyrosine kinase inhibitors) both in first line and in resistant CLL considerably increases the probability of achieving long-term remission. The present paper provides the comparative analysis of clinical and hematological efficacy and tolerability of ibrutinib in first-line CLL therapy of high-risk patients as well as second- and third-line therapies of resistant CLL. Ibrutinib shows high efficacy and low toxicity. First-line ibrutinib treatment results in a faster response and effectively reduces the probability of CLL progression. Second- and third-line ibrutinib treatment allows to overcome CLL resistance without impairing patients’ quality of life.

Diverse B-cell tumors associated with t(14;19)(q32;q13)/IGH::BCL3 identified by G-banding and fluorescence in situ hybridization.

We characterized 5 B-cell tumors carrying t(14;19)(q32;q13) that creates the IGH::BCL3 fusion gene. The patients' ages ranged between 55 and 88 years. Two patients presented with progression or recurrence of B-cell chronic lymphocytic leukemia (B-CLL)/small lymphocytic lymphoma (SLL), two with diffuse large B-cell lymphoma (DLBCL) of non-germinal center B-like phenotype, and the remaining one with composite angioimmunoblastic T-cell lymphoma and Epstein-Barr virus-positive DLBCL. The presence of t(14;19)(q32;q13) was confirmed by fluorescence in situ hybridization (FISH), showing colocalization of 3' IGH and 3' BCL3 probes on der(14)t(14;19) and 5' BCL3 and 5' IGH probes on der(19)t(14;19). One B-CLL case had t(2;14)(p13;q32)/IGH::BCL11A, and 2 DLBCL cases had t(8;14)(q24;q32) or t(8;11;14)(q24;q11;q32), both of which generated IGH::MYC by FISH, and showed nuclear expression of MYC and BCL3 by immunohistochemistry. The IGH::BCL3 fusion gene was amplified by long-distance polymerase chain reaction in 2 B-CLL/SLL cases and the breakpoints occurred immediately 5' of BCL3 exon 1 and within the switch region associated with IGHA1. The 5 cases shared IGHV preferentially used in B-CLL cells, but the genes were unmutated in 2 B-CLL/SLL cases and significantly mutated in the remaining 3. B-cell tumors with t(14;19)(q32;q13) can be divided into B-CLL/SLL and DLBCL groups, and the anatomy of IGH::BCL3 in the latter may be different from that of the former.

Demyelinating Polyradiculoneuropathy in Chronic Lymphocytic Leukemia: A Case Report on BTKis versus Venetoclax-Rituximab

The dysregulation of the immune system in Chronic Lymphocytic Leukemia (CLL) often allows for the development of immune-mediated diseases. Among them, autoimmune cytopenias are the most common, but cases of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) have been reported. We herein report on a patient who developed a CIDP while undergoing ibrutinib treatment for CLL, prompting drug discontinuation. Steroid treatment and a rituximab course proved to be ineffective at obtaining long-term control of CIDP, but therapy with venetoclax and rituximab, which was started due to CLL progression, led to the progressive amelioration of the symptoms up to complete remission of the neurological disease.

PRMT1 Promotes Chronic Lymphocytic Leukemia Progression Via Modulating Methylation of MAST1

Introduction As a member of the protein arginine N-methyltransferase family, protein arginine methyltransferase 1 (PRMT1) plays a crucial regulatory role in the etiology of malignant neoplasms. PRMT1-mediated protein methylation promoted the maintenance of acute myeloid leukemia, which revealed the promising potency of PRMT1 inhibitors. Recent study suggested that dysregulation of PRMT5 promotes Richter's transformation in chronic lymphocytic leukemia (CLL). However, the effects of PRMT1 in the tumorigenesis and progression of CLL still remained ill-defined. Hence, the aim of this study was to investigate the clinical significance and mechanisms of PRMT1 underlying the development of CLL. Methods Peripheral blood samples were collected from 79 newly diagnosed CLL patients (47 males and 32 females; age range 39-85 years, median 63 years) in Shandong Provincial Hospital CLL (SPHCLL) cohort with informed consent. CRISPR-Cas9 technology was used to stably knockout PRMT1 in CLL cells. A label free quantitative proteomics analysis was implemented to reveal the protein methylation mediated by PRMT1. Assessment of cell viability, apoptosis and cell cycle were analyzed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. This study was approved by the Medical Ethics Committee of Shandong Provincial Hospital. Results This study examined the expression of PRMT1 in SPHCLL and GEO databases, and discovered the upregulation of PRMT1 mRNA in CLL cells. Aberrantly elevated expression of PRMT1 was observed in a cohort of newly diagnosed CLL patients than healthy donors in mRNA level (donors vs. CLL patients, 0.07±0.06 vs. 0.20 ±0.21, p=0.013). Furthermore, increased expression of PRMT1 was correlated with inferior prognosis in two long-term follow-up cohorts of CLL patients (HR=2.80, p=0.002, and HR=1.551, p=0.009). To elucidate the functional significance of PRMT1 in CLL, we established stable PRMT1 knockdown cells using lentiviral shRNAs and PRMT1 knockout cells using CRISPR/Cas9 technology. The suppression of PRMT1 remarkably inhibited cell proliferation, induced cell apoptosis and blocked cell cycle at G1/S phase in CLL cells. Furthermore, it was observed that C7280948, a selective inhibitor of PRMT1, resulted in the defective proliferation of CLL primary cells in a dose-dependent manner. We subsequently performed pre-clinical investigations of C7280948 in the orthotopic CLL xenograft murine model. Notably, the administration of C7280948 significantly diminished the leukemia burden and induced the lessened degree of splenomegaly in comparison to the control group (Fig. 1A). Moreover, a reduction in the population of CLL cells was detected in bone marrow and spleen of mice treated with C7280948. To decipher the role of PRMT1 in the pathogenesis of CLL, the label free quantitative proteomics analysis was conducted on PRMT1-deficent and control MEC1 cells. Knockdown of PRMT1 diminished asymmetric dimethylarginine of some proteins in CLL cells. In accordance with the results of proteomics analysis, microtubule associated serine/threonine kinase 1 (MAST1) was selected as a candidate target for further investigation. The methylation site of MAST1 protein was identified as R802 (Fig. 1B). Increased expression of MAST1 was correlated with inferior prognosis of CLL patients ( p<0.001), indicating the essential role of MAST1 in the progression of CLL. These results supported the hypothesis that PRMT1 interacted with MAST1 and enhanced the activity of MAST1 via methylating this oncoprotein in CLL cells. Conclusion The present study provides robust evidence for the oncogenic function of PRMT1 in the pathogenesis of CLL, highlighting the potential therapeutic efficacy of the selective PRMT1 inhibitor C7280948. Collectively, the targeted inhibition of PRMT1 exhibits promising prospects for the management of CLL patients.

Skewing Towards Effector Memory Cells in CLL Is Associated with IGHV -Mutation Status and IFNγ and IL-4

Memory T cells are a small, heterogenous population of T cells that remain following pathogen clearance and provide long-lasting protection. They are generally characterized as either central memory (T CM) or effector memory (T EM) based on differences in their surface and intracellular markers, anatomic location, effector function, and cytokine production. It is well known that patients with chronic lymphocytic leukemia (CLL) display differences in both T cell function and population compared to healthy individuals. Previous studies have shown that CLL patients skew towards the CD4 + memory compartment and among these, a skewing toward T EM is especially associated with unmutated IGHV and more progressive disease. Moreover, T EM cells appear to protect CLL cells, possibly via IFNγ and IL-4 mediated pathways. To understand this crosstalk between memory T cells and CLL B cells, we co-cultured FACS-purified T CM (CD3 +CD4 +CD19 -CCR7 +CD45RA -CD45RO +CD62L +) and T EM (CD3 +CD4 +CD19 -CCR7 -CD45RA -CD45RO +CD62L -) cells with CLL B cells and assessed shifts in the memory population as well as cytokines produced. Furthermore, as leukemic B-cell subpopulations differing in time since cell birth/division display distinct functional abilities to present antigen, we also investigated the effect of B-cell activation on this crosstalk. FACS-purified peripheral blood memory T cells and CLL B cells were obtained from treatment-naïve patient samples and co-cultured for 7 days to determine if the presence of CLL B cells, either resting (B rest) or pre-activated with CpG-ODN2006 and IL-15 (B act), would alter the T-cell population distribution. In the presence of either B rest or unmanipulated PBMC, overall, there were no significant changes in population of T EM or T CM. However, the distribution of T EM with B rest was bimodal and when grouped based on IGHV mutation status, we found significantly reduced counts of T EM in co-cultures with M-CLL (14.50% T EM, 11,019 cells) as compared to U-CLL (26.68% T EM, 20,648 cells) based on relative percentages and absolute cell counts ( P<0.001). Conversely, we saw an increase of T CM in M-CLL B rest co-cultures that correlated with decrease of T EM ( P<0.01). We then analyzed supernatants taken at the end of the 7-day co-culture period for IFNγ, IL-4, IL-17A, and IL-17F. We found elevated concentrations of IFNγ in co-cultures with both mutated-CLL and M- CLL, but significantly higher concentrations ( P<0.01) were found with U-CLL (312pg/ml) than M-CLL (173pg/ml). Similarly, IL-4 was elevated but more so (p<0.001) in U-CLL (88pg/ml) than M-CLL (44pg/ml). Of note, IFNγ and IL-4 have been implicated in progression of CLL with literature suggesting their role in prolonging CLL B cell survival. IL-17A and IL-17F were not elevated above baseline and no differences were found. Consistent with literature, the relative fraction of T EM in our patients negatively correlated with TTFT. We then repeated the experiments with pre-activated CLL B cells. When B act were co-cultured with memory T cells, we found significantly ( P<0.001) elevated absolute number of T EM and T CM. This indicated heightened proliferation of the memory population, with a greater degree of increase in the T EM population, resulting in a skewing toward T EM. Subsequent experiments found that co-culture with B act led to significantly elevated IL-4 and IFNγ levels. Interestingly, the effects mediated by B act, on both memory cell proliferation and cytokine production were markedly greater than with B rest. Moreover, unlike B rest, the effects of B act did not depend on IGHV mutation status but rather showed uniform increase in both T EM proliferation and IFNγ and IL-4 levels. Lastly, we analyzed the survival of CLL B cells at the end of the co-cultures. Overall, the addition of memory T cells significantly ( P<0.001) enhanced survival of CLL B cells and this effect was more prominent in B act (U-CLL: 51.1%, M-CLL: 40.3% survival) than B rest (U-CLL: 34.8%, M-CLL: 23.5% survival). Together, our in vitro results suggest crosstalk between memory T cells and CLL B cells in which CLL B cells, especially U-CLL, promote the expansion of T EM which is associated with shorter TTFT and more progressive disease. T EM, in turn, promote the survival of CLL B cells possibly through increased production of IFNγ and IL-4.

ZAP-70 Integrates Tonic B Cell Receptor and Chemokine Signaling in IGHV unmutated Chronic Lymphocytic Leukemia

The importance of B cell receptor (BCR) signaling for the progression of Chronic Lymphocytic Leukemia (CLL) has impressively been demonstrated through the clinical success of small molecule inhibitors interfering with this pathway, providing effective treatment options for a vast majority of patients. The scientific evidence which supported the development of drugs like BTK and PI3K inhibitors largely originate from studies of antigen-induced BCR-signaling. Notably, the presence of a constitutive, so-called tonic BCR-signal was long postulated to co-exist besides antigen-driven activation and is based on the identification of constitutively activated signaling molecules down-stream of the BCR. We here provide direct evidence for the presence of a tonic BCR-signal, which critically relies on the expression of the tyrosine kinase ZAP-70. We have previously established a method to inhibit ZAP-70 expression in primary CLL cells usingRNA-interference in combination with an extended co-culture system. Our previous work demonstrated that ZAP-70 is required for the constitutive gene expression of T cell chemokines CCL3 and CCL4 and contributes to the dysregulation of global protein synthesis and MYC expression in CLL ( Blood, 2021). Applying this method, we here provide evidence for the presence of a constitutive tonic BCR signal, which is dependent on ZAP-70 expression and exclusively found in IGHV unmutated CLL, but not in IGHV mutated cases discordantly expressing ZAP-70. We demonstrate that ZAP-70 is directly involved in the constitutive activation of AKT and GSK-3beta. Furthermore, ZAP-70 expression increases steady state activation of CD19 and the downstream kinase SRC, providing direct evidence for the existence of a ZAP-70 dependent tonic BCR signal in I GHV unmutated CLL only. To identify the underlying signaling events driving this signal, we performed mass spectrometry in combination with proximity ligation assays on CLL cells in the absence of an induced BCR-signal. Unexpectedly, we identified that the majority of ZAP-70 binding proteins do not related to BCR-signaling components, but to cytoskeleton organizing proteins involved in cell migration. Indeed, migration assays demonstrated that the loss of ZAP-70 directly and significantly impaired cell migration induced by CCL19 and CCL21, in keeping with previous data indication an association between ZAP-70 and cell migration. These results were confirmed using a switchable model of ZAP-70, which allowed us to toggle ZAP-70 expression on and off at any given time. To further define the mechanisms of ZAP-70 mediated cell migration, we studied CCL19 and CCL21 signaling in ZAP-70 proficient and deficient cells. Unexpectedly, we identified that ZAP-70 is required for chemokine-mediated activation of AKT. In summary, we here demonstrate that a tonic BCR-signal is present only in IGHV-unmutated CLL and dependent on the expression of ZAP-70. Furthermore, ZAP-70 converges two seemingly independent signaling events, namely chemokine- and tonic BCR signaling. Our data provide further explanations for the more aggressive clinical course of ZAP-70 positive CLL.

CD49d Shapes B Cell Receptor Responsiveness and Tissue Distribution in an Aggressive Chronic Lymphocytic Leukemia Murine Model

Background: High expression of the very late antigen-4 (VLA-4) integrin subunit CD49d predicts disease progression in chronic lymphocytic leukemia (CLL). We previously demonstrated that B cell receptor (BCR) signals induce activation of VLA-4 (Tissino et al., J Exp Med. 2018) and characterized Eµ-TCL1-transgenic (TCL1-tg) mice as an adequate model for the human high-risk CD49d-high CLL patient subgroup, with VLA-4 inhibition hampering the disease (Szenes et al., Leukemia 2020). Here, we address the consequences of genetic B cell-specific ablation of CD49d on disease development, engraftment capacity and treatment susceptibility in a novel CLL mouse model. Methods: We crossed ITGA4fl/fl mice, kindly provided by T. Papayannopoulou, University of Washington, with TCL1-tg mice and CD19 Cre mice, subsequently named CD49d∆B TCL1-tg. Tumor development was continuously monitored by regular blood checks and mice were sacrificed at 10 months. Tumor infiltration was quantified in peritoneal cavity (PC), spleen and bone marrow by flow cytometry using anti-CD5/CD19 antibodies. Integrin and chemokine receptor expression was profiled. In vitro responsiveness to BCR stimulation of CD49d-deficient and proficient leukemic cells was assessed using phospho-specific flow cytometry. Furthermore, purified leukemic cells derived from PC or spleen were subjected to RNA-seq analyses. CD49d-deficient or proficient leukemic cells were transplanted into wild-type C57BL/6J recipients in short and long term experiments. Ongoing: CD49d-deficient or proficient leukemic cells were transplanted competitively into C57BL/6J recipients and are receiving continuous Bruton's tyrosine kinase (BTK)-inhibitor (acalabrutinib) treatment upon tumor onset. Results: CD49d∆B TCL1-tg mice showed a B cell-specific integrin loss in all organs. Blood tumor burden was measured monthly. At months seven and eight, leukemic cells of CD49d∆B TCL1-tg mice were reduced by approx. 25% compared to TCL1-tg littermates (Fig. 1A). This trend was reversed at month nine, with approx. 45% increased lymphocytosis in CD49d∆B TCL1-tg mice. CD49d expression in leukemic cells of TCL1-tg mice was unaffected by disease progression. At sacrifice, CD49d∆B TCL1-tg mice showed an 80% ( p=<0.0001) reduction of tumor burden in the bone marrow (Fig. 1B), but comparable leukemic cell numbers in PC and spleen. Leukemic splenocytes derived from CD49d∆B TCL1-tg mice displayed 38% ( p=0.032) higher IgM and 33% higher CD44 expression but 43% ( p=0.015) reduced expression of CXCR4. BCR signaling was modulated by the integrin loss and BCR stimulation with anti-IgM induced 43% ( p=0.022) higher PLCγ2 and 45% ( p=0.022) higher SYK phosphorylation in leukemic cells derived from CD49d∆B TCL1-tg mice compared to littermates, but comparable BTK phosphorylation. RNA-seq analysis revealed strong differences in leukemic cell transcriptomes between PC and spleen, indicating tissue dependent effects on leukemic cells. Particularly, TNF-α signaling via NF-κB, MTORC1, MAPK signaling and JAK-STAT signaling pathways were upregulated in leukemic cells derived from spleen compared to PC irrespective of CD49d expression, while chemokine signaling pathways were downregulated in PC. When comparing CD49d deficient and proficient leukemic cells, the gene set MYC targets v1 was enriched in deficient cells. When injecting CD49d-deficient leukemic splenocytes intravenously, more cells were recovered after 5 days from blood ( p=0.048) and spleen compared to CD49d-proficient cells, but did not engraft in bone marrow or peritoneum. When injected intraperitonally, CD49d-deficient leukemic cells were found in peritoneum in contrast to TCL1-tg littermates ( p=<0.0001). Conclusion: The TCL1 mouse model resembles a human high-risk CLL (e.g. IGHV unmutated, CD49d-expressing), and thus allows to investigate the impact of CD49d expression in this CLL subset. Loss of CD49d expression resulted in an altered redistribution of leukemic cells in tissue sites with a significant reduction of leukemic cells in the bone marrow. Lack of integrin signals resulted in increased antigen-induced BCR signaling and IgM expression, as well as a B cell receptor activation phenotype associated with MYC gene set enrichment, indicating a feedback loop from integrins to the BCR. Currently, the susceptibility of CD49d∆B TCL1-tg mice to BTK inhibitors is under investigation.

Chronic Obstructive Pulmonary Disease and Previous Infections Have Impact on Infectious Complications in Patients with Chronic Lymphocytic Leukemia Treated with Venetoclax: A Multicentre Seifem Study

Introduction. Infections are a major source of morbidity and mortality in patients with Chronic Lymphocytic Leukemia (CLL). The development of targeted agents decreased the rate of these complications in comparison to standard chemoimmunotherapy regimens. However, these patients often elderly, with other comorbidities, heavily treated, experienced serious infections. The aim of our study was to evaluate the incidence of clinically or microbiologically documented bacterial, fungal and viral infectious complications in CLL patients treated with venetoclax. Methods. The retrospective multicenter study included CLL patients treated since 2017 with venetoclax single agent until progression or toxicity or venetoclax plus anti-CD20 antibody (mainly rituximab as part of VR protocol for 24 months or obinutuzumab as part of VO protocol for 12 months). Results. A total of 287 patients with CLL received venetoclax during the study period from 16 different institutions: 151 patients (52.6%) as monotherapy and 136 (47.4%) associated to anti-CD20 antibody. Basal characteristics of the whole population and of the two groups are summarized in Table 1. Patients of the first group were older, more frequently had del17/TP53mut, renal impairment and lower basal levels of IgG. They also showed more previous infections in the 12 months before the beginning of the treatment with venetoclax. We registered 284 infections of any grade. When comparing time of first infection between the patients treated with venetoclax and those treated with venetoclax plus anti-CD20 antibody, we registered a trend toward a higher rate of infection in the latter group after the first year (p=0.066). This difference was not confirmed when we focused on infections of grade 3-4 (p=0.521). One-hundred eighty-one infections of grade 1-2 developed in 114 patients (39.7%) during the study. Most of the infections involved the respiratory tract (106 events, 58.6%), followed by genitourinary tract (23, 12.7%) and gastrointestinal one (16, 8.8%). Pathogens implicated in the infections were isolated only in 57 (31.5%) cases: 36 viral, 18 bacterial and 3 fungal. We recorded 103 episodes of infections of grade 3-4, occurred in 73 patients (25.4%). The most common site of infection involved the respiratory tract (71 events, 68.9%), then we registered sepsis (13, 12.6%) and gastrointestinal tract infections (7 events, 6.8%). Of 103 severe infections, 64 (62.1%) were microbiologically proven, of whom 40 were viral, 21 bacterial and 3 fungal. When comparing patients with and without infection, COPD (p<0.001, OR 3.75), previous infections in the last 12 months (p<0.001, OR 3.15), renal impairment CrCl<70 (p = 0.049, OR 1.62), previous treatments (p=0.023; OR 1.196) and stage A (p=0.001; OR 0.2) were more frequently associated with infection in univariate analysis. In multivariate analysis COPD (p <0.001, OR 5.39) and previous infections (p=0.001, OR 2.57) resulted significant. Stratifying patients according to COPD and previous infections in the last 12 months we obtained 3 groups significantly different in terms of infective risk (p <0.001; figure 1). When considering only grade 3-4 infections, risk factors significant in the univariate analysis were COPD (p <0.001, OR 3.23), smoke (p=0.033, OR 1.98) and previous infections (p=0.020, OR 1.91). COPD was the unique significant variable in multivariate analysis (p=0.008, OR 2.62). Treatment was withdrawn for infections in 80 patients (27.9%): in 58 (20.2%) treatment was temporarily discontinued, while in 22 (7.7%) discontinuation was permanent. The infections that caused definitive withdrawals were mainly pneumonia (12 cases, 6 of whom from SarS-CoV2 infection) and sepsis (8 cases, 5 of whom after a SarS-CoV2 infection). A total of 83 patients (28.9%) died and the median OS was 55 months. The main causes of death were CLL progression in 36 cases and infection in 22 cases. Conclusions. This is a real-life study on 287 patients affected by LLC treated with venetoclax with the aim to describe the infectious complications in such population in routine clinical practice. The analysis found a significant rate of infections, most of grade 1-2: 39.7% of the patients experienced a grade 1-2 infection; 25.4% a grade 3-4 infection. The identification of additional infectious risk factors found a role of comorbidities such as COPD and previous infections; COPD resulted a risk factor also for infections of grade 3-4.

Outcomes of Venetoclax Retreatment after MRD-Guided Venetoclax +/- Ibrutinib: Results of the Improve Study

Data from small cohorts of patients (pts) with chronic lymphocytic leukemia (CLL) relapsing after venetoclax-based fixed-duration regimens suggest that venetoclax retreatment may be a feasible option. Information on the efficacy and tolerability of venetoclax retreatment in this setting is still scanty, making it worth exploring venetoclax retreatment in prospective cohorts of well-characterized pts with CLL. We analyzed the efficacy and safety of venetoclax retreatment in pts with relapsed/refractory CLL enrolled in our clinical trial IMPROVE (NCT04754035). BTK- and BCL2-inhibitor naïve pts enrolled into the study were treated with venetoclax single-agent for 12 months; those with undetectable measurable residual disease (uMRD) discontinued therapy while those with detectable MRD received the combination of ibrutinib and venetoclax until uMRD, toxicity or for up to 12 months. Pts with detectable MRD at the end the combination therapy stopped venetoclax and continued with ibrutinib only. In pts who experienced progressive disease (PD) requiring treatment, venetoclax single-agent was restarted according to the standard schedule and continued until PD or unacceptable toxicity. MRD was tested every 3 months in peripheral blood using the 6-color flow cytometry panel validated by the ERIC (European Research Initiative on CLL). After a median follow-up of 58 months (5-67) from C1D1, 20/38 (52.6%) pts experienced PD requiring treatment after discontinuing therapy, including 19 upon reaching confirmed uMRD (11 treated with venetoclax single-agent, 8 with the combination of venetoclax+ibrutinib), 1 who discontinued both venetoclax and ibrutinib in partial response (PR) with detectable MRD (dMRD) due to an unrelated adverse event. Two out of 20 had Richter transformation (RT), 2 developed autoimmune hemolytic anemia, 2 received other treatments (BTK inhibitors) per investigator's decision and all 6 went off study. The remaining 14 pts (1 with detectable MRD, 7 who previously achieved uMRD with venetoclax monotherapy, 6 who were treated with the combination of venetoclax+ibrutinib) received venetoclax retreatment at a median of 32.5 months (12-44) after the end of initial therapy (Table 1); to note, the pt who stopped treatment in PR with dMRD restarted venetoclax after only 13 months. All 13 uMRD subjects experienced MRD relapse at a median of 5 months (0-41) after stopping treatment, 26 (0-37) months before disease progression requiring therapy. At the time of venetoclax initiation tumor lysis syndrome (TLS) risk was high in 6, medium in 3, low in 5. Best overall response rate was 79% (11/14), all partial responses (PR), 2 pts experienced stable disease (on treatment for 2 and 24 months). With a median time on treatment of 10 months, 2 pts achieved uMRD after 3 and 6 months, the second one maintaining uMRD for 6 months and then becoming MRD positive again (Figure 1). Five out of 14 pts progressed after 2, 7, 9 and 11 (n=2) months on venetoclax retreatment, 3/5 received next-line treatment with ibrutinib, 2/5 received non-covalent BTK inhibitor and all achieved a PR. At last follow-up, 57% of pts remain on venetoclax; 12/14 patients undergoing venetoclax retreatment were alive (1 death due to RT on ibrutinib, 1 due to CLL progression on non-covalent BTK inhibitor). Progression-free survival from venetoclax restart to PD on venetoclax retreatment, last follow-up or death was not reached (0-28 months). Venetoclax retreatment was very well tolerated with no patients experiencing TLS, 6/14 having grade 3-4 neutropenia (only in 1 case leading to treatment discontinuation), 8/14 infections (2 grade 1, 4 grade 2, 2 grade 3). No other grade 3/4 hematologic or non-hematologic toxicities were observed. Venetoclax retreatment was effective and well-tolerated in a cohort of pts with relapsed/refractory CLL who have previously received time limited, MRD-guided treatment with venetoclax+/-ibrutinib. MRD relapse occurred at a median of about 2 years before clinical progression requiring treatment. Venetoclax retreatment was started at a median of 32.5 months after the end of initial therapy. Nearly 80% of patients achieved at least a partial response with venetoclax retreatment, 14% of pts achieved uMRD and 57% remain on venetoclax, with a median time on treatment of 10 months.

Characterization of Three Somatic Mutations in the 3'UTR of RRAS2 and Their Inverse Correlation with Lymphocytosis in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a hematologic malignancy characterized by progressive accumulation of a rare population of CD5+ B-lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. CLL exhibits remarkable clinical heterogeneity, with some patients presenting with indolent disease and others progressing rapidly to aggressive CLL. The significant heterogeneity of CLL underscores the importance of identifying novel prognostic markers. Recently, the RAS-related gene RRAS2 has emerged as both a driver oncogene and a potential marker for CLL progression, with higher RRAS2 expression associated with poorer disease prognosis. Although missense somatic mutations in the coding sequence of RRAS2 have not been described in CLL, this study reports the frequent detection of three somatic mutations in the 3' untranslated region (3'UTR) affecting positions +26, +53, and +180 downstream of the stop codon in the mRNA. An inverse relationship was observed between these three somatic mutations and RRAS2 mRNA expression, which correlated with lower blood lymphocytosis. These findings highlight the importance of RRAS2 overexpression in CLL development and prognosis and point to somatic mutations in its 3'UTR as novel mechanistic clues. Our results may contribute to the development of targeted therapeutic strategies and improved risk stratification for CLL patients.

Combined Ibrutinib and Venetoclax for First-Line Treatment of Patients with Chronic Lymphocytic Leukemia (CLL): 5-Year Follow-up Data

Background: Ibrutinib (IBR) and venetoclax (VEN) combination is an effective therapy for patients (pts) with CLL. We previously reported results of the first-line cohort of a phase II trial of combined IBR and VEN for high-risk pts with CLL (Jain, NEJM 2019, JAMA Oncology 2021). Here we report updated data for 120 pts (80 pts in the original published cohort and 40 pts in an expansion cohort) with a median follow-up of 61.5 months. Methods: Pts with previously untreated CLL meeting IWCLL treatment criteria were enrolled. All pts had at least one high-risk feature: del(17p), mutated TP53, del(11q), unmutated IGHV, or age ≥65 years. Pts received IBR 420 mg daily for 3 cycles followed by addition of VEN (weekly dose-escalation to 400mg daily). Combined therapy was given for 24 cycles (28 days/cycle). Pts with bone marrow (BM) undetectable MRD (U-MRD) (flow cytometry; sensitivity 10 -4) at 24 cycles of combined therapy discontinued both VEN and IBR; MRD+ pts continued IBR. A trial amendment allowed an additional 12 cycles of combined VEN and IBR for pts who remained BM MRD+ after Cycle 24. Response assessments included BM and CT studies (2008 IWCLL criteria). U-MRD was defined as <0.01%; low MRD+ 0.01% to <1%; high MRD+ ≥1%. Progression-free survival (PFS) was assessed as the time from the start of study drug to CLL progression, Richter transformation, or death from any cause. Blood MRD was monitored every 6 months after active therapy. Results: Between August 2016 and February 2019, a total of 120 pts were enrolled. Median age was 64.5 years (range, 26-88 years). 86% had IGHV-unmutated CLL. 23% had del(17p)/ TP53 mutation. The median follow-up is 61.5 months. Six pts came off study during 1 st 3 cycles of IBR monotherapy; 114 pts initiated VEN. After 12 cycles of the combination, 62/120 (52%) achieved BM U-MRD remission; 43/120 (36%) were BM MRD-positive (low MRD+, n=35; high MRD+, n=8). After 24 cycles of the combination, 77/120 (64%) achieved BM U-MRD remission; 24/120 (20%) were BM MRD+ (low MRD+, n=23; high MRD+, n=1). Overall, 86/120 (72%) achieved BM U-MRD as the best response. One pt had DLBCL transformation and 1 pt had CLL progression during the first 2 year of therapy (details below). The 5-year PFS is 90.1% and 5-year OS is 95.6% (Figure 1). The 5-year PFS for pts with del(17p)/ TP53 mutation (n=27) is 86.1%. Of the 77 pts who were BM U-MRD at the end of cycle 24 of the combination, 73 discontinued all therapy, 4 pts continued IBR per treating physician discretion. Among these 77 pts, with a median follow-up of 40 months post Cycle 24, 22 pts had recurrence of blood MRD (defined as MRD ≥0.01% in 2 consecutive visits). Of the 22 pts with MRD recurrence, 5 pt had CLL progression at a median of 18 months (range, 6-30 months) from MRD recurrence (details below); 16 are being monitored without any active therapy for CLL and without clinical disease progression; 1 pt died from mesothelioma. Among the 55 pts who remain in blood U-MRD remission, 53 remain in long-term follow-up in U-MRD remission; 1 pt had B-PLL transformation and 1 patient died (found unresponsive at home; was off therapy for 2 years). There were 24 pts who were BM MRD+ at the end of cycle 24 of the combination (low MRD+, n=23; high MRD+, n=1). The only pt with high-MRD+ at end of cycle 24 was noted to have Richter transformation at that time. The remaining 23 pts (all low MRD+ in BM, range 0.01-0.95%) continued IBR monotherapy. With a trial amendment, MRD+ pts after Cycle 24 could get 12 additional cycles of VEN; 18/23 pts have resumed VEN. 11/18 (61%) pts achieved U-MRD remission during the third year of combined therapy. Only 2/23 pts are still receiving IBR (remaining pts have d/c IBR; all pts have d/c venetoclax); none of these 23 pts had clinical relapse. A total of 6 pts had CLL progression (1 during the first 2 yr of therapy; remaining 5 during off-therapy phase). 3/6 had evaluation of BTK, PLGG2 and BCL2 mutations at the time of relapse and none were detected. 5 pts have started subsequent therapy (acalabrutinib, n=4; ibrutinib, n=1; all are clinically responding); 1 pt has not yet started therapy. Conclusions: We report long term follow-up of combined IBR and VEN in first-line CLL (n=120) with a 5-year PFS of 90.1%. The 5-year PFS for pts with del(17p)/ TP53 mutation is 86.1%. Retreatment with BTK inhibitor appears effective for pts with disease relapse.