Kay, Neil E.

Abstract Introduction: A subset of patients with chronic lymphocytic leukemia (CLL) experiences disease progression from CLL to Richter transformation (RT), characterized by the occurrence of a high-grade lymphoma. The outcomes of patients with RT are typically dismal and identifying better therapeutic strategies remains critically important for these patients. To discern the biologic underpinnings driving this transformation, we studied immune changes and genetic evolution in samples from CLL patients who developed RT. Methods: We identified 18 patients with CLL who developed RT; and performed single cell (sc) RNA-sequencing(seq) on sorted B and T cells from peripheral blood mononuclear cells (PBMCs) at the time of RT (n=18) and at the earlier time of CLL diagnosis (n=11). The median time between the CLL sample and the one closest to RT was 4.3 years (range, 0.2-7.3); and patients received a median of 1 CLL directed treatment (range, 0 – 7). A total of 749,906 cells and 36,601 genes were captured during scRNAseq and 720,580 cells and 21,892 genes were retained for downstream analysis, respectively. Analysis was performed using the immunopipe (https://github.com/pwwang/immunopipe) that wraps around Seurat (v5.1.0) and identified enriched pathways using Enrichr service. The sc-T cell receptor (TCR)-repertoire(R)-seq data was analyzed and visualized using scplotter (). The whole exome sequencing (WES) analysis with tumor B-cells from same patients of CLL & RT stages were done using the Agilent sureselect Human all exon v5 + UTR capture kit. Reads from paired end sequencing were processed with the sarek pipeline. Results: Analysis of 720,580 cells from 11 CLL and 18 RT samples showed 16 distinct cell types- 3 B and 13 T cell subtypes. We observed an increasing trend of CD8 TEM [mean 0.548 vs. 0.384, p = 0.12 (Wilcoxon test)] but a decreasing trend of CD4 TCM (mean 0.236 vs. 0.337, p = 0.08) and B-memory (mean 0.0085 vs. 0.0195, p = 0.51) subsets in RT compared to their CLL stage. When comparing transcriptional changes, we found that among the top 18 highly variable genes, the expression of FP236383.3 (lncRNA) and TCL1A was significantly higher (p<0.0001) in the B cell subsets of patients with RT vs. CLL stage. Additionally, in T cell subsets—primarily dnT, γδT, and CD8/CD4 naïve cells—the expression of FP236383.3, NKG7, GNLY, and CCL5 was significantly higher (p<0.0001), while the expression of LDHB, GZMK, and IL32 was significantly lower (p<0.0001) in RT vs. CLL stage samples. Comparison of specific gene set enrichment analysis showed significantly increased expression of GZMB in gdT (p<0.001) and decreased expression of GZMA, GZMK, TIGIT and TCF7 in dnT and CD8 Naïve cells (p<0.001) for RT vs. CLL stage. The top 10 differentially expressed pathways in RT included oxidative phosphorylation, allograft rejection, IFN-α/-γ responses, TNF-α signaling via NF-κβ, complement, Myc targets V1, mTORC1, hypoxia, and p53 signaling across various B and T cell subsets compared to the CLL stage. Initial analysis of TCR-R data showed that patients with RT stage have lower number of unique clones (p<0.5) and reduced clonal diversity (p<0.5), and a higher number of large and hyperexpanded clones compared to their CLL stage. Additionally, WES data indicated that the most frequently mutated gene is TP53 in tumor B cells of both CLL and RT stages. However, a higher percentage of RT-stage samples carry mutations in MGA, ATM, and IRF2BP2 genes compared to their CLL-stage. WES data also showed a trend toward differing frequencies of copy number amplification at 19q12 (p=0.10) and 4q13.1 (p=0.52) between RT and CLL stages. Conclusions: The main findings of our study are as follows:The transcriptional landscape showed significant changes in multiple genes and pathway enrichment related to cell growth, differentiation and immune response in RT vs. CLL stage.A decreased fraction of CD4 TCM cells and reduced TCR clonal diversity were observed in samples at the RT stage and may indicate a poor prognosis for the patients at this stage. WES data of tumor B cells also indicated possible genetic alterations in the RT vs. CLL stage. Data from in-depth analyses on understanding of the dynamics of T cell response and additional experiments on these patients are expected to provide valuable insights into tumor genetic heterogeneity, transcriptional and immune regulation in blood-based immune subsets.

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is marked by impaired T cell responses that limit the efficacy of immunotherapies. CD19-directed chimeric antigen receptor T cell therapy (CART19) received FDA approval to treat CLL in 2024. However, itsefficacy in CLL patients remains low, with durable remission rate of ~20%. Ibrutinib, a Bruton tyrosine kinase inhibitor, has shown synergy with CART19 in preclinical and early clinical studies. We aimed to study the impact of ibrutinib on CART19 utilizing clinical samples from the pivotal clinical trial E1912 (NCT02048813) of ibrutinib/rituximab (IR) vs fludarabine/cyclophosphamide/rituximab (FCR). This trial led to the FDA approval of first line ibrutinib in CLL. Methods: We generated CART19 incorporating either CD28 (28ζ) or 41BB (BBζ) costimulatory domains from T cells isolated from patients with CLL treated with IR or FCR on the E1912 trial. IR samples were collected at baseline (pretreatment), 6- or 12-months during treatment, or at progression. FCR samples were collected at baseline, 15-18 months during treatment, or at progression. For animal experiments, NOD-SCID-γ-/- (NSG) mice were intravenously (i.v.) engrafted with 1×10⁶ luciferase⁺ CD19⁺ JeKo-1 cells. Upon tumor establishment (~10⁸ photons/sec by bioluminescence imaging), mice were randomized and treated i.v. with 1×10⁶ 28ζ or BBζ derived from longitudinal IR- or FCR-treated patient samples. To further assess the impact of prolonged ibrutinib exposure on CART19 function, we chronically stimulated healthy donor-derived 28ζ or BBζ in vitro for 22 days with JeKo-1 cells and 100nM ibrutinib or vehicle control and then tested CART19 functions. Finally, we performed single cell RNA sequencing (scRNAseq) of longitudinal patient T cell samples treated with IR. Results: Ex vivo T cell expansion was significantly enhanced following IR compared to FCR. BBζ generated from IR-treated patients after 6 months of ibrutinib exhibited enhanced in vitro expansion (p=0.016) and antigen-specific proliferation (p=0.038) in the presence of JeKo-1 cells. This corresponded with significantly improved antitumor efficacy and prolonged survival in vivo compared to cells generated at disease progression (p=0.017). In contrast, 28ζ showed no significant differences in efficacy across IR studied treatment timepoints. BBζ from FCR-treated patients did not show differences in tumor control based on treatment timepoints but demonstrated shorter survival compared to BBζ from IR-treated patients. 28ζ from FCR-treated patients generated at disease progression demonstrated significantly reduced tumor control and shorter survival compared to 28ζ produced at baseline (p=0.0002), indicating detrimental effects of FCR treatment on the therapeutic potential of CART products. To further investigate the impact of prolonged ibrutinib exposure, we utilized in vitro models of CART exhaustion. We found that, after repeated antigen stimulation with JeKo-1 cells, ibrutinib-treated BBζ had significantly increased antigen-specific degranulation (p=0.005), and cytokine production (IL-2, p=0.045; TNF-α, p=0.003; IFN-γ, p=0.001), compared to BBζ treated with vehicle control. In contrast, 28ζ did not show improved degranulation or cytokine production in cocultures with ibrutinib. Prolonged ibrutinib exposure led to downregulation of FOXO3, a transcription factor associated with T cell exhaustion which has been shown to cause dysfunction specifically in BBζ. These results further indicate that ibrutinib may be more beneficial to BBζ. Indeed, scRNAseq revealed that after ≥ 6 months of ibrutinib, T cells showed downregulation of inflammatory, oxidative stress, and apoptosis pathways, paralleled by modest activation of MYC and E2F targets. However, at disease progression, T cells under ibrutinib treatment displayed marked upregulation of interferon signaling, inflammation responses, and stress-related programs, indicating a shift toward immune reactivation or dysfunction.Conclusion: Our study shows that BBζ, but not 28ζ, generated 6 months during ibrutinib treatment has superior antitumor activity and persistence compared to similar later timepoints or FCR-treated counterparts. Prolonged ibrutinib exposure enhances BBζ function by modulating stress and apoptotic pathways. These findings highlight the importance of pre-CART treatment timing and support ibrutinib as a potential enhancer of CART cell fitness in CLL.

Abstract Recent studies have highlighted the important but pleiotropic role of IL-4 in response to chimeric antigen receptor T (CART) cell therapy. IL-4 was identified as a regulator of dysfunction in CD19-targeting CART cells with a CD28 costimulatory domain (CART19-28ζ) through a genome-wide CRISPR knockout screen as well as RNA and ATAC sequencing of baseline and exhausted healthy donor CART19-28ζ cells and pre-infusion CART19-28ζ cell products from responders and non-responders in the ZUMA-1 trial (Stewart et al, Nat Commun., 2024). However, interrogation of a single-cell atlas fromactivated pre-infusion CART cell products from patients with acute lymphoblastic leukemia revealed a beneficial role for IL-4 and other type 2 cytokines in the promotion of long-term response (Bai et al, Nature, 2024). In this report, we aim to further understand how IL-4 can be detrimental to CART cell therapy. To study the regulation of CART19-28ζ cells by IL-4, we performed RNA sequencing on CART19-28ζ cells that were stimulated with CD19+target cells in the presence of either 20 ng/mL hrIL-4 or diluent for 24 hours, one week, or two weeks in an in vitro model for exhaustion as previously described (Stewart et al, Nat Commun., 2024). We observed that hrIL-4 treatment drove the development of a distinct transcriptomic profile after chronic stimulation for one week (up genes = 3253 and down genes = 3645) or two weeks (up genes = 6435 and down genes = 6224) but not following 24 hours of activation (up genes = 0 and down genes = 0). Gene set enrichment analysis on the differentially expressed genes following two weeks of chronic stimulation showed that hrIL-4 treatment pushes CART cells to a more exhausted and less naïve phenotype (FDR = 0.015) and leads to a broad downregulation in cytokine signaling (FDR = 0.012). To investigate transcription factors associated with IL-4-induced CART cell dysfunction, we used BETA analysis with publicly available transcription factor binding sites for STAT6, GATA3, and EOMES and our differential expression data. EOMES (p = 0.039), but not STAT6 (p = 0.999) nor GATA3 (p = 0.999) significantly regulated the differentially expressed genes. In support of these findings, the addition of hrIL-4 also significantly upregulated EOMES expression after one (p < 0.0001) and two (p < 0.0001) weeks of chronic stimulation and downregulated the expression of TBX21 (p < 0.0001), a binding competitor of EOMES, following two weeks of chronic stimulation. To evaluate if EOMES is critical for IL-4-induced CART cell dysfunction, we treated either control or EOMES CRISPR knockdown CART cells with 20ng/mL hrIL-4 during chronic stimulation. EOMES knockdown reduced IL-4-induced CART19-28ζ cell dysfunction as seen by an increase in antigen-specific expansion (p = 0.046) and an increase in cytokine production including IL-2 (p < 0.0001), TNF-α (p = 0.006), and IFN-γ (p = 0.020). Having identified that EOMES is at least partially responsible for IL-4-induced CART cell dysfunction, we evaluated EOMES binding sites in human CART19-28ζ cells with CUT&RUN sequencing. Our analysis revealed EOMES binding at hypersensitivity site 2 (HS2) in the IL-4 locus which, in a previous study, was more accessible in both exhausted healthy donor CART19-28ζ cells and pre-infusion CART cell products from non-responders in the ZUMA-1 clinical trial (Stewart et al, Nat Commun., 2024). To determine how HS2 can impact CART cell functions, we generated CART19-28ζ cells from either wildtype (HS2+/+) or HS2-deleted (HS2-/-) murine T-cells and tested them in a syngeneic model of lymphoma generated by engrafting C57BL6 mice with luciferase+ TBL12 cells (1x106, tail vein injection on Day -3) following preconditioning (250mg/kg cyclophosphamide, intraperitoneal injection on Day -4). Following randomization of mice via tumor burden (n = 5 per group), mice were treated with either HS2+/+ or HS2-/- CART19-28ζ cells (1x106 cells, tail vein injection). In this model, HS2-/- CART cells showed enhanced expansion (p = 0.022), increased anti-tumor activity (p = 0.018), and improved overall survival (p = 0.003). Together, our study shows that IL-4 induces a distinct cellular fate in CART19-28ζ cells in the presence of chronic antigen-specific stimulation at least partially through the upregulation of EOMES. Further, the epigenetic regulation of CART cells by the IL-4 enhancer HS2 can significantly influence CART cell activity, and deletion of HS2 can improve CART cell therapy.

Abstract Introduction FD Ibr+Ven is approved as first-line (1L) treatment (tx) for CLL/SLL in 78 countries across Asia, Europe, the Middle East, and South America, plus Australia, Canada, and New Zealand. Clinical trials with up to 5.5 years (yrs) of follow-up have demonstrated progression-free and overall survival benefits of 1L FD Ibr+Ven in pts with CLL/SLL (Wierda WG, ASH 2024; Jacobs R, EHA 2024). Despite established clinical benefits, there is a lack of RW evidence on the effectiveness and tolerability of FD Ibr+Ven outside of clinical trial settings. Here we present pooled data from REALITY-WW and REALITY-2 to understand the usage, factors for therapy decision, clinical response, and safety of FD Ibr+Ven in routine clinical practice. Methods REALITY-WW and REALITY-2 are prospective observational cohort studies. REALITY-WW is an international study; REALITY-2 is conducted in Germany. Both included previously untreated pts with CLL/SLL from hospitals and medical institutions where Ibr+Ven was routinely used. The decision to start FD Ibr+Ven was made prior to and independent of pt enrollment. Pts aged ≥18 yrs with a confirmed diagnosis of CLL/SLL requiring 1L tx per iwCLL 2018 criteria received 3 cycles of Ibr, followed by 12 cycles of FD Ibr+Ven orally (Ibr, 420 mg/d; Ven, 5-week ramp-up to 400 mg/d). Primary end point was overall response rate (ORR) per iwCLL 2018 criteria. Secondary end points and outcomes of interest included tx-emergent adverse events (TEAEs), tumor lysis syndrome (TLS) risk, and factors associated with physician decision to initiate FD Ibr+Ven in routine clinical practice. Results At data cutoff (REALITY-WW: May 2025; REALITY-2: March 2025), 129 pts from both studies received FD Ibr+Ven (REALITY-WW, 81; REALITY-2, 48). The median (range) time on study was 6.4 (0.1-19.0) months (mo). Median (range) age was 65.0 (37.0-87.0) yrs, with 46.5% of pts aged <65 yrs and 53.5% of pts aged ≥65 yrs; 62.8% were male. Median (range) time from diagnosis to FD Ibr+Ven initiation was 38.1 mo (0.1-238.0) and median (range) duration of tx was 6.0 mo (0.1-15.9) for Ibr and 3.2 mo (0.1-8.4) for Ven. A Cumulative Illness Rating Scale score of >6 was observed in 12.6% of pts, with a higher score in pts aged ≥65 vs <65 yrs (18.0% vs 6.9%). TP53 mutations were seen in 7.4% of pts; 52.8% had an unmutated IGHV status. ORR was assessed in the 44 pts with ≥ 1 post-baseline assessment in REALITY-WW. Of these, 39 showed a response by the end of 6 tx cycles (3 cycles of Ibr + 3 cycles of FD Ibr+Ven) and 5 had stable disease. ORR at 6 mo per iwCLL 2018 criteria (partial response [PR] with lymphocytosis [PR-L] or better) was 88.6% (95% CI, 79.3-98.0), including complete responses in 29.5%, PR in 34.1%, and PR-L in 25.0% pts. Among 129 treated pts, TEAEs were reported in 61.2%; diarrhea was the most common TEAE (14.0%). Serious TEAEs and grade 3/4 TEAEs were reported in 12.4% and 8.5% of pts, respectively. The most common grade 3/4 TEAE was myocardial infarction (1.6%). Dose reduction was observed in 13.2% (Ibr) and 2.3% (Ven) of pts, mainly due to tx-associated TEAE/toxicity/TLS. Rates of TEAEs leading to discontinuation, dose reduction, and interruption of ≥1 study tx were 4.7%, 10.9%, and 20.9%, respectively. At baseline (n=129), 18.6%, 45.0%, and 14.7% of pts had high, intermediate, and low TLS risk, respectively, while 20.2% were not assessed and 1.6% had missing data. After the 3-cycle Ibr lead-in, the distribution shifted to 9.3%, 24.8%, and 24.0%, respectively, with 14.7% not assessed and 27.1% missing. There were no TEAEs of TLS and no hospitalizations due to TLS. Seven pts (6 aged ≥65 yrs and 1 <65 yrs) were hospitalized prophylactically for TLS. Prophylactic hospitalization was observed in 8.5% (11/129) of pts with high or medium TLS risk and impaired renal function for whom the Ven ramp-up was initiated; no pts were hospitalized due to laboratory/clinical TLS during the Ven ramp-up period. Physician questionnaire responses (n=126) indicated the most common factors associated with decision to initiate FD Ibr+Ven were administration route advantage (91.3%), overall health status (87.3%), anticipated superior efficacy of tx (84.1%), and general risk factors such as high tumor burden/genetic risk factors (81.0%). Conclusions Overall findings suggest that FD Ibr+Ven is an effective and safe tx for pts with CLL/SLL in a RW population, including those aged ≥65 yrs. Data with longer follow-up will be presented at the meeting.

Abstract Background: The synergistic mechanisms of BCL-2 inhibitors and CD20-directed antibodies in the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) often lead to deep and durable responses. Although the use of venetoclax-obinutuzumab (Ven-O) in the frontline setting (1L) for CLL/SLL has led to a paradigm shift in the disease management, there are limited data on its efficacy in the relapsed/refractory (R/R) setting. In this study, we evaluated the efficacy and safety of Ven-O in both 1L and R/R CLL among patients treated outside the context of clinical trials at our institution. Methods We used the Mayo Clinic CLL Database to identify patients with CLL who were treated with Ven-O, both in 1L and R/R settings. Patients seen at Mayo Clinic Rochester between 2017 and 2025 were included. Overall survival (OS) was calculated from date of Ven-O initiation until last known alive date. Event-free survival (EFS) was calculated from date of Ven-O initiation until the next line of treatment or last known alive date. Kaplan-Meier methods estimated EFS and OS rates at 1, 3, and 5 years. Cox proportional hazards models were used to explore the association between baseline factors and EFS and OS; results are reported as hazard ratios (HR) and 95% confidence intervals (95%CI). Finally, we compared the OS to the age- and sex-matched general population of the state of Minnesota. Results A total of 154 patients (91 1L, 63 R/R) were included in the study. The median age was 68 years (69 1L, 68 R/R), and 68% were male (64% 1L, 73% R/R). Overall, TP53 disruptions (either del17p by FISH or TP53 mutation) were noted in 22 (15%) patients (9% 1L, 23% R/R). CLL FISH findings demonstrated del11q in 24 (19%) patients (19% 1L, 18% R/R), del13q in 46 (36%) patients (36% 1L, 36% R/R), and trisomy 12 in 21 (16%) patients (16% 1L, 18% R/R). IGHV genes were unmutated in 95 (68%) patients (60% 1L, 80% R/R). The CLL-IPI risk score was high in 54 (51%) patients (51% 1L, 53% R/R) and very-high in 18 (17%) patients (8% 1L, 34% R/R). A total of 121 (89%) patients completed planned treatment (1 year of venetoclax in 73 [90%] 1L and 2 years of venetoclax in 48 [87%] R/R). Median follow-up for the entire cohort was 1.8 years (2.2 1L, 1.5 R/R). Neither median EFS nor OS was reached for the overall cohort or within the 1L and R/R subgroups. The 1, 3, and 5-year EFS estimates for the overall cohort were 91% (86-96%), 85% (78-92%), and 67% (51-89%), respectively; corresponding EFS values for the 1L subgroup were 95% (91-100%), 89% (82-98%), and 67% (47-95%), respectively; and for the R/R subgroup were 85% (76-95%), 77% (65-92%), and 77% (65-92%), respectively. The 1, 3, and 5-year OS estimates were 98% (95-100%), 95% (91-99%), and 88% (76%-100%), respectively; corresponding OS values for the 1L subgroup were 99% (96-100%), 97% (94-100%), and 97% (94-100%) and for the R/R subgroup were 96% (91-100%), 91% (82-100%), and 61% (27-100%), respectively. Univariable analyses showed that TP53 disruption was associated with shorter EFS (HR 3.8, 95%CI 1.4-10.2, p=0.01), although IGHV mutation status was not associated with shorter EFS. A higher level of serum beta-2 microglobulin (HR 1.27, 95%CI 1.01-1.59, p=0.04) and R/R status (HR 4.9, 95%CI 0.9-25.8, p=0.06) were associated with a shorter OS. Interestingly, TP53 mutation status and IGHV mutation status were not associated with inferior OS. There was no difference in the observed and expected OS for both the 1L (p-0.23) and R/R (p=0.14) Ven-O treated cohorts compared to the age- and sex-matched general population of the state of Minnesota. Conclusions Our study shows that Ven-O is a well-tolerated regimen with demonstration of durable treatment responses both in the 1L and R/R groups, when used outside the context of a clinical trial. To the best of our knowledge, these data for the first time demonstrate no difference in OS of patients treated with Ven-O compared to age- and sex-matched general population, in both the 1L and R/R settings.

Abstract Measurable residual disease (MRD)-testing is often used to evaluate therapy efficacy in chronic lymphocytic leukaemia (CLL), with undetectable MRD (uMRD) often serving as a surrogate for progression-free survival (PFS). However, its accuracy in predicting PFS with Bruton tyrosine kinase inhibitors (BTKis), with or without venetoclax, remains controversial. We searched PubMed, Web of Science, Embase, and Cochrane Library databases up to February 8, 2025 using terms chronic lymphocytic leukemiaOR small lymphocytic lymphomaAND measurable residual diseaseAND progression-free survival. Bayesian Weibull accelerated failure time models were used for individual-level analyses, while trial-level correlations used weighted Spearman rank tests and weighted least squares regression. A total of 41 trials (9,569 subjects) were included. Therapies tested included BTKis (11 trials), venetoclax (12), the combination (5), and other therapies like chemoimmunotherapy and monoclonal antibodies (23). MRD was assessed in blood (21 trials), bone marrow (7), or both (9). At the individual level, subjects with detectable MRD (dMRD) had a higher progression and/or death risk compared to uMRD (Hazard Ratio [HR] = 3.63; 95% Credibility Interval [CrI] 3.30-4.00; P < 0.01). In sensitivity analysis, MRD-testing showed reduced predictive accuracy for PFS in subjects receiving BTKi-based therapy (HR = 1.52, 1.02–2.19) compared to venetoclax-based (HR = 4.46, 3.61–5.50) or other therapies (HR = 3.86, 3.45–4.34). Across subgroups, dMRD remained consistently associated with increased risk. In subjects with del(17p)/TP53 variants, dMRD predicted greater (HR = 6.52; 3.47–12.79) versus those without (HR = 4.34; 3.24–5.81). By IGHV status, dMRD conferred a 3.54-fold risk in IGHV-mutated (95% CrI: 2.42-5.34) and 2.64-fold in unmutated subjects (95% CrI: 2.10-3.34). Among response categories, risk was higher in partial responders (HR = 2.39; 1.70–3.43) than complete responders (HR = 1.64; 1.24–2.17). dMRD also predicted increased risk in relapsed/refractory (R/R) CLL subjects (HR = 4.11, 2.54-6.84) and untreated subjects (HR = 3.59, 3.25-3.98). Bone marrow MRD testing proved a stronger predictor of PFS than blood (HR = 4.27, 3.42-5.35 versus HR = 3.52, 3.17-3.92). The highest risk was seen with MRD testing 12–32 months post-treatment (HR = 5.37, 4.56-6.40), though differences across timepoints were modest after adjusting for analytical method. At the trial level, correlations between MRD and PFS were weak (Spearman's rho [R] = -0.35; Determination Coefficients [R²] = 0.02). In sensitivity analyses, correlations remained low for BTKi-based therapy (R = -0.19 [-0.73, 0.50]; R² = 0.04) and venetoclax-based therapy (R = -0.32 [-0.85, 0.45]; R² = 0.14), but were stronger for other therapies (R = -0.80; R² = 0.58). For other therapies, MRD tests of bone marrow samples showed higher predictive value than blood (R = -0.94; R² = 0.86 versus R = -0.81; R² = 0.63). Despite blood samples and testing at 9-months of other therapies meeting the validity threshold (R² = 0.63, P = 0.21; R² = 0.63, P = 0.06), the associated P-values preclude definitive conclusions. Additional analyses examining sample source, time points and testing method within treatment classes suggest limited accuracy of predicting PFS in heterogeneous conditions (all R < -0.85, R² < 0.60). In subgroup analyses, untreated trials showed weak associations (R = -0.30 [-0.77, 0.13]; R²= 0.02), while R/R CLL trials were insufficient for analysis. MRD-testing of blood showed lower PFS predictive accuracy than bone marrow(R = -0.26 [-0.71, 0.20]; R² = 0.03 versus R = -0.37 [-1.00, 0.62]; R² = 0.03). MRD-testing at 15 months demonstrated slightly better accuracy than 9-month testing (R = -0.59 [-1.00, 0.61]; R² = 0.27 versus R = -0.26 [-0.88, 0.41]; R² = 0.04).MRD-testing by NGS outperformed MPFC (R = -0.54 [-1.00, 1.00]; R² = 0.45 versus R = -0.30 [-0.76, 0.13]; R² = 0.01), though both methods remained below the pre-specified determination coefficient threshold. Our data indicate accuracy of results of MRD-testing to predict PFS depends on whether used at the subject or trial-level, type of therapy and sample tested. Importantly MRD-testing is not an accurate predictor of PFS for BTKis with or without venetoclax. We suggest results of MRD-testing as a surrogate for PFS should not be used as a surrogate endpoint for studies of BTKis with or without venetoclax.

Abstract BACKGROUND Whilst the broad clonal architecture of naturally progressing chronic lymphocytic leukemia (CLL) has been described, a comprehensive picture of how chemotherapy and targeted agents reshape that landscape is lacking. Here we integrate clone-specific growth kinetics with mutational profiles, transcriptomic subtypes, and CpG-methylation-based epigenetic classes to capture the multidimensional evolutionary responses of CLL under native conditions and during treatment. METHODS We previously reported a systematic whole-exome sequencing (WES) analysis of 417 leukemia–germline pairs from 169 treatment-naïve patients ≥ 65 y (Karisani et al., Blood 2024). The treatment arm comprised 83 patients who received ibrutinib and 39 who received chlorambucil in the RESONATE-2 trial (NCT01722487). Forty-seven age-matched “watch-and-wait” patients from the CLL Research Consortium served as untreated controls. Peripheral-blood samples were collected at baseline and again ~300 d and ~600 d after therapy initiation (or diagnosis in the watch-and-wait cohort), enabling longitudinal clonal tracking. WES data were processed with PhylogicNDT to reconstruct clonal architectures, define phylogenies, and identify significant clonal shifts. MutSig tools identified mutations enriched in expanding or contracting clones. Pretreatment RNA-sequencing profiles were grouped into transcriptional subtypes by consensus clustering based on the Louvain algorithm. Differentially methylated regions plus epitypes were called from reduced representation bisulfite sequencing data. RESULTS Across the 169 patients with WES, we resolved 579 subclonal clusters. Significant clonal shifts (defined as a distribution shift of cancer cell fraction in a subclone of >95% between two timepoints) were uncommon during watch-and-wait (36%) but markedly higher after therapy — chlorambucil 67% (q = 0.01), and ibrutinib 77% (q < 10-4). In ibrutinib-treated CLL, regressing subclones were enriched for KRAS (q<10-3) and SF3B1 mutations (q<10-3), whereas expanding subclones preferentially harboured mutations in BIRC3 (q<10-4), NOTCH1 (q <10-3), TP53 (q=0.02), and POT1 (q=0.098). In chlorambucil-treated patients, no specific mutations were found to be enriched in expanding or regressing subclones. Using the recently defined CLL expression clusters (ECs) in patients treated with chemoimmunotherapy as defined by Knisbacher et al. (Nat Genet 2022), we were unable to identify any relationship between ECs and outcomes. We therefore sought to identify de novo ECs with prognostic relevance in the context of BTK-inhibition (BTKi-ECs). We identified a transcriptionally defined patient subgroup (C4, n=20) associated with inferior progression-free survival (median PFS = 63 months, log-rank q=0.05), independent of known prognostic clinical and molecular features, including epitypes, in multivariate analysis (Cox HR=3.7, 95% CI: 1.6-8.6, q=0.02). Gene-set enrichment in this cluster highlighted TNF-ɑ signalling via NF-κB (REL, TNFAIP3, NFKB2) and G2M checkpoint pathways (SMC4, EZH2). We also identified a distinct transcriptionally defined patient subgroup (C3, n=18) that was enriched for patients harboring at least one contracting subclone and no expanding subclones (q=0.005). This cluster showed a relative depletion of mutations within the chromatin modification pathway (6% vs 50%, q<0.01). Gene-set enrichment in this cluster highlighted a downregulation of genes in the TNF-ɑ signalling via NF-κB pathway (SOCS3, ICOSLG, NFKB2). CONCLUSION BTK inhibition and chemotherapy drive distinct evolutionary trajectories in CLL. Multi-omics profiling reveals mutation-defined subclones with divergent sensitivity to targeted therapy and delineates two informative expression states: a high-risk NF-κB/inflammatory cluster (C4; poor PFS) and a cluster enriched with contracting subclones with depleted TNF-ɑ signalling via NF-κB (C3). Integrating clonal growth kinetics with genomic, transcriptomic, and methylation data can potentially identify patients likely to progress on BTK inhibitors and identify actionable pathways for adaptive or combination therapy.

Abstract Introduction: Although therapies targeting Bruton Tyrosine Kinase (BTK), either as monotherapy or in combination have revolutionized the treatment in chronic lymphocytic leukemia (CLL), the disease remains incurable. Continuous treatment with these agents frequently leads to the development of resistance and disease progression, mostly associated with mutations in BTK or PLCG2. However, a significant fraction of BTKi-resistant patients does not harbor mutations in these genes, revealing that multiple distinct mechanisms of resistance can independently develop in these patient cohorts. Identifying novel molecular pathways associated with drug resistance in the relapsed/refractory (RR) setting of CLL requires more exploration. Materials and Methods: Using the Mayo Clinic CLL Database and tissue bank, we identified 30 RR patients, who had had disease progressionon a BTKi. The median lines of prior therapies were four. The median age at start of treatment was 57.9 (range, 36-78), 77% were male. Rai stage at diagnosis was 0 in 10% of patients, stage I in 37%, stage II in 20%, stage III in 6%, and stage IV in 27%. IGHV unmutated was observed in 87% of cases. Five patients subsequently evolved to Richter's Transformation (RT). A total of 64 blood samples were collected at different timepoints: baseline (before BTKi therapy), during treatment and at disease progression from BTKi (Ibrutinib/Acalabrutinib/Pirtobrutinib/Nemtabrutinib). The CD5+/CD19+ tumor population was enriched by flow sorting. We performed whole genome sequencing (WGS) in tumor/germline pairs with an average sequencing depth of 60X. Copy number variants (CNVpytor), structural variants – SV (Manta and DELLY2), single nucleotide variants (SNV) and indels (MuTect2, Strelka) were calculated. Mutational signature analysis was conducted independently on each disease timepoint with sigProfiler. De novo signatures were extracted with non-negative matrix factorization, then decomposed and mapped to known signatures from the COSMIC Human Cancer Signatures database. Results: Prior to start of BTKi treatment, the most common abnormality was del13q (65%), followed by abnormalities in TP53 (mutation and/or del17p) in 59% of patients, del8p, del11q (23% each), and mutations in SF3B1 (35%), NFKIE, NOTCH1 and MAPK signaling pathway (BRAF, KRAS, NRAS, PTPN11) in 23% of cases, each. Complex karyotype, defined as >3 copy number abnormalities was found in 59% of cases. Disease progression was observed 4.4 years (median; range 1.9-10.8 yrs) after starting BTKi therapy. At disease progression, there was an overall increase in genomic complexity. Overall, 17 cases (56%) showed mutations in BTK (p.C481S, p.C481Y, p.L528W, p.A428D) and/or PLCG2 (p.P844H). Four cases showed mutations in both genes or two or more independent mutations in BTK. Del17p and/or TP53 mutations were found in 20 (67%) cases, 9 of them with biallelic abnormalities. A third of patients (N=10) were found to have MAPK mutations, and 9 (30%) cases had mutations in the epigenetic regulators CREBBP, SETD2, SETD1A andKMT2D. Loss of heterozygosity, including 11q and 17p was found in 13% of cases. Chromothripsis was found in 23% of cases. In patients without BTK or PLCG2 mutations (N=13), TP53 abnormalities were present in 92% of cases and mutations in the MAPK pathway in 46% of these patients. Finally, we identified several SVs involving driver genes at disease progression, including inversions in RB1 (N=1), NOTCH1 (N=1) and translocations in MYC (N=1) and IGH (N=2). Analyzing the mutational signatures, baseline timepoints were dominated heavily by aging signatures (SBS5, SBS1). However, in cases that progressed after therapy, we found enrichment of mutational signatures associated with defective DNA mismatch repair (SBS44, 6%), chemotherapy treatment (SBS25, 14%), homologous and nucleotide excision repair deficiency (SBS8, 48%), and indirect effects of AID-induced somatic mutagenesis (SBS85, 7%). Additionally, the Melphalan signature (SBS99, 10%) was found in patients treated alkylating agents. Conclusions: Disease progression of CLL on BTKi confirmed the existence of multiple driver genes beyond BTK, PLCG2 andTP53, including the high prevalence of mutations in MAPK pathway and epigenetic regulators, and increased genomic complexity. Ongoing efforts are focused on integrating these findings with clinical demographics, epigenetic modifications, and preclinical drug sensitivity profiles.

SummaryIn individuals with high‐count monoclonal B‐cell lymphocytosis (MBL), we investigated if lymphadenopathy or splenomegaly found by imaging was associated with shorter time to first chronic lymphocytic leukaemia (CLL) therapy (TTFT) and overall survival (OS). Individuals with MBL seen at Mayo Clinic (2002–2019) were retrospectively divided into three cohorts based on imaging studies within 1 year of diagnosis: no imaging studies (Cohort A); imaging with no evidence of lymphadenopathy/splenomegaly (Cohort B); imaging with evidence of lymphadenopathy/splenomegaly (Cohort C). We compared baseline characteristics, TTFT and OS across the MBL cohorts to a cohort of individuals with small lymphocytic lymphoma (SLL). A total of 1078 patients were included: 640 with MBL and 438 with SLL. Compared to Cohort B, individuals in Cohort C were more likely to have unmutated immunoglobulin heavy chain variable region (IGHV) (43% vs. 25% p = 0.016), high‐risk fluorescence in situ hybridization (FISH) (del17p and del11q in 15% vs. 4%, p = 0.038) and higher expression of CD38 (31% vs. 16%; p = 0.011). After adjusting for sex and CLL‐International Prognostic Index (IPI), lymphadenopathy/splenomegaly was associated with a shorter TTFT (hazard ratio [HR] = 2.04, 95% confidence interval [CI]: 1.02–4.04, p = 0.042) but not OS (HR = 1.09, 95% CI: 0.62–1.92, p = 0.775). Lymphadenopathy/splenomegaly on imaging in individuals with high‐count MBL is associated with a more unfavourable risk profile and shorter time to first CLL‐directed therapy.